WO2024047096A1

WO2024047096A1 - Process for purification of an oligosaccharide

Info

Publication number: WO2024047096A1
Application number: PCT/EP2023/073771
Authority: WO
Inventors: Jordy BAUWELINCK; Joeri Beauprez; Gert PETERS; Dries VAN HERPE
Original assignee: Inbiose N.V.
Priority date: 2022-08-30
Filing date: 2023-08-30
Publication date: 2024-03-07

Abstract

The present invention relates to processes for the purification of an oligosaccharide from a solution, a product of such processes, and the use of a product of such processes.

Description

Process for purification of an oligosaccharide

Field of the invention

Background

To date oligosaccharides are gaining more and more attention. Oligosaccharides are very diverse in chemical structure and are composed of a diverse number of monosaccharides, such as e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N- acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, galacturonic acid. Oligosaccharides are widely distributed in all living organisms. Often present as glycoconjugated forms to proteins and lipids, oligosaccharides play important roles in a variety of physiological and pathological processes, such as differentiation, development and biological recognition processes related to the development and progress of fertilization, embryogenesis, cell metastasis, signal transduction, intercellular adhesion, inflammation, host-pathogen adhesion, and immune response. Oligosaccharides can also be present as unconjugated glycans in body fluids and human milk wherein they also modulate important developmental and immunological processes (Bode, Early Hum. Dev. 1-4 (2015); Reily et al., Nat. Rev. Nephrol. 15, 346-366 (2019); Varki, Glycobiology 27 , 3-49 (2017)). Economical production of these oligosaccharides is of utmost importance to fully benefit of their biological advantages. An example of such oligosaccharides are milk oligosaccharides (MOs) in mammalian milk, called mammalian milk oligosaccharides or MMOs, and in human milk, called human milk oligosaccharides (HMOs) (Usashima T. et al., 2011). Many of these MOs contain a fucose residue, a galactose residue, a N-acetylglucosamine or an N-acetylneuraminic acid residue at their non-reducing end. An important MO is sialyllacto-N-tetraose c (LSTc; Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc). The importance of MOs for animal and human infant nutrition is directly linked to their biological activities including protection of the neonate from pathogens, supporting development of the infant's immune system and cognitive abilities. In addition, MOs serve as a substrate for beneficial bacteria like Bifidobacteria or Lactobacilli. MOs are further known to act as decoys to reduce the risk of infections by bacterial and viral pathogens which adhere to human cells by binding to these cells' surface glycoproteins. Additionally, various MOs possess an antiinflammatory effect and act as immunomodulators (e.g., reducing the risk of developing food allergies).

A wide variety of synthesis methods have been developed already, ranging from extraction over chemical synthesis to enzymatic synthesis. These methods are currently least applied, biotechnological fermentative production is nowadays pursued and commercialized. Methods for the production of oligosaccharides are reviewed by Lu et al (2021), Faijes et al (2019), Kruschitz et al (2020), Ghosh et al (2020), Vera et al (2021), Walsh et al (2020), Li et al (2020), Li and Ye (2020) and are well known for a person skilled in the art. For all production methods the final oligosaccharide is preferably purified before it is to be added in the respective application.

To take advantage of the positive effects of specific oligosaccharides, individual oligosaccharides are being added to nutritional compositions, cosmetics, pharmaceutical compositions and plant protection products. In some instances, supplementing with a combination of different oligosaccharides is more convenient, as such compositions e.g., more closely resemble the natural source of the oligosaccharides in case the oligosaccharide mixture is a mixture of mammalian milk oligosaccharides. In other cases, a mix of specific oligosaccharides is produced more efficiently in a simpler manner by producing the mixture of oligosaccharides in one fermentation and purifying the mixture of oligosaccharides all together from the biomass, medium components and contaminants, without separating the different oligosaccharides from each other.

The oligosaccharides are nowadays purified by means of pressure-driven processes comprising microfiltration (MF), ultrafiltration (UF) and nanofiltration. These processes are typically applied when the removal of suspended solids and bacteria are the primary goals. The removal of ionic substances is also possible, but at a much lower efficiency. Hence, pressure-driven processes are combined with cation and anion exchange chromatography or with mixed bed ion exchange, which both require regeneration with high dosed chemicals such as sodium hydroxide or sulphuric acid. In the case of a stepwise cation and anion removal, the salts are less efficiently removed compared to mixed bed ion exchange. Mixed bed ion exchange on the other hand is more difficult to regenerate after use. Alternatively, electrically driven approaches aim at the removal of ions through the selective control and transport of ionic species. The fundamental principle behind electrically driven processes is the passage of ions through a selective barrier (ion exchange membrane) due to a gradient or driving force (electric field). Electrodialysis is the most popular technology for electrically driven processes in industry, as it separates undesired ions from aqueous solutions at low operational cost and with the advantage that it does not generate residues. This technology, which combines the principles of dialysis and electrolysis, was first applied for the demineralization of simple sugar syrups (e.g., monosaccharide syrups) and has been earlier applied in the purification of biotechnologically produced oligosaccharides (e.g., WO09039653, WO15106943, WO20233958). When ions are separated from the feed solution, there is an inherent drawback in which a phenomenon known as concentration polarization develops, whereby a high cumulative resistance within the cell is built up, which decreases cell efficiency. To overcome this problem, the electrodialysis principle was modified with a solid conductive ion medium that was introduced into the dilute compartment in the form of ion exchange resins, resulting in a new ion removal technology, named electrodeionization (EDI) (Alvarado and Chen, Electrochim. Acta 132, 583-597 (2014)). EDI or continuous electrodeionization (CEDI) has always been used for the production of low conductivity water, mainly in laboratory environments, and has also been applied in the removal of toxic metals in water streams. The challenge to apply this technology on an oligosaccharide product stream is the compatibility of the ion exchange resin, which is generally mixed bed, in the system and the compatibility of the membranes with the ionic load in the product stream. In particular, the presence of certain divalent ions, such as magnesium ions and calcium ions, is detrimental for the efficiency of the EDI or CEDI system. Hence, to date, EDI is mainly used in water purification processes or in processes with a low so-called hardness in the liquid (low concentrations of magnesium and calcium).

Description

Summary of the invention

It is an object of the present invention to provide for methods by means of which an oligosaccharide can be purified from a solution, preferably in an efficient, time and cost-effective way and which yields a high quality, high purity and good yield of the desired oligosaccharide.

According to the invention, this and other objects are achieved by providing a process for the purification of an oligosaccharide from a solution. The process comprises pH adjustment of the solution comprising the oligosaccharide to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing the pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form. Preferably, said cationic ion exchange resin in said mixed bed ion exchange and/or in said cationic ion exchange, when present, is in Na+ form.

It is also an object of the present invention to provide for methods by means of which a negatively charged, preferably sialylated oligosaccharide, can be purified from a solution. This and other objects are achieved by providing a process for the purification of a negatively charged, preferably sialylated, oligosaccharide from a solution. The process comprises pH adjustment of the solution comprising the negatively charged, preferably sialylated, oligosaccharide to a pH ranging from 2 to 5, preferably from 3 to 5, more preferably from 4 to 5, and passing the pH adjusted solution through a mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In addition, it is an object of the present invention to provide for methods by means of which sialyllacto- N-tetraose c (LSTc; Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) can be purified from a solution comprising LSTc and a sialyllactose, preferably in an efficient, time and cost-effective way and which yields a high quality, high purity and good yield of the desired oligosaccharide. According to the invention, this and other objects are achieved by providing a process for the purification of LSTc from a solution comprising LSTc and a sialyllactose. The process comprises pH adjustment of the solution comprising LSTc and a sialyllactose to a pH ranging from 4 to 7, preferably from 5 to 7, even more preferably from 6 to 7, preferably to a pH of 6.5, and passing the pH adjusted solution through a mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. Furthermore, the present invention shows that it is also possible to purify liquid streams, preferably from biotechnological production processes, containing high amounts of magnesium and calcium with an EDI or CEDI system, more specifically the purification technique of EDI or CEDI can be used for the purification of molecules sensitive to high and low pH conditions such as oligosaccharides. This and other objects are achieved by providing a process for the purification of an oligosaccharide from a solution wherein the process comprises electrodeionization (EDI).

The solution comprising the oligosaccharide, like e.g., the negatively charged, preferably sialylated, oligosaccharide like e.g., LSTc and a sialyllactose is any one of a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation or a process stream of the above-referenced process wherein the oligosaccharide is produced by the biocatalysis reaction solution, the chemical synthesis solution, or by a cell cultivated in the cell cultivation. Preferably, the cell cultivation is a fermentation. This invention also provides a purified oligosaccharide, like e.g., a purified negatively charged, preferably sialylated, oligosaccharide like e.g., LSTc by the above-referenced process. Furthermore, this invention provides a purified oligosaccharide mixture comprising a purified oligosaccharide by the above-referenced process. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention.

Definitions

The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well- known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

In the specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps, unless specifically stated otherwise.

Throughout the application, unless explicitly stated otherwise, the features "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively. Throughout the application, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e., "expresses" is preferably replaced with "capable of expressing". Throughout this document and in its claims, the verbs "to comprise", "to have" and "to contain" and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb "to consist essentially of" means that a solution or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. Said additional compound(s) might be inevitable by-product(s), for example, generated during production of the oligosaccharide, the negatively charged, preferably sialylated, oligosaccharide, LSTc and/or a sialyllactose, or the oligosaccharide mixture of present invention like e.g., an oligosaccharide mixture comprising LSTc and a sialyllactose as well as compound(s) that were introduced into a process stream from which the oligosaccharide or the oligosaccharide mixture is recovered but which could not have been removed therefrom. The term "consisting essentially of" with respect to spray-dried powders includes spray-dried powders containing with respect to the dry matter of the spray-dried powder at least 80 %-wt., at least 85 %-wt., at least 90 % -wt., at least 93 %-wt., at least 95 %-wt. or at least 98 %-wt. of the oligosaccharide mixture. The term "consisting essentially of" is used likewise with respect to spray-dried powders, process streams and solutions containing the oligosaccharide mixture. Throughout this document and in its claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain”, and their conjugations, may be preferably replaced by "to consist of" (and its conjugations) or "to consist essentially of" (and its conjugations) and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Throughout the document and in the claims, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least one", more preferably "at least two", even more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two". The word "about" or "approximately" when used in association with a numerical value (e.g., "about 10") or with a range (e.g., "about x to approximately y") preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margins are specified, the expression "about" or "approximately" when used in association with a numerical value is interpreted as having the same round-off as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein x is the lower value of the range and y is the upper value of the range. Herein, x and y are also included in the range.

The terms "sialic acid", "N-acetylneuraminate", "N-acylneuraminate", "N-acetylneuraminic acid" and "Neu(n)Ac molecule" are used interchangeably and refer to an acidic sugar with a nine-carbon backbone comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4 and Neu4,5,7,8,9Ac5 and Neu5Gc. Neu4Ac is also known as 4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2- ulopyranosonic acid or 4-O-acetyl neuraminic acid and has C11H19NO9 as molecular formula. Neu5Ac is also known as 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5- acetamido-3,5-dideoxy-D-galacto-non-2-ulo-pyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D- galacto-2-nonulopyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-nonulosonic acid or 5-(acetylamino)-3,5- dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid and has C11H19NO9 as molecular formula. Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 4- O-acetyl-N-acetylneuraminate, 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 4- acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate, 4-acetate 5-acetamido-3,5- dideoxy-D-glycero-D-galacto-nonulosonic acid or 4-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D- galacto-2-nonulosonic acid and has C13H21NO10 as molecular formula. Neu5,7Ac2 is also known as 7-0- acetyl-N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-0-acetyl-N-acetylneuraminate, 7- acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 7-acetate 5-(acetylamino)-3,5- dideoxy-D-glycero-D-galacto-2-nonulosonate, 7-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulosonic acid or 7-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid and has C13H21NO10 as molecular formula. Neu5,8Ac2 is also known as 5-n-acetyl-8-o-acetyl neuraminic acid and has C13H21NO10 as molecular formula. Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-0-acetylsialic acid, 9-0-acetyl-N-acetylneuraminic acid, 5-n-acetyl-9-O-acetyl neuraminic acid, N,9-0-diacetylneuraminate or N,9-O-diacetylneuraminate and has C13H21NO10 as molecular formula. Neu4,5,9Ac3 is also known as 5-N-acetyl-4,9-di-O-acetylneuraminic acid. Neu5,7,9Ac3 is also known as 5-N-acetyl-7,9-di-O-acetylneuraminic acid. Neu5,8,9Ac3 is also known as 5-N-acetyl-8,9-di-O- acetylneuraminic acid. Neu4,5,7,9Ac4 is also known as 5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid. Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7,8,9-tri-O-acetylneuraminic acid. Neu4,5,7,8,9Ac5 is also known as 5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acid. Neu5Gc is also known as N-glycolyl- neuraminic acid, N-glycolylneuraminicacid, N-glycolylneuraminate, N-glycoloyl-neuraminate, N-glycoloyl- neuraminic acid, N-glycoloylneuraminic acid, 3,5-dideoxy-5-((hydroxyacetyl)amino)-D-glycero-D-galacto- 2-nonulosonic acid, 3,5-dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-2-nonulopyranosonic acid, 3,5- dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-non-2-ulopyranosonic acid, 3,5-dideoxy-5- [(hydroxyacetyl)amino]-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5-glycolylamido-3,5- dideoxy-D-galacto-non-2-ulo-pyranosonic acid and has C11H19NO10 as molecular formula.

The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed as an aldose, a ketose, a deoxysugar, a deoxy-aminosugar, a uronic acid, an aldonic acid, a ketoaldonic acid, an aldaric acid or a sugar alcohol, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar. Examples of monosaccharides comprise Hexose, D-Glucopyranose, D-Galactofuranose, D-Galactopyranose, L- Galactopyranose, D-Mannopyranose, D-Allopyranose, L-Altropyranose, D-Gulopyranose, L-ldopyranose, D-Talopyranose, D-Ribofuranose, D-Ribopyranose, D-Arabinofuranose, D-Arabinopyranose, L- Arabinofuranose, L-Arabinopyranose, D-Xylopyranose, D-Lyxopyranose, D-Erythrofuranose, D- Threofuranose, Heptose, L-glycero-D-manno-Heptopyranose (LDmanHep), D-glycero-D-manno- Heptopyranose (DDmanHep), 6-Deoxy-L-altropyranose, 6-Deoxy-D-gulopyranose, 5-Deoxy-D- talopyranose, 6-Deoxy-D-galactopyranose, 6-Deoxy-L-galactopyranose, 6-Deoxy-D-mannopyranose, 6- Deoxy-L-mannopyranose, 6-Deoxy-D-glucopyranose, 2-Deoxy-D-arabino-hexose, 2-Deoxy-D-erythro- pentose, 2,6-Dideoxy-D-arabino-hexopyranose, 3,6-Dideoxy-D-arabino-hexopyranose, 3,6-Dideoxy-L- arabino-hexopyranose, 3,6-Dideoxy-D-xylo-hexopyranose, 3,6-Dideoxy-D-ribo-hexopyranose, 2,6- Dideoxy-D-ribo-hexopyranose, 3,6-Dideoxy-L-xylo-hexopyranose, 2-Amino-2-deoxy-D-glucopyranose, 2- Amino-2-deoxy-D-galactopyranose, 2-Amino-2-deoxy-D-mannopyranose, 2-Amino-2-deoxy-D- allopyranose, 2-Amino-2-deoxy-L-altropyranose, 2-Amino-2-deoxy-D-gulopyranose, 2-Amino-2-deoxy-L- idopyranose, 2-Amino-2-deoxy-D-talopyranose, 2-Acetamido-2-deoxy-D-glucopyranose, 2-Acetamido-2- deoxy-D-galactopyranose, 2-Acetamido-2-deoxy-D-mannopyranose, 2-Acetamido-2-deoxy-D- allopyranose, 2-Acetamido-2-deoxy-L-altropyranose, 2-Acetamido-2-deoxy-D-gulopyranose, 2- Acetamido-2-deoxy-L-idopyranose, 2-Acetamido-2-deoxy-D-talopyranose, 2-Acetamido-2,6-dideoxy-D- galactopyranose, 2-Acetamido-2,6-dideoxy-L-galactopyranose, 2-Acetamido-2,6-dideoxy-L- mannopyranose, 2-Acetamido-2,6-dideoxy-D-glucopyranose, 2-Acetamido-2,6-dideoxy-L-altropyranose, 2-Acetamido-2,6-dideoxy-D-talopyranose, D-Glucopyranuronic acid, D-Galactopyranuronic acid, D- Mannopyranuronic acid, D-Allopyranuronic acid, L-Altropyranuronic acid, D-Gulopyranuronic acid, L- Gulopyranuronic acid, L-ldopyranuronic acid, D-Talopyranuronic acid, sialic acid, 5-Amino-3,5-dideoxy-D- glycero-D-galacto-non-2-ulosonic acid, 5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, 5-Glycolylamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, Erythritol, Arabinitol, Xylitol, Ribitol, Glucitol, Galactitol, Mannitol, D-ribo-Hex-2-ulopyranose, D-arabino-Hex-2-ulofuranose (D- fructofuranose), D-arabino-Hex-2-ulopyranose, L-xylo-Hex-2-ulopyranose, D-lyxo-Hex-2-ulopyranose, D- threo-Pent-2-ulopyranose, D-altro-Hept-2-ulopyranose, 3-C-(Hydroxymethyl)-D-erythofuranose, 2,4,6- Trideoxy-2,4-diamino-D-glucopyranose, 6-Deoxy-3-O-methyl-D-glucose, 3-O-Methyl-D-rhamnose, 2,6- Dideoxy-3-methyl-D-ribo-hexose, 2-Amino-3-O-[(R)-l-carboxyethyl]-2-deoxy-D-glucopyranose, 2- Acetamido-3-O-[(R)-carboxyethyl]-2-deoxy-D-glucopyranose, 2-Glycolylamido-3-O-[(R)-l-carboxyethyl]- 2-deoxy-D-glucopyranose, 3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid, 3-Deoxy-D-manno-oct-2- ulopyranosonic acid, 3-Deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9- tetradeoxy-L-glycero-L-manno-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-L-glycero-L- altro-non-2-ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2- ulopyranosonic acid, 5,7-Diamino-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulopyranosonic acid, 2- acetamido-2,6-dideoxy-L-arabino-4-hexulose, 2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, N-acetyl-L- rhamnosamine, N-acetyl-D-fucosamine, N-acetyl-L-pneumosamine, N-acetylmuramic acid, N-acetyl-L- quinovosamine, glucose (Glc), galactose (Gal), N-acetylglucosamine (GIcNAc), glucosamine (Glen), mannose (Man), xylose (Xyl), N-acetylmannosamine (ManNAc), N-glycolylneuraminic acid, N- acetylgalactosamine (GalNAc), galactosamine (Gain), fucose (Fuc), rhamnose (Rha), glucuronic acid, gluconic acid, fructose (Fru) and polyols. With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example, glycerol, sorbitol, or mannitol.

The term "phosphorylated monosaccharide" as used herein refers to one of the above listed monosaccharides which is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-l-phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-1- phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-1- phosphate, glucosamine-6-phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose-l-phosphate. Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharide.

The terms "activated monosaccharide", "nucleotide-activated sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used herein interchangeably and refer to activated forms of monosaccharides. Examples of activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2- acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2- acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L- QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-sialic acid (CMP-Neu5Ac or CMP-N-acetylneuraminic acid), GDP-fucose (GDP-Fuc), GDP-rhamnose and UDP-xylose. Nucleotidesugars act as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions that are catalysed by glycosyltransferases.

The term "glycosyltransferase" as used herein refers to an enzyme capable to catalyse the transfer of a sugar moiety of a donor to a specific acceptor, forming glycosidic bonds. Said donor can be a precursor as defined herein. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.or ). As used herein the glycosyltransferase can be selected from the list comprising but not limited to: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

The term "disaccharide" as used herein refers to a saccharide polymer containing two simple sugars, i.e., monosaccharides. Such disaccharides contain monosaccharides preferably selected from the list of monosaccharides as used herein above. Examples of disaccharides comprise lactose (Gal-bl,4-Glc), lacto- N-biose (Gal-bl,3-GlcNAc), N-acetyllactosamine (Gal-bl,4-GlcNAc), LacDiNAc (GalNAc-bl,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-bl,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and fucopyranosyl- (l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc).

"Oligosaccharide" as the term is used herein and as generally understood in the state of the art, refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides. Preferably the oligosaccharide as described herein contains monosaccharides selected from the list as used herein above. The oligosaccharide as used in the present invention can be a linear structure or can include branches. The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. For example, the terms "Gal-bl,4-Glc", "Gal-pi,4-Glc", "b-Gal-(l->4)-Glc", "P-Gal-(l->4)-Glc", "Galbetal-4-Glc", "Gal-b(l-4)-Glc" and "Gal-P(l-4)-Glc" have the same meaning, i.e. a beta-glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form). Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2- >1, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->6. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha-glycosidic or only beta-glycosidic bonds. The term "polysaccharide" refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically. Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (EGA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O- glycans, a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharide, neutral oligosaccharide, fucosylated oligosaccharide, N-acetyllactosamine containing fucosylated oligosaccharide, N-acetyllactosamine non-fucosylated oligosaccharide, lacto-N- biose containing fucosylated oligosaccharide, lacto-N-biose containing non-fucosylated oligosaccharide, N-acetyllactosamine containing negatively charged oligosaccharide and lacto-N-biose containing negatively charged oligosaccharide.

The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having one or more sialic acid residue(s). It has an acidic nature. Some examples are 3'SL (3'-sialyllactose), 3'- sialyllactosamine, 6'SL (6'sialyllactose), 8'SL (8'sialyllactose), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac- a2,6)-Gal-bl,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-bl,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,4-Glc), 6'-sialyllactosamine, oligosaccharides comprising 6'sialyllactose, SGG hexasaccharide (Neu5Aca-2,3Gaip -l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, LSTc, LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a (LSTa), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose and oligosaccharides bearing one or several sialic acid residue(s), including but not limited to: oligosaccharide moieties of the gangliosides selected from GM3 (3'sialyllactose, Neu5Aca-2,3Gaip-4Glc) and oligosaccharides comprising the GM3 motif, GD3 Neu5Aca-2,8Neu5Aca-2,3Gaip-l,4Glc GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca- 2,3Gaip-l,4Glc); GM2 GalNAc -l,4(Neu5Aca-2,3)Gaip-l,4Glc, GM1 Gaip-l,3GalNAcP-l,4(Neu5Aca-

2,3)Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GTla Neu5Aca-

2,8Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GD2 GalNAcP-l,4(Neu5Aca- 2,8Neu5Aca2,3)Gaip-l,4Glc, GT2 GalNAc -l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GDlb, Gaip-l,3GalNAcP-l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GTlb Neu5Aca-2,3Gaip-l,3GalNAcP- l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GQlb Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc p - l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GTlc Gaip-l,3GalNAcp-l,4(Neu5Aca-2,8Neu5Aca- 2,8Neu5Aca2,3)Gaip-l,4Glc, GQlc Neu5Aca-2,3Gaip-l,3GalNAc P -l,4(Neu5Aca-2,8Neu5Aca- 2,8Neu5Aca2,3)Gaip-l,4Glc, GPlc Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc P -l,4(Neu5Aca- 2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3(Neu5Aca-2,6)GalNAcP -l,4Gaip- l,4Glc, Fucosyl-GMl Fuca-l,2Gaip-l,3GalNAcp -l,4(Neu5Aca-2,3)Gal p -l,4Glc; all of which may be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthetizing the above oligosaccharides on a ceramide.

The terms "3' sialyllactose", "3'-sialyllactose", "alpha-2, 3-sialyllactose", "alpha 2,3 sialyllactose", "a-2,3- sialyllactose", "a 2,3 sialyllactose", "3SL", "3'SL" or "Neu5Ac-a2,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably. The terms "6' sialyllactose", "6' -sialyllactose", "alpha-2, 6- sialyllactose", "alpha 2,6 sialyllactose", "a-2,6-sialyllactose", "a 2,6 sialyllactose", "6SL", "6'SL" or "Neu5Ac-a2,6-Gal-pi,4-Glc" as used in the present invention, are used interchangeably. The terms "8' sialyllactose", "8'-sialyllactose", "alpha-2, 8-sialyllactose", "alpha 2,8 sialyllactose", "a-2,8-sialyllactose", "a 2,8 sialyllactose", "8SL", "8'SL" or "Neu5Ac-a2,8-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.

The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto-/V-triose II", "lacto-N-triose", "lacto-M-triose" or "GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNT", "lacto-N-tetraose", "lacto-A/-tetraose" or "Gaipi-3GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LNnT", "lacto-N-neotetraose", "lacto-/V-neotetraose", "neo-LNT" or "Gaipi-4GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably. The terms "LSTa", "LS-Tetrasaccharide a", "Sialyl-lacto-N-tetraose a", "sialyllacto-N-tetraose a" or "Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably. The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl-lacto-N-tetraose b", "sialyllacto-N- tetraose b" or "Gal-bl,3-(Neu5Ac-a2,6)-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably. The terms "LSTc", "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", "sialyllacto- N-tetraose c", "sialyllacto-N-neotetraose c" or "Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably. The terms "LSTd", "LS-Tetrasaccharide d", "Sialyl- lacto-N-tetraose d", "sialyllacto-N-tetraose d", "sialyllacto-N-neotetraose d" or "Neu5Ac-a2,3-Gal-bl,4- GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably. The terms "DSLNnT" and "Disialyllacto-N-neotetraose" are used interchangeably and refer to Neu5Ac-a2,6-Gal-bl,4- GlcNAc-bl,3-[Neu5Ac-a2,6]-Gal-bl,4-Glc. The terms "DSLNT", "DS-LNT" and "Disialyllacto-N-tetraose” are used interchangeably and refer to Neu5Ac-a2,3-Gal-bl,3-[Neu5Ac-a2,6]-GlcNAc-bl,3-Gal-bl,4-Glc.

"Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N- glycolylneuraminic acid (Neu5Gc), glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid). Neu5Gc is a derivative of sialic acid, which is formed by hydroxylation of the N-acetyl group at C5 of Neu5Ac. In contrast, neutral oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Other examples of charged oligosaccharides are sulphated chitosans and deacetylated chitosans.

The terms 'neutral oligosaccharide' and 'non-charged' oligosaccharide as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group. Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyl lactose (3FL), 2', 3-difucosyllactose (diFL), lacto-N-triose II (LN3), lacto- N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto- N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N- neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaose, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose.

A 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue. Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose. A fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more. A fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages. Examples comprise 2'-fucosyl lactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), lacto-N- fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose I, lacto-N- difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), Monofucosyllacto-N-hexaose III (MFLNH III), Difucosyllacto-N-hexaose (DFLNHa), difucosyl-lacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.

The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNFPI", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1" and "Blood group H antigen pentaose type 1" are used interchangeably and refer to Fuc- al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "GalNAc-LNFPT and "blood group A antigen hexaose type I" are used interchangeably and refer to GalNAc-al,3-(Fuc-al,2)-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "LNFP-II" and "lacto-N-fucopentaose II" are used interchangeably and refer to Gal-bl,3-[Fuc- al,4]-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "LNFP-III", "LNFP III", "LNFPIII" and "lacto-N-fucopentaose III" are used interchangeably and refer to Gal-bl,4-(Fuc-al,3)-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "LNFP-V", "LNFP V", "LNFPV" and "lacto-N-fucopentaose V" are used interchangeably and refer to Gal-bl,3-GlcNAc- bl,3-Gal-bl,4-(Fuc-al,3)-Glc. The terms "LNFP-VI", "LNFP VI", "LNnFP V" and "lacto-N-neofucopentaose V" are used interchangeably and refer to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc. The terms "LNnFP I" and "Lacto-N-neofucopentaose I" are used interchangeably and refer to Fuc-al,2-Gal-bl,4-GlcNAc- bl,3-Gal-bl,4-Glc. The terms "LNDFH I", "Lacto-N-difucohexaose I", "LNDFH-I", "LDFH I", "Le^b-lactose" and "Lewis-b hexasaccharide" are used interchangeably and refer to Fuc-al,2-Gal-bl,3-[Fuc-al,4]- GlcNAc-bl,3-Gal-bl,4-Glc. The terms "LNDFH II", "Lacto-N-difucohexaose II", "LNDFH-II", "Lewis a-Lewis x" and "LDFH II" are used interchangeably and refer to Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-Gal-bl,4-(Fuc- al,3)-Glc. The terms "LNnDFH II", "Lacto-N-neodifucohexaose II", "LNDFH III", "Lewis x hexaose" and "LeX hexaose" are used interchangeably and refer to Gal-bl,4-(Fuc-al,3)-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc. The terms "alpha-tetrasaccharide" and "A-tetrasaccharide" are used interchangeably and refer to GalNAc- al,3-(Fuc-al,2)-Gal-bl,4-Glc. The terms "LNH" and "lacto-N-hexaose" are used interchangeably and refer to Gal-bl,3-GlcNAc-bl,3-(Gal-bl,4-GlcNAc-bl,5)-Gal-bl,4-Glc. The terms "para-LNH", "pLNH" and "para- lacto-N-hexaose" are used interchangeably and refer to Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal- bl,4-Glc. The terms "LNnH" and "lacto-N-neohexaose" are used interchangeably and refer to Gal-bl,4- GlcNAc-bl,3-[Gal-bl,4-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "para-LNnH", "pLNnH" and "para-lacto-N- neohexaose" are used interchangeably and refer to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal- bl,4-Glc.

The terms "F-LNH I", "FLNH I" and "fucosyllacto-N-hexaose I" are used interchangeably and refer to Fuc- al,2-Gal-bl,3-GlcNAc-bl,3-[Gal-bl,4-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "F-LNH-II", "FLNH II" and "fucosyllacto-N-hexaose II" are used interchangeably and refer to Gal-bl,3-GlcNAc-bl,3-[Gal-bl,4-[Fuc- al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "DF-LNH I", "difucosyllacto-N-hexaose I", "DF-LNH a", "DFLNH a", "difucosyllacto-N-hexaose a" and "2,3-Difucosyllacto-N-hexaose" are used interchangeably and refer to Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-[Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "DF-LNH II", "DF-LNH b", "DFLNH b" and "difucosyllacto-N-hexaose II" are used interchangeably and refer to Gal-bl,3- [Fuc-al,4]-GlcNAc-bl,3-[Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "DFLNH c", "DF-LNH c" and "difucosyllacto-N-hexaose c" are used interchangeably and refer to Fuc-al,2-Gal-bl,3-[Fuc-al,4]- GlcN Ac-bl,3-[Gal-bl,4-GlcN Ac-bl,6]-Gal-bl,4-Glc. The terms "DF-LNnH" and "difucosyllacto-N- neohexaose" are used interchangeably and refer to Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,3-[Gal-bl,4-[Fuc- al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc.

The terms "DF-para-LNH", "DF-p-LNH", "DF-pLNH" and "difucosyl-para-lacto-N-hexaose" are used interchangeably and refer to Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,3-Gal-bl,4- Glc. The terms "DF-para-LNnH", "DF-p-LNnH" and "difucosyl-para-lacto-N-neohexaose" are used interchangeably and refer to Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,3-Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,3-Gal-bl,4- Glc. The terms "TF-LNH" and "trifucosyllacto-N-hexaose" are used interchangeably and refer to Fuc-al,2- Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-[Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc.

The terms "F-LST a", "F-LSTa", "S-LNF II" and "fucosyl-sialyllacto-N-tetraose a" are used interchangeably and refer to Neu5Ac-a2,3-Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "F-LST b", "F-LSTb", "S-LNF I" and "fucosyl-sialyllacto-N-tetraose b" are used interchangeably and refer to Fuc-al,2-Gal-bl,3- (Neu5Ac-a2,6)-GlcNAc-bl,3-Gal-bl,4-Glc. The terms "F-LST c", "F-LSTc" and "fucosyl-sialyllacto-N- neotetraose" are used interchangeably and refer to Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-[Fuc- al,3]-Glc.

The terms "FS-LNH" and "fucosyl-sialyllacto-N-hexaose" are used interchangeably and refer to Fuc-al,2- Gal-bl,3-GlcNAc-bl,3-(Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,6)-Gal-bl,4-Glc. The terms "FS-LNnH I" and "fucosyl-sialyllacto-N-neohexaose I" are used interchangeably and refer to Neu5Ac-a2,6-Gal-bl,4-GlcNAc- bl,3-[Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc. The terms "FDS-LNH II" and "fucosyldisialyllacto-N- hexaose II" are used interchangeably and refer to Neu5Ac-a2,3-Gal-bl,3-[Neu5Ac-a2,6]-GlcNAc-bl,3- [Gal-bl,4-[Fuc-al,3]-GlcNAc-bl,6]-Gal-bl,4-Glc.

The terms "alpha-tetrasaccharide" and "A-tetrasaccharide" are used interchangeably and refer to GalNAc- al,3-(Fuc-al,2)-Gal-bl,4-Glc.

The terms "Fuc-al,2-Gal-bl,3-GlcNAc", "2-fucosyllacto-N-biose", "2FLNB", "2 FLNB", "2-FLNB", "2'-FLNB" and "2'FLNB" are used interchangeably and refer to a trisaccharide wherein a fucose residue is linked to the galactose residue of lacto-N-biose (LNB, Gal-bl,3-GlcNAc) in an alpha-1,2 linkage. The terms "Gal- pi,3-[Fuc-al,4]-GlcNAc", "4-fucosyllacto-N-biose", "4FLNB", "4 FLNB" and "4-FLNB" are used interchangeably and refer to a trisaccharide wherein a fucose residue is linked to the N-acetylglucosamine residue of lacto-N-biose (LNB, Gal-pi,3-GlcNAc) in an alpha-1,4 linkage. The terms "Gal-pi,4-[Fuc-al,3]- GIcNAc", "3-fucosyl-N-acetyllactosamine", "3-FLacNAc", "3FLacNAc" and "3 FLacNAc" are used interchangeably and refer to a trisaccharide wherein a fucose residue is linked to the GIcNAc residue of N-acetyllactosamine (LacNAc, Gal-pi,4-GlcNAc) in an alpha-1, 3-linkage.

As used herein the term "Lewis-type antigens" comprise the following oligosaccharides: Hl antigen, which is Fucal-2Gaipi-3GlcNAc, or in short 2'FLNB; Lewisa, which is the trisaccharide Gaipi-3[Fucal-4]GlcNAc, or in short 4-FLNB; Lewisb, which is the tetrasaccharide Fucal-2Gaipi-3[Fucal-4]GlcNAc, or in short DiF- LNB; sialyl Lewisa which is 5-acetylneuraminyl-(2-3)-galactosyl-(l-3)-(fucopyranosyl-(l-4))-N- acetylglucosamine, or written in short Neu5Aca2-3Gaipi-3[Fucal-4]GlcNAc; H2 antigen, which is Fucal- 2Gaipi-4GlcNAc, or otherwise stated 2'fucosyl-N-acetyl-lactosamine, in short 2'FLacNAc; Lewisx, which is the trisaccharide Gaipi-4[Fucal-3]GlcNAc, or otherwise known as 3-Fucosyl-N-acetyl-lactosamine, in short 3-FLacNAc, Lewisy, which is the tetrasaccharide Fucal-2Gaipi-4[Fucal-3]GlcNAc and sialyl Lewisx which is 5-acetylneuraminyl-(2-3)-galactosyl-(l-4)-(fucopyranosyl-(l-3))-N-acetylglucosamine, or written in short Neu5Aca2-3Gaipi-4[Fucal-3]GlcNAc.

As used herein, the term "O-antigen" refers to the repetitive glycan component of the surface lipopolysaccharide (LPS) of Gram-negative bacteria. The term "lipopolysaccharide" or "LPS" refers to glycolipids found in the outer membrane of Gram-negative bacteria which are composed of a lipid A, a core oligosaccharide and the O-antigen. The term "enterobacterial common antigen" or "EGA" refers to a specific carbohydrate antigen built of repeating units of three amino sugars, i.e., N-acetylglucosamine, N- acetyl-d-mannosaminuronic acid and 4-acetamido-4,6-dideoxy-d-galactose, which is shared by all members of the Enterobacteriaceae, and which is located in the outer leaflet of the outer membrane and in the periplasm. The term "capsular polysaccharides" refers to long-chain polysaccharides with oligosaccharide repeat structures that are present in bacterial capsules, the latter being a polysaccharide layer that lies outside the cell envelope. The terms "peptidoglycan" or "murein" refers to an essential structural element in the cell wall of most bacteria, being composed of sugars and amino acids, wherein the sugar components consist of alternating residues of beta-1,4 linked GIcNAc and N-acetylmuramic acid. The term "amino-sugar" as used herein refers to a sugar molecule in which a hydroxyl group has been replaced with an amine group. As used herein, an antigen of the human ABO blood group system is an oligosaccharide. Such antigens of the human ABO blood group system are not restricted to human structures. Said structures involve the A determinant GalNAc-alphal,3(Fuc-alphal,2)-Gal-, the B determinant Gal-alphal,3(Fuc-alphal,2)-Gal- and the H determinant Fuc-alphal,2-Gal- that are present on disaccharide core structures comprising Gal-betal,3-GlcNAc, Gal-betal,4-GlcNAc, Gal-betal,3-GalNAc and Gal-betal,4-Glc.

Mammalian milk oligosaccharides comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans and mammals including but not limited to cows (Bos Taurus), sheep (Ov/'s aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Cams lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). As used herein, "mammalian milk oligosaccharide" or "MMO" refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2' ,2- difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'- disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose 11, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto- N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylrnonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N- hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto- N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharides and/or sialylated oligosaccharides.

The terms "human milk oligosaccharide" or "HMO" refer to oligosaccharides found in human breast milk, including preterm human milk, colostrum and term human milk. HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides (see e.g., Chen X., Chapter Four: Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis in Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Examples of HMOs comprise 3- fucosyllactose, 2'-fucosyl lactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N- tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexaose, lacto-N-difucohexaose I, lacto-N- difucohexaose II, disialyllacto-N-tetraose, fucosyllacto-N-hexaose, difucosyllacto-N-hexaose, fucodisialyllacto-N-hexaose, disialyllacto-N-hexaose.

"Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated. The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell" or a sequence "foreign to said location or environment in said cell"). Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or metabolically engineered cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; and related techniques. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell. The terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g., knocking-out genes).

Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g., the Universal Protein Resource (UniProt) (www.uniprot.or ) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifiers (UniProt ID) are unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is represented by an UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021 03 and consulted on 09 June 2021. It should be understood for those skilled in the art that for the databases used herein, comprising UniProt, the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art.

As used herein, the term "cell productivity index (CPI)" refers to the mass of the oligosaccharide produced by the cells divided by the mass of the cells produced in the culture.

As used herein, the term "mammary cell(s)" generally refers to mammalian mammary epithelial cell(s), mammalian mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof. As used herein, the term "mammary-like cell(s)" generally refers to mammalian cell(s) having a phenotype/genotype similar (or substantially similar) to natural mammalian mammary cell(s) but is/are derived from mammalian non-mammary cell source(s). Such mammalian mammary-like cell(s) may be engineered to remove at least one undesired genetic component and/or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell. Non-limiting examples of mammalian mammary-like cell(s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof. Further nonlimiting examples of mammalian mammary-like cell (s) may include mammalian cell(s) having a phenotype similar (or substantially similar) to natural mammalian mammary cell (s), or more particularly a phenotype similar (or substantially similar) to natural mammalian mammary epithelial cell(s). A mammalian cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammalian mammary cell or a mammalian mammary epithelial cell may comprise a mammalian cell (e.g., derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component. As used herein, the term "nonmammary cell(s)" may generally include any mammalian cell of non-mammary lineage. In the context of the invention, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non-mammary cell(s) include hepatocyte(s), blood cell (s), kidney cell(s), cord blood cell(s), epithelial cell (s), epidermal cell(s), myocyte(s), fibroblast(s), mesenchymal cell(s), or any combination thereof. In some instances, molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.

The terms "cultivation", "cell cultivation" and "incubation" are used interchangeably; the terms comprise the culture medium wherein the cell is cultivated, or fermented, or incubated, medium components, the cell itself, and an oligosaccharide, like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g. LSTc and a sialyllactose, that is/are produced by the cell in whole broth, i.e., inside (intracellularly) as well as outside (extracellularly) of the cell.

The terms "biocatalysis reaction solution" and "enzymatic synthesis reaction" are used interchangeably and refer to a mixture wherein an oligosaccharide, like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g., LSTc and a sialyllactose is/are produced in an enzymatic way. Said mixture can comprise one or more enzyme(s), one or more precursor(s) and one or more acceptor(s) as defined herein present in a buffered solution and incubated for a certain time at a certain temperature enabling production of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g. LSTc and a sialyllactose catalysed by said one or more enzyme(s) using said one or more precursor(s) and said one or more acceptor(s) in said mixture. Said mixture can also comprise i) a cell producing one or more enzyme(s), one or more precursor(s) and/or one or more acceptor(s) as defined herein and used in said biocatalysis reaction for production of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g. LSTc and a sialyllactose and ii) a buffered solution or the culture or incubation medium wherein said cell was cultivated or incubated.

The term "chemical synthesis solution" is to be understood as a mixture wherein an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g., LSTc and a sialyllactose is/are produced in a chemical way. Said chemical synthesis solution can comprise one or more reactant(s), one or more intermediate chemical compound(s) and one or more by-product(s) that are incubated for a certain time at a certain temperature enabling production of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g., LSTc and a sialyllactose via one or more chemical reaction(s) in said solution. Said chemical synthesis solution can also comprise one or more catalyst(s) that speed up or slow down the synthesis reaction(s) in said chemical synthesis solution.

The term "any process stream" is to be understood as any solution that occurs or that is used or that is created at any step throughout the purification process of present invention. Examples of said process streams comprise but are not limited to an inlet solution, outlet solution, influent, effluent, eluent, eluate, flow, waste solution, buffer, solvent, alcohol, acid, base, lysate, filtrate, extract. The terms "reactor" and "incubator" refer to the recipient filled with the cultivation, incubation, chemical synthesis solution or biocatalysis reaction solution. Examples of reactors and incubators comprise but are not limited to microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators. Said reactor and incubator can each vary from lab-scale dimensions to large-scale industrial dimensions.

The term "purified" refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99.0 % pure as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy. Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and/or endotoxins which can be present in an aqueous medium like e.g., a cultivation, an incubation, a chemical synthesis solution or a biocatalysis reaction solution.

The term "clarifying" as used herein refers to the act of treating an aqueous medium like e.g., a cultivation, an incubation, a chemical synthesis solution or a biocatalysis reaction solution to remove suspended particulates and contaminants from the production process, like e.g., cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the oligosaccharide solution, oligosaccharide or oligosaccharide mixture. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a Celite™ filter aid).

The terms "protein-free oligosaccharide solution" as used herein means an oligosaccharide solution from a cultivation, an incubation, a chemical synthesis solution or a biocatalysis reaction solution, which has been treated to remove substantially all the proteins, as well as any related impurities, such as amino acids, peptides, endotoxins, glycolipids, RNA and DNA, from the process that could interfere with the eventual purification of the oligosaccharide solution from the process. Such removal of proteins, preferably substantially all proteins, can be accomplished in a conventional manner by ion exchange chromatography, affinity chromatography, ultrafiltration, and size exclusion chromatography. Preferably, a protein-free oligosaccharide solution is a clarified oligosaccharide solution. The terms "purification of an oligosaccharide solution from a cultivation" according to the present invention mean harvesting, collecting or retrieving the oligosaccharide solution from the cells and/or the medium of its growth.

A "purified oligosaccharide solution" comprises one oligosaccharide or a mixture of oligosaccharides dissolved in an aqueous medium. An aqueous medium is a solvent comprising water. In some embodiments, the aqueous medium is pure water. In other embodiments, the medium comprises water with a trace amount of one or more organic solvents. In some such embodiments, the medium comprises less than 1%-wt. (percent by weight) organic solvent. In some embodiments, the medium comprises less than 0.1%-wt. organic solvent. In some embodiments, the medium comprises less than 0.01%-wt. organic solvent. In some embodiments, the medium comprises less than 0.001%-wt. organic solvent. In some embodiments, the medium comprises less than 0.0001%-wt. organic solvent.

In some embodiments, the oligosaccharide solution comprises a trace amount of one or more organic solvents. In some such embodiments, the purified oligosaccharide solution comprises less than 1%-wt. organic solvent. In some embodiments, the purified oligosaccharide solution comprises less than 0.1%- wt. organic solvent. In some embodiments, the purified oligosaccharide solution comprises less than 0.01%-wt. organic solvent. In some embodiments, the purified oligosaccharide solution comprises less than 0.001%-wt. organic solvent. In some embodiments, the purified oligosaccharide solution comprises less than 0.0001%-wt. organic solvent.

The term "precursor" as used herein refers to substances which are taken up or synthetized by the cell for the specific production of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g., LSTc and/or a sialyllactose according to the present invention. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of one or more biochemical synthesis route(s) of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide, like e.g., LSTc and/or a sialyllactose. The term "precursor" as used herein is also to be understood as a chemical compound that participates in a chemical synthesis reaction, an incubation or a biocatalysis reaction (i.e., enzymatic reaction) to produce another compound like e.g., an intermediate or an acceptor as defined herein, as part in one or more metabolic pathway(s) of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide like e.g. LSTc and/or a sialyllactose. The term "precursor" as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in one or more metabolic pathway(s) of an oligosaccharide like e.g., a negatively charged, preferably sialylated oligosaccharide like e.g., LSTc and/or a sialyllactose. Examples of such precursors comprise the acceptors as defined herein, and/or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-1- phosphate, galactose-l-phosphate, glucose-6-phosphate, fructose-5-phosphate, fructose-1,6- bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3- phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-l-phosphate, N-acetylneuraminic acid-9- phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g. UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP-fucose.

Optionally, the cell used to produce the oligosaccharide like e.g., the negatively charged, preferably sialylated oligosaccharide, like e.g. LSTc and a sialyllactose is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the oligosaccharide of present invention.

The term "acceptor" as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase. Examples of such acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N- neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, and oligosaccharide containing 1 or more N-acetyllactosamine units and/or 1 or more lacto-N-biose units or an intermediate into oligosaccharide, fucosylated and sialylated versions thereof, ceramide, N-acylated sphingoid, glucosylceramide, lactosylceramide, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with beta-GIcNAc-Asn residues, glycoproteins with terminal GIcNAc and Gal residues, immunoglobulins.

As used herein a "Brix value" indicates the sugar content of an aqueous solution. A Brix value can be expressed as a percentage (percent Brix) or as "degrees Brix". Strictly, a Brix value is the percentage by weight of sucrose in a pure water solution, and so does not apply to solutions comprising other solutes and/or solvents. However, a Brix value is simple to measure, and, therefore, is commonly used in the art as an approximation of the total saccharide content of sugar solutions other than pure sucrose solutions. As used herein, the "Brix value" indicates the combined sugar content of the aqueous solution, when the purified oligosaccharide solution comprises two or more different oligosaccharides. Techniques for measuring a Brix value are well known in the art. Dissolution of sugar in an aqueous solution changes the refractive index of the solution. Accordingly, an appropriately calibrated refractometer can be used to measure a Brix value of a solution. Alternatively, the density of a solution may be measured and converted to a Brix value. A digital density meter can perform this measurement and conversion automatically, or a hydrometer or pycnometer may be used.

The terms "dry solid" and "dry matter" as used herein are used interchangeably and are further described in Example 1.

The term "flow rate" as used herein refers to how fast a solution, like e.g., a solution comprising an oligosaccharide of present invention, is being passed over a resin in e.g., an ion exchange, a cationic ion exchange, an anionic ion exchange, a mixed bed ion exchange. The flow rate is expressed in BV/h. The terms "bed volume / hour", "bed volume / h", "BV / hour" and "BV/h" are used interchangeably. The terms "bed volume" or "BV" are used interchangeably and refer to the volume of the resin (in m³) used in ion exchange, ion exchange chromatography, mixed bed ion exchange, cationic ion exchange, anionic ion exchange. The term "bed volume" as used herein is also to be understood as the minimum volume of solvent necessary to wet the defined quantity of sorbent within the column. This can vary depending on the nature of the sorbent.

The terms "electrodialysis" or "ED" are used interchangeably and refer to an electrically driven process that combines dialysis and electrolysis for the separation of ions from an aqueous solution like e.g., a solution comprising an oligosaccharide of present invention.

The terms "electrodeionization" or "EDI" are used interchangeably and refer to an ED process that is modified with a solid conductive ion medium that is introduced into the dilute compartment of the ED in the form of ion exchange resins to overcome the phenomenon of concentration polarization that is present in ED. EDI is used for the separation of ions from an aqueous solution like e.g., a solution comprising an oligosaccharide of present invention.

The terms "conductivity" or "electrical conductivity" are used interchangeably and are to be understood as a measure of a material's ability to carry an electrical current. Said material can be a solution. Conductivity is expressed in S (Siemens)/ m, mostly in mS/cm or pS/cm. Conductivity can be measured by applying a known DC voltage across a pair of parallel electrodes immersed in the solution, measuring the current produced and calculating the resistance of the solution. The conductivity of a solution is determined mainly by the charged species present, particularly the salts.

As used herein, the term "bulk density" is the weight of the particles of a particulate solid (such as a powder) in a given volume and is expressed in grams per liter (g/L). The total volume that the particles of a particulate solid occupy depends on the size of the particles themselves and the volume of the spaces between the particles. Entrapped air between and inside the particles also can affect the bulk density. Thus, a particulate solid consisting of large, porous particles with large inter-particulate spaces will have a lower bulk density than a particulate solid consisting of small, non-porous particles that compact closely together. Bulk density can be expressed in two forms: "loose bulk density" and "tapped bulk density". Loose bulk density (also known in the art as "freely settled" or "poured" bulk density) is the weight of a particulate solid divided by its volume where the particulate solid has been allowed to settle into that volume of its own accord (e.g., a powder poured into a container).

Closer compaction of a particulate solid within a container may be achieved by tapping the container and allowing the particles to settle more closely together, thereby reducing volume while weight remains the same. Tapping therefore increases bulk density. Tapped bulk density (also known in the art as "tamped" bulk density) is the weight of a particulate solid divided by its volume where the particulate solid has been tapped and allowed to settle into the volume a precise number of times. The number of times the particulate solid has been tapped is typically when stating the tapped bulk density. For example, "lOOx tapped bulk density" refers to the bulk density of the particulate solid after it has been tapped 100 times. Techniques for measuring bulk density are well known in the art. Loose bulk density may be measured using a measuring cylinder and weighing scales: the particulate solid is poured into the measuring cylinder and the weight and volume of the particulate solid; weight divided by volume gives the loose bulk density. Tapped bulk density can be measured using the same technique, with the addition of tapping the measuring cylinder a set number of times before measuring weight and volume. Automation of tapping ensures the number, timing and pressure of individual taps is accurate and consistent. Automatic tapping devices are readily available, an example being the Jolting Stampfvolumeter (STAV 203) from J. Englesmann AG.

The ash content is a measure of the total amount of minerals present within a food or ingredients such as oligosaccharides, whereas the mineral content is a measure of the amount of specific inorganic components present within a food, such as Ca, Na, K, Mg, phosphate, sulphate and Cl. Determination of the ash and mineral content of foods or oligosaccharides is important for a number of reasons: I) Nutritional labeling. The concentration and type of minerals present must often be stipulated on the label of a food or ingredient such as oligosaccharides. The quality of many foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture and stability. II) Microbiological stability. High mineral contents are sometimes used to retard the growth of certain microorganisms. Ill) Nutrition. Some minerals are essential to a healthy diet (e.g., calcium, phosphorous, potassium and sodium) whereas others can be toxic (e.g., lead, mercury, cadmium and aluminium). IV) Processing. It is often important to know the mineral content of foods/products during processing because this affects the physicochemical properties of foods or ingredient such as oligosaccharides. Ash is the inorganic residue remaining after the water and organic matter have been removed by heating in the presence of oxidizing agents, which provides a measure of the total amount of minerals within a food. Analytical techniques for providing information about the total mineral content are based on the fact that the minerals (the analyte) can be distinguished from all the other components (the matrix) within a food or ingredient in some measurable way. The most widely used methods are based on the fact that minerals are not destroyed by heating, and that they have a low volatility compared to other food components. The three main types of analytical procedure used to determine the ash content of foods are based on this principle: dry ashing, wet ashing and low temperature plasma dry ashing. The method chosen for a particular analysis depends on the reason for carrying out the analysis, the type of food or ingredient analyzed and the equipment available. Ashing may also be used as the first step in preparing samples for analysis of specific minerals, by atomic spectroscopy or the various traditional methods described below. For the sample preparation a sample whose composition represents that of the ingredient is selected to ensure that its composition does not change significantly prior to analysis. For instance, a dry oligosaccharide sample is generally hygroscopic, and the selected sample should be kept under dry conditions avoiding the absorption of water. Typically, samples of 1-10 gram are used in the analysis of ash content. Solid ingredients are finely ground and then carefully mixed to facilitate the choice of a representative sample. Before carrying out an ash analysis, samples that are high in moisture or in solution are generally dried to prevent spattering during ashing. Other possible problems include contamination of samples by minerals in grinders, glassware or crucibles which come into contact with the sample during the analysis. For the same reason, deionized water is used when preparing samples and the same is used in the blank sample. Dry ashing procedures use a high temperature muffle furnace capable of maintaining temperatures of between 500 and 600 °C. Water and other volatile materials are vaporized and organic substances are burned in the presence of the oxygen in air to COZ, H2O and N2. Most minerals are converted to oxides, sulphates, phosphates, chlorides or silicates. Although most minerals have fairly low volatility at these high temperatures, some are volatile and may be partially lost, e.g., iron, lead and mercury, for these minerals ICP-MS analysis of the product is more appropriate for quantification.

Detailed description of the invention

According to a first aspect, the present invention provides a process for the purification of an oligosaccharide from a solution, wherein the solution comprising said oligosaccharide is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process and wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation. The process comprises i) pH adjustment of the solution comprising said oligosaccharide to a pH ranging from 2 to 7 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form.

In a preferred embodiment, the process comprises i) pH adjustment of the solution comprising said oligosaccharide to a pH ranging from 3 to 7 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and an anionic ion exchange resin in OH- form.

In a more preferred embodiment, the process comprises i) pH adjustment of the solution comprising said oligosaccharide to a pH ranging from 3 to 6 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form.

In an even more preferred embodiment, the process comprises i) pH adjustment of the solution comprising said oligosaccharide to a pH ranging from 3 to 5 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form.

In a most preferred embodiment, the process comprises i) pH adjustment of the solution comprising said oligosaccharide to a pH ranging from 3 to 4 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, the cationic ion exchange resin present in said mixed bed ion exchange is in Na⁺ form.

In another and/or additional embodiment, said anionic ion exchange is preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. It is to be understood that when said anionic ion exchange is present in a process of present invention wherein said anionic ion exchange is preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, the pH adjusted solution comprising an oligosaccharide to be purified by a process of present invention is first passed through said cationic ion exchange prior to passage through said anionic ion exchange. Preferably, when present said cationic ion exchange is in Na⁺ form.

In a preferred embodiment of the process of present invention, the process comprises pH adjustment of a solution comprising an oligosaccharide to be purified by present invention to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing said pH adjusted solution through an anionic ion exchange using an anionic ion exchange resin in OH- form.

In another preferred embodiment of the process of present invention, the process comprises pH adjustment of a solution comprising an oligosaccharide to be purified by present invention to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing said pH adjusted solution through a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na⁺ form, and an anionic ion exchange resin in OH- form.

In another preferred embodiment of the process of present invention, the process comprises pH adjustment of a solution comprising an oligosaccharide to be purified by present invention to a pH ranging from 2 to 7 , preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing said pH adjusted solution through an anionic ion exchange using an anionic ion exchange resin in OH- form and through a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na⁺ form, and an anionic ion exchange resin in OH- form. Herein, said anionic ion exchange and said mixed bed ion exchange can be performed in any order. In a preferred embodiment, said anionic ion exchange is preceding said mixed bed ion exchange. In a more preferred embodiment, said anionic ion exchange is immediately preceding said mixed bed ion exchange without another method being performed after said anionic ion exchange and before said mixed bed ion exchange.

In another preferred embodiment, said mixed bed ion exchange is preceding said anionic ion exchange.

In a more preferred embodiment, said mixed bed ion exchange is immediately preceding said anionic ion exchange without another method being performed after said mixed bed ion exchange and before said anionic ion exchange.

In another preferred embodiment of the process of present invention, the process comprises pH adjustment of a solution comprising an oligosaccharide to be purified by present invention to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing said pH adjusted solution through a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na⁺ form, prior to an anionic ion exchange using an anionic ion exchange resin in OH- form.

In another preferred embodiment of the process of present invention, the process comprises pH adjustment of a solution comprising an oligosaccharide to be purified by present invention to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and passing said pH adjusted solution through 1) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na⁺ form, and an anionic ion exchange resin in OH- form and 2) through a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na⁺ form, prior to an anionic ion exchange using an anionic ion exchange resin in OH- form. Herein, said mixed bed ion exchange can be performed before said cationic ion exchange or after said anionic ion exchange. In a preferred embodiment, said mixed bed ion exchange is immediately preceding said cationic ion exchange. In another preferred embodiment, said mixed bed ion exchange is immediately succeeding said anionic ion exchange.

According to another aspect, the present invention provides a process for the purification of an oligosaccharide from a solution wherein the process comprises electrodeionization (EDI) of said solution. The solution comprising said oligosaccharide to be purified by a process of present invention is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution and a cell cultivation, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation. Within the context of the present invention, said solution comprising said oligosaccharide is produced by incubation in a reactor or incubator as defined herein. Said reactor or incubator can vary from small-scale dimensions (lab-scale) to large-scale dimensions (industrial set-up).

In a preferred embodiment of present invention, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH form.

In a more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺ form. In an even more preferred embodiment, said cationic ion exchange resin is provided in H⁺ form by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin is not provided in H⁺ form but in another form like e.g., Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ by a supplier and is regenerated upon use in said EDI into H⁺ form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in Na⁺ form. In an even more preferred embodiment, said cationic ion exchange resin is provided in Na⁺ form by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin is not provided in Na⁺ form but in another form like e.g., H⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ by a supplier and is regenerated upon use in said EDI into Na⁺ form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. In an even more preferred embodiment, said cationic ion exchange resin is provided in the desired form, which is K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively, by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin is not provided in the desired form but in another ion form by a supplier and is regenerated upon use in said EDI into the desired form by method(s) known by the person skilled in the art.

In another and/or additional preferred embodiment, said EDI comprises an anionic ion exchange using an anionic ion exchange resin in OH", Cl’ or SO₃ ²' form.

In a more preferred embodiment, said EDI comprises an anionic ion exchange using an anionic ion exchange resin in OH' form. In an even more preferred embodiment, said anionic ion exchange resin is provided in OH form by a supplier. In an alternative more preferred embodiment, said anionic ion exchange resin is not provided in OH' form but in another form like e.g., Cl' or SO₃ ²' by a supplier and is regenerated upon use in said EDI into OH' form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises an anionic ion exchange using an anionic ion exchange resin in Cl' or SO₃ ²' form. In an even more preferred embodiment, said anionic ion exchange resin is provided the desired form, which is Cl' or SO₃ ²' form, respectively by a supplier. In an alternative more preferred embodiment, said anionic ion exchange resin is not provided in the desired form but in another ion form by a supplier and is regenerated upon use in said EDI into the desired form by method(s) known by the person skilled in the art.

In another and/or additional preferred embodiment, said EDI comprises a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, and an anionic ion exchange using an anionic ion exchange resin in OH', Cl' or SO₃ ²' form.

In a more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is in H⁺ form. In an even more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is provided in H⁺ form by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is not provided in H⁺ form but in another form like e.g., Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ by a supplier and is regenerated upon use into H⁺ form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is in Na⁺ form. In an even more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is provided in Na⁺ form by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is not provided in Na⁺ form but in another form like e.g., H⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ by a supplier and is regenerated upon use into Na⁺ form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. In an even more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is provided in the desired form, which is K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively, by a supplier. In an alternative more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is not provided in the desired form but in another ion form by a supplier and is regenerated upon use into the desired form by method(s) known by the person skilled in the art.

In another and/or additional more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is in OH' form. In an even more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is provided in OH' form by a supplier. In an alternative more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is not provided in OH' form but in another form like e.g., Cl' or SO₃ ²' by a supplier and is regenerated upon use into OH' form by method(s) known by the person skilled in the art.

In another and/or additional more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is in Cl or SO₃ ² form. In an even more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is provided in the desired form, which is O' or SO₃ ²'form, respectively by a supplier. In an alternative more preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is not provided in the desired form but in another ion form by a supplier and is regenerated upon use into the desired form by method(s) known by the person skilled in the art. In another and/or additional more preferred embodiment, said EDI comprises a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH ' form. Herein, said cationic ion exchange resin in said mixed bed ion exchange is provided in H⁺ or Na⁺ form by a supplier. Alternatively, said cationic ion exchange resin in said mixed bed ion exchange is not provided in H⁺ or Na⁺ form but in another form like e.g., K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ by a supplier and is regenerated upon use into H⁺ or Na⁺ form by method(s) known by the person skilled in the art. Additionally and/or alternatively, said anionic cationic ion exchange resin in said mixed bed ion exchange is provided in OH' form by a supplier. Alternatively, said anionic ion exchange resin in said mixed bed ion exchange is not provided in OH' form but in another form like e.g., Cl or SO₃ ² by a supplier and is regenerated upon use into OH form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH', Cl' or SOs²' form. In even more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form and an anionic ion exchange using an anionic ion exchange resin in OH' form. If the resin(s) of said cationic ion exchange and/or said anionic ion exchange is/are not in the desired form(s) or is not provided in the desired form(s) by the supplier, the resin(s) is/are to be regenerated upon use into the desired form by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH', Cl' or SOs²' form. In an even more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form and an anionic ion exchange using an anionic ion exchange resin in OH' form. If the resin(s) in said cationic ion exchange and/or mixed bed ion exchange is/are not in the desired form(s) or is/are not provided in the desired form(s) by the supplier, said resin(s) is/are to be regenerated upon use into the desired form(s) by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises an anionic ion exchange using an anionic ion exchange resin in OH', Cl' or SO₃ ²' form and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH', Cl' or SOa²' form. In an even more preferred embodiment, said EDI comprises an anionic ion exchange using an anionic ion exchange resin in OH' form and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form and an anionic ion exchange using an anionic ion exchange resin in OH- form. If the resin(s) in said anionic ion exchange and/or mixed bed ion exchange is/are not in the desired form(s) or is/are not provided in the desired form(s) by the supplier, said resin(s) is/are to be regenerated upon use into the desired form(s) by method(s) known by the person skilled in the art.

In another more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, an anionic ion exchange using an anionic ion exchange resin in OH", Cl' or SO₃ ²’ form and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH", Cl’ or SO₃ ²’ form. In an even more preferred embodiment, said EDI comprises a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form, an anionic ion exchange using an anionic ion exchange resin in OH form and a mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺ or Na⁺ form and an anionic ion exchange using an anionic ion exchange resin in OH’ form. If the resin(s) in said cationic ion exchange, anionic ion exchange and/or mixed bed ion exchange is/are not in the desired form(s) or is/are not provided in the desired form(s) by the supplier, said resin(s) is/are to be regenerated upon use into the desired form(s) by method(s) known by the person skilled in the art.

In another and/or additional preferred embodiment, said process comprises pH adjustment of said solution. In another and/or additional preferred embodiment, said process comprises pH adjustment of said solution to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, prior to passing said solution onto said EDI. Said pH adjustment of said solution to a pH ranging from 2 to 7 is to be understood as a pH adjustment of said solution to a pH of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,

6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 2 and 7, including 2 and 7. In a more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 3 and 7, including 3 and 7. In an even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or to any pH value between 3 and 6, including 3 and 6. In another even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5,

3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or to any pH value between 3 and 5, including 3 and 5. In a most preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4 or to any pH value between 3 and 4, including 3 and 4.

According to another aspect, the present invention provides a process for the purification of a negatively charged oligosaccharide from a solution. The process comprises i) pH adjustment of the solution comprising said negatively charged oligosaccharide to a pH ranging from 2 to 5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In a preferred embodiment, the process comprises i) pH adjustment of the solution comprising said negatively charged oligosaccharide to a pH ranging from 3 to 5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In an alternative preferred embodiment, the process comprises i) pH adjustment of the solution comprising said negatively charged oligosaccharide to a pH ranging from 4 to 5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In a preferred embodiment, the solution comprising a negatively charged, preferably sialylated, oligosaccharide to be purified by a process of present invention is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process. Within the context of the present invention, said negatively charged, preferably sialylated, oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation.

According to another aspect of present invention, the present invention provides a process for the purification of sialyllacto-N-tetraose c (LSTc; Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) from a solution comprising LSTc and a sialyllactose. The process comprises i) pH adjustment of the solution comprising said LSTc and a sialyllactose to a pH ranging from 4 to 7 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In a preferred embodiment, the process comprises i) pH adjustment of the solution comprising said LSTc and sialyllactose to a pH ranging from 5 to 7 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In a more preferred embodiment, the process comprises i) pH adjustment of the solution comprising said LSTc and sialyllactose to a pH ranging from 6 to 7 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In an even more preferred embodiment, the process comprises i) pH adjustment of the solution comprising said LSTc and sialyl lactose to a pH of 6.5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, the solution comprising said LSTc and a sialyllactose wherein said LSTc is to be purified by a process of present invention is a solution chosen from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of the process of present invention for the purification of said LSTc from said sialyllactose. Within the context of the present invention, said LSTc and sialyllactose are produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation.

The present invention concerns a process for the purification of an oligosaccharide or a negatively charged, preferably sialylated, oligosaccharide that is provided in a solution comprising said oligosaccharide or negatively charged, preferably sialylated, oligosaccharide, respectively. The present invention also concerns a process for the purification of LSTc that is provided in a solution comprising said LSTc and a sialyllactose. In a preferred embodiment, said sialyllactose is chosen from the list comprising 3' -sialyllactose (3'SL, Neu5Ac-a2,3-Gal-pi,4-Glc), 6' -sialyllactose (6'SL, Neu5Ac-a2,6-Gal-pi,4-Glc) and 8'- sialyllactose (8'SL, Neu5Ac-oc2,8-Gal-pi,4-Glc). In a more preferred embodiment, said sialyllactose is 6'SL.

In a preferred embodiment, the oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N- acetylglucosamine containing neutral (non-charged) oligosaccharide, N-acetyllactosamine containing oligosaccharide, lacto-N-biose containing oligosaccharide, a galactose containing oligosaccharide, non- fucosylated neutral (non-charged) oligosaccharide, chitosan, chitosan comprising oligosaccharide, heparosan, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, chondroitin sulphate, dermatan sulphate, hyaluronan or hyaluronic acid, keratan sulphate, a milk oligosaccharide, O-antigen, enterobacterial common antigen (EGA), the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan, an amino-sugar, an antigen of the human ABO blood group system, an animal oligosaccharide, a plant oligosaccharide, erlose (Glc-al,4-Glc-al,2-Fru), lactul-N-triose II (GlcNAc-bl,3- Gal-bl,4-Fru), lactul-N-tetraose, lactul-N-neotetraose, globotriose. Preferably, the milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO). Preferably, the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans. Preferably, the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans. In the context of present invention, N-glycans and O-glycans refer to the oligosaccharide structures as known by the person skilled in the art wherein said structures are not attached to a protein or a peptide. In an additional preferred embodiment, the fucosylated oligosaccharide is selected from the list comprising 2'-fucosyl lactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3- Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose V, lacto- N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal- bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3- )GlcNAc-bl,3-Gal-bl,4-Glc, Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4- (Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,4-(Fuc-al,2-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc- al,3-)Glc, monofucosyllacto-N-hexaose-lll, difucosyllacto-N-hexaose (a), difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc- al,2-)Gal-bl,4-(Fuc-al,3-)Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3- fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4- fucosyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3- )Glc.

In another and/or additional preferred embodiment, the neutral (non-charged) oligosaccharide is a milk oligosaccharide. In a more preferred embodiment, the oligosaccharide is a mammalian milk oligosaccharide (MMO). In an even more preferred embodiment, the oligosaccharide is a human milk oligosaccharide (HMD), selected from the group comprising 2'-fucosyllactose, 3-fucosyllactose, 2',3- difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N- neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- neofucopentaose V, lacto-N- difucohexaose I, lacto-N-neodifucohexaose, lacto-N-difucohexaose II, monofucosyllacto-n-hexaose III, difucosyllacto-N-hexaose a, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose and combinations thereof.

In another and/or additional preferred embodiment, the N-acetylglucosamine containing neutral (noncharged) oligosaccharide is selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 4'-galactosyllactose, 3'-galactosyllactose, GlcNAc-bl,6- Gal-bl,4-Glc, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (pLNH), para- lacto-N-neohexaose (pLNnH), GlcNAc-bl,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, lacto-N-pentaose (LN5), lacto-N- neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N- heptaose (LN7), lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto- N-neooctaose, para lacto-N-neooctaose (pLNnO), iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N- nonaose (LN9), lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, para lacto-N-neodecaose (pLNnD), al,3-galactosyllacto-N-neotetraose, GlcNAc-bl,3-Gal-bl,3-GlcNAc-bl,3- Gal-bl,4-Glc, GlcNAc-bl,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc and GlcNAc-bl,6-(Gal-bl,3-GlcNAc-bl,3- )Gal-bl,4-Glc.

In an alternative and/or additional preferred embodiment the negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid group selected from the list comprising Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO). In a more preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide having one Neu5Ac (neuraminic acid) group. In another more preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide having two sialic acid groups. In another more preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide having two identical sialic acid groups. In an even more preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide having two Neu5Ac groups. In another more preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide having three or more sialic acid groups.

In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide selected from the list comprising a negatively charged, preferably sialylated, milk oligosaccharide; O-antigen; the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide; a negatively charged, preferably sialylated, plant oligosaccharide; N-acetyllactosamine containing negatively charged, preferably sialylated, oligosaccharide and lacto-N-biose containing negatively charged, preferably sialylated, oligosaccharide. In an even more preferred embodiment, the sialylated oligosaccharide is a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO). In another even more preferred embodiment, the sialylated oligosaccharide is a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO). Preferably, the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans. Preferably, the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.

In another more preferred embodiment, the negatively charged oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac- 2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3- disialyllactose (Neu5Ac-ot2,8-Neu5Ac-a2,3-Gal- i,4-Glc), 6'-sialyllactosamine, 3'-sialyllactosamine, sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto- N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N- neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal, Neu5Ac-a2,3-Gal-bl,3- GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-6'sialyllactose, 3'KDO-8-sialyllactose, KDO- 2,3Gaip-l,3GalNacP-l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNacP-l,3Gaip-l,4Glc, KDO-2,3Gal - l,4GlcNacP-l,3Gaip-l,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3- Gal, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'-fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-Sialyl-3- fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-bl,4-Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc-al,4- (Neu5Ac-a2,3-Gal-bl,3-)GlcNAc, 6'-Sialyllacto-N-biose, 3'-Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-bl,3- )Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4-(Fuc- al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4- Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4- (Fuc-al,3-)Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc- bl,3-Gal-bl,4-Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac- a2,6-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3- GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-Glc and combinations thereof.

In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is an oligosaccharide with a degree of polymerization (DP) of at least 3. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is an oligosaccharide with a DP chosen from the list comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is an oligosaccharide with a DP chosen from the list comprising 3, 4, 5, 6, 7, 8, 9 and 10. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a trisaccharide. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a tetrasaccharide. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a pentasaccharide. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a hexasaccharide. In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a heptasaccharide.

In another preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a linear oligosaccharide. In an alternative preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a branched oligosaccharide. The oligosaccharide or negatively charged, preferably sialylated, oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide does not contain any protective group.

In another preferred embodiment, the oligosaccharide or the negatively charged, preferably sialylated, oligosaccharide is an oligosaccharide that comprises one or more sialic acid groups and one or more monosaccharide building blocks chosen from the list comprising fucose, galactose, glucose, xylose, mannose, N-acetylglucosamine, N-acetylgalactosamine, rhamnose, glucuronate, galacturonate, and N- acetylmannosamine. In a more preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a sialylated oligosaccharide that is also fucosylated. In another more preferred embodiment, the oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a sialylated oligosaccharide that is not fucosylated.

Within the context of present invention, the solution comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc and a sialyllactose wherein said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or said LSTc to be purified by a process of present invention is a solution chosen from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process. Within the context of the present invention, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose is/are produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation. Within the context of the present invention, said solution comprising said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose is produced by incubation in a reactor or incubator as defined herein. Said reactor or incubator can vary from small-scale dimensions (lab-scale) to large-scale dimensions (industrial set-up).

In a preferred embodiment, the purity of said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc in said solution is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process.

In another and/or additional preferred embodiment, the solution comprising an oligosaccharide to be purified by a process of present invention is a cell cultivation using a cell that produces said oligosaccharide and comprising said oligosaccharide, biomass, medium components and contaminants. In a more preferred embodiment, the purity of said oligosaccharide in said cell cultivation is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process.

In another and/or additional preferred embodiment, the solution comprising a negatively charged, preferably sialylated, oligosaccharide to be purified by a process of present invention is a cell cultivation using a cell that produces said negatively charged, preferably sialylated, oligosaccharide and comprising said negatively charged, preferably sialylated, oligosaccharide, biomass, medium components and contaminants. In a more preferred embodiment, the purity of said negatively charged, preferably sialylated, oligosaccharide in said cell cultivation is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process.

In another and/or additional preferred embodiment, the solution comprising LSTc and a sialyllactose, wherein said LSTc is to be purified by a process of present invention is a cell cultivation using a cell that produces said LSTc and sialyllactose and comprising said LSTc, sialyllactose, biomass, medium components and contaminants. In a more preferred embodiment, the purity of said LSTc and sialyllactose in said cell cultivation is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process.

In another and/or additional more preferred embodiment, the biomass, when present, that is separated during the process, is optionally recycled to the cell cultivation.

In another and/or additional preferred embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose is/are accompanied in said solution by sialic acid as defined herein; ashes, preferably, said ashes comprise sulphates, phosphates, sodium, chloride, potassium, heavy metals like e.g., ammonium, lead, arsenic, cadmium, mercury; one or more monosaccharide(s) like e.g., fucose (Fuc), galactose (Gal), glucose (Glc), N-acetylglucosamine (GIcNAc), N- acetylgalactosamine (GalNAc), mannose (Man), N-acetylmannosamine (ManNAc); one or more activated monosaccharide(s) like e.g., UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP- GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N₃, CMP-Neu4,5Ac₂, CMP-Neu5,7Ac₂, CMP-Neu5,9Ac₂, CMP- Neu5,7(8,9)Ac₂, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc); one or more phosphorylated monosaccharide(s) like e.g., fructose-6-phosphate, glucose-6-phosphate, glucose-l-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylglucosamine-l-phosphate, galactose-l-phosphate, N-acetylmannosamine-6-phosphate, mannose-6-phosphate, mannose-1- phosphate, fructose-l-phosphate, fructose-l,6-bisphosphate, glycerol-3-phosphate, glyceraldehyde-3- phosphate, dihydroxyacetone-phosphate; one or more disaccharide(s) like e.g., lactose (Gal-bl,4-Glc), lacto-N-biose (Gal-bl,3-GlcNAc), N-acetyllactosamine (Gal-bl,4-GlcNAc), and/or one or more other oligosaccharide(s) that is/are selected from the list comprising a neutral (non-charged) oligosaccharide, a negatively charged oligosaccharide, a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); O-antigen; enterobacterial common antigen (EGA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an aminosugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; erlose (Glc-al,4- Glc-al,2-Fru); lactul-N-triose II (GlcNAc-bl,3-Gal-bl,4-Fru); lactul-N-tetraose; lactul-N-neotetraose; globotriose; fucosylated oligosaccharide preferably selected from the list comprising Z'-fucosyllactose (2'FL), 3-fucosyl lactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP l)_; lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose V, lacto-N-difucohexaose I (LNDFH- I), lacto-N-difucohexaose II (LNDFH-II), Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2- Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal- bl,4-(Fuc-al,3-)Glc, Fuc-al,4-(Fuc-al,2-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, monofucosyllacto-N-hexaose-lll, difucosyllacto-N-hexaose (a), difucosyl-lacto-N-hexaose, difucosyl-lacto- N-neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc-al,2-)Gal-bl,4- (Fuc-al,3-)Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N- acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllacto-N- biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc; sialylated oligosaccharide preferably selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(Neu5Ac-<x2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4- Glc), 6'-sialyllactosamine, 3'-sialyllactosamine, sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'- sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto- N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal- bl,4-GlcNAc-bl,3-Gal, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'- KDO-6'sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Gai -l,3GalNac -l,3Gala-l,4Gai -l,4Gal, KDO- 2,3Gaip-l,3GlcNac -l,3Gaip-l,4Glc, KDO-2,3Gai -l,4GlcNac -l,3Gai -l,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'- fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-Sialyl-3-fucosyllactose, Neu5Ac-a2,5-(Neu5Ac-a2,3-)Gal-bl,4- Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc, 6'-Sialyllacto-N-biose, 3'- Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-(Fuc-al,3-)GlcNAc- bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac- a2,6-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,5-Gal-bl,3-GlcNAc-bl,3-Gal- bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2- )Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc- bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac- a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3- )Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc and combinations thereof; N- acetylglucosamine containing neutral (non-charged) oligosaccharide preferably selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 4'-galactosyllactose, 3'-galactosyllactose, GlcNAc-bl,6-Gal-bl,4-Glc, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (pLNH), para-lacto-N-neohexaose (pLNnH), GlcNAc-bl,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, lacto-N-pentaose (LN5), lacto-N-neopentaose, para lacto-N- pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-heptaose (LN7), lacto-N- neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N- neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose (pLNnO), iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose (LN9), lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, para lacto-N- neodecaose (pLNnD), a 1,3-galactosyl lacto-N-neotetraose, GlcNAc-bl,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4- Glc, GlcNAc-bl,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc and GlcNAc-bl,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4- Glc; N-acetyllactosamine containing oligosaccharide; lacto-N-biose containing oligosaccharide; non- fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; chondroitin sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

In another and/or additional preferred embodiment, said solution comprises two or more oligosaccharides.

In another and/or additional preferred embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose is/are accompanied in said solution by one or more other oligosaccharide(s) wherein at least one of said other oligosaccharides has the same degree of polymerization (DP) as said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc. In a more preferred embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose is/are accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have the same DP as said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc. In an alternative preferred embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose is/are accompanied in said solution by one or more other oligosaccharide(s) wherein at least one of said other oligosaccharides has a different degree of polymerization (DP) as said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc. In a more preferred alternative embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyl lactose is/are accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have a different DP as said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc.

In another and/or additional preferred embodiment, LSTc and sialyllactose are accompanied in said solution by one or more other oligosaccharide(s) wherein said one or more other oligosaccharide(s) has/have a degree of polymerization (DP) of at least 3. In a more preferred embodiment, said LSTc and sialyllactose are accompanied in said solution by one or more other oligosaccharide(s) with a DP of at least 4. In an even more preferred embodiment, said LSTc and sialyllactose are accompanied in said solution by one or more other oligosaccharide(s) with a DP of at least 5. In another even more preferred embodiment, said LSTc and sialyllactose are accompanied in said solution by one or more other oligosaccharide(s) with a DP of at least 6. In other words, in a preferred embodiment said LSTc and sialyllactose are accompanied in said solution by a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, and/or an oligosaccharide comprising more than 7 monosaccharide subunits. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two trisaccharides or by more than two trisaccharides. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two tetrasaccharides or by more than two tetrasaccharides. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two pentasaccharides or by more than two pentasaccharides. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two hexasaccharides or by more than two hexasaccharides. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two heptasaccharides or by more than two heptasaccharides. It is also possible that said LSTc and sialyllactose are accompanied in said solution by two oligosaccharides comprising more than 7 monosaccharide subunits or by more than two of said oligosaccharides comprising more than 7 monosaccharide subunits. In a more preferred embodiment, said LSTc and sialyllactose are accompanied in said solution by one or more trisaccharide(s), one or more tetrasaccharide(s), one or more pentasaccharide(s), one or more hexasaccharide(s), one or more heptasaccharide(s), and/or one or more oligosaccharide(s) comprising more than 7 monosaccharide subunits.

In another preferred embodiment, the solution comprises 6'SL, lactose and sialic acid. In another preferred embodiment, the solution comprises 3'SL, lactose and sialic acid. In another preferred embodiment, the solution comprises 3'SL and 6'SL. In another preferred embodiment, the solution comprises sialic acid, lactose, 3'SL and 6'SL.

In another preferred embodiment, the solution comprises sialic acid, LSTc and 6'SL. In another preferred embodiment, the solution comprises sialic acid, lactose, LSTc and 6'SL. In another preferred embodiment, the solution comprises LSTc, 3'SLand sialic acid. In another preferred embodiment, the solution comprises LSTc, 3'SL, lactose and sialic acid. In another preferred embodiment, the solution comprises LSTc, 6'SL and 3'SL. In another preferred embodiment, the solution comprises LSTc, 6'SL, 3'SL and sialic acid. In another preferred embodiment, the solution comprises LSTc, 6'SL, 3'SL, sialic acid and lactose. In another preferred embodiment, the solution comprises LSTc, 6'SL and LN3. In another preferred embodiment, the solution comprises LSTc, 6'SL, LN3 and LNnT. In another preferred embodiment, the solution comprises LSTc, 6'SL, LN3, LNnT and lactose. In another preferred embodiment, the solution comprises sialic acid, LN3, LNnT, LSTc and 6'SL. In another preferred embodiment, the solution comprises sialic acid, lactose, LN3, LNnT, LSTc and 6'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNnT, LSTc and 6'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNnT, lacto-N-hexaoses, LSTc and 6'SL.

In another preferred embodiment, the solution comprises LSTc, 3'SL and LN3. In another preferred embodiment, the solution comprises LSTc, 3'SL, LN3 and LNnT. In another preferred embodiment, the solution comprises LSTc, 3'SL, LN3, LNnT and lactose. In another preferred embodiment, the solution comprises LSTc, 3'SL, LN3, LNnT, lactose and sialic acid. In another preferred embodiment, the solution comprises LSTc, 3'SL, LN3, LNnT and sialic acid. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNnT, LSTc and 3'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNnT, lacto-N-hexaoses, LSTc and 3'SL. In another preferred embodiment, the solution comprises LSTc, 3'SL, LN3 and LNT.

In another preferred embodiment, the solution comprises sialic acid, LSTa and 3'SL. In another preferred embodiment, the solution comprises sialic acid, lactose, LSTa and 3'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, LNT, LSTa and 3'SL. In another preferred embodiment, the solution comprises sialic acid, lactose, LN3, LNT, LSTa and 3'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNT, LSTa and 3'SL. In another preferred embodiment, the solution comprises sialic acid, LN3, sialylated LN3, LNT, lacto-N-hexaoses, LSTa and 3'SL.

In another preferred embodiment, the solution comprises 2’FL, 3-FL, 3'SL and 6'SL. In another preferred embodiment, the solution comprises 2'FL, 3-FL, 3'SL, 6'SL, and lactose. In another preferred embodiment, the solution comprises 2'FL, 3-FL, 3'SL, 6'SL, sialic acid and lactose. In another preferred embodiment, the solution comprises 2'FL, LNFP-I, 3'SL and LSTa. In another preferred embodiment, the solution comprises 3-FL, LNFP-III, 6'SL and LSTc. In another preferred embodiment, the solution comprises 2'FL, 3-FL, DiFL, 3'SL, 6'SL, LNT and LNnT. In another preferred embodiment, the solution comprises LSTc and LSTa.

In another preferred embodiment, the solution comprises 2'FL, 3-FL, 3'SL, 6'SL and LSTc. In another preferred embodiment, the solution comprises 2'FL, LNFP-I, 3'SL, LSTa, 6'SLand LSTc. In another preferred embodiment, the solution comprises 3-FL, LNFP-III, 6'SL and LSTc. In another preferred embodiment, the solution comprises 2'FL, 3-FL, DiFL, 3'SL, 6'SL, LNT, LNnT and LSTc. In another preferred embodiment, the solution comprises LSTc, 6'SL and LSTa. In another preferred embodiment, the solution comprises LSTc, 3'SL and LSTa. In another preferred embodiment, the solution comprises LSTc, 3'SL and LSTd. In another preferred embodiment, the solution comprises only one or more neutral (non-charged) fucosylated oligosaccharide(s) as defined herein. In another preferred embodiment, the solution comprises one or more neutral (non-charged) fucosylated oligosaccharide(s) and one or more charged fucosylated oligosaccharide(s). A charged fucosylated oligosaccharide is to be understood as an oligosaccharide comprising at least one fucose residue and at least one sialic acid residue as defined herein. In another preferred embodiment, the solution comprises only one or more neutral (non-charged) oligosaccharide(s). In another preferred embodiment, the solution comprises one or more neutral oligosaccharides and one or more charged oligosaccharide(s).

In a preferred embodiment, the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose wherein the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is to be purified by a process of present invention is/are produced by a cell that is cultured in a cell cultivation. Within the context of present invention, the cell cultivation comprises in vitro and/or ex vivo cultivation of cells. In another preferred embodiment of present invention, the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli \N, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobaci II ial es, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales with members such as from the genus Bacillus, such as Bacillus subtilis or, 8. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae. The latter bacterium belonging to the phylum Proteobacteria, preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g. Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (like e.g. Yarrowia lipolytica) or Starmerella (like e.g. Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc. Preferably, said plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. The latter animal cell is preferably derived from non-human mammals (e.g. cattle, buffalo, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur), dog, cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), birds (e.g. chicken, duck, ostrich, turkey, pheasant), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g. lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g. snake, alligator, turtle), amphibians (e.g. frogs) or insects (e.g. fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably chosen from the list comprising an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g. HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g. an N20, SP2/0 or YB2/0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO21067641, preferably mesenchymal stem cell or derivates thereof as described in WO21067641, a lactocyte derived from mammalian induced pluripotent stem cells, preferably human induced pluripotent stem cells, a lactocyte as part of mammary-like gland organoids, a post-parturition mammary epithelium cell, a polarized mammary cell, preferably a polarized mammary cell selected from the group comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof as well-known to the person skilled in the art from e.g. WO2021/219634, WO 2022/054053, WO 2021/141762, WO 2021/142241, WO 2021/067641 and WO2021/242866. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila S2 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell. In another and/or additional preferred embodiment, the cell is an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.

In another and/or additional more preferred embodiment, the cell is a metabolically engineered cell. In another and/or additional more preferred embodiment, the cell has been metabolically engineered to produce any one or more compound(s) that is/are not (a) oligosaccharide(s). In another and/or additional more preferred embodiment, the cell has been metabolically engineered to produce an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or the LSTc and sialyllactose. In an even more preferred embodiment, the cell has been metabolically engineered to produce two or more oligosaccharides or negatively charged, preferably sialylated, oligosaccharides.

In another and/or additional preferred embodiment, the cell produces an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose and any one or more of sialic acid as defined herein; one or more monosaccharide(s); one or more activated monosaccharide(s); one or more phosphorylated monosaccharide(s); one or more disaccharide(s) and/or one or more other oligosaccharide(s), as described herein. In another and/or additional preferred embodiment, the cell has been metabolically engineered to produce an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose and any one or more of sialic acid as defined herein; one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s) and/or one or more other oligosaccharide(s) as described herein.

In another and/or additional preferred embodiment, the solution is a cell cultivation using at least one cell that has been metabolically engineered to produce said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc and sialyllactose and one or more of i) sialic acid, ii) one or more monosaccharide(s), iii) one or more activated monosaccharide(s), iv) one or more phosphorylated monosaccharide(s), v) one or more disaccharide(s) and/or vi) one or more other oligosaccharides.

In a more preferred embodiment, the cell comprises a sialyation pathway. A sialylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising an L-glutamine— D-fructose-6-phosphate aminotransferase, a phosphoglucosamine mutase, an N-acetylglucosamine-6-P deacetylase, an N-acylglucosamine 2-epimerase, a UDP-N- acetylglucosamine 2-epimerase, an N-acetylmannosamine-6-phosphate 2-epimerase, a UDP-GIcNAc 2- epimerase/kinase, a glucosamine 6-phosphate N-acetyltransferase, an N-acetylglucosamine-6-phosphate phosphatase, a phosphoacetylglucosamine mutase, an N-acetylglucosamine 1-phosphate uridylyltransferase, a glucosamine-l-phosphate acetyltransferase, an Neu5Ac synthase, an N- acetylneuraminate lyase, an N-acylneuraminate-9-phosphate synthase, an N-acylneuraminate-9- phosphatase, a sialic acid transporter, a cytidine monophosphate (CMP) kinase and a CMP-sialic acid synthase, combined with a sialyltransferase leading to any one or more of a 2,3; a 2,6 and/or a 2,8 sialylated oligosaccharides.

In an even more preferred embodiment, the cell is metabolically engineered to comprise a sialylation pathway. In another even more preferred embodiment, the cell has been metabolically engineered to comprise a sialylation pathway wherein any one or more of the genes chosen from the list comprising L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2- epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine- 1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9- phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase and sialyltransferase has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises a fucosylation pathway. A fucosylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase combined with a fucosyltransferase leading to a 1,2; a 1,3; a 1,4 and/or a 1,6 fucosylated oligosaccharides.

In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a fucosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a fucosylation pathway wherein any one or more of the genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase and fucosyltransferase has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises a galactosylation pathway. A galactosylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase combined with a galactosyltransferase leading to a galactosylated compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound galactose on any one or more of the 2, 3, 4 and 6 hydroxyl group of said mono-, di-, or oligosaccharide. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a galactosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a galactosylation pathway wherein any one or more of the genes chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises an 'N-acetylglucosaminylation' pathway. An N-acetylglucosaminylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose-6- phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase combined with a glycosyltransferase leading to a GIcNAc-modified compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound N-acetylglucosamine (GIcNAc) on any one or more of the 3, 4 and 6 hydroxyl group of said mono-, di- or oligosaccharide.

In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N- acetylglucosaminylation pathway wherein any one or more of the genes chosen from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l- phosphate acetyltransferase and a glycosyltransferase transferring GIcNAc has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises an 'N-acetylgalactosaminylation' pathway. An N-acetylgalactosaminylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and/or UDP-N-acetylgalactosamine pyrophosphorylase combined with a glycosyltransferase leading to a GalNAc-modified compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound N-acetylgalactosamine on said mono-, di- or oligosaccharide.

In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N- acetylgalactosaminylation pathway wherein any one or more of the genes chosen from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-N- acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and/or UDP-N- acetylgalactosamine pyrophosphorylase and a glycosyltransferase transferring GalNAc has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises a 'mannosylation' pathway. A mannosylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase and/or mannose-l-phosphate guanylyltransferase combined with a mannosyltransferase leading to a mannosylated compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound mannose on said mono-, di- or oligosaccharide.

In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a mannosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a mannosylation pathway wherein any one or more of the genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase and/or mannose-l-phosphate guanylyltransferase and mannosyltransferase has/have a modified and/or enhanced expression.

In another and/or additional preferred embodiment, the cell comprises an 'N-acetylmannosaminylation' pathway. An N-acetylmannosaminylation pathway is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose-6- phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-5-phosphate deacetylase, glucosamine 5-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-GIcNAc 2-epimerase and/or ManNAc kinase combined with a glycosyltransferase leading to a Ma nN Ac-modified compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound N-acetylmannosamine on said mono-, di- or oligosaccharide.

In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylmannosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N- acetylmannosaminylation pathway wherein any one or more of the genes chosen from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-GIcNAc 2-epimerase and/or ManNAc kinase and a glycosyltransferase transferring ManNAc has/have a modified and/or enhanced expression. In another and/or additional preferred embodiment, the cell is metabolically engineered for an enhanced production of an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of an oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose, a better efflux of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g. ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.

In another and/or additional preferred embodiment, the cell produces said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose from one or more internalized precursor(s) as defined herein. Preferably, said precursor is fed to the cell from the culture medium or the incubation. In a more preferred embodiment, the cell synthesizes one or more precursor(s) that is/are involved in the production of said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose. In another preferred embodiment, the precursor(s) that is/are used by the cell for the production of said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose is/are completely converted into said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose. In another preferred embodiment, the precursor(s) that is/are used in said solution for the production of said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose is/are completely converted into said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, LSTc and/or sialyllactose.

In another and/or additional more preferred embodiment, the cell cultivation is a fermentation.

In an alternative and/or additional more preferred embodiment, the cell is cultivated or incubated in a reactor as defined herein. In an alternative and/or additional more preferred embodiment, the cell is cultivated or incubated in an incubator as defined herein.

In another and/or additional preferred embodiment, the cell is cultivated in culture medium comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract. Preferably, said carbon source is selected from the list comprising glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. In a more preferred embodiment, the culture medium is a chemically defined medium. In an additional preferred embodiment, the culture medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.

In another and/or additional preferred embodiment, the solution comprising an oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose wherein said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is be purified by a process of present invention further comprises phosphate, N-cyclohexyl-3-aminopropanesulonic acid (CAPS), ethylenediaminetetraacetic acid (EDTA), Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES), bicarbonate, taurine, glycine, glycerol, sorbitol, sulfonic acid, tris(hydroxymethyl)aminomethane (Tris), a zwitterionic agent, polyaminosaccharide, carboxymethyl chitosan (CM-CS), CM-CS-HCI, CM-CS-hydroacetic acid, N, N-Bis-(2- hydroxyethyl)-2-aminoethanesulphonic acid (BES), 3-(N-morpholino)propanesulphonic acid (MOPS), dimethyl sulfoxide (DMSO), 2-(N-morpholino)ethanesulfonic acid (MES), albumin, amino acid residue, sulphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.

In another and/or additional preferred embodiment, the solution comprising an oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose wherein said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is be purified by a process of present invention further comprises a glycosyl donor like e.g. a halide, a hemiacetal, a peracetate, a thioglycoside, an 1,2-orthoester, an O-imidate, a thio-imidate, a glycosyl fluoride, a glycosyl ester, a glycosyl carbonate, a thiocyanate, a diazirine, a xanthate, a glycal, a phosphite, a sulfoxide, a sulfone, a selenium glycoside, an alkenyl glycoside, a heteroaryl glycoside, a glycosyl iodide, a glycosyl phosphate, a glycosyldisulfide, a Te-glycoside, a glycosyl sulfonylcarbamate, a 2-(hydroxycarbonyl)benzyl glycoside.

In another and/or additional preferred embodiment, the solution comprising an oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose wherein said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is be purified by a process of present invention further comprises a polar reaction solvent, CH2CI2, CICH2CH2CI, toluene, an ethereal solvent, a nitrile solvent, benzene, o-dichlorobenzene, urea, (thio)urea, K2CO3, Tris(2,4,6- trimethoxyphenyl)phosphine (TTMPP), isobutylene oxide, trimethylsilyl trifluoromethanesulfonate (TMSOTf), N-iodosuccinimide (NIS) and/or trifluoromethanesulfonic acid (TfOH).

In a specific embodiment of present invention, said solution is used in a process of the invention for the purification of an oligosaccharide from said solution wherein said process comprises i) pH adjustment of said solution to a pH ranging from 2 to 7 , preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4 and ii) passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na⁺ form and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na⁺ form, and an anionic ion exchange resin in OH- form.

In another specific embodiment of present invention, said solution is used in a process of the invention for the purification of a negatively charged, preferably sialylated, oligosaccharide from said solution wherein said process comprises i) pH adjustment of said solution to a pH ranging from 2 to 5, preferably from 3 to 5, more preferably from 4 to 5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another specific embodiment of present invention, said solution is used in a process of the invention for the purification of LSTc from said solution comprising LSTc and a sialyllactose wherein said process comprises i) pH adjustment of said solution to a pH ranging from 4 to 7, preferably from 5 to 7, more preferably from 6 to 7, even more preferably to a pH of 6.5 and ii) passing said pH adjusted solution through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In a preferred embodiment, said pH adjustment of said solution is obtained by any one or more of addition of an acidic agent, an alkaline agent and/or a buffered solution; filtration; nanofiltration; dialysis; electrodialysis; electrodeionization; ion exchange; mixed bed ion exchange; ion exchange chromatography; reverse osmosis; use of activated carbon or charcoal.

In another and/or additional preferred embodiment, said pH adjustment of said solution is an active step performed after the production of said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose in said solution or of said solution comprising said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose. Said pH adjustment of said solution is not obtained during and/or by synthesis of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose in said solution. Said active step involves any one or more of addition of any one or more of an acidic agent, an alkaline agent, a buffered solution, use of activated carbon or charcoal, use of an operational unit and/or an operational act comprising filtration; nanofiltration; dialysis; electrodialysis; electrodeionization; ion exchange; mixed bed ion exchange; ion exchange chromatography; reverse osmosis. For example, in the case that said solution is a cell cultivation wherein said cell produces said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose said pH adjustment does not comprise the acidification of said solution due to cell growth, cell lysis and/or the net negative charge of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, or LSTc and sialyllactose produced by the cell growing and being cultivated in said cultivation.

In another and/or additional preferred embodiment, said pH adjustment of said solution is obtained by addition of an acidic agent selected from the list comprising but not limited to phosphoric acid, hydrochloric acid, sulphuric acid, acetic acid, lactic acid, citric acid, tartaric acid, malic acid, succinic acid and fumaric acid. In another and/or additional preferred embodiment, said pH adjustment of said solution is obtained by addition of an alkaline agent selected from the list comprising but not limited to sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia. In another and/or additional preferred embodiment, said pH adjustment of said solution is obtained by addition of any one or more of phosphoric acid, hydrochloric acid, sulphuric acid, acetic acid, lactic acid, citric acid, tartaric acid, malic acid, succinic acid, fumaric acid, sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia. In another and/or additional preferred embodiment, said pH adjustment of said solution is obtained by passing said solution through a mixed bed ion exchange, a cationic ion exchange, an anionic ion exchange and/or an ion exchange chromatography step. In a more preferred embodiment, said pH adjustment of said solution is obtained by passing said solution through a mixed bed ion exchange to obtain a lowered pH.

In another and/or additional preferred embodiment, said pH adjustment of said solution to a pH ranging from 2 to 7 is to be understood as a pH adjustment of said solution to a pH of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,

2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 2 and 7, including 2 and 7. In a more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,

4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,

6.7, 6.8, 6.9, 7 or to any pH value between 3 and 7, including 3 and 7. In an even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2,

5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 or to any pH value between 3 and 6, including 3 and 6. In another even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or to any pH value between 3 and 5, including 3 and 5. In a most preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4 or to any pH value between 3 and 4, including 3 and 4.

In another and/or additional preferred embodiment, said pH adjustment of said solution to a pH ranging from 2 to 5 is to be understood as a pH adjustment of said solution to a pH of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,

2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or to any pH value between 2 and 5, including 2 and 5. In a more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 3, 3.1, 3.2, 3.3, 3.4, 3.5,

3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or to any pH value between 3 and 5, including 3 and 5. In an even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or to any pH value between 4 and 5, including 4 and 5.

In another and/or additional preferred embodiment, said pH adjustment of said solution to a pH ranging from 4 to 7 is to be understood as a pH adjustment of said solution to a pH of 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,

4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 4 and 7, including 4 and 7. In a more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 5 and 7, including 5 and 7. In an even more preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 or to any pH value between 6 and 7, including 6 and 7. In a most preferred embodiment, said pH adjustment of said solution is to be understood as a pH adjustment of said solution to a pH of 6.5.

In an additional specific embodiment, said pH adjusted solution is passed through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, and/or ii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an anionic ion exchange resin in OH- form.

In a preferred embodiment, said anionic ion exchange resin in said anionic ion exchange is provided in OH- form by a supplier. In an alternative preferred embodiment, said anionic ion exchange resin in said anionic ion exchange is not provided in OH- form but in another form, like e.g., Cl', SO₃ ²' by a supplier and is regenerated upon use in said process into OH- form by method(s) known by the person skilled in the art.

In an alternative and/or additional preferred embodiment, the anionic ion exchange used in the anionic ion exchange step is present in a single vessel like e.g., a column, in a small-scale (lab model) or large-scale (industrial scale) set-up.

In another and/or additional preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is in Na⁺ form. In a more preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is provided in Na⁺ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is not provided in Na⁺ form but in another ion form, like e.g., H⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ by a supplier and is regenerated upon use in said process into Na⁺ form by method(s) known by the person skilled in the art.

In another preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. In a more preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is provided in the desired form, which is K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively, by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said cationic ion exchange, when present in i), is not provided in the desired form but in another ion form by a supplier and is regenerated upon use in said process into the desired form by method(s) known by the person skilled in the art.

In another and/or additional preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is in Na⁺ form. In a more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is provided in Na⁺ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is not provided in Na⁺ form but in another ion form, like e.g., H⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ by a supplier and is regenerated upon use in said process into Na⁺ form by method(s) known by the person skilled in the art.

In another preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. In a more preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is provided in the desired form, which is K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively, by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said mixed bed ion exchange is not provided in the desired form but in another ion form by a supplier and is regenerated upon use in said process into the desired form by method(s) known by the person skilled in the art.

In an alternative and/or additional preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is provided in OH- form by a supplier. In an alternative and/or additional preferred embodiment, said anionic ion exchange resin in said mixed bed ion exchange is not provided in OH- form but in another form, like e.g., Cl’, SO₃ ²' by a supplier and is regenerated upon use in said process into OH- form by method(s) known by the person skilled in the art.

In an additional specific embodiment, said pH adjusted solution is passed through a mixed bed ion exchange, wherein the mixed bed ion exchange comprises a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. In a preferred embodiment, said cationic ion exchange resin is provided in H+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin is not provided in H+ form but in another ion form, like e.g., Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ by a supplier and is regenerated upon use in said mixed bed ion exchange of said process into H+ form by method(s) known by the person skilled in the art. In an alternative and/or additional preferred embodiment, said anionic ion exchange resin is provided in OH- form by a supplier. In an alternative and/or additional preferred embodiment, said anionic ion exchange resin is not provided in OH- form but in another form, like e.g., Cl’, SO₃ ^2- by a supplier and is regenerated upon use in said mixed bed ion exchange of said process into OH- form by method(s) known by the person skilled in the art.

In an alternative and/or additional preferred embodiment, the mixed bed ion exchange is present in a single vessel like e.g., a column, in a small-scale (lab model) or large-scale (industrial scale) set-up.

In another and/or additional preferred embodiment, the mixed bed ion exchange comprises an ion exchange column packed with a mixture of said cationic ion exchange resin and said anionic ion exchange resin in any volume ratio. In another and/or alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed before packing into a mixed bed ion exchange column. In another and/or alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed in said mixed bed ion exchange in a uniform mixture. In an alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed in said mixed bed ion exchange, wherein said cationic ion exchange resin is present in a selected volume ratio in said mixture and wherein said anionic ion exchange resin is present in a selected volume ratio in said mixture. In another preferred embodiment, the mixed bed ion exchange contains more of said anionic ion exchange resin than of said cationic ion exchange resin. In another preferred embodiment, the volume ratio of said anionic ion exchange resin to said cationic ion exchange resin in said mixed bed ion exchange is about 90:10, about 80:20, about 70:30, about 65:35, about 60:40, about 55:45. In an alternative preferred embodiment, the volume ratio of said cationic ion exchange resin to said anionic ion exchange resin in said mixed bed ion exchange is about 50:50.

In another and/or additional preferred embodiment, the mixed bed ion exchange comprises an ion exchange column packed with alternating layers of said cationic ion exchange resin and said anionic ion exchange resin. In a more preferred embodiment, each layer has the same volume. In an alternative more preferred embodiment, the layers have different volumes. In a more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 6 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 10 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 30 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 100 or more alternating layers.

In another and/or additional preferred embodiment, the total ion exchange capacity of said anionic ion exchange resin is equal to the total ion exchange capacity of said cationic ion exchange resin in said mixed bed ion exchange.

In another and/or additional preferred embodiment, the mixed bed ion exchange in said EDI comprises a mixture of said cationic ion exchange resin and said anionic ion exchange resin in any volume ratio. In another and/or alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed before packing into said EDI. In another and/or alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed in said mixed bed ion exchange in a uniform mixture. In an alternative preferred embodiment, said cationic ion exchange resin and said anionic ion exchange resin are mixed in said mixed bed ion exchange, wherein said cationic ion exchange resin is present in a selected volume ratio in said mixture and wherein said anionic ion exchange resin is present in a selected volume ratio in said mixture. In another preferred embodiment, the mixed bed ion exchange contains more of said anionic ion exchange resin than of said cationic ion exchange resin. In another preferred embodiment, the volume ratio of said anionic ion exchange resin to said cationic ion exchange resin in said mixed bed ion exchange is about 90:10, about 80:20, about 70:30, about 65:35, about 60:40, about 55:45. In an alternative preferred embodiment, the volume ratio of said cationic ion exchange resin to said anionic ion exchange resin in said mixed bed ion exchange is about 50:50.

In another and/or additional preferred embodiment, the mixed bed ion exchange comprises alternating layers of said cationic ion exchange resin and said anionic ion exchange resin. In a more preferred embodiment, each layer has the same volume. In an alternative more preferred embodiment, the layers have different volumes. In a more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 6 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 10 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 30 or more alternating layers. In another more preferred embodiment, the cationic ion exchange resin and anionic ion exchange resin are packed in the mixed bed ion exchange in 100 or more alternating layers.

In another and/or additional preferred embodiment, said EDI comprises a cationic ion exchange and/or a mixed bed ion exchange as described herein, wherein said cationic ion exchange resin in said cationic ion exchange and/or said mixed bed ion exchange is selected from the group comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin.

In another and/or additional preferred embodiment, said EDI comprises an anionic ion exchange and/or a mixed bed ion exchange as described herein, wherein said anionic ion exchange resin in said anionic ion exchange and/or said mixed bed ion exchange is selected from the group comprising a weak base anion (WBA) exchange resin, a strong base anion (SBA) exchange resin Type 1 and an SBA exchange resin Type 2.

In another and/or additional preferred embodiment, the cationic ion exchange resin present in said cationic ion exchange, when present, and/or in said mixed bed ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin. In another and/or additional preferred embodiment, the anionic ion exchange resin present in said anionic ion exchange and/or in said mixed bed ion exchange is chosen from the list comprising a weak base anion (WBA) exchange resin, a strong base anion (SBA) exchange resin Type 1 and an SBA exchange resin Type 2. The terms "WAC", "SAC", "WBA" and "SBA" are generally known in the art. A strong ion exchange resin will not significantly lose the charge on its matrix once the ion exchange resin is equilibrated and so a wide pH range can be used. A weak ion exchange resin has a more specific pH range in which it will maintain its charge: usually an acidic to about neutral pH in the case of a WBA, respectively, an alkaline to about neutral pH in the case of a WAC.

In another and/or additional preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBAType 2 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in Na+ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in Na+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in Na+ form and an SBA Type 2 in OH- form.

In another and/or additional preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBAType 1 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBAType 2 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBAType 2 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in H+ form and a WBA in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in H+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, the mixed bed ion exchange comprises an SAC in H+ form and an SBA Type 2 in OH- form.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55:45. In another preferred embodiment, the mixed bed ion exchange comprises a WAC in H+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 55:45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type

1 in OH- form in an SBA:SAC volume ratio of about 55:45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type

2 in OH- form in an SBA:SAC volume ratio of about 55:45. In another preferred embodiment, the mixed bed ion exchange comprises a SAC in H+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional more preferred embodiment, when present in said EDI the cationic ion exchange comprises a WAC in any one of H+, Na+, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form. In another and/or additional more preferred embodiment, when present in said EDI the cationic ion exchange comprises a SAC in any one of H+, Na+, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form.

In another and/or additional more preferred embodiment, when present in said EDI the anionic ion exchange comprises a WBA in any one of OH", Cl’ or SO₃ ²' form. In another and/or additional more preferred embodiment, when present in said EDI the anionic ion exchange comprises an SBA Type 1 in any one of OH', Cl' or SO₃ ²' form. In another and/or additional more preferred embodiment, when present in said EDI the anionic ion exchange comprises an SBA Type 2 in any one of OH , Cl or SO₃ ² form.

In another and/or additional preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na-i- form and a WBA in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and a WBA in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and an SBA Type 2 in OH- form.

In another and/or additional preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form.

In another and/or additional preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl' or SO₃ ²' form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in CT or SO₃ ²' form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and a WBA in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and an SBA Type 1 in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in H+ or Na+ form and an SBA Type 2 in Cl or SO₃ ² form.

In another and/or additional preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBAType 1 in Cl’ or SO₃ ²’ form. In an alternative preferred embodiment, when present in said EDI the mixed bed ion exchange comprises an SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl’ or SO₃ ²’ form.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in OH- form in a WBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBAType 1 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBAType 1 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBAType 1 in OH- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na-i- form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na-i- form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in OH- form in a WBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in OH- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and a WBA in OH- form in a WBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 50:50. In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in OH- form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 55:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ^2- form in a WBA:WAC volume ratio of about 50:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ² form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:WAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ² form in an SBA:WAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ² form in an SBA:WAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a WAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:WAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 55:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 50:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ² form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and a WBA in Cl- or SO₃ ^2- form in a WBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- form in a WBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ^2- form in a WBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 50:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ²' form in a WBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and a WBA in Cl- or SO₃ ^2- form in a WBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 1 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 1 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in H+ or Na+ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 50:50.

In another and/or additional preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SOa^2- form in an SBA:SAC volume ratio of about 90:10. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 80:20. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SOa^2- form in an SBA:SAC volume ratio of about 70:30. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ² form in an SBA:SAC volume ratio of about 65:35. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 60:40. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ^2- form in an SBA:SAC volume ratio of about 55: 45. In another preferred embodiment, when present in said EDI, the mixed bed ion exchange comprises a SAC in K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form and an SBA Type 2 in Cl- or SO₃ ²' form in an SBA:SAC volume ratio of about 50:50.

Ion exchange resins can e.g., be provided in packed columns, as membranes, as charge-modified depth filter cartridges or used as a material suspended or fluidized in a liquid that is to be treated with the ion exchange resin. Ion exchange materials typically comprise a matrix provided with fixed functional groups differing between cationic ion exchange materials and anionic ion exchange materials. Examples of suitable ion exchange materials include fibrous gels, microcrystalline gels, or beaded gels. These may be made of e.g., any of the materials selected from polysaccharide-based materials (e.g., agaroses, sepharoses, celluloses; silica-based materials, and organic polymeric matrix material (e.g., polyacrylamides, polystyrenes); that are derivatised to carry anionic or cationic groups.

In another and/or additional preferred embodiment, the cationic ion exchange resin present in said cationic ion exchange, when present, and/or in said mixed bed ion exchange is in a gel-type version, a porous-type version or in a highly porous-type version. In another and/or additional preferred embodiment, the anionic ion exchange resin present in said anionic ion exchange and/or in said mixed bed ion exchange is in a gel-type version, a porous-type version or in a highly porous-type version. In another and/or additional preferred embodiment, the cationic ion exchange resin present in said cationic ion exchange, when present, and/or in said mixed bed ion exchange has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix. In another and/or additional preferred embodiment, the anionic ion exchange resin present in said anionic ion exchange and/or in said mixed bed ion exchange has an acrylic based, a styrene based or a polystyrene based matrix.

In another and/or additional preferred embodiment, the matrix used in said cationic ion exchange resin in said cationic ion exchange, when present, and/or said mixed bed ion exchange and/or in said anionic ion exchange resin in said anionic ion exchange and/or said mixed bed ion exchange further comprises divinylbenzene (DVB). In a more preferred embodiment, DVB is cross-linked to a styrene or a polystyrene based matrix used in said cationic ion exchange resin in said cationic ion exchange, when present, and/or said mixed bed ion exchange and/or in said anionic ion exchange resin in said anionic ion exchange and/or said mixed bed ion exchange.

Examples of cationic ion exchange resins that can be used comprise an Amberlite FPC22H strong acid cation exchange resin (Dupont), a Diaion PK228 strong acid cation exchange resin (Mitsubishi), Amberlite FPC11 Na (Dupont), AmberLite FPC88 (Dupont), AmberLite HPR1100 Na (Dupont), AmberLite HPR2900 Na (Dupont), AmberLite IRC200 Na (Dupont), Diaion SK110L (Mitsubishi), Diaion UBK08 (Mitsubishi), Diaion PK216 (Mitsubishi), a Diaion PK216 strong acid cation exchange resin (Mitsubishi). If not already present in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, the cationic ion exchange resin needs to be regenerated by means known by the person skilled to obtain a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH/form, respectively.

Examples of anionic ion exchange resins that can be used comprise an Amberlite FPA90 (OH-) strong base anion exchange resin (Dupont), an Amberlite FPA51 anion exchange resin (Dupont), an Amberlite FPA77 resin (Dupont), an Amberlite FPA98 resin (Dupont), DOWEX 1x8 200-400 resin (Dow), a Diaion SA20A resin (Mitsubishi) or a Diaion HPA25 strong acid cation exchange resin (Mitsubishi). If not already present in OH- form, the anionic ion exchange resin needs to be regenerated by means known by the person skilled to obtain an anionic ion exchange resin in OH- form.

Mixed bed resins that can be used in a process of present invention comprise but are not limited to Amberlite MB20 resin (Dupont), AmberTec MR-300 UPW (DuPont), AmberTec MR-450 UPW (DuPont), AmberTec UP6040 (DuPont), AmberTec UP6150 (DuPont), DOWEX 50 WX2 200-400 (Dow), DOWEX 50 WX4200-400 (Dow), DOWEX 50 WX8 200-400 (Dow), Resinex NC-3010 (Resinex), Resinex MX-1 (Resinex), MB3710 H/OH (Polysciences). Alternatively, mixed bed resins used in a process of present invention can be made by packing a cationic ion exchange resin and an anionic ion exchange resin in a volume ratio as described herein in a single mixed bed ion exchange column. If not already present in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, the cationic ion exchange resin present in said mixed bed ion exchange needs to be regenerated by means known by the person skilled to obtain a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively. If not already present in OH- form, the anionic ion exchange resin present in said mixed bed ion exchange needs to be regenerated by means known by the person skilled to obtain an anionic ion exchange resin in OH- form. Alternatively, mixed bed resins used in a process of present invention can be made by packing a cationic ion exchange resin as described herein and an anionic ion exchange resin as described herein in a desired volume ratio in a single mixed bed ion exchange column. If not already present in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, the cationic ion exchange resin needs to be regenerated by means known by the person skilled to obtain a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, respectively. If not already present in OH- form, the anionic ion exchange resin needs to be regenerated by means known by the person skilled to obtain an anionic ion exchange resin in OH- form. If not already present in H+ form, the cationic ion exchange resin needs to be regenerated by means known by the person skilled to obtain a cationic ion exchange resin in H+ form. If not already present in OH- form, the anionic ion exchange resin needs to be regenerated by means known by the person skilled to obtain an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or said mixed bed ion exchange is at least 0.5 bed volume / hour (BV/h). In a more preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or said mixed bed ion exchange is at least 1 BV/h. In an even more preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or said mixed bed ion exchange is at least 1.5 BV/h. In an even more preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or said mixed bed ion exchange is at least 2 BV/h. In an even more preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or said mixed bed ion exchange is at least 2.5 BV/h. In a most preferred embodiment, the flow rate through said anionic ion exchange, said cationic ion exchange when present, and /or mixed bed ion exchange is at least 3 BV/h.

In another and/or additional preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step performed on said pH adjusted solution in the process and/or EDI is/are performed at a temperature ranging from 0°C to 80°C, including 0°C and 80°C in the range. In a more preferred embodiment, the cationic ion exchange when present, anionic ion exchange, mixed bed ion exchange step and/or EDI is/are performed at a temperature ranging from 4°C to 60°C, including 4°C and 60°C in the range. In an even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange, mixed bed ion exchange step and/or EDI is/are performed at a temperature ranging from 4°C to 40°C, including 4°C and 40°C in the range. In another even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange, mixed bed ion exchange step and/or EDI is/are performed at a temperature ranging from 4°C to 20°C, including 4°C and 20°C in the range. In another even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange, mixed bed ion exchange step and/or EDI is/are performed at a temperature ranging from 10°C to 20°C, including 10°C and 20°C in the range. In an even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at a temperature ranging from 10°C to 37°C, including 10°C and 37°C in the range. In an even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at a temperature ranging from 20°C to 30°C, including 20°C and 30°C in the range. In an even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at a temperature ranging from 20°C to 25°C, including 20°C and 25°C in the range. In an even more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at a temperature ranging from 22°C to 24°C, including 22°C and 24°C in the range. In a most preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at a temperature ranging from 23°C to 24°C, including 23°C and 24°C in the range. In another more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step is/are performed at room temperature. In another more preferred embodiment, the cationic ion exchange when present, anionic ion exchange and /or mixed bed ion exchange step are performed at a temperature chosen from the list comprising about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, H°C, 12°C, 13°C, 14°C, 15’C, 16°C, 17°C, 18°C, 19°C, 20°C,

21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C,

39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51’C, 52°C, 53°C, 54°C, 55°C, 56°C,

57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C,

75°C, 76°C, 77°C, 78°C, 79°C and 80°C.

In the context of present invention, performing a cationic ion exchange step at a specific temperature is to be understood as that the temperature of the cationic ion exchanger used in said cationic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said cationic ion exchange is adjusted to said specific temperature. Temperature adjustment of a cationic ion exchanger can be performed by temperature adjustment of e.g., the resin, the jacket surrounding the cationic ion exchanger, and/or the environment wherein the cationic ion exchanger is being used.

In the context of present invention, performing an anionic ion exchange step at a specific temperature is to be understood as that the temperature of the anionic ion exchanger used in said anionic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said anionic ion exchange is adjusted to said specific temperature. Temperature adjustment of an anionic ion exchanger can be performed by temperature adjustment of e.g., the resin, the jacket surrounding the anionic ion exchanger, and/or the environment wherein the anionic ion exchanger is being used.

In the context of present invention, performing a mixed bed ion exchange step at a specific temperature is to be understood as that the temperature of the mixed bed ion exchanger used in said mixed bed ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said mixed bed ion exchange is adjusted to said specific temperature. Temperature adjustment of a mixed bed ion exchanger can be performed by temperature adjustment of e.g., the resins, the jacket surrounding the mixed bed ion exchanger, and/or the environment wherein the mixed bed ion exchanger is being used.

In the context of present invention, performing EDI at a specific temperature is to be understood as that the temperature of the EDI is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said EDI is adjusted to said specific temperature. Temperature adjustment of an EDI can be performed by temperature adjustment of e.g., the resin(s) present in the EDI, the jacket surrounding the EDI, and/or the environment wherein the EDI is being used.

In another and/or additional preferred embodiment, the conductivity of said solution after treatment with said EDI is reduced by at least 60%, preferably at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 87.5%, even more preferably at least 90%, even more preferably at least 92.5%, even more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%. At least 60% should be understood as 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 70% should be understood as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 75% should be understood as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 80% should be understood as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 85% should be understood as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 87.5% should be understood as 87.5%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 90% should be understood as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 92.5% should be understood as 92.5%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 95% should be understood as 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 97% should be understood as 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 98% should be understood as 98%, 98.5%, 99%, 99.5% or 100%. At least 99% should be understood as 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

In another and/or additional preferred embodiment, an ash content of said solution after treatment with said EDI is obtained of < 10% on total dry solid, preferably < 9% on total dry solid, more preferably < 8% on total dry solid, even more preferably < 7% on total dry solid, even more preferably < 6% on total dry solid, even more preferably < 5% on total dry solid, even more preferably < 4% on total dry solid, even more preferably < 3% on total dry solid, even more preferably < 2% on total dry solid, even more preferably < 1% on total dry solid, even more preferably < 0.5% on total dry solid, most preferably < 0.1% on total dry solid.

In another and/or additional preferred embodiment, the process further comprises any one or more of the methods selected from the list comprising homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, electrodialysis, electrodeionization, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange, ion exchange chromatography, mixed bed ion exchange, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, anion exchange adsorbent resin, use of an adsorbent material, use of ion exchange resin, evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying in any order. In a more preferred embodiment, any or more of said methods is performed more than one time during said process.

In an additional preferred embodiment, any one or more of said method(s) precede(s) said i) when present, pH adjustment, ii) when present, anionic ion exchange, iii) when present, cationic ion exchange, and/or iv) when present, mixed bed ion exchange. In another and/or additional preferred embodiment, any one or more of said method(s) succeed(s) said i) when present, pH adjustment, ii) when present, anionic ion exchange, iii) when present, cationic ion exchange, and/or iv) when present, mixed bed ion exchange.

In another and/or additional preferred embodiment, any one or more of said method(s) succeed(s), when present, said pH adjustment and precede(s) said i) when present, anionic ion exchange, ii) when present, cationic ion exchange, and/or iii) when present, mixed bed ion exchange. In an alternative preferred embodiment, no one of said method(s) is performed succeeding, when present, said pH adjustment and preceding said i) when present, anionic ion exchange, ii) when present, cationic ion exchange, and/or iii) when present, mixed bed ion exchange. In other words, when present, said pH adjustment of said solution is performed immediately before passing said pH adjusted solution through said i) when present, anionic ion exchange, ii) when present, cationic ion exchange and/or iii) when present, mixed bed ion exchange. In an additional preferred embodiment, any one or more of said method(s) precede(s) said EDI in said process and/or any one or more of said method(s) succeed(s) said EDI in said process.

In another preferred embodiment of present invention, said process comprises EDI wherein said EDI is combined with nanofiltration and/or electrodialysis. Herein, said EDI is combined with said nanofiltration and/or said electrodialysis in any order. In a more preferred embodiment, said nanofiltration and/or electrodialysis is performed twice in said process. In another and/or additional preferred embodiment, said process comprises EDI wherein said EDI is combined with two consecutive steps of nanofiltration. Herein, said EDI can be performed before or after said two consecutive steps of nanofiltration. In a more preferred embodiment, said two consecutive steps of nanofiltration are performed in said process before said EDI.

In another and/or additional preferred embodiment, said process comprises EDI wherein said EDI is combined with two consecutive steps of electrodialysis. Herein, said EDI can be performed before or after said two consecutive steps of electrodialysis. In a more preferred embodiment, said two consecutive steps of electrodialysis are performed in said process before said EDI.

In another and/or additional preferred embodiment, said process comprises two consecutive steps of ultrafiltration prior to EDI. Herein, the membrane molecular weight cut-off of the membrane used in the first ultrafiltration step is higher than the membrane molecular weight cut-off of the membrane used in the second ultrafiltration step.

In another and/or additional preferred embodiment of present invention, the process comprises a first step of ultrafiltration, a second step of nanofiltration and a third step of pH adjustment of said solution in said order before passing said pH adjusted solution in a fourth step through said EDI.

In another and/or additional preferred embodiment, the concentration of magnesium ions, when present, in said solution is reduced below 1000 ppm prior to passing said solution onto said EDI, preferably the concentration of magnesium ions, when present, in said solution is reduced below 500 ppm, more preferably below 400 ppm, more preferably below 300 ppm, more preferably below 200 ppm, more preferably below 100 ppm, more preferably below 50 ppm, more preferably below 10 ppm, preferably by means of any one or more of nanofiltration, electrodialysis, diafiltration, cationic ion exchange.

In another and/or additional preferred embodiment, the concentration of calcium ions, when present, in said solution is reduced below 200 ppm prior to passing said solution onto said EDI, preferably the concentration of calcium ions, when present, in said solution is reduced below 100 ppm, more preferably below 50 ppm, more preferably below 20 ppm, more preferably below 10 ppm, more preferably below 5 ppm, more preferably below 2 ppm, more preferably below 1 ppm, more preferably below 0.5 ppm, more preferably below 0.1 ppm, preferably by means of any one or more of nanofiltration, electrodialysis, diafiltration, cationic ion exchange.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, 4) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, monosaccharide and disaccharide removal through nanofiltration, 4) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) spray drying, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) lyophilization, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, 4) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, monosaccharide and disaccharide removal through nanofiltration, 4) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) lyophilization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, 4) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, 5) concentrating to a syrup of at least 20% dry matter, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, monosaccharide and disaccharide removal through nanofiltration, 4) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) concentrating to a syrup of at least 20% dry matter, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) crystallization, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, 4) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, 6) crystallization, in said order. In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2)

Activated Charcoal treatment, 3) EDI, 4) concentration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) nanofiltration, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) nanofiltration, 3) Activated Charcoal treatment, 4) EDI, 5) cation exchange, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) electrodialysis, 4) Activated Charcoal treatment, 5) EDI, 6) concentration, monosaccharide and disaccharide removal through nanofiltration, 7) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) electrodialysis, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration,

5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) EDI, 3) concentration, monosaccharide and disaccharide removal through nanofiltration, 4) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through microfiltration, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through centrifugation, 2) ultrafiltration, 3) Activated Charcoal treatment, 4) EDI, 5) concentration, monosaccharide and disaccharide removal through nanofiltration, 6) crystallization, in said order.

In another preferred embodiment, the process for the purification of an oligosaccharide from a solution as described herein comprises the following steps: 1) broth clarification through ultrafiltration, 2) Activated Charcoal treatment, 3) EDI, 4) concentration, monosaccharide and disaccharide removal through nanofiltration, 5) crystallization, in said order.

In another and/or additional preferred embodiment, the temperature of the solution is adjusted to a temperature of from 36°C to 65°C, wherein said temperature is within 5°C of a temperature at which the solution exhibits maximum turbidity. Said temperature adjustment can be performed at any time during said process. In a more preferred embodiment, said temperature adjustment is combined with a filtration step. In another more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 36°C to 60°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 40°C to 55°C. In a most preferred embodiment, the temperature of the solution is adjusted to a temperature of from 40°C to 45°C. A temperature of from 36°C to 65°C should be understood as a temperature of 36’C, 37°C, 38°C, 39’C, 40°C, 41°C, 42°C, 43’C, 44°C, 45’C, 46°C, 47°C, 48°C, 49°C, 50’C, 51’C, 52’C, 53°C, 54’C, 55’C, 56°C, 57°C, 58°C, 59°C, 60°C, 61’C, 62°C, 63°C, 64°C or 65°C. A temperature of from 36°C to 50°C should be understood as a temperature of 36°C, 37’C, 38°C, 39’C, 40°C, 41’C, 42’C, 43°C, 44’C, 45°C, 46’C, 47°C, 48’C, 49’C, 50°C, 51’C, 52°C, 53’C, 54°C, 55’C, 56°C, 57’C, 58°C, 59°C or 60°C. A temperature of from 40°C to 55’C should be understood as a temperature of 40°C, 41’C, 42’C, 43°C, 44’C, 45°C, 46’C, 47°C, 48’C, 49°C, 50°C, 51°C, 52°C, 53’C, 54°C or 55°C. A temperature of from 40°C to 45°C should be understood as a temperature of 40°C, 41°C, 42°C, 43°C, 44’C or 45°C.

In another and/or additional preferred embodiment, the temperature of the solution is adjusted to a temperature of from 0°C to 122°C. Said temperature adjustment can be performed at any time during said process. A temperature of from O’C to 122°C should be understood as a temperature of 0°C, l’C, 2°C, 3°C, 4°C, 5’C, 6°C, 7’C, 8°C, 9’C, 10’C, ll’C, 12’C, 13’C, 14’C, 15’C, 16’C, 17°C, 18’C, 19°C, 20°C, 21’C, 22°C, 23’C, 24°C, 25’C, 26°C, 27°C, 28’C, 29°C, 30’C, 31°C, 32’C, 33°C, 34’C, 35°C, 36°C, 37’C, 38°C, 39’C,

40°C, 41’C, 42°C, 43’C, 44°C, 45°C, 46’C, 47°C, 48’C, 49°C, 50’C, 51°C, 52’C, 53°C, 54°C, 55°C, 56°C, 57’C,

58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C,

76°C, 77’C, 78°C, 79’C, 80°C, 81’C, 82’C, 83°C, 84’C, 85°C, 86’C, 87°C, 88’C, 89°C, 90°C, 91’C, 92°C, 93’C,

94°C, 95’C, 96°C, 97°C, 98’C, 99°C, 100’C, 101’C, 102’C, 103°C, 104°C, 105’C, 106°C, 107’C, 108°C, 109°C, 110’C, lire, 112’C, 113°C, 114’C, 115’C, 116°C, 117’C, 118°C, 119°C, 120’C, 121°C or 122°C. In a more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 2°C to 80°C. A temperature of from 2°C to 80°C should be understood as a temperature of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10’C, ll’C, 12°C, 13°C, 14°C, 15°C, 16’C, 17’C, 18’C, 19°C, 20’C, 21°C, 22°C, 23’C, 24°C, 25’C, 26°C, 27’C, 28°C, 29’C, 30°C, 31°C, 32’C, 33°C, 34’C, 35°C, 36’C, 37°C, 38’C, 39’C, 40°C, 41°C, 42°C, 43’C, 44°C, 45’C, 46°C, 47’C, 48’C, 49°C, 50’C, 51’C, 52’C, 53°C, 54’C, 55°C, 56’C, 57’C, 58°C, 59’C, 60°C, 61’C, 62°C, 63’C, 64°C, 65’C, 66°C, 67’C, 68°C, 69’C, 70°C, 71°C, 72’C, 73°C, 74’C, 75°C, 76’C, 77°C, 78°C, 79°C or 80°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 4°C to 60°C. A temperature of from 4°C to 60°C should be understood as a temperature of 4°C, 5’C, 6°C, 7’C, 8°C, 9’C, 10°C, ll’C, 12°C, 13°C, 14’C, 15°C, 16’C, 17’C, 18°C, 19’C, 20°C, 21’C, 22°C, 23’C, 24’C, 25°C, 26’C, 27°C, 28’C, 29°C, 30’C, 31°C, 32’C, 33’C, 34°C, 35’C, 36°C, 37’C, 38°C, 39’C, 40°C, 41’C, 42’C, 43°C, 44’C, 45°C, 46’C, 47°C, 48’C, 49°C, 50’C, 51’C, 52°C, 53°C, 54°C, 55’C, 56°C, 57°C, 58°C, 59°C or 60°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 10°C to 55°C. A temperature of from 10°C to 55°C should be understood as a temperature of 10°C, ll’C, 12’C, 13°C, 14’C, 15°C, 16’C, 17’C, 18°C, 19’C, 20°C, 21’C, 22°C, 23’C, 24°C, 25’C, 26’C, 27°C, 28’C, 29°C, 30’C, 31°C, 32’C, 33°C, 34’C, 35’C, 36°C, 37’C, 38°C, 39’C, 40°C, 41’C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51’C, 52°C, 53°C, 54°C or 55°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 20°C to 45°. A temperature of from 20°C to 45°C should be understood as a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 21°C to 40°C. A temperature of from 21°C to 40°C should be understood as a temperature of 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27’C, 28’C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 22°C to 37°C. A temperature of from 22°C to 37°C should be understood as a temperature of 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C. In an even more preferred embodiment, the temperature of the solution is adjusted to a temperature of from 25°C to 30°C. A temperature of from 25°C to 30°C should be understood as a temperature of 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

In another and/or additional preferred embodiment of present invention, the process comprises a first step of ultrafiltration, a second step of nanofiltration and a third step of pH adjustment of said solution comprising an oligosaccharide in said order before passing said pH adjusted solution in a fourth step through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, ii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na+ form, and succeeding said anionic ion exchange and/or iii) a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺, preferably in Na+ form, and an anionic ion exchange resin in OH- form.

In another preferred embodiment of present invention, the process comprises a first step of ultrafiltration, a second step of nanofiltration and a third step of pH adjustment of said solution comprising a negatively charged, preferably sialylated, oligosaccharide in said order before passing said pH adjusted solution in a fourth step through said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another preferred embodiment of present invention, the process comprises a first step of ultrafiltration, a second step of nanofiltration and a third step of pH adjustment of said solution comprising LSTc and sialyllactose in said order before passing said pH adjusted solution in a fourth step through said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another preferred embodiment, the process further comprises ultrafiltration, nanofiltration and electrodialysis. In another preferred embodiment, the process further comprises ultrafiltration, nanofiltration and electrodeionization. In another preferred embodiment, the process further comprises ultrafiltration, nanofiltration, electrodialysis and electrodeionization. In another preferred embodiment, the process does not further comprise electrodialysis. In another preferred embodiment, the process does not further comprise electrodeionization. In another preferred embodiment, the process further comprises mixed bed ion exchange comprising a cationic ion exchange resin and an anionic ion exchange resin, wherein said cationic ion exchange resin is in any form selected from the list comprising Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ and wherein said anionic ion exchange resin is in any form selected from the list comprising OH-, Cl’ and SO₃ ²’. In another preferred embodiment, the process comprises two mixed bed ion exchanges wherein the cationic ion exchange resins present in both mixed bed ion exchanges are in H+ form and wherein the anionic ion exchange resins present in both mixed bed ion exchanges are in OH- form.

In another and/or additional preferred embodiment, the process comprises EDI of said solution and wherein said EDI is combined in the process with nanofiltration and/or electrodialysis. In a more preferred embodiment, the nanofiltration and/or electrodialysis is performed twice in the process.

In another and/or additional preferred embodiment, the process further comprises ultrafiltration, nanofiltration and electrodialysis. In another and/or additional preferred embodiment, the process further comprises ultrafiltration, nanofiltration and electrodeionization. In another and/or additional preferred embodiment, the process further comprises ultrafiltration, nanofiltration, electrodialysis and electrodeionization. In another and/or additional preferred embodiment of present invention, the process comprises two consecutive steps of nanofiltration. In another and/or additional preferred embodiment of present invention, the process comprises two consecutive steps of electrodialysis. In another and/or additional preferred embodiment, the process comprises two consecutive ultrafiltration steps wherein the membrane molecular weight cut-off used in the first ultrafiltration step is higher than that used in the second ultrafiltration step. In another and/or additional preferred embodiment, the process does not comprise electrodialysis. In another and/or additional preferred embodiment, the process does not comprise ion exchange. In another and/or additional preferred embodiment, the process does not comprise ion exchange chromatography. In another and/or additional preferred embodiment, the process does not comprise electrodeionization. In another and/or additional preferred embodiment, the solution is subjected to two consecutive ultrafiltration steps wherein the membrane molecular weight cut-off used in the first ultrafiltration step is higher than that used in the second ultrafiltration step.

In another and/or additional preferred embodiment, the process further comprises mixed bed ion exchange comprising a cationic ion exchange resin and an anionic ion exchange resin, wherein said cationic ion exchange resin is in any form chosen from the list comprising H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ and wherein said anionic ion exchange resin is in any form chosen from the list comprising OH-, Cl' and SO₃ ²'. In another and/or additional preferred embodiment, the process comprises two mixed bed ion exchanges wherein the cationic ion exchange resins present in both mixed bed ion exchanges are in Na+ form and wherein the anionic ion exchange resins present in both mixed bed ion exchanges are in OH- form. In another and/or additional preferred embodiment, the process comprises two consecutive steps of ultrafiltration prior to said i) pH adjustment, ii) anionic ion exchange using an anionic ion exchange resin in OH- form, iii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺ form, preferably in Na+ form, iv) mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺, NH₄ ⁺, preferably in Na+ form, and an anionic ion exchange resin in OH- form, and/or v) said EDI, respectively, and wherein the membrane molecular weight cut-off of the membrane used in the first ultrafiltration step is higher than the membrane molecular weight cut-off of the membrane used in the second ultrafiltration step.

In another and/or additional preferred embodiment, the process further comprises clarification, preferably wherein said clarification is performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration.

In another and/or additional preferred embodiment, the process further comprises use of a cation exchange adsorbent resin, an anion exchange adsorbent resin and/or use of an adsorbent material.

In another and/or additional preferred embodiment, the process further comprises drying, preferably wherein said drying is chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and agitated thin film drying.

In another and/or additional preferred embodiment, the process further comprises filtration, preferably wherein said filtration is performed by use of a filtration aid and/or flocculant. Preferably said filtration aid is an adsorbing agent, more preferably said filtration aid is active carbon.

In another and/or additional preferred embodiment, the process further comprises ultrafiltration, preferably wherein said ultrafiltration has a molecular weight cut-off equal to or higher than 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa.

In another and/or additional preferred embodiment, the solution is subjected to two consecutive ultrafiltration steps, preferably wherein the membrane molecular weight cut-off used in the first ultrafiltration step is higher than that used in the second ultrafiltration step.

In another and/or additional preferred embodiment, the process further comprises nanofiltration, preferably wherein the nanofiltration membrane used in said nanofiltration has a size exclusion limit of < 20 A, in other words said nanofiltration has a size exclusion limit of 1 A, 2 A, 3 A, 4 A, 5 A, 6 A, 7 A, 8 A, 9 A, 10 A, 11 A, 12 A, 13 A, 14 A, 15 A, 16 A, 17 A, 18 A, 19 A or 20 A.

In another and/or additional preferred embodiment, the process further comprises diafiltration, preferably wherein said diafiltration is performed until a conductivity is reached of < 15 mS/cm, preferably < 10 mS/cm, < 5 mS/cm, < 1 mS/cm, < 0.1 mS/cm, < 0.01 mS/cm, < 0.001 mS/cm. In a more preferred embodiment, the process further comprises diafiltration, wherein said diafiltration is performed on the solution until a conductivity is reached of any one of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mS/cm. In another and/or additional preferred embodiment, the process further comprises microfiltration, preferably wherein the pore openings in the membrane used in the microfiltration are ranging from 0.1 to 1 pm (micron).

In another and/or additional preferred embodiment, the process further comprises ultrafiltration, preferably wherein the pore openings in the membrane used in the ultrafiltration are ranging from 0.01 to 0.1 pm (micron).

In another and/or additional preferred embodiment, the process further comprises nanofiltration, preferably wherein the pore openings in the membrane used in the nanofiltration are ranging from 0.001 to 0.01 pm (micron).

In another and/or additional preferred embodiment, the process further comprises reverse osmosis, preferably wherein the pore openings in the membrane used in the reverse osmosis are ranging from 0.0001 to 0.001 pm (micron).

In another and/or additional preferred embodiment, the process further comprises nanofiltration, preferably wherein said nanofiltration is performed at a pressure ranging from 5 to 20 bar. In other words, said nanofiltration is performed at a pressure of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bar.

In another and/or additional preferred embodiment, the process further comprises an enzymatic treatment, preferably wherein the enzymatic treatment comprises incubation of the solution with one or more enzymes selected from the group comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.

In another and/or additional preferred embodiment, the process further comprises an enzymatic treatment, preferably wherein the enzymatic treatment converts lactose, sucrose, maltooligosaccharides, maltotriose, sorbitol, trehalose, starch, cellulose, hemi-cellulose, lignocellulose, molasses, corn-steep liquor and/or high-fructose syrup to monosaccharides.

In another and/or additional preferred embodiment, the process further comprises a mixed bed ion exchange that is performed at a temperature ranging from 0°C to 80°C, including 0°C and 80°C in the range. In a more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 4°C to 60°C, including 4°C and 60°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 4°C to 40°C, including 4°C and 40°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 10°C to 37°C, including 10°C and 37°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 20°C to 30°C, including 20°C and 30°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 20°C to 25°C, including 20°C and 25°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 22°C to 24°C, including 22°C and 24°C in the range. In an even more preferred embodiment, the mixed bed ion exchange step is performed at a temperature ranging from 23°C to 24°C, including 23°C and 24°C in the range. In another more preferred embodiment, the mixed bed ion exchange step is performed at a temperature selected from the list comprising about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, H°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C,

21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31’C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C,

75°C, 76°C, 77°C, 78°C, 79°C and 80°C.

In another and/or additional preferred embodiment, the process is a batch process. In an alternative and/or additional preferred embodiment, the process is a continuous process.

In another and/or additional preferred embodiment, the solution has an ash content of > 10 % on total dry solid before purification by the process as described herein. In the context of present invention, the ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

In another and/or additional preferred embodiment, the solution comprises a lead content > 0.1 mg/kg dry solid before purification by the process as described herein.

In another and/or additional preferred embodiment, the solution comprises an arsenic content > 0.2 mg/kg dry solid before purification by the process as described herein.

In another and/or additional preferred embodiment, the solution comprises a cadmium content > 0.1 mg/kg dry solid before purification by the process as described herein.

In another and/or additional preferred embodiment, the solution comprises a mercury content > 0.5 mg/kg dry solid before purification by the process as described herein.

In another and/or additional preferred embodiment, the oligosaccharide is accompanied in a solution by sialic acid and/or ashes and the oligosaccharide is purified from said sialic acid and/or said ashes by a process of present invention. In a more preferred embodiment, the oligosaccharide is accompanied in a solution by sialic acid and/or ashes and the oligosaccharide is purified from said sialic acid and/or said ashes by a process of present invention, the process comprising: pH adjustment of said solution comprising said oligosaccharide and said sialic acid and/or ashes to a pH of about 3, and passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, ii) when present, a cationic ion exchange using a cationic ion exchange resin Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na+ form, and/or iii) a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na+ form, and an anionic ion exchange resin in OH- form. In another and/or additional preferred embodiment, the oligosaccharide is accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and the oligosaccharide and the one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by a process of present invention. In a more preferred embodiment, the oligosaccharide is accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and the oligosaccharide and the one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by a process of present invention, the process comprising: pH adjustment of said solution comprising said i) oligosaccharide and one or more other oligosaccharide(s) and ii) said sialic acid and/or ashes to a pH of about 3, and passing said pH adjusted solution through i) an anionic ion exchange using an anionic ion exchange resin in OH- form, ii) when present, a cationic ion exchange using a cationic ion exchange resin Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na+ form, and/or iii) a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH form, preferably in Na-i- form, and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, the negatively charged, preferably sialylated, oligosaccharide is accompanied in a solution by sialic acid and/or ashes and the negatively charged, preferably sialylated, oligosaccharide is purified from said sialic acid and/or said ashes by a process of present invention.

In a more preferred embodiment, the negatively charged, preferably sialylated, oligosaccharide is accompanied in a solution by sialic acid and/or ashes and the negatively charged, preferably sialylated, oligosaccharide is purified from said sialic acid and/or said ashes by a process of present invention, the process comprising: pH adjustment of said solution comprising said negatively charged, preferably sialylated, oligosaccharide and said sialic acid and/or ashes to a pH of about 3, and passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, the negatively charged, preferably sialylated, oligosaccharide is accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and the negatively charged, preferably sialylated, oligosaccharide and the one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by a process of present invention.

In a more preferred embodiment, the negatively charged, preferably sialylated, oligosaccharide is accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and the negatively charged, preferably sialylated, oligosaccharide and the one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by a process of present invention, the process comprising: pH adjustment of said solution comprising said i) negatively charged, preferably sialylated, oligosaccharide and one or more other oligosaccharide(s) and ii) said sialic acid and/or ashes to a pH of about 3, and passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, said LSTc and sialyllactose are accompanied in a solution by sialic acid, ashes, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) and said LSTc is purified i) from said sialyllactose and ii) from said sialic acid, ashes, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) by a process of present invention.

In a more preferred embodiment, said LSTc and sialyllactose are accompanied in a solution by sialic acid, ashes, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) and said LSTc is purified i) from said sialyllactose and ii) from said sialic acid, ashes, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) by a process of present invention, the process comprising: pH adjustment of said solution to a pH of about 6.5, and passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment, said LSTc and sialyllactose are accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and said LSTc and the one or more other oligosaccharide(s) are purified from i) said sialyllactose and ii) said sialic acid and/or said ashes by a process of present invention.

In a more preferred embodiment, said LSTc and sialyllactose are accompanied in a solution by i) one or more other oligosaccharide(s) and ii) sialic acid and/or ashes and said LSTc and the one or more other oligosaccharide(s) are purified from i) said sialyllactose and ii) said sialic acid and/or said ashes by a process of present invention, the process comprising: pH adjustment of said solution comprising said i) LSTc and one or more other oligosaccharide(s) and ii) said sialyllactose and iii) said sialic acid and/or ashes, to a pH of about 6.5, and passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

In another and/or additional preferred embodiment of the present invention, the purity of the oligosaccharide, the negatively charged, preferably sialylated, oligosaccharide or of LSTc obtained in the purified oligosaccharide solution at the end of the process is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% on total dry solid. At least 70% should be understood as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 75% should be understood as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 80% should be understood as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 85% should be understood as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 90% should be understood as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 95% should be understood as 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 97% should be understood as 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 98% should be understood as 98%, 98.5%, 99%, 99.5% or 100%. At least 99% should be understood as 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

In another and/or additional preferred embodiment of the present invention, the yield of purification of the oligosaccharide, the negatively charged, preferably sialylated, oligosaccharide or of LSTc obtained in the purified oligosaccharide solution at the end of the process is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. At least 60% should be understood as 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% ,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 65% should be understood as 65%, 66%, 67%, 68% ,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 70% should be understood as 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 75% should be understood as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 80% should be understood as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 85% should be understood as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 90% should be understood as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. At least 95% should be understood as 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 97% should be understood as 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%. At least 98% should be understood as 98%, 98.5%, 99%, 99.5% or 100%. At least 99% should be understood as 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

In another and/or additional preferred embodiment of the present invention, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium. In another and/or additional preferred embodiment of the present invention, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid, preferably < 9% on total dry solid, more preferably < 8% on total dry solid, even more preferably < 7% on total dry solid, even more preferably < 6% on total dry solid, even more preferably < 5% on total dry solid, even more preferably < 4% on total dry solid, even more preferably < 3% on total dry solid, even more preferably < 2% on total dry solid, even more preferably < 1% on total dry solid, most preferably < 0.5% on total dry solid. In a more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has an ash content of any one of 10%, 9%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% on total dry solid.

In another and/or additional preferred embodiment of the present invention, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid, preferably with a lead content lower than 0.1 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid and/or a mercury content lower than 0.5 mg/kg dry solid.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has a lead content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably below 0.01 mg/kg dry solid. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has an arsenic content lower than 0.2 mg/kg dry solid, more preferably lower than 0.1 mg/kg, even more preferably lower than 0.05 mg/kg dry solid, even more preferably lower than 0.02 mg/kg dry solid. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has a cadmium content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably lower than 0.01 mg/kg dry solid. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has a mercury content lower than 0.5 mg/kg dry solid, more preferably lower than 0.2 mg/kg dry solid, even more preferably below 0.1 mg/kg, even more preferably lower than 0.005 mg/kg dry solid.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is filter-sterilized. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is subjected to endotoxin removal. Preferably, endotoxin removal is performed by filtration through a 3 kDa filter, i.e., filtration with a membrane having a molecular weight cut-off of 3 kDa.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has a protein content below 100 mg per kg dry solid, a DNA content below 10 ng per gram dry solid and/or an endotoxin content below 10000 EU per gram dry solid. In a more preferred Ill embodiment, the purified oligosaccharide solution obtained at the end of the process is free of DNA, proteins, and/or recombinant genetic material.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further concentrated. Concentration can be performed by means of one or more of nanofiltration, diafiltration, reverse osmosis, evaporation, wiped film evaporation, and falling film evaporation. In another and/or additional preferred embodiment, the process further comprises any one or more of nanofiltration, diafiltration, reverse osmosis, evaporation, wiped film evaporation, and falling film evaporation, wherein one or more of said nanofiltration, diafiltration, reverse osmosis, evaporation, wiped film evaporation, and falling film evaporation is performed more than one time during the process. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further concentrated to a syrup of at least 30% dry matter. In a more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further concentrated to a syrup of at least 40% dry matter.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is first subjected to a polishing step prior to concentration. For this polishing step, an adsorbent material, such as activated carbon or charcoal, a cation exchange adsorbent resin, an anion exchange adsorbent resin or a charge-modified depth filter can be used.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further crystallised. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further dried to a powder. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further granulated.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is further concentrated by a method to an oligosaccharide concentration of > 100 g/L, preferably > 200 g/L, more preferably > 300 g/L, more preferably > 400 g/L, more preferably > 500 g/L, more preferably > 600 g/L, most preferably between 300 g/L and 650 g/L. Preferably, said concentration is performed at a temperature of < 80°C, preferably < 60°C, more preferably < 50°C, more preferably 20°C to 50°C, even more preferably 30°C to 45°C. Herein, 20°C to 50°C is to be understood as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. Herein, 30°C to 45°C is to be understood as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C. Preferably, any of said concentration method is chosen from the list comprising using vacuum evaporation or reverse osmosis or nanofiltration. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process comprises an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, or LSTc which is concentrated to a concentration of > 1.5 M and cooled to a temperature < 25 °C, more preferably < 8 °C, to obtain crystalline material of the oligosaccharide, the negatively charged, preferably sialylated, oligosaccharide, or LSTc. A temperature < 25°C is to be understood as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15’C, 16°C, 17’C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or a temperature below 0°C. A temperature of < 8°C is to be understood as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C or a temperature below 0°C.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process has a Brix value of from about 8 to about 75%, preferably the purified oligosaccharide solution has a Brix value of from about 30 to about 65%.

In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is dried by any one or more of drying steps selected from the list comprising spray drying, lyophilization, evaporation, precipitation, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying and agitated thin film drying. In a more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is dried by spray-drying, freeze-drying or agitated thin film drying. In an additional more preferred embodiment, the pH of the purified oligosaccharide solution is ranging from 2 to 5. In other words, the pH of the purified oligosaccharide solution is any one of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5. In an additional even more preferred embodiment, the pH of the purified oligosaccharide solution is ranging from 3 to 5; in other words, the pH of the purified oligosaccharide solution is any one of 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5. In an additional even more preferred embodiment, the pH of the purified oligosaccharide solution is ranging from 4 to 5; in other words, the pH of the purified oligosaccharide solution is any one of 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5. In another more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is dried by spraydrying, particularly spray-dried at an oligosaccharide solution concentration of 20-60 (w/v), preferably 30- 50 (w/v), more preferably 35-45 (w/v), with a nozzle temperature of 110-150°C, preferably 120-140°C, more preferably 125-135°C and/or an outlet temperature of 60-80°C, preferably 65-70°C.

According to another aspect, the present invention provides a purified oligosaccharide solution, a purified oligosaccharide, a purified negatively charged, preferably sialylated, oligosaccharide, purified LSTc or a purified oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, or LSTc, respectively, obtainable, preferably obtained, by a process as described herein. The purified oligosaccharide solution can comprise one purified oligosaccharide or a purified oligosaccharide mixture. Alternatively, the purified oligosaccharide solution can comprise one purified negatively charged, preferably sialylated, oligosaccharide or a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide. Alternatively, the purified oligosaccharide solution can comprise purified LSTc or a purified oligosaccharide mixture comprising LSTc.

In a preferred and/or additional embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is dried. In a more preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is spray- dried. In an alternative more preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is lyophilized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is crystallized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides a purified oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is concentrated to a syrup of at least 40% dry matter.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is dried. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is spray-dried. In an alternative more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is lyophilized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is crystallized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising an oligosaccharide is concentrated to a syrup of at least 40% dry matter.

In another preferred and/or additional embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is dried. In a more preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is spray-dried. In an alternative more preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is lyophilized.

In another and/or additional preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is crystallized.

In another and/or additional preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides a purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 40% dry matter.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is dried. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is spray-dried. In an alternative more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is lyophilized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is crystallized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is concentrated to a syrup of at least 40% dry matter.

In another preferred and/or additional embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is dried. In a more preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is spray-dried. In an alternative more preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is lyophilized.

In another and/or additional preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is crystallized.

In another and/or additional preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides purified LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified LSTc is concentrated to a syrup of at least 40% dry matter.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising LSTc is dried. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is spray-dried. In an alternative more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is dried via an agitated thin film dryer.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is lyophilized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is crystallized.

In another and/or additional preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is concentrated to a syrup of at least 20% dry matter. In a more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is concentrated to a syrup of at least 30% dry matter. In an even more preferred embodiment, the present invention provides a purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process as described herein, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising said LSTc is concentrated to a syrup of at least 40% dry matter.

In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sia lylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein and that has an ash content of < 10% on total dry solid after said process. In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sia lylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein and that has an ash content of < 10% on total dry solid after said process, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium. An ash content of < 10% on total dry solid is to be understood as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than 0.5% ash on total dry solid or less than 0.1% ash on total dry solid. Preferably, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, is produced through cell cultivation.

In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a lead content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably below 0.01 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has an arsenic content lower than 0.2 mg/kg dry solid, more preferably lower than 0.1 mg/kg, even more preferably lower than 0.05 mg/kg dry solid, even more preferably lower than 0.02 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a cadmium content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably lower than 0.01 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a mercury content lower than 0.5 mg/kg dry solid, more preferably lower than 0.2 mg/kg dry solid, even more preferably below 0.1 mg/kg, even more preferably lower than 0.005 mg/kg dry solid after said process. According to a next aspect, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc or a spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein and wherein said spray-dried oligosaccharide or spray-dried oligosaccharide mixture obtained after said process has an ash content of < 10% on total dry solid. In a preferred embodiment, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc or a spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein and wherein said spray-dried oligosaccharide or spray-dried oligosaccharide mixture obtained after said process has an ash content of < 10% on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium. An ash content of < 10% on total dry solid is to be understood as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than 0.5% ash on total dry solid or less than 0.1% ash on total dry solid. Preferably, said spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or said spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, is produced through cell cultivation.

In another and/or additional preferred embodiment, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a lead content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably below 0.01 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has an arsenic content lower than 0.2 mg/kg dry solid, more preferably lower than 0.1 mg/kg, even more preferably lower than 0.05 mg/kg dry solid, even more preferably lower than 0.02 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a cadmium content lower than 0.1 mg/kg dry solid, more preferably lower than 0.05 mg/kg dry solid, even more preferably below 0.02 mg/kg dry solid, even more preferably lower than 0.01 mg/kg dry solid after said process.

In another and/or additional preferred embodiment, the present invention provides a spray-dried oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or spray-dried oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, that is purified according to a process as described herein, and that has a mercury content lower than 0.5 mg/kg dry solid, more preferably lower than 0.2 mg/kg dry solid, even more preferably below 0.1 mg/kg, even more preferably lower than 0.005 mg/kg dry solid after said process.

According to another aspect, the present invention provides a dried powder of purified oligosaccharide solution obtained from a process as described herein, wherein said dried powder contains < 15%-wt. of water. A dried powder containing < 15%-wt. of water is to be understood as a dried powder containing 15%-wt., 14%-wt., 13%-wt., 12%-wt., 11%-wt., 10%-wt., 9%-wt., 8%-wt., 7%-wt., 6%-wt., 5%-wt., 4%-wt., 3%-wt., 2%-wt., 1%-wt., 0.5%-wt., 0.4%-wt., 0.3%-wt., 0.2%-wt., 0.1%-wt. or 0%-wt. of water. In a preferred embodiment, said powder contains < 10%-wt. of water; in other words, said powder contains 10%-wt., 9%-wt., 8%-wt., 7%-wt., 6%-wt., 5%-wt., 4%-wt., 3%-wt., 2%-wt., 1%-wt., 0.5%-wt., 0.4%-wt., 0.3%-wt., 0.2%-wt., 0.1%-wt. or 0%-wt. of water. In a more preferred embodiment, said powder contains < 7%-wt. of water; in other words, said powder contains 7%-wt., 6%-wt., 5%-wt., 4%-wt., 3%-wt., 2%-wt., 1%-wt., 0.5%-wt., 0.4%-wt., 0.3%-wt., 0.2%-wt., 0.1%-wt. or 0%-wt. of water. In a most preferred embodiment, said powder contains < 5%-wt. of water; in other words, said powder contains 5%-wt., 4%- wt., 3%-wt., 2%-wt., 1%-wt., 0.5%-wt., 0.4%-wt., 0.3%-wt., 0.2%-wt., 0.1%-wt. or 0%-wt. of water.

According to another aspect, the present invention provides a dried powder, preferably a spray-dried powder, of purified oligosaccharide solution obtained from a process as described herein, wherein said dried powder, preferably spray-dried powder, has a mean particle size of 50 to 250 pm as determined by laser diffraction. In a preferred embodiment, said dried powder, preferably spray-dried powder, has a mean particle size of 95 to 120 pm as determined by laser diffraction. In a more preferred embodiment, said dried powder, preferably spray-dried powder, has a mean particle size of 110 to 120 pm as determined by laser diffraction.

In a preferred embodiment, the present invention provides dried powder of purified oligosaccharide solution obtained from a process as described herein, wherein said powder exhibits a loose bulk density of from about 500 to 700 g/L, a lOOx tapped bulk density of from about 600 to about 850 g/L, a 625x tapped bulk density of from about 600 to about 900 g/L, and/or a 1250x tapped bulk density of from about 650 to about 900 g/L

In another preferred embodiment, the present invention provides dried powder of purified oligosaccharide solution obtained from a process as described herein, wherein said powder exhibits a loose bulk density of from about 600 to 700 g/L, a lOOx tapped bulk density of from about 750 to about 850 g/L, a 625x tapped bulk density of from about 750 to about 850 g/L, and/or a 1250x tapped bulk density of from about 850 to about 900 g/L

In another preferred embodiment, the present invention provides dried powder of purified oligosaccharide solution obtained from a process as described herein, wherein said powder exhibits a loose bulk density of from about 500 to 600 g/L, a lOOx tapped bulk density of from about 600 to about 700 g/L, a 625x tapped bulk density of from about 700 to about 800 g/L, and/or a 1250x tapped bulk density of from about 750 to about 800 g/L

In another and/or additional embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH between 4 and 7. In other words, said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH of 4, 4.5, 5, 5.5, 6, 6.5 or 7. In a preferred embodiment, said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH between 4 and 6, i.e. with a pH of 4, 4.5, 5, 5.5 or 6. In a more preferred embodiment, said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH between 4 and 5, i.e. with a pH of 4, 4.5 or 5. In an even more preferred embodiment, said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH between 5 and 6, i.e. with a pH of 5, 5.5 or 6.

In another and/or additional embodiment, the present invention provides for a purified oligosaccharide or negatively charged, preferably sialylated, oligosaccharide as described herein wherein any one or more of said purified oligosaccharide or negatively charged, preferably sialylated, oligosaccharide is a milk oligosaccharide. In another embodiment, the present invention provides for a purified oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc as described herein wherein said purified oligosaccharide mixture comprises a milk oligosaccharide. In a preferred embodiment, the milk oligosaccharide is a mammalian milk oligosaccharide (MMO). In a more preferred embodiment, the milk oligosaccharide is a human milk oligosaccharide (HMO). In an additional preferred embodiment, the milk oligosaccharide a neutral (non-charged) milk oligosaccharide, preferably a neutral (non-charged) human milk oligosaccharide (HMO), selected from the list comprising 2'- fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N- neotetraose, lacto-N-fucopentaose I, lacto-N neofucopentaose, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose and any combination thereof. In an alternative preferred embodiment, the milk oligosaccharide is a sialylated milk oligosaccharide, preferably a sialylated human milk oligosaccharide (HMO), selected from the list comprising 3'sialyllactose, 6'sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyl lacto-N- tetraose c, sialyllacto-N-tetraose d, disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto- N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucod isialyllacto- N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof.

In another and/or additional embodiment, the present invention provides for a purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or purified oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc as described herein, wherein the purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, or purified oligosaccharide mixture a) has a conductivity of less than 10 mS/cm at a 300 g/L solution; b) is free of recombinant DNA material, optionally free of any DNA; and/or c) is free of proteins derived from the recombinant micro-organism, optionally free of any proteins.

For identification of said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc as described herein, the monomeric building blocks (e.g. the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion- Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, methods such as e.g. acid-catalysed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC/MS (gas-liquid chromatography coupled with mass spectrometry). To determine the glycan sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alpha-glucosidase, etc., and NMR may be used to analyse the products.

In another aspect, the present invention provides for a purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc or purified oligosaccharide mixture comprising an oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide or LSTc as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

In another aspect, the present invention provides use of a purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc obtained by a process as described herein in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient. In some embodiments, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. Said purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications. In another aspect, the present invention provides use of milk oligosaccharide as described herein as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food. In the context of present invention, the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement and the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed. In another preferred embodiment, a preparation is provided that further comprises at least one probiotic microorganism. In another preferred embodiment of present invention, said preparation is a nutritional composition. In a more preferred embodiment, said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula. A "prebiotic" is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including said oligosaccharide being a prebiotic purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and 8. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, said oligosaccharide produced and/or purified by a process of this specification is orally administered in combination with such microorganism. Examples of further ingredients for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.

In some embodiments, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc purified by a process as described herein is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, said oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc purified by a process as described herein is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, said purified oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs). In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil. In some embodiments, the concentration of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc in the infant formula is approximately the same concentration as the concentration of the oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, generally present in human breast milk. In some embodiments, an oligosaccharide, negatively charged, preferably sialylated, oligosaccharide or LSTc purified by a process as described herein is added to the infant formula with a concentration that is approximately the same concentration as the concentration of the compound generally present in human breast milk.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

Further advantages follow from the specific embodiments and the examples. It goes without saying that the abovementioned features and the features which are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of the present invention.

Moreover, the present invention relates to the following specific embodiments:

1. A process for purification of an oligosaccharide from a solution, wherein said solution comprising said oligosaccharide is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation, the process comprising: i) pH adjustment of said solution to a pH ranging from 2 to 7, preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and ii) passing said pH adjusted solution through: an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably said cationic ion exchange resin is in Na⁺ form, and/or a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and an anionic ion exchange resin in OH- form, preferably said cationic ion exchange resin in said mixed bed ion exchange is in Na⁺ form.

2. A process for purification of an oligosaccharide from a solution, wherein said solution comprising said oligosaccharide is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution and a cell cultivation, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation, characterized in that said process comprises electrodeionization (EDI) of said solution.

3. Process according to embodiment 2, wherein said EDI comprises: cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in H⁺ or Na⁺ form, anionic ion exchange using an anionic ion exchange resin in OH Cl- or SO₃ ²’ form, preferably in OH" form, and/or mixed bed ion exchange comprising a cationic ion exchange using a cationic ion exchange resin in H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in H⁺ or Na⁺ form, and an anionic ion exchange using an anionic ion exchange resin in OH; Cl’ or SO₃ ²’ form, preferably in OH’ form. Process according to any one of embodiment 2 or 3, wherein the pH of said solution is adjusted to a pH ranging from 2 to 7 , preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, prior to passing said solution onto said EDI. Process according to any one of previous embodiments, wherein said oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, negatively charged, preferably sialylated, oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing neutral (non-charged) oligosaccharide, N-acetyllactosamine containing oligosaccharide, lacto-N-biose containing oligosaccharide, a galactose containing oligosaccharide, non-fucosylated neutral (non-charged) oligosaccharide, chitosan, chitosan comprising oligosaccharide, heparosan, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, chondroitin sulphate, dermatan sulphate, hyaluronan or hyaluronic acid, keratan sulphate, a milk oligosaccharide, O-antigen, enterobacterial common antigen (ECA), the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan, an aminosugar, an antigen of the human ABO blood group system, an animal oligosaccharide, a plant oligosaccharide, erlose (Glc-al,4-Glc-al,2-Fru), lactul-N-triose II (GlcNAc-bl,3-Gal-bl,4-Fru), lactul-N- tetraose, lactul-N-neotetraose, globotriose; preferably wherein said milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); preferably wherein said animal oligosaccharide is selected from the list consisting of N-glycans and O- glycans; preferably wherein said plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans; preferably wherein said fucosylated oligosaccharide is selected from the list comprising 2'-fucosyl lactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto- N-neofucopentaose V, lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), Fuc- al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc- al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3- Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,4- (Fuc-al,2-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, monofucosyllacto-N-hexaose-lll, difucosyllacto-N-hexaose (a), difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3- )Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N- acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllacto-N- biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc; preferably wherein said neutral (non-charged) oligosaccharide is a milk oligosaccharide, preferably a neutral (non-charged) mammalian milk oligosaccharide (MMO), more preferably a neutral (noncharged) human milk oligosaccharide (HMD), selected from the list comprising 2'-fucosyllactose, 3- fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N- fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-neofucopentaose V, lacto-N- difucohexaose I, lacto-N-neodifucohexaose, lacto-N-difucohexaose II, monofucosyllacto-n-hexaose III, difucosyllacto-N-hexaose a, 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose and combinations thereof; preferably wherein the N-acetylglucosamine containing neutral (non-charged) oligosaccharide is selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 4'-galactosyllactose, 3'-galactosyllactose, GIcNAc- bl,6-Gal-bl,4-Glc, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (pLNH), para-lacto-N-neohexaose (pLNnH), GlcNAc-bl,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, lacto-N-pentaose (LN5), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-heptaose (LN7), lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N- neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose (pLNnO), iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose (LN9), lacto-N-decaose, iso lacto-N-decaose, novo lacto-N- decaose, lacto-N-neodecaose, para lacto-N-neodecaose (pLNnD), al,3-galactosyllacto-N- neotetraose, GlcNAc-bl,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, GlcNAc-bl,6-(Gal-bl,4-GlcNAc-bl,3- )Gal-bl,4-Glc and GlcNAc-bl,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc. A process for purification of a negatively charged, preferably sialylated, oligosaccharide from a solution, the process comprising: i) pH adjustment of said solution to a pH ranging from 2 to 5, preferably from 3 to 5, more preferably from 4 to 5, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. Process according to embodiment 6, wherein said solution comprising said negatively charged, preferably sialylated, oligosaccharide is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process, wherein said negatively charged, preferably sialylated, oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation.

8. A process for purification of sialyllacto-N-tetraose c (LSTc; Neu5Ac-oc2,6-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc) from a solution comprising LSTc and a sialyllactose, the process comprising: i) pH adjustment of said solution to a pH ranging from 4 to 7, preferably from 5 to 7, more preferably from 6 to 7, even more preferably to a pH of 6.5, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

9. Process according to embodiment 8, wherein said solution comprising said LSTc and sialyllactose is a solution selected from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process, wherein said LSTc and sialyllactose are produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation.

10. Process according to any one of embodiment 8 or 9, wherein said sialyllactose is selected from the list comprising 3' -sialyllactose (3'SL, Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactose (6'SL, Neu5Ac-a2,6- Gal-pi,4-Glc) and 8' -sialyllactose (8'SL, Neu5Ac-<x2,8-Gal-pi,4-Glc), preferably said sialyllactose is 6'SL.

11. Process according to any one of embodiments 1, 4 to 6, 8 to 10, wherein said pH adjustment is obtained by any one or more of addition of an acidic agent, an alkaline agent and/or a buffered solution; filtration; nanofiltration; dialysis; electrodialysis; electrodeionization; ion exchange; mixed bed ion exchange; ion exchange chromatography; reverse osmosis; use of activated carbon or charcoal.

12. Process according to any one of embodiments 1, 3 to 11, wherein said cationic ion exchange resin in said cationic ion exchange, when present, and/or said mixed bed ion exchange: is selected from the list consisting of a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin, and/or has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix.

13. Process according to any one of embodiments 1, 3 to 12, wherein said anionic ion exchange resin in said anionic ion exchange and/or said mixed bed ion exchange: is selected from the list consisting of a weak base anion (WBA) exchange resin, a strong base anion (SBA) exchange resin Type 1 and an SBA exchange resin Type 2 and/or has an acrylic based, a styrene based or a polystyrene based matrix.

14. Process according to any one of embodiments 12 or 13, wherein said matrix further comprises divinylbenzene (DVB). 15. Process according to any one of embodiments 1, 3 to 14, wherein said mixed bed ion exchange comprises an ion exchange column packed with: a mixture of said cationic ion exchange resin and said anionic ion exchange resin in any volume ratio and/or alternating layers of said cationic ion exchange resin and said anionic ion exchange resin.

16. Process according to any one of embodiments 1, 3 to 15, wherein in said mixed bed ion exchange the total ion exchange capacity of said anionic ion exchange resin is equal to the total ion exchange capacity of said cationic ion exchange resin.

17. Process according to any one of embodiments 1, 3 to 16, wherein said mixed bed ion exchange contains more of said anionic ion exchange resin than of said cationic ion exchange resin.

18. Process according to any one of embodiments 1, 3 to 17, wherein the volume ratio of said anionic ion exchange resin to said cationic ion exchange resin in said mixed bed ion exchange is about 90:10, about 80:20, about 70:30, about 65:35, about 60:40, about 55:45.

19. Process according to any one of embodiments 1 to 16, wherein the volume ratio of said cationic ion exchange resin to said anionic ion exchange resin in said mixed bed ion exchange is about 50:50.

20. Process according to any one of embodiments 1, 3 to 19, wherein the flow rate through said anionic ion exchange, said cationic ion exchange, when present, and/or said mixed bed ion exchange is at least 0.5 bed volume / hour (BV/h), preferably at least 1 BV/h, more preferably at least 2 BV/h, more preferably at least 2.5 BV/h, most preferably at least 3 BV/h.

21. Process according to any one of embodiments 1, 3 to 20, wherein said anionic ion exchange, said cationic ion exchange, when present, and /or said mixed bed ion exchange is/are performed at a temperature ranging from 0°C to 80°C, preferably from 4°C to 60°C, more preferably from 4°C to 40°C, even more preferably from 4°C to 20°C, even more preferably from 10°C to 20°C, even more preferably from 10°C to 37°C, even more preferably from 20°C to 30°C, even more preferably from 20°C to 25°C, even more preferably from 22°C to 24°C, even more preferably from 23°C to 24°C.

22. Process according to any one of embodiments 2 to 5, 11 to 21, wherein the conductivity of said solution after treatment with said EDI is reduced by at least 60%, preferably at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 87.5%, even more preferably at least 90%, even more preferably at least 92.5%, even more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, most preferably at least 99%.

23. Process according to any one of embodiments 2 to 5, 11 to 22, wherein an ash content of said solution after treatment with said EDI is obtained of < 10% on total dry solid, preferably < 9% on total dry solid, more preferably < 8% on total dry solid, even more preferably < 7% on total dry solid, even more preferably < 6% on total dry solid, even more preferably < 5% on total dry solid, even more preferably < 4% on total dry solid, even more preferably < 3% on total dry solid, even more preferably < 2% on total dry solid, even more preferably < 1% on total dry solid, even more preferably < 0.5% on total dry solid, most preferably < 0.1% on total dry solid. Process according to any one of embodiments 2 to 5, 11 to 23, wherein the concentration of: magnesium ions, when present, in said solution is reduced below 1000 ppm prior to passing said solution onto said EDI, preferably the concentration of magnesium ions, when present, in said solution is reduced below 500 ppm, more preferably below 400 ppm, more preferably below 300 ppm, more preferably below 200 ppm, more preferably below 100 ppm, more preferably below 50 ppm, more preferably below 10 ppm, preferably by means of any one or more of nanofiltration, electrodialysis, diafiltration, cationic ion exchange, and/or calcium ions, when present, in said solution is reduced below 200 ppm prior to passing said solution onto said EDI, preferably the concentration of calcium ions, when present, in said solution is reduced below 100 ppm, more preferably below 50 ppm, more preferably below 20 ppm, more preferably below 10 ppm, more preferably below 5 ppm, more preferably below 2 ppm, more preferably below 1 ppm, more preferably below 0.5 ppm, more preferably below 0.1 ppm, preferably by means of any one or more of nanofiltration, electrodialysis, diafiltration, cationic ion exchange. Process according to any one of previous embodiments, wherein said process further comprises any one or more of the methods selected from the list comprising homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, electrodialysis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange, ion exchange chromatography, mixed bed ion exchange, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, anion exchange adsorbent resin, use of an adsorbent material, use of ion exchange resin, evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying in any order, preferably wherein any one or more of said methods is performed more than one time during said process. Process according to embodiments 1, 5 to 21, 25, wherein said process further comprises electrodeionization. Process according to embodiment 25 or 26, wherein (a) any one or more of said method(s) precede(s) said i) when present, pH adjustment, ii) when present, anionic ion exchange, iii) when present, cationic ion exchange, and/or iv) when present, mixed bed ion exchange and/or (b) any one or more of said method(s) succeed(s) said i) when present, pH adjustment, ii) when present, anionic ion exchange, iii) when present, cationic ion exchange, and/or iv) when present, mixed bed ion exchange. 28. Process according to any one of embodiments 25 to 27, wherein any one or more of said method(s) succeed(s), when present, said pH adjustment and precede(s) said i) when present, anionic ion exchange, ii) when present, cationic ion exchange, and/or iii) when present, mixed bed ion exchange.

29. Process according to embodiment 25, wherein any one or more of said method(s) precede(s) said EDI in said process and/or wherein any one or more of said method(s) succeed(s) said EDI in said process.

30. Process according to any one of previous embodiments, wherein the temperature of said solution is adjusted to a temperature of: from 36°C to 65°C, wherein said temperature is within 5°C of a temperature at which the solution exhibits maximum turbidity, preferably from 36°C to 60°C, more preferably from 40°C to 55°C, even more preferably from 40°C to 45°C, or from 0°C to 122°C, preferably from 2°C to 80°C, more preferably from 4°C to 60°C, even more preferably from 10°C to 55°C, even more preferably 20°C to 45°C, even more preferably from 21°C to 40°C, even more preferably from 22°C to 37°C, even more preferably from 25°C to 30°C.

31. Process according to any one of embodiments 25, 27 to 30, wherein said process comprises EDI of said solution and wherein said EDI is combined in said process with nanofiltration and/or electrodialysis, preferably wherein said nanofiltration and/or electrodialysis is performed twice in said process.

32. Process according to any one of embodiments 25 to 31, wherein said process: comprises two consecutive steps of nanofiltration, comprises two consecutive steps of electrodialysis, and/or two consecutive ultrafiltration steps wherein the membrane molecular weight cut-off used in the first ultrafiltration step is higher than that used in the second ultrafiltration step.

33. Process according to any one of embodiments 25 to 32, wherein said process does not comprise electrodialysis.

34. Process according to any one of embodiments 25, 27 to 33, wherein said process does not comprise electrodialysis, ion exchange and/or ion exchange chromatography.

35. Process according to any one of embodiments 25 to 34, wherein said solution is subjected to two consecutive ultrafiltration steps wherein the membrane molecular weight cut-off used in the first ultrafiltration step is higher than that used in the second ultrafiltration step.

36. Process according to any one of embodiments 25 to 35, wherein said process comprises two consecutive steps of ultrafiltration prior to said i) pH adjustment, ii) anionic ion exchange using an anionic ion exchange resin in OH- form, iii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably in Na+ form, iv) mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺, preferably in Na+ form, and an anionic ion exchange resin in OH- form and/or v) said EDI, respectively, and wherein the membrane molecular weight cut-off of the membrane used in the first ultrafiltration step is higher than the membrane molecular weight cut-off of the membrane used in the second ultrafiltration step. Process according to any one of embodiments 25 to 36, wherein said process comprises: clarification performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration, drying selected from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and agitated thin film drying, filtration performed by use of a filtration aid and/or flocculant, preferably said filtration aid is an adsorbing agent, more preferably active carbon, ultrafiltration wherein said ultrafiltration has a molecular weight cut-off equal to or higher than 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, nanofiltration wherein the nanofiltration membrane used in said nanofiltration has a size exclusion limit of < 20 A, and/or diafiltration until a conductivity is reached of < 15 mS/cm, preferably < 10 mS/cm, < 5 mS/cm, < 1 mS/cm, < 0.1 mS/cm, < 0.01 mS/cm, < 0.001 mS/cm. Process according to any one of embodiments 25 to 37, wherein said process comprises enzymatic treatment: comprising incubation of said solution with one or more enzymes selected from the group comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase, and/or converting lactose, sucrose, malto-oligosaccharides, maltotriose, sorbitol, trehalose, starch, cellulose, hemi-cellulose, lignocellulose, molasses, corn-steep liquor and/or high-fructose syrup to monosaccharides. Process according to any one of embodiments 25 to 38, wherein said process comprises a mixed bed ion exchange that is performed at a temperature ranging from 0°C to 80°C, preferably from 4°C to 60°C, more preferably from 4°C to 40°C, even more preferably from 10°C to 37°C, even more preferably from 20°C to 30°C, even more preferably from 20°C to 25°C, even more preferably from 22°C to 24°C, most preferably from 23°C to 24°C. Process according to any one of previous embodiments, wherein said process is a batch or continuous process. Process according to any one of embodiments 5 to 7, 11 to 40, wherein said negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid group selected from the list comprising Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3- deoxymanno-octulonic acid (KDO). Process according to any one of embodiments 5 to 7 , 11 to 41, wherein said sialylated oligosaccharide is selected from the list comprising a negatively charged, preferably sialylated, milk oligosaccharide; preferably a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO); more preferably a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO); O-antigen; the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an aminosugar; Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a negatively charged, preferably sialylated, plant oligosaccharide, preferably selected from the list consisting of N- glycans and O-glycans; sialylated oligosaccharide; N-acetyllactosamine containing negatively charged, preferably sialylated, oligosaccharide and lacto-N-biose containing negatively charged, preferably sialylated, oligosaccharide; preferably said negatively charged oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac- <x2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, 3'- sialyllactosamine, sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-bl,4-GlcNAc- bl,3-Gal, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO- 6'sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Gai -l,3GalNac -l,3Gala-l,4Gai -l,4Gal, KDO-

2,3Gai -l,3GlcNac -l,3Gai -l,4Glc, KDO-2,3Gai -l,4GlcNac -l,3Gai -l,4Glc, 3'-KDO-3- fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-Sialyl-2'-fucosyllactose, 6'- Sialyl-2'-fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-Sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac- a2,3-)Gal-bl,4-Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc, 6'- Sialyllacto-N-biose, 3'-Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6- (Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal- bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6- (Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal- bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,5-(Fuc- al,2-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4- Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal- bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc- bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-Glc and combinations thereof. Process according to any one of embodiments 1 to 5, 11 to 42, wherein the purity of said oligosaccharide in said solution is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %>, < 10 % on total dry solid before purification by said process. Process according to any one of embodiments 6, 7, 11 to 42, wherein the purity of said negatively charged, preferably sialylated, oligosaccharide in said solution is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process. Process according to any one of embodiments 8 to 42, wherein the purity of said LSTc in said solution is < 70 %, < 60 %, < 50 %>, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process. Process according to any one of previous embodiments, wherein said solution is a cell cultivation using a cell, preferably a metabolically engineered cell, wherein said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, is produced by said cell, the cell cultivation comprising i) a) said oligosaccharide, b) said negatively charged, preferably sialylated, oligosaccharide or c) said LSTc and sialyllactose, respectively, and ii) biomass, medium components and contaminants, wherein the purity of said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide, or said LSTc and sialyllactose, respectively, in said cell cultivation is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process. Process according to any one of previous embodiments, wherein said solution is a cell cultivation using a cell, preferably a metabolically engineered cell, wherein said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, is/are produced by said cell, the cell cultivation comprising i) said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, and ii) biomass, medium components and contaminants, wherein, when present, biomass separated during said process is optionally recycled to said cell cultivation. Process according to any one of previous embodiments, wherein said solution is a cell cultivation using at least one cell that has been metabolically engineered to produce said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively. Process according to any one of previous embodiments, wherein said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, is /are accompanied in said solution by sialic acid, ashes, preferably, said ashes comprise sulphates, phosphates, sodium, chloride, potassium, heavy metals, preferably said heavy metals comprise ammonium, lead arsenic, cadmium and/or mercury, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) selected from the list comprising a neutral (noncharged) oligosaccharide, a negatively charged oligosaccharide, a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O- glycans; a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; erlose (Glc-al,4-Glc-al,2-Fru); lactul-N-triose II (GlcNAc-bl,3-Gal-bl,4-Fru); lactul- N-tetraose; lactul-N-neotetraose; globotriose; fucosylated oligosaccharide preferably selected from the list comprising 2'-fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (di FL), lacto-N-fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3- (Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N- fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto-N-neofucopentaose V, lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), Fuc-al,2-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3- )Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Gal-bl,4-(Fuc-al,3-)GlcNAc- bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3- )Glc, Fuc-al,4-(Fuc-al,2-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, monofucosyllacto-N- hexaose-lll, difucosyllacto-N-hexaose (a), difucosyl-lacto-N-hexaose, difucosyl-lacto-N- neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc-al,2- )Gal-bl,4-(Fuc-al,3-)Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N- acetyllactosamine, 4-fucosyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc; sialylated oligosaccharide preferably selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6- disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6- Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, 3'-sialyllactosamine, sialyllacto-N-tetraose a (LSTa), sialyllacto-N- tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto- N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose

I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose

II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3- Gal, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO- 6'sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Gai -l,3GalNac -l,3Gala-l,4Gai -l,4Gal, KDO-2,3Gai -l,3GlcNac -l,3Gai -l,4Glc, KDO-2,3Gaip-l,4GlcNacP-l,3Gaip-l,4Glc, 3'-KDO- 3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-Sialyl-2'- fucosyllactose, 6'-Sialyl-2'-fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-Sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-bl,4-Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc-al,4-(Neu5Ac- a2,3-Gal-bl,3-)GlcNAc, 6'-Sialyllacto-N-biose, 3'-Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc- bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac- a2,3-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4- (Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc- bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-Gal- bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal- bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3- GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6- Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc and combinations thereof; N-acetylglucosamine containing neutral (non-charged) oligosaccharide preferably selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 4'-galactosyllactose, 3'-galactosyllactose, GlcNAc-bl,6-Gal-bl,4-Glc, lacto- N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (pLNH), para-lacto-N- neohexaose (pLNnH), GlcNAc-bl,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, lacto-N-pentaose (LN5), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N- novopentaose I, lacto-N-heptaose (LN7), lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose (pLNnO), iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose (LN9), lacto-N- decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, para lacto-N- neodecaose (pLNnD), al,3-galactosyllacto-N-neotetraose, GlcNAc-bl,3-Gal-bl,3-GlcNAc- bl,3-Gal-bl,4-Glc, GlcNAc-bl,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc and GlcNAc-bl,6-(Gal- bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc; N-acetyllactosamine containing oligosaccharide; lacto-N- biose containing oligosaccharide; non-fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

50. Process according to any one of previous embodiments, wherein said solution is a cell cultivation using at least one cell that has been metabolically engineered to produce a) said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, and b) one or more of i) sialic acid, ii) one or more monosaccharide(s), iii) one or more activated monosaccharide(s), iv) one or more phosphorylated monosaccharide(s), v) one or more disaccharide(s) and/or vi) one or more other oligosaccharides.

51. Process according to any one of previous embodiments, wherein said cell produces said oligosaccharide, said negatively charged, preferably sialylated, oligosaccharide or said LSTc and sialyllactose, respectively, from one or more internalized precursor(s).

52. Process according to any one of previous embodiments, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, preferably, said bacterium belongs to a phylum selected from the list consisting of Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family selected from the list comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is selected from the list comprising an Escherichia coli strain, a Bacillus subtilis strain, a Vibrio natriegens strain; even more preferably said Escherichia coli strain is a K-12 strain, most preferably said Escherichia coli K-12 strain is E. coli MG1655, preferably, said fungus belongs to a genus selected from the list comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus selected from the list comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the list consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii, preferably, said plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, preferably, said animal cell is derived from insects, amphibians, reptiles, invertebrates, fish, birds or mammalian cells excluding human embryonic stem cells, more preferably said mammalian cell is selected from the list comprising an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a lactocyte derived from mammalian induced pluripotent stem cells, more preferably said mammalian induced pluripotent stem cells are human induced pluripotent stem cells, a post-parturition mammary epithelium cell, a polarized mammary cell, more preferably said polarized mammary cell is selected from the list comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof, more preferably said insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster, preferably, said protozoan cell is a Leishmania tarentolae cell.

53. Process according to any one of embodiments 1 to 5, 7, 9 to 52, wherein said cell is an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.

54. Process according to any one of embodiments 1 to 5, 7, 9 to 53, wherein said cell is cultivated in culture medium comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, said carbon source is selected from the list comprising glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high- fructose syrup, acetate, citrate, lactate and pyruvate; preferably, said culture medium is a chemically defined medium.

55. Process according to embodiment 54, wherein said culture medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.

56. Process according to any one of previous embodiments, wherein said solution is a cell cultivation and said cell cultivation is a fermentation.

57. Process according to any one of embodiments 1 to 7, 11 to 44, 46 to 56, wherein said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively, is accompanied in said solution by one or more other oligosaccharide(s) wherein at least one of said other oligosaccharides has the same degree of polymerization (DP) as said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively, preferably wherein all of said other oligosaccharides have the same DP as said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively.

58. Process according to any one of embodiments 1 to 7, 11 to 44, 46 to 56, wherein said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively, is accompanied in said solution by one or more other oligosaccharide(s) wherein at least one of said other oligosaccharides has a different degree of polymerization (DP) as said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively preferably wherein all of said other oligosaccharides have a different DP as said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively.

59. Process according to any one of embodiments 8 to 40, 45 to 56, wherein said LSTc and sialyllactose are accompanied in said solution by one or more other oligosaccharide(s) wherein said one or more other oligosaccharide(s) has/have a degree of polymerization (DP) of at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6.

60. Process according to any one of previous embodiments, wherein said solution has an ash content of > 10% on total dry solid before purification by said process, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

61. Process according to any one of previous embodiments, wherein said solution comprises a lead content > 0.1 mg/kg dry solid, an arsenic content > 0.2 mg/kg dry solid, a cadmium content > 0.1 mg/kg dry solid, and/or a mercury content > 0.5 mg/kg dry solid before purification by said process.

62. Process according to any one of embodiments 49 to 58, 60, 61, wherein said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively, is purified from said sialic acid and/or said ashes by said process.

63. Process according to embodiment 62, wherein said oligosaccharide, which is accompanied by said sialic acid and/or said ashes in said solution, is purified from said sialic acid and/or ashes by said process comprising: i) pH adjustment of said solution comprising said oligosaccharide and said sialic acid and/or ashes to a pH of about 3, and ii) passing said pH adjusted solution through: an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably said cationic ion exchange resin is in Na⁺ form, and/or a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably said cationic ion exchange resin in said mixed bed ion exchange is in Na⁺ form, and an anionic ion exchange resin in OH- form.

54. Process according to any one of embodiments 49 to 58, 60, 51, wherein i) said oligosaccharide or said negatively charged, preferably sialylated, oligosaccharide, respectively, and ii) said one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by said process.

55. Process according to embodiment 64, wherein said oligosaccharide is accompanied by i) said one or more other oligosaccharide(s) and ii) by said sialic acid and/or said ashes in said solution and wherein said oligosaccharide and said one or more other oligosaccharide(s) is/are purified from said sialic acid and/or said ashes by said process comprising: i) pH adjustment of said solution comprising i) said oligosaccharide and said one or more other oligosaccharide(s) and ii) said sialic acid and/or ashes to a pH of about 3, and ii) passing said pH adjusted solution through: an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺or NH₄ ⁺ form, preferably said cationic ion exchange resin is in Na⁺ form, and/or a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably said cationic ion exchange resin in said mixed bed ion exchange is in Na+ form and an anionic ion exchange resin in OH- form.

56. Process according to embodiment 64, wherein said negatively charged, preferably sialylated oligosaccharide, which is accompanied by said sialic acid and/or said ashes in said solution, is purified from said sialic acid and/or ashes by said process comprising: i) pH adjustment of said solution comprising said negatively charged, preferably sialylated, oligosaccharide and said sialic acid and/or ashes to a pH of about 3, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

67. Process according to any one of embodiments 64, wherein said negatively charged, preferably sialylated, oligosaccharide is accompanied by i) said one or more other oligosaccharide(s) and ii) by said sialic acid and/or said ashes in said solution and wherein said negatively charged, preferably sialylated, oligosaccharide and said one or more other oligosaccharide(s) is/are purified from said sialic acid and/or said ashes by said process comprising: i) pH adjustment of said solution comprising i) said negatively charged, preferably sialylated, oligosaccharide and said one or more other oligosaccharide(s) and ii) said sialic acid and/or ashes to a pH of about 3, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form. 68. Process according to any one of embodiments 49 to 56, 59 to 61, wherein said LSTc is purified i) from said sialyllactose and ii) from said sialic acid, ashes, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) by said process.

69. Process according to embodiment 68, wherein said LSTc is purified by said process comprising: i) pH adjustment of said solution to a pH of about 6.5, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

70. Process according to any one of embodiments 49 to 56, 59 to 61, wherein said LSTc and said one or more other oligosaccharide(s) are purified i) from said sialyllactose and ii) from said sialic acid and/or said ashes by said process.

71. Process according to embodiment 70, wherein said LSTc and said one or more other oligosaccharide(s) are purified from i) said sialyllactose and ii) said sialic acid and/or said ashes present in said solution by a process comprising: i) pH adjustment of said solution to a pH of about 6.5, and ii) passing said pH adjusted solution through a mixed bed ion exchange, said mixed bed ion exchange comprising a cationic ion exchange resin in H+ form and an anionic ion exchange resin in OH- form.

72. Process according to any one of previous embodiments, wherein the purity of the oligosaccharide, of the negatively charged, preferably sialylated, oligosaccharide or of LSTc obtained in the purified oligosaccharide solution at the end of said process is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% on total dry solid.

73. Process according to any one of previous embodiments, wherein the yield of purification of the oligosaccharide, of the negatively charged, preferably sialylated, oligosaccharide or of LSTc, respectively, obtained in the purified oligosaccharide solution at the end of said process is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%.

74. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process has an ash content of < 10% on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

75. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process has an ash content of < 10% on total dry solid, preferably < 9% on total dry solid, more preferably < 8% on total dry solid, even more preferably < 7% on total dry solid, even more preferably < 6% on total dry solid, even more preferably < 5% on total dry solid, even more preferably < 4% on total dry solid, even more preferably < 3% on total dry solid, even more preferably < 2% on total dry solid, even more preferably < 1% on total dry solid, most preferably < 0.5% on total dry solid.

76. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process has an ash content of < 10% on total dry solid, preferably with a lead content lower than 0.1 mg/kg dry solid, more preferably a lead content lower than 0.02 mg/kg dry solid, even more preferably a lead content lower than 0.01 mg/kg dry solid; an arsenic content lower than 0.2 mg/kg dry solid, more preferably an arsenic content lower than 0.05 mg/kg dry solid, even more preferably an arsenic content lower than 0.02 mg/kg dry solid; a cadmium content lower than 0.1 mg/kg dry solid, more preferably a cadmium content lower than 0.01 mg/kg dry solid and/or a mercury content lower than 0.5 mg/kg dry solid, more preferably a mercury content lower than 0.1 mg/kg dry solid, even more preferably a mercury content lower than 0.005 mg/kg dry solid.

77. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process is filter-sterilized and/or subjected to endotoxin removal, preferably by filtration through a 3 kDa filter.

78. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process has a protein content below 100 mg per kg dry solid, a DNA content below 10 ng per gram dry solid and/or an endotoxin content below 10000 EU per gram dry solid, preferably the purified oligosaccharide solution is free of DNA, proteins, and/or recombinant genetic material.

79. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process is further i) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter; ii) crystallised; iii) dried to a powder or iv) granulated.

80. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process is further concentrated to an oligosaccharide concentration of > 100 g/L, preferably > 200 g/L, more preferably > 300 g/L, more preferably > 400 g/L, more preferably > 500 g/L, more preferably > 600 g/L, most preferably between 300 g/L and 650 g/L and/or at a temperature of < 80°C, preferably < 60°C, more preferably < 50°C, more preferably 20°C to 50°C, even more preferably 30°C to 45°C, preferably concentrated by a method comprising using vacuum evaporation or reverse osmosis or nanofiltration.

81. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process comprises an oligosaccharide or a negatively charged, preferably sialylated, oligosaccharide or LSTc, respectively, which is concentrated to a concentration of > 1.5 M and cooled to a temperature < 25 °C, more preferably < 8 °C, to obtain crystalline material of the oligosaccharide or of the negatively charged, preferably sialylated, oligosaccharide, or LSTc, respectively. Process according to any one of embodiments 1 to 80, wherein the purified oligosaccharide solution obtained at the end of said process is dried by any one or more of drying steps selected from the list comprising spray drying, lyophilization, evaporation, precipitation, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying and agitated thin film drying. Process according to embodiment 82, wherein the purified oligosaccharide solution obtained at the end of said process is dried by spray-drying, freeze-drying or agitated thin film drying and preferably wherein the pH of said purified oligosaccharide solution is ranging from 2 to 5, preferably from 3 to 5, more preferably from 4 to 5. Process according to any one of embodiment 82 or 83, wherein the purified oligosaccharide solution obtained at the end of said process is dried by spray-drying, preferably particularly spray-dried at an oligosaccharide solution concentration of 20-60 (w/v), preferably 30-50 (w/v), more preferably 35-45 (w/v), with a nozzle temperature of 110-150°C, preferably 120-140°C, more preferably 125-135°C and/or an outlet temperature of 60-80°C, preferably 65-70°C. The purified oligosaccharide solution, the purified oligosaccharide, the purified negatively charged, preferably sialylated, oligosaccharide, the purified LSTc, the purified oligosaccharide mixture, the purified oligosaccharide mixture comprising a negatively charged, preferably sialylated oligosaccharide, the purified LSTc or the purified oligosaccharide mixture comprising LSTc, respectively, obtainable, preferably obtained, by a process according to any one of previous embodiments. Purified oligosaccharide obtainable, preferably obtained, by a process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is i) dried, preferably spray-dried or dried via an agitated thin film dryer; ii) lyophilized, iii) crystallized or iv) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Purified negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84, wherein the purified oligosaccharide solution comprising said purified negatively charged, preferably sialylated, oligosaccharide is i) dried, preferably spray-dried or dried via an agitated thin film dryer; ii) lyophilized; iii) crystallized or iv) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Purified LSTc obtainable, preferably obtained, by a process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84, wherein the purified oligosaccharide solution comprising said purified LSTc is i) dried, preferably spray-dried or dried via an agitated thin film dryer; ii) lyophilized; iii) crystallized or iv) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Purified oligosaccharide mixture obtainable, preferably obtained, by a process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture is i) dried, preferably spray- dried or dried via an agitated thin film dryer; ii) lyophilized; iii) crystallized or iv) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide obtainable, preferably obtained, by a process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is i) dried, preferably spray-dried or dried via an agitated thin film dryer; ii) lyophilised; iii) crystallized or iv) concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Purified oligosaccharide mixture comprising LSTc obtainable, preferably obtained, by a process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84, wherein the purified oligosaccharide solution comprising said purified oligosaccharide mixture comprising LSTc is dried, preferably spray-dried or dried via an agitated thin film dryer, lyophilised or crystallized or concentrated to a syrup of at least 20% dry matter, preferably at least 30% dry matter, more preferably at least 40% dry matter. Oligosaccharide purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein the purified oligosaccharide obtained after said process has an ash content of < 10 % on total dry solid, preferably wherein said oligosaccharide is produced through cell cultivation. Oligosaccharide purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein the purified oligosaccharide obtained after said process has an ash content of < 10 % on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium. Oligosaccharide purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein the purified oligosaccharide obtained after said process has a lead content < 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably < 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid.

95. Negatively charged, preferably sialylated, oligosaccharide purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein the purified negatively charged, preferably sialylated, oligosaccharide obtained after said process has an ash content of < 10 % on total dry solid, preferably wherein said negatively charged, preferably sialylated, oligosaccharide is produced through cell cultivation.

96. Negatively charged, preferably sialylated, oligosaccharide purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein the purified negatively charged, preferably sialylated, oligosaccharide obtained after said process has an ash content of < 10 % on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

97. Negatively charged, preferably sialylated, oligosaccharide purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein the purified negatively charged, preferably sialylated, oligosaccharide obtained after said process has a lead content < 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably

< 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid.

98. LSTc purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84 and wherein the purified LSTc obtained after said process has an ash content of

< 10 % on total dry solid, preferably wherein said LSTc is produced through cell cultivation.

99. LSTc purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84and wherein the purified LSTc obtained after said process has an ash content of

< 10 % on total dry solid, wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

100. LSTc purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84 and wherein the purified LSTc obtained after said process has a lead content

< 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably < 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid. 101. Spray-dried oligosaccharide or oligosaccharide mixture, wherein said oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein said spray-dried oligosaccharide or oligosaccharide mixture obtained after said process has an ash content of < 10 % on total dry solid, preferably wherein said oligosaccharide or oligosaccharide mixture is produced through cell cultivation.

102. Spray-dried oligosaccharide or oligosaccharide mixture, wherein said oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein said spray-dried oligosaccharide or oligosaccharide mixture obtained after said process has an ash content of < 10 % on total dry solid wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

103. Spray-dried oligosaccharide or oligosaccharide mixture, wherein said oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 1 to 5, 11 to 21, 25 to 43, 46 to 58, 60 to 65, 72 to 84 and wherein said spray-dried oligosaccharide or oligosaccharide mixture obtained after said process has a lead content < 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably < 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid.

104. Spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide, wherein said negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein said spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture obtained after said process has an ash content of < 10 % on total dry solid, preferably wherein said negatively charged, preferably sialylated, oligosaccharide or said oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide is produced through cell cultivation.

105. Spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide, wherein said negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein said spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture obtained after said process has an ash content of < 10 % on total dry solid wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

106. Spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide, wherein said negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture is purified according to the process according to any one of embodiments 6, 7, 11 to 21, 25 to 42, 44, 46 to 58, 60 to 67, 72 to 84 and wherein said spray-dried negatively charged, preferably sialylated, oligosaccharide or oligosaccharide mixture obtained after said process has a lead content < 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably < 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid.

107. Spray-dried LSTc or oligosaccharide mixture comprising LSTc, wherein said LSTc or oligosaccharide mixture comprising LSTc is purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84 and wherein said spray-dried LSTc or oligosaccharide mixture comprising LSTc obtained after said process has an ash content of < 10 % on total dry solid, preferably wherein said LSTc or oligosaccharide mixture comprising LSTc is produced through cell cultivation.

108. Spray-dried LSTc or oligosaccharide mixture comprising LSTc, wherein said LSTc or oligosaccharide mixture comprising LSTc is purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84 and wherein said spray-dried LSTc or oligosaccharide mixture comprising LSTc obtained after said process has an ash content of < 10 % on total dry solid wherein said ash comprises any one or more of a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.

109. Spray-dried LSTc or oligosaccharide mixture comprising LSTc, wherein said LSTc or oligosaccharide mixture comprising LSTc is purified according to the process according to any one of embodiments 8 to 21, 25 to 40, 45 to 56, 59 to 62, 68 to 84 and wherein said spray-dried LSTc or oligosaccharide mixture comprising LSTc obtained after said process has a lead content < 0.1 mg/kg dry solid, preferably < 0.02 mg/kg dry solid, more preferably < 0.01 mg/kg dry solid; an arsenic content < 0.2 mg/kg dry solid, preferably < 0.05 mg/kg dry solid, more preferably < 0.02 mg/kg dry solid; a cadmium content < 0.1 mg/kg dry solid, preferably < 0.01 mg/kg dry solid and/or a mercury content < 0.5 mg/kg dry solid, preferably < 0.1 mg/kg dry solid, more preferably < 0.005 mg/kg dry solid.

110. Dried powder of purified oligosaccharide solution obtained from a process according to any one of embodiments 82 to 84, wherein said dried powder: contains < 15%-wt. of water, preferably < 10%-wt. of water, more preferably < 7%-wt. of water, most preferably < 5%-wt. of water, and/or has a mean particle size of 50 to 250 pm, preferably of 95 to 120 pm, more preferably of 110 to 120 pm, wherein said particle size is determined by laser diffraction, preferably said powder is a spray-dried powder. . Dried powder of purified oligosaccharide solution obtained from a process according to any one of embodiments 82 to 84, wherein said powder exhibits: a loose bulk density of from about 500 to 700 g/L, a lOOx tapped bulk density of from about 600 to about 850 g/L a 625x tapped bulk density of from about 600 to about 900 g/L, and/or a 1250x tapped bulk density of from about 650 to about 900 g/L. . Dried powder according to embodiment 111, wherein said powder exhibits: a) a loose bulk density of from about 600 to 700 g/L, a lOOx tapped bulk density of from about 750 to about 850 g/L a 625x tapped bulk density of from about 750 to about 850 g/L, and/or a 1250x tapped bulk density of from about 850 to about 900 g/L, or b) a loose bulk density of from about 500 to 600 g/L, a lOOx tapped bulk density of from about 600 to about 700 g/L a 625x tapped bulk density of from about 700 to about 800 g/L, and/or a 1250x tapped bulk density of from about 750 to about 800 g/L. . Dried powder according to any one of embodiments 86, 89, 92 to 94, 101 to 103, 110 to 112, wherein said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with a pH between 4 and 7, preferably with a pH between 4 and 6, more preferably with a pH between 4 and 5, even more preferably with a pH between 5 and 6. . Purified oligosaccharide according to any one of embodiments 85, 86, 92 to 94, 101 to 103, 110 to 113, wherein any one or more of said oligosaccharide is a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO), even more preferably said milk oligosaccharide is:

1) a neutral (non-charged) milk oligosaccharide, preferably a neutral (non-charged) human milk oligosaccharide (HMO), selected from the list comprising 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N- fucopentaose I, lacto-N neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N- neohexaose and any combination thereof, or 2) a sia lylated milk oligosaccharide, preferably a sialylated human milk oligosaccharide (HMO), selected from the list comprising 3'sialyllactose, 6'sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, sialyllacto-N-tetraose d, disialyllacto-N- tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof.. Purified negatively charged, preferably sialylated, oligosaccharide according to any one of embodiments 85, 87, 90, 95 to 97, 104 to 106, 110 to 113, wherein any one or more of said negatively charged, preferably sialylated, oligosaccharide is a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO), even more preferably said milk oligosaccharide is a sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N- hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N- neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N- neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof. . Purified oligosaccharide mixture according to any one of embodiments 85, 89, 101 to 103, 110 to 113, wherein said mixture comprises a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO), even more preferably said milk oligosaccharide is:

1) a neutral (non-charged) milk oligosaccharide, preferably a neutral (non-charged) human milk oligosaccharide (HMO), selected from the list comprising 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N- fucopentaose I, lacto-N neofucopentaose, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose and lacto-N- neohexaose and any combination thereof, or

2) a sialylated milk oligosaccharide, preferably a sialylated human milk oligosaccharide (HMO), selected from the list comprising 3'sialyllactose, 6'sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, sialyllacto-N-tetraose d, disialyllacto-N- tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof.

117. Purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide according to any one of embodiments 85, 90, 104 to 106, 110 to 113, wherein said purified oligosaccharide mixture comprises a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO), even more preferably said milk oligosaccharide is a sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N- hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N- neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N- neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof.

118. Purified oligosaccharide mixture comprising LSTc according to any one of embodiments 85, 88, 91, 98 to 100, 107 to 113, wherein said oligosaccharide mixture comprising LSTc further comprises a milk oligosaccharide, preferably said milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably said milk oligosaccharide is a human milk oligosaccharide (HMO), even more preferably said milk oligosaccharide is a sialylated milk oligosaccharide selected from the list comprising sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto- N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N- neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose , disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose and any combination thereof.

119. Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 85, 86, 89, 92 to 94, 101 to 103, 110 to 114, 116, wherein the purified oligosaccharide or purified oligosaccharide mixture a) has a conductivity of less than 10 mS/cm at a 300 g/L solution; b) is free of recombinant DNA material, optionally free of any DNA; and/or c) is free of proteins derived from the recombinant micro-organism, optionally free of any proteins.

120. Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 85, 86, 89, 92 to 94, 101 to 103, 110 to 114, 116, 119 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

121. Purified negatively charged, preferably sialylated, oligosaccharide or purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide according to any one of embodiments 85, 87, 90, 95 to 97, 104 to 106, 110 to 113, 115, 117, wherein the purified oligosaccharide or purified oligosaccharide mixture a) has a conductivity of less than 10 mS/cm at a 300 g/L solution; b) is free of recombinant DNA material, optionally free of any DNA; and/or c) is free of proteins derived from the recombinant micro-organism, optionally free of any proteins.

122. Purified negatively charged, preferably sialylated, oligosaccharide or purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide according to any one of embodiments 85, 87, 90, 95 to 97, 104 to 106, 110 to 113, 115, 117, 121 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

123. Purified LSTc or purified oligosaccharide mixture comprising LSTc according to any one of embodiments 85, 88, 91, 98 to 100, 107 to 113, 118, wherein said LSTc or oligosaccharide mixture comprising LSTc a) has a conductivity of less than 10 mS/cm at a 300 g/L solution; b) is free of recombinant DNA material, optionally free of any DNA; and/or c) is free of proteins derived from the recombinant cell, optionally free of any proteins.

124. Purified LSTc or purified oligosaccharide mixture comprising LSTc according to any one of embodiments 85, 88, 91, 98 to 100, 107 to 113, 118, 123 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

The invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting.

Examples

Example 1. Materials and Methods

A. Escherichia coli

Media

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH₄CI, 5.00 g/L (NE hSO^ 2.993 g/L KH2PO4, 7.315 g/L K₂HPO₄, 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO₄.7H₂O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 pil/L molybdate solution, and 1 mL/L selenium solution. As specified in the respective examples, 0.30 g/L sialic acid, 0.30 g/L GIcNAc, 20 g/L lactose, 20 g/L LacNAc (Gaipi-4GlcNAc), 20 g/L LNB (Gaipi-3GlcNAc), 20 g/L LN3 (GlcNAcpi-3Gaipi-4Glc), 20 g/L LNT (Gaipi- 3GlcNAcpi-3Gaipi-4Glc) and/or 20 g/L LNnT (Gaipi-4GlcNAcpi-3Gaipi-4Glc) were additionally added to the medium. The minimal medium was set to a pH of 7 with IM KOH. Vitamin solution consisted of 3.6 g/L FeCI₂.4H₂O, 5.0 g/L CaCI₂.2H₂O, 1.3 g/L MnCI₂.2H₂O, 0.38 g/L CuCI₂.2H₂O, 0.5 g/L CoCI₂.6H₂O, 0.94 g/L ZnCI₂, 0.0311 g/L H3BO4, 0.4 g/L Na₂EDTA.2H₂O and 1.01 g/L thiamine.HCl. The molybdate solution contained 0.967 g/L NaMoO .2H₂O. The selenium solution contained 42 g/L Seo₂. The minimal medium for fermentations contained 6.75 g/L NH₄CI, 1.25 g/L (NH₄)₂SO₄, 2.93 g/L KH₂PO₄ and 7.31 g/L KH₂PO₄, 0.5 g/L NaCI, 0.5 g/L MgSO₄.7H₂O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 p.L/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 0.30 g/L sialic acid, 0.30 g/L GIcNAc, 20 g/L lactose, 20 g/L LacNAc, 20 g/L LNB, 20 g/L LN3, 20 g/L LNT and/or 20 g/L LNnT were additionally added to the medium.

Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).

Strains and mutations

Escherichia coli K12 MG1655 [A-, F, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). Genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier. Proteins described in present disclosure are summarized in Table 1. All strains were stored in cryovials at -80°C (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

Cultivation conditions

A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 pL LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96- well square microtiter plate, with 400 pL minimal medium by diluting 400x. These final 96-well culture plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 15 min at 60°C before spinning down the cells (= average of intra- and extracellular sugar concentrations).

A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 m L or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37°C on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition were set to 37 °C, and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

Optical density

Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland). The maximum growth speed (mumax) was calculated based on the observed optical densities at 600nm using the R package grofit.

B. Saccharomyces cerevisiae

Media

Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura, 0.77 g/L CSM-Trp, or 0.77 g/L CSM-His (MP Biomedicals).

Strains

S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).

Plasmids

In an example to produce sialic acid and CMP-sialic acid, a yeast expression plasmid derived from the pRS420-plasmid series (Christianson et aL, 1992, Gene 110: 119-122) containing the TRP1 selection was modified as described in e.g., WO22034067. In an example to produce GDP-fucose, the yeast expression plasmid p2a_2p_Fuc (Chan 2013, Plasmid 70, 2-17) comprising an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2p yeast ori and the Ura3 selection marker for selection and maintenance in yeast was modified as described in e.g., WO22034067. In an example to produce UDP-galactose, a yeast expression plasmid derived from the pRS420-plasmid series (Christianson et aL, 1992, Gene 110: 119-122) containing the HIS3 selection marker was modified as described e.g., in WO22034067. In an example to produce LN3, said plasmid was further modified with transcriptional units encoding a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6) and a lactose permease like e.g., LAC12 from K. lactis (UniProt ID P07921). In another example, the 5. cerevisiae strain engineered for LN3 production is further modified with a transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 or with an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis to produce lacto-N-tetraose (LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) or lacto-N- neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), respectively. Cultivations conditions

In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30°C. Starting from a single colony, a preculture was grown over night in 5 mL at 30°C, shaking at 200 rpm. Subsequent 125 L shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30°C with an orbital shaking of 200 rpm. Gene expression promoters

Genes were expressed using synthetic constitutive promoters, as described by e.g., Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact.2021, 20, 148) and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).

C. Bacillus subtilis

Media

Two media are used to cultivate B. subtilis: i.e., a complex medium like a rich Luria Broth (LB) and a minimal medium for shake flask cultures. The LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco) added. The minimal medium contained 2.00 g/L (NH4)2SO4, 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO4.7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL/L trace element mix and 10 mL/L Fe-citrate solution. The medium was set to a pH of 7 with 1 M KOH. Depending on the experiment lactose is added as a precursor. The trace element mix consisted of 0.735 g/L CaCI2.2H2O, 0.1 g/L MnCI2.2H20, 0.033 g/LCuCI2.2H2O, 0.06 g/LCoCI2.6H2O, 0.17 g/L ZnCI2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.2H2O and 0.06 g/L Na2MoO4. The Fe-citrate solution contained 0.135 g/L FeCI3.6H2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887). Complex medium, e.g., LB, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic.

Strains, plasmids and mutations

B. subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA). Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from B. subtilis 168 and modified as described e.g., in WO22034067. To allow sialylated oligosaccharide production, the mutant B. subtilis strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. The strain could additionally be modified to comprise a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920).

In an example for fucosylated oligosaccharide production, the mutant strain was derived from B. subtilis 168 and modified as described e.g., in WO22034069 to comprise an alpha-1,2- and/or alpha-1, 3- fucosyltransferase expression construct. Additionally, the mutant strain could be further modified with a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920) if necessary.

In an example for LN3 production, the mutant strain was derived from B. subtilis 168 and modified as described e.g., in WO22034069 to comprise a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). In an example for LNT or LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 or an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively.

In an example to produce one or more fucosylated non-charged oligosaccharide(s), a B. subtilis strain is modified for production of GDP-fucose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a B. subtilis strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a B. subtilis strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

Cultivation conditions

B. subtilis strains were initially grown on LB agar to obtain single colonies. These plates were grown over night at 37°C. Starting from a single colony, a preculture was grown over night in 5 mL at 37°C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90°C or for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations). D. Corynebacterium glutamicum

Media

Two different media are used, namely complex medium like e.g., a rich tryptone-yeast extract (TY) medium, and a minimal medium for shake flask (MMsf). The minimal medium uses a lOOOx stock trace element mix. Trace element mix consisted of 10 g/L CaCI2, 10 g/L FeSO4.7H2O, 10 g/L MnSO4.H2O, 1 g/L ZnSO4.7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCI2.6H2O, 0.2 g/L biotin (pH 7) and 0.03 g/L protocatechuic acid. The minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH4)2SO4, 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4.7H2O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix. Depending on the experiment lactose, LNB, and/or LacNAc could be added to the medium. The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added. Complex medium, e.g., TY, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic.

Strains and mutations

Corynebacterium glutamicum was used as available at the American Type Culture Collection (ATCC 13032). Integrative plasmid vectors were made using the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. BiotechnoL, 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (BiotechnoL Bioeng., 2013 Nov, 110(ll):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. glutamicum and modified as described e.g., in WO22034067. To allow sialylated oligosaccharide production, the mutant C. glutamicum strain producing CMP-sialic acid was further modified with one or more transcriptional unit(s) encoding one or more sialyltransferases. The strain could additionally be modified to comprise a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920).

In an example forfucosylated oligosaccharide production, the mutant strain is derived from C. glutamicum and modified as described e.g., in WO22034069 to comprise an alpha-1,2- and/or alpha-1, 3- fucosyltransferase expression construct. Additionally, the mutant strain could be further modified with a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920) if necessary.

In an example for LN3 production, the mutant strain was derived from C. glutamicum and modified as described e.g., in WO22034069 to comprise a transcriptional unit for a lactose permease like e.g., E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). In an example for LNT or LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 or an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively.

In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. glutamicum strain is modified for production of GDP-fucose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. glutamicum strain is modified for production of CMP-sialic acid, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. glutamicum strain is modified for production of CMP-sialic acid, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

Cultivation conditions

A preculture was started from a cryovial or a single colony from a TY plate, in 6 mL TY and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL MMsf medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90°C or for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).

E. Chlamydomonas reinhardtii

Media

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7). The TAP medium uses a lOOOx stock Hutner's trace element mix. Hutner's trace element mix consisted of 50 g/L Na2EDTA.H2O (Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCI2.4H2O, 5 g/L FeSO4.7H2O, 1.6 g/L CoCI2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3. The TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid. The salt stock solution consisted of 15 g/L NH4CL, 4 g/L MgSO4.7H2O and 2 g/L CaCI2.2H2O. As precursor(s) and/or acceptor(s) for saccharide synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB could be added. Medium was sterilized by autoclaving (121°C, 21 min). For stock cultures on agar slants TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm 2). Strains, plasmids and mutations

C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from the Chlamydomonas Resource Center (https://www.chlamycollection.org) (University of Minnesota, U.S.A) were used. Expression plasmids originated from pSH03, as available from the Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210) and as described like e.g., in WO22034067 or in WO22034069.

In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localised sialyltransferase chosen from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.

In an example for GDP-fucose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for fucosylation, C. reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1, 2-fucosyltransferase like e.g., HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1, 3-fucosyltransferase like e.g., HpFucT from H. pylori (UniProt ID 030511).

In an example for UDP-galactose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.

In an example for LN3 production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067 to comprise a transcriptional unit for a galactoside beta-1, 3-N- acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). In an example for LNT or LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli O55:H7 or an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively.

In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose,

LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).

Cultivation conditions

Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23 +/- 0.5°C under 14/10 h I ight/dark cycles with a light intensity of 8000 Lx. Cells were analysed after 5 to 7 days of cultivation. For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. TechnoL 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).

F. Animal ceils

Isolation of mesenchymal stem cells from adipose tissue of different animals

Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2. The initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics. The culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage. For example, Ahmad and Shakoori (2013, Stem Cell Regen. Med. 9(2): 29-36), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

Isolation of mesenchymal stem cells from milk

This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein. An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min. The cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.

Differentiation of stem cells using 2D and 3D culture systems

The mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191 -199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348 - C355; each of which is incorporated herein by reference in their entireties for all purposes.

For 2D culture, the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium.

For 3D culture, the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra- low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin. At confluence, cells were fed with growth medium supplemented with 2% foetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h. To induce differentiation, the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium.

Method of making mammary-like cells

In a next step, the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, epigenetic remodelling is performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes. In an example for production of one or more oligosaccharide(s), isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W022034070 and WO22034075.

Cultivation

Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, and 5 pg/mL hydrocortisone. Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, 5 pg/mL hydrocortisone, and 1 pg/mL prolactin (5ug/mL in Hyunh 1991). Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing. Within about a week, the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media. A desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration. A desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media. Hormones and other growth factors used can be selectively extracted by resin purification, for example the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.

G. Chemical synthesis

Chemical synthesis of an oligosaccharide like e.g., a milk oligosaccharide can be performed as described e.g., by Aly et al. (Carbohydr. Res. 1999, 316(1-4), 121-132), Bandara et al. (J. Org. Chem. 2019, 84(24), 16192-19198), Bandara et al. (Org. Biomol. Chem. 2020, 18, 1747-1753), Craft and Townsend (Carbohydr. Res. 2017, 440-441, 43-50), Crich and Wu (Org. Lett. 2008, 10(18), 4033-4035), Kiefel and von Itzstein (Chem. Rev., 2002, 102(2), 471-490), Miermont et al. (J. Org. Chem., 2007, 72(23), 8958-8961), Pistorio et al. (J. Org. Chem. 2016, 81(19), 8796-8805), Shirakawa et al. (Angewandte Chemie, 2021, 60(46), 24686- 24693), Tanaka et al. (J. Am. Chem. Soc., 2006, 128(22), 7124-7125).

H. General

Heterologous and homologous expression

Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT or Twist Bioscience. Proteins described in present disclosure are summarized in Table 1. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database version release 2021_03 of 09 June 2021. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

Table 1. Overview of proteins with corresponding UniProt IDs described in present disclosure

Analytical analysis

Standards such as but not limited to sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.

Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection. A volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm. The column temperature was 50 °C. The mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added. The method was isocratic with a flow of 0.130 mL/min. The ELSD detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps. The temperature of the Rl detector was set at 35 °C.

Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. A volume of 0. 5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50 °C. The mobile phase consisted of a mixture of 70 % acetonitrile, 26 % ammonium acetate buffer (150 mM) and 4 % methanol to which 0.05 % pyrrolidine was added. The method was isocratic with a flow of 0.150 mL/min. The temperature of the Rl detector was set at 35 °C.

Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. A volume of 0.5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50°C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the Rl detector was set at 35°C.

For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 °C, a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 pm) on 35 °C. A gradient was used wherein eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid. The oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min. As a washing step 100 % of eluent B was used for 5 min. For column equilibration, the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.

Both neutral and sialylated sugars at low concentrations (below 50 mg/L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 pL of sample was injected on a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200 guard column 4 x 50 mm. The column temperature was set to 30 °C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and

4 % of eluent C, a second increase from 4 to 12 % of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63 % of eluent A and 12 % of eluent C and a third increase from 12 to 48 % of eluent C over

5 min. As a washing step 48 % of eluent C was used for 3 min. For column equilibration, the initial condition of 75 % of eluent A and 0 % of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min.

Ash content measurement

The ash content within a sample can be measured by methods like e.g., dry ashing, wet ashing or low temperature plasma dry ashing. The sample is weighed before and after ashing to determine the concentration of ash present. The ash content can be expressed on dry basis and is calculated by dividing the mass of the ashed material by the mass of the dry material before ashing. Multiplied with 100, this gives the percentage of ash in the material. In a similar way the wet ash percentage can be determined for liquid products, wherein the mass of the liquid before and after ashing is used instead of the mass of the dry material.

In examples further described, the ash content was determined gravimetrically. For each sample, a porcelain crucible was pre-heated at 500°Cfor 30 minutes. Afterwards, it was cooled to room temperature in a desiccator containing anhydrous silica. When cooled, the crucible was weighed with 0.1 mg accuracy. Next, 5 g of oligosaccharide (e.g., HMO) powder was weighed in the crucible and charred using a Bunsen burner. Afterwards, the charred crucibles were put in a muffle furnace at 500°C for 4 hours. They were then again cooled to room temperature and weighed. The ash content was determined according the equation = (mass of crucible - mass of charred crucible)/(mass of oligosaccharide powder) * 100%. Heavy metal determination

A robust general inductively coupled plasma-mass spectrometry (ICP-MS) based method was used for the detection and quantitation for each of the following elements: arsenic (As), selenium (Se), cadmium (Cd), tin (Sn), lead (Pb), silver (Ag), palladium (Pd), platinum (Pt), mercury (Hg), molybdenum (Mo), sodium (Na), potassium (K), Calcium (Ca), Magnesium (Mg), Iron (Fe), zinc (Zn), manganese (Mn), Phosphorus (P), selenium (Se).

Nitric acid (> 65%, Sigma-Aldrich) was used for microwave digestion and standard/sample preparation. All dilutions were done using 18.2 MQ'cm (Millipore, Bedford, MA, USA) de-ionized water (DIW). About 0.2 g of each sample were digested in 5 mL of HNO3 using the microwave digestion (CEM, Mars 6) program 15 minutes (min) ramping time and 15 min holding time at 100W and 50°C followed by 15 min ramping time and 20 min holding time at 1800 W and 210°C. The samples were cooled after digestion for 30 minutes. The fully digested samples were then diluted to 50 mL with DIW.

Analyses were carried out using a standard Agilent 7800 ICP-MS, which includes the fourth-generation ORS cell system for effective control of polyatomic interferences using helium collision mode (He mode). The ORS controls polyatomic interferences using He to reduce the transmission of all common matrixbased polyatomic interferences. Smaller, faster analyte ions are separated from larger, slower interference-ions using kinetic energy discrimination (KED). All elements, except Se, were measured in He mode with a flow rate of 5 mL/min. Se was measured in High Energy He (HEHe) mode, using a cell gas flow rate of 10 mL/min. The 7800 ICP-MS was configured with the standard sample introduction system consisting of a MicroMist glass concentric nebulizer, quartz spray chamber, quartz torch with 2.5 mm i.d. injector, and nickel interface cones. The ICP-MS operating conditions are: 1550 W RF power, 8mm sampling depth, 1.16 l/min nebulizing gas, autotuned lens tuning, 5 or 10 ml/min helium gas flow, 5 V KED.

Dry matter and moisture content quantification

Sartorius MA150 Infrared Moisture Analyzer is used to determine the dry matter content of the oligosaccharide(s). 0.5 g of oligosaccharide is weighed on an analytical balance and is dried in the infrared moisture analyzer until the weight of the sample is stable. The mass of the dried sample divided by the mass of the sample before drying gives the dry matter content (in percent) of the oligosaccharide(s) or sample including oligosaccharide(s). In a similar way a liquid sample is weighed, however, the amount of liquid weighed is adapted to the expected amount of dry matter in the liquid, so the mass of the dry matter is properly measurable on an analytical balance.

A moisture analyzer measures the dry matter, but not the water content. Karl Fisher titration is used to determine the amount of water present in a powder, ingredient of food. The KF titration is carried out with a Karl Fischer titrator DL31 from Mettler Toledo using the two-component technique with Hydra- Point Solvent G and Hydra-Point titrant (5 mg H2O/mL), both purchased from J.T. Baker (Deventer, Holland). The polarising current for bipotentiometric end-point determination was 20 pA and the stop voltage 100 mV. The end-point criterion was the drift stabilisation (15 pg H2O /min) or maximum titration time (10 min).

The moisture content (MC) of sample was calculated using the following equation: MC = V_KF W_eq 100/ W_sample ; where V_KF is the consumption of titrant in mL, W_eq the titer of titrant in mg H2O/mL and W_sample the weight of sample in mg.

Biomass dry mass content (cell dry mass)

Cell dry weight was obtained by centrifugation (15 min, 5000 g) of 20 g broth in pre-dried (70°C overnight) and weighted falcons. The pellets were subsequently washed once with 20 mL physiological solution (9 g/L NaCI) and dried at 70 °C to a constant weight. The final weight was corrected for the added sodium chloride to the sample.

Protein quantification

For protein quantification a method is used that is compatible with reducing agents, such as reducing sugars or oligosaccharides with a reducing end. To this end, a Bradford assay (Thermo Scientific, Pierce) was used with a linear range between 1 and 1500 pg/mL. The assay was calibrated with a standard curve of BSA. The protein content of dried oligosaccharide products was quantified by dissolving a pre-weighed quantity in 18.2 MQ-cm (Millipore, Bedford, MA, USA) de-ionized water (DIW) up to a quantity of 50% (m/v). The amount of protein is measured at 595 nm and converted to concentration with the calibration curve based on BSA.

DNA quantification

Production host specific DNA residue is quantified by RT-qPCR, for which specific primers on the host are designed so that residual DNA of the production host is amplified. The RT-qPCR was performed according to the standard protocol of a kit obtained from Sigma and was based on SYBR Green detection.

Total DNA is measured by means of a Threshold assay (Molecular Devices), based on an immunoassay allowing to measure as low as 2 pg of DNA in a sample in solution. Double stranded DNA is measured by means of the SpectraMax® Quant™ AccuBlue™ Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.

Endotoxin measurement

Endotoxin in the liquid was measured by means of a limulus amebocyte lysate (LAL) test like e.g., from Lonza; Genscript or ThermoFisher according to the protocol as set out by the manufacturer.

Laser diffraction

The powder particle size can be assessed by laser diffraction. The system detects scattered and diffracted light by an array of concentrically arranged sensor elements. The software-algorithm is then approximating the particle counts by calculating the z-values of the light intensity values, which arrive at the different sensor elements. The analysis can be executed using a SALD-7500 Aggregate Sizer (Shimadzu Corporation, Kyoto, Japan) quantitative laser diffraction system (qLD).

A small amount (spatula tip) of each sample can be dispersed in 2 mL isooctane and homogenized by ultrasonication for five minutes. The dispersion will then be transferred into a batch cell filled with isooctane and analyzed in manual mode. Data acquisition settings can be as follows: Signal Averaging Count per Measurement: 128, Signal Accumulation Count: 3, and Interval: 2 seconds.

Prior to measurement, the system can be blanked with isooctane. Each sample dispersion will be measured 3 times, and the mean values and the standard deviation will be reported. Data can be evaluated using software WING SALD II version V3.1. When the refractive index of the sample is unknown, the refractive index of sugar (disaccharide) particles (1.530) can be used for determination of size distribution profiles. Size values for mean and median diameter are reported. The mean particle sizes for all samples are very similar due to the spray dryer settings used. In addition, the particle size distribution will show the presence of one main size population for all the samples.

Color determination

Color was determined by filtering 1 mL of an oligosaccharide solution of 10 Brix over a 0.45 pm syringe filter and afterwards measuring the absorbance of this solution at a wavelength of 430 nm.

Example 2. Production of 6'SL or 3'SL with a modified E. coli host in fed-batch fermentations

An E. coli strain engineered for production of 6'SL or 3'SL as described in WO2018122225 was used in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed. Regular broth samples were taken at several time points during the fermentation process and the 6'SL or 3'SL produced, respectively, was measured using UPLC as described in Example 1.

Example 3. Production of 2'FL with a modified E. coli host in fed-batch fermentation

An E. coli strain producing 2-fucosyllactose as described in WO21122708 was used in a fed batch fermentation as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed until the lactose concentration in the supernatant was lower than 5 g/L. The amounts of the salts present in the minimal medium fermentation was reduced by exclusion of NH₄CI and (NH₄)₂SO₄ and by using Na₂SO₄ instead of NaCI. Regular broth samples were taken at several time points during the fermentation process and the 2'FL produced was measured using UPLC as described in Example 1.

Example 4. Production of an oligosaccharide mixture comprising 2'FL, 3-FL and DiFL with a modified E. coli host in fed-batch fermentations

An E. coli strain engineered for production of an oligosaccharide mixture comprising 2'FL (Fuc-ocl,2-Gal- pi,4-Glc), 3-FL (Gal-pi,4-[Fuc-al,3]-Glc) and DiFL (Fuc-al,2-Gal-pi,4-[Fuc-ccl,3]-Glc) as described e.g., in WO22034067 was evaluated in a batch and in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale (5 and 30L) were performed as described in Example 1. In these examples, sucrose was used as a carbon source and lactose was added in the batch medium as a precursor. Regular broth samples were taken and the production of 2'FL, 3-FL and DiFL was measured using UPLC as described in Example 1. The experiment demonstrated that broth samples taken at the end of batch phase comprised an oligosaccharide mixture of 2'FL and 3-FL together with unmodified lactose, whereas broth samples taken at the end of the fed-batch phase comprised an oligosaccharide mixture of 2'FL, 3-FL and DiFL. As the ratios of lactose, 2'FL, 3-FL and DiFL changed over time during fed-batch, they could be manipulated during the fermentation process by discontinuation of the fermentation process at a desired time in fed- batch phase.

Example 5. Production of LNT or LNnT with a modified E. coli host in fed-batch fermentations

An E. coli K12 MG1655 strain modified for production of lacto-N-triose (LN3, GlcNAc-pi,3-Gal-pi,4-Glc) as described e.g., in WO22034075 was further modified to express 1) the N-acetylglucosamine beta-1, 3- galactosyltransferase WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 to produce lacto-N-tetraose (LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis to produce lacto-N- neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), respectively. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed. Regular broth samples were taken at several time points during the fermentation process and the LNT or LNnT produced, respectively, was measured using UPLC as described in Example 1.

Example 6. Production of an oligosaccharide mixture comprising LNT, LNnT and poly-galactosylated structures with a modified E. coli host in fed-batch fermentations

An E. coli K12 MG1655 strain modified for production of lacto-N-triose (LN3, GlcNAc-pi,3-Gal-pi,4-Glc) as described e.g., in WO22034075 is further modified with a constitutive transcriptional unit for the N- acetylglucosamine beta-1, 4-galactosyltransferase (LgtB) from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000) in one or more copies. To enhance UDP-galactose production, the ushA and galT genes are knocked out. Furthermore, the mutant strain is modified with a genomic knock- in of a constitutive transcriptional unit for the UDP-glucose-4-epimerase gene (galE) from E. coli (UniProt ID P09147), the phosphoglucosamine mutase (glmM) from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007) and the N-acetylglucosamine-l-phosphate uridyltransferase / glucosamine-l-phosphate acetyltransferase (glmU) from E. coli (UniProt ID P0ACC7). The mutant strain is further engineered for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter (CscB) from E. coli\N (UniProt ID E0IXR1), a fructose kinase (Frk) originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase (BaSP) originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). This strain is further modified with genomic knock-ins of constitutive transcriptional units for the N-acetylglucosamine beta-1, 3-galactosyltransferase (WbgO) from E. coli O55:H7 (UniProt ID D3QY14). Fed-batch fermentations at bioreactor scale (5L and 30L) are performed as described in Example 1. Sucrose is used as a carbon source and lactose is added in the batch medium. During fed-batch, sucrose is added via an additional feed. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para- Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, betafl, 3)galactosyl-para-Lacto-N-neopentaose and beta-(l,4)galactosyl-para-Lacto-N-pentaose.

Example 7. Production of an oligosaccharide mixture comprising 2'FL, 3-FL, Di FL, LN3, LNT and LNFP-I with a modified E. coli host in fed-batch fermentations

An E. coli strain modified for GDP-fucose as described in Example 1 is further modified with a genomic knock-out of the LacZ, LacY and LacA genes and with addition of transcriptional units to express the lactose permease LacY from E. coli (UniProt ID P02920), the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the galactoside beta- 1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N- acetylglucosamine beta-1, 3-galactosyltransferase (WbgO) from E. coli 055:1-17 (UniProt ID D3QY14), the alpha-1, 2-fucosyltransferase HpFutC from Helicobacter pylori (UniProt ID Q9X435) and the alpha-1, 3- fucosyltransferase HpFucT from H. pylori (UniProt ID 030511). Fed-batch fermentations at bioreactor scale (5Land 30L) are performed as described in Example 1. Sucrose is used as a carbon source and lactose is added in the batch medium. During fed-batch, sucrose is added via an additional feed. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, LN3, LNT and LNFP-I (Fuc-al,2-Gal- bl,3-GlcNAc-bl,3-Gal-bl,4-Glc).

Example 8. Production of an oligosaccharide mixture comprising LN3, sialylated LN3, LNT, LSTa and 3'SL with a modified E. coli host when evaluated in a fed-batch fermentation process with glycerol as carbon source, and sialic acid and lactose as precursors

An E. coli K12 MG1655 strain modified for production of LN3 as described e.g., in WO22034075 was further modified to express the N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 to produce LNT. Said strain was further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID A0A849CI62) and the a-2,3-sialyltransferase from P. multocida (UniProt ID Q9CLP3). Fed-batch fermentations at bioreactor scale (5L and 30L) are performed as described in Example 1. Glycerol is used as a carbon source and lactose is added in the batch medium. During fed-batch, glycerol and sialic acid are added via additional feeds. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-bl,3-Gal-bl,4-Glc), LNT, LSTa (Neu5Ac-a2,3-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-Glc) and 3'SL.

Example 9. Production of an oligosaccharide mixture comprising 6'SL and LSTc with a modified E. coli host in fed-batch fermentations

An E. coli strain engineered for production of 6'SL as described in WO2018122225 was further modified with transcriptional units encoding LgtA from N. meningitidis (UniProt ID Q9JXQ6) and LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). The mutant strain thus obtained was used in a fed-batch fermentation process. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed. Regular broth samples were taken at several time points during the fermentation process and the production of an oligosaccharide mixture comprising 6'SL and LSTc was measured using UPLC as described in Example 1.

Example 10. Production of an oligosaccharide mixture comprising LN3, sialylated LN3, LNnT, para-lacto- N-neohexaose, di-sialylated LNnT, LSTc and 6'SL with a modified E. coli host when evaluated in a fed- batch fermentation process

An E. coli K12 MG1655 strain modified for production of sialic acid as described e.g., in WO2018122225 was further modified with genomic knock-ins of constitutive transcriptional units the galactoside beta- 1,3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6) and the N- acetylglucosamine beta-1, 4-galactosyltransferase (LgtB) from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000) to allow production of LNnT. In a next step, the novel strain was further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid having constitutive transcriptional units for the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62) and a P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of the a-2,6-sialyltransferase P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) having beta-galactoside alpha-2, 6-sialyltransferase activity. The mutant strain was further engineered for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), a fructose kinase (Frk) originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase (BaSP) originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). Fed-batch fermentations at bioreactor scale (5L and 30L) were performed as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed. Regular broth samples were taken at several time points during the fermentation process and evaluated via UPLC. UPLC analysis showed that fermentation broth of the selected strain taken after the batch phase comprised lactose, LN3, 6'SL, and LNnT, whereas fermentation broth of the selected strain taken after the fed-batch phase comprised an oligosaccharide mixture comprising LN3, 6'SL, and LNnT, 6' -sialylated LN3 (Neu5Ac-a-2,6-(GlcNAc-b-l,3)-Gal-b-l,4-Glc), LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), para-lacto-N-neohexaose and di-sialylated LNnT.

Example 11. Production of an oligosaccharide mixture comprising LN3, sialylated LN3, LNnT, LSTc and 6'SL with a modified E. coli host when evaluated in a fed-batch fermentation process with glycerol as carbon source, and sialic acid and lactose as precursors

An E. coli K12 MG1655 strain modified for production of LN3 as described e.g., in WO22034075 was further modified to express the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) to produce LNnT. Said strain was further modified with a genomic knock-out of the E. coli lacZ gene and transformed with an expression plasmid containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID A0A849CI62) and the alpha-2, 6-sialyltransferase (PdST6) from Photobacterium damselae (UniProt ID 066375). Fed-batch fermentations at bioreactor scale (5L and 30L) are performed as described in Example 1. Glycerol is used as a carbon source and lactose is added in the batch medium. During fed-batch, glycerol and sialic acid are added via additional feeds. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising LN3, 3'-sialylated LN3 (Neu5Ac-a2,3-GlcNAc-bl,3- Gal-bl,4-Glc), LNnT, LSTc and 6'SL.

Example 12. Production of an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide structures with a modified E. coli host when evaluated in a fed-batch fermentation process

An E. coli strain modified for production of sialic acid as described e.g., in WO2018122225 is further modified with genomic knock-outs of the E. coli LacZ, LacY, LacA, wcaJ, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for the UDP-glucose-4-epimerase (galE) from E. coli (UniProt ID P09147), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 3-galactosyltransferase (WbgO) (Uniprot ID D3QY14) from E. coli O55:H7 and the lactose permease LacY from E. coli (UniProt ID P02920). In a next step, the novel strain is transformed with two compatible expression plasmids wherein a first plasmid contained constitutive expression units for the H. pylori alpha-1, 2-fucosyltransferase HpFutC (UniProt ID Q9X435) and the H. pylori alpha-1, 3-fucosyltransferase HpFucT (UniProt ID 030511), and wherein a second plasmid contained constitutive expression units for the alpha-2, 3-sialyltransferase (PmultST3) from P. multocida (UniProt ID Q9CLP3), the alpha-2, 6-sialyltransferase (PdST6) from Photobacterium damselae (UniProt ID 066375) and the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62). The mutant strain is further engineered for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), a fructose kinase (Frk) originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase originating from B. adolescentis (UniProt ID A0ZZH6). Fed-batch fermentations at bioreactor scale (5L and 30L) are performed as described in Example 1. Sucrose is used as a carbon source and lactose is added in the batch medium. During fed-batch, sucrose was added via an additional feed. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, 3'SL, 6'SL, 3'S-2'FL, 3'S-3- FL, 6'S-2'FL, 6'S-3-FL, LNB, 2'FLNB, 4-FLNB, difucosylated LNB, 3'SLNB, 6'SLNB, LN3, sialylated LN3, LNT, LNFP-I and LSTa.

Example 13. Production of an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide structures with a modified E. coli host when evaluated in a fed-batch fermentation process

An E. coli strain modified for production of sialic acid as described e.g., in WO2018122225 is further modified with genomic knock-outs of the E. coli LacZ, LacY, LacA, wcaJ, ushA and galT genes and genomic knock-ins of constitutive expression cassettes for the UDP-glucose-4-epimerase (galE) from E. coli (UniProt ID P09147), the galactoside beta-1, 3-N-acetylglucosaminyltransferase (LgtA) from N. meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 4-galactosyltransferase (LgtB) from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) and the lactose permease LacY from E. coli (UniProt ID P02920). In a next step, the novel strain is transformed with an expression plasmid comprising constitutive expression units for the H. pylori alpha-1, 3-fucosyltransferase HpFucT (UniProt ID 030511), the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) and the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62). The mutant strain is further engineered for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), a fructose kinase (Frk) originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase (BaSP) originating from B. adolescentis (UniProt ID A0ZZH6). Fed-batch fermentations at bioreactor scale (5L and 30L) are performed as described in Example 1. Sucrose is used as a carbon source and lactose is added in the batch medium. During fed- batch, sucrose is added via an additional feed. Regular broth samples are taken at several time points during the fermentation process and evaluated via UPLC for production of an oligosaccharide mixture comprising 3-FL, 6'SL, LN3, sialylated LN3, LNnT, LNFP-III and LSTc. Example 14. Production of one or more sialylated oligosaccharide(s) with an engineered S. cerevisiae host

In an example a S. cerevisiae strain is engineered for production of 3'SL as described in Example 1 with a compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169 (sequence version 04, 23 Jan 2007), by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the phosphatase SurE from E. coli (UniProt ID P0A840), the N-acylglucosamine 2-epimerase AGE from B. ovatus (UniProt ID A7LVG6), the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4), the N- acylneuraminate cytidylyltransferase NeuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of 3'SL.

In another example a S. cerevisiae strain is engineered as described in Example 1 with a first compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169 (sequence version 04, 23 Jan 2007), by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the phosphatase SurE from E. coli (UniProt ID P0A840), the N-acylglucosamine 2-epimerase AGE from B. ovatus (UniProt ID A7LVG6), the N- acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4), the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375), and with a second compatible yeast expression plasmid comprising constitutive transcriptional units for the UDP-glucose-4-epimerase galE from E. coli (UniProt ID P09147), the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA from N. meningitidis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 6'SL, LN3, LNnT and LSTc.

Example 15. Production of one or more fucosylated oligosaccharide(s) with an engineered S. cerevisiae host

In an example to produce 3-FL, a S. cerevisiae strain was transformed with an expression plasmid that was modified for production of GDP-fucose as described in Example 1 and that further comprised transcriptional units encoding the lactose permease LAC12 from K. lactis (UniProt ID P07921) and the H. pylori alpha-1, 3-fucosyltransferase HpFucT (UniProt ID 030511). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of 3-FL.

In another example a 5. cerevisiae strain is engineered as described in Example 1 with a first compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88), the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055, sequence version 02, 01 Nov 1997) and the a-l,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and with a second compatible yeast expression plasmid comprising constitutive transcriptional units for the UDP-glucose-4- epimerase galE from E. coli (UniProt ID P09147), the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N. meningitidis (UniProt ID Q9JXQ6) and the N- acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E. coli 055:1-17 (Uniprot ID D3QY14). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose and LacNAc (Gal-pi,4-GlcNAc). Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 2'FL, Di FL, LN3, LNT, lacto-N-fucopentaose I (LNFP-I, Fuc-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) and 2'FLacNAc (Fuc- al,2-Gal-pi,4-GlcNAc).

Example 16. Production of an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide structures with an engineered S. cerevisiae host

In another example a S. cerevisiae strain is engineered as described in Example 1 with a first compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88), the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055, sequence version 02, 01 Nov 1997), the a-l,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and the alpha-1, 3- fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) and with a second compatible yeast expression plasmid comprising constitutive transcriptional units for the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169 (sequence version 04, 23 Jan 2007), by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the phosphatase SurE from E. coli (UniProt ID P0A840), the N-acylglucosamine 2-epimerase AGE from B. ovatus (UniProt ID A7LVG6), the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4), the N-acylneuraminate cytidylyltransferase NeuA from P. multocida (UniProt ID A0A849CI62), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) and the alpha-2, 6- sialyltransferase (PdST6) from P. damselae (UniProt ID 066375). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, 3'SL and 6'SL. Example 17. Production of 6'SL with a modified B. subtilis host

A wild-type B. subtilis strain modified for production of sialic acid and CMP-sialic acid as described e.g., in WO22034067 is further modified with transcriptional units encoding the lactose permease LacY (UniProt ID P02920) from E. coli and the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production 6'SL.

Example 18. Production of an oligosaccharide mixture comprising 6'SL and LSTc with a modified B. subtilis host

In another example, the B. subtilis host modified for production of 6'SL as described in Example 17 is further modified with transcriptional units encoding LgtA from N. meningitidis (UniProt ID Q9JXQ6) and LgtB from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using appropriate selective medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising LN3, LNnT, 6'SL and LSTc.

Example 19. Production of an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, 3'SL and 6'SL with a modified C. glutamicum host

A C. glutamicum strain modified for production of sialic acid and CMP-sialic acid as described e.g., in WO22034067 is further modified with transcriptional units encoding the lactose permease LacY (UniProt ID P02920) from E. coli, the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417), the sucrose phosphorylase from B. adolescentis (UniProt ID A0ZZH6), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3), the alpha- 2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375), the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, 3'SL and 6'SL.

Example 20. Production of an oligosaccharide mixture comprising 3'SL and 6'SL with a modified C. glutamicum host

A C. glutamicum strain modified for production of sialic acid and CMP-sialic acid as described e.g., in WO22034067 is further modified with transcriptional units encoding the lactose permease LacY (UniProt ID P02920) from E. coli, the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417), the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3), and the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 3'SL and 6'SL.

Example 21. Production of an oligosaccharide mixture comprising LSTc and 6'SL with a modified C. glutamicum host

A C. glutamicum strain modified for production of sialic acid and CMP-sialic acid as described e.g., in WO22034067 is further modified with transcriptional units encoding the lactose permease LacY (UniProt ID P02920) from E. coli, the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417), the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6), LgtA from N. meningitidis (UniProt ID Q9JXQ6), LgtB from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000), and the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising LSTc and 6'SL.

Example 22. Production of an oligosaccharide mixture comprising 3-FL, LNFP-III, LNnT and LN3 with a modified C. glutamicum host

A C. glutamicum strain modified for production of LN3 as described e.g., in WO22034069 is further modified for LNnT production with a transcriptional unit encoding the N-acetylglucosamine beta-1, 4- galactosyltransferase LgtB from N. meningitidis (Uniprot ID Q51116, sequence version 02, 01 Dec 2000). In a next step, the mutant strain is further modified with transcriptional units encoding the sucrose transporter (CscB) from E. coli \N (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6) enabling the strain to grow on sucrose. In a final step, the mutant strain is modified with a transcriptional unit encoding the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511). Cultivation of the novel strain is performed according to the culture conditions provided in Example 1 using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising 3-FL, LN3, LNnT and LNFP-III.

Example 23. Production of LSTc with modified C. reinhardtii cells

C. reinhardtii cells modified for production of UDP-galactose as described e.g., in WO22034067 are further modified for CMP-sialic acid synthesis with genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GN E from Homo sapiens (UniProt ID Q9Y223) differing from the native polypeptide with a R263L mutation, the N-acylneuraminate-9-phosphate synthetase NANS from H. sapiens (UniProt ID Q9NR45), the N- acylneuraminate cytidylyltransferase CMAS from H. sapiens (UniProt ID Q8NFW8) and the CMP-sialic acid transporter CST from Mus musculus (UniProt ID Q61420). In a final step, the engineered cells are modified with an expression plasmid comprising constitutive transcriptional units comprising the alpha-2, 6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375), the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N. meningitis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). Cultivation of the engineered cells is performed according to the culture conditions provided in Example 1 using TAP medium comprising galactose, glucose and GIcNAc. Regular samples are taken and evaluated via UPLC for production of LSTc.

Example 24. Production of 6'SL with modified C. reinhardtii cells

C. reinhardtii cells modified for production of UDP-galactose as described e.g., in WO22034067 are further modified for CMP-sialic acid synthesis with genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GN E from Homo sapiens (UniProt ID Q9Y223) differing from the native polypeptide with a R263L mutation, the N-acylneuraminate-9-phosphate synthetase NANS from H. sapiens (UniProt ID Q9NR45), the N- acylneuraminate cytidylyltransferase CMAS from H. sapiens (UniProt ID Q8NFW8) and the CMP-sialic acid transporter CST from Mus musculus (UniProt ID Q61420). In a final step, the engineered cells are modified with an expression plasmid comprising a constitutive transcriptional unit comprising the alpha-2, 6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375). Cultivation of the engineered cells is performed according to the culture conditions provided in Example 1 using TAP medium comprising galactose and glucose. Regular samples are taken and evaluated via UPLC for production of 6'SL.

Example 25. Production of an oligosaccharide mixture comprising LSTc and 6'SL with modified C. reinhardtii cells

C. reinhardtii cells modified for production of UDP-galactose as described e.g., in WO22034067 are further modified for CMP-sialic acid synthesis with genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GN E from Homo sapiens (UniProt ID Q9Y223) differing from the native polypeptide with a R263L mutation, the N-acylneuraminate-9-phosphate synthetase NANS from H. sapiens (UniProt ID Q9NR45), the N- acylneuraminate cytidylyltransferase CMAS from H. sapiens (UniProt ID Q8NFW8) and the CMP-sialic acid transporter CST from Mus musculus (UniProt ID Q61420). In a final step, the engineered cells are modified with an expression plasmid comprising constitutive transcriptional units comprising the alpha-2,6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375), the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N. meningitis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). Cultivation of the engineered cells is performed according to the culture conditions provided in Example 1 using TAP medium comprising galactose, glucose and GIcNAc. Regular samples are taken and evaluated via UPLC for production of an oligosaccharide mixture comprising LN3, LNnT, 6'SL and LSTc.

Example 26. Production of 3'SL using animal cells that are re-programmed into mammary-like cells

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 1 are modified via CRISPR-CAS to express the GlcN6P synthase GFPT1 from H. sapiens (UniProt ID Q06210), the glucosamine 6-phosphate N-acetyltransferase GNA1 from H. sapiens (UniProt ID Q96EK6), the phosphoacetylglucosamine mutase PGM3 from H. sapiens (UniProt ID 095394), the UDP-N- acetylhexosamine pyrophosphorylase UAP1 from H. sapiens (UniProt ID Q16222), the N-acylneuraminate cytidylyltransferases NeuA from M. musculus (UniProt ID Q99KK2) and the CMP-N-acetylneuraminate- beta-l,4-galactoside alpha-2, 3-sialyltransferase ST3GAL3 from H. sapiens (UniProt ID Q11203). Cells are seeded at a density of 20,000 cel Is/cm 2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 1, cells are subjected to UPLC to analyse for production of 3'SL.

Example 27. Production of one or more oligosaccharide(s) using mammalian cell cultures

In another example, one or more oligosaccharide(s) are produced by means of mammalian cell cultures. These cell cultures are well known to a person skilled in the art; the cultivation and differentiation of said cell cultures is, e.g., described by Qu et al. (Stem Cell Report 2017, 8, 205-215). The primary cell supernatant of said cell cultures contains lactose or human milk oligosaccharides (HMOs), like e.g., fucosylated and sialylated HMOs. To simplify the separation of cells and the produced cell-cultured milk, the cells are immobilized on typical carriers. Microcarriers are for instance Cytodex 3, Cytopore 1 (cyteva), BioNOC II (Cesco), allowing the use of stirred bioreactors for the production of cellular milk.

Example 28. Production of an oligosaccharide mixture comprising 6'SL and LSTc using animal cells that are re-programmed into mammary-like cells

Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 1 are modified via CRISPR-CAS to express the GlcN6P synthase GFPT1 from H. sapiens (UniProt ID Q06210), the glucosamine 6-phosphate N-acetyltransferase GNA1 from H. sapiens (UniProt ID Q96EK6), the phosphoacetylglucosamine mutase PGM3 from H. sapiens (UniProt ID 095394), the UDP-N- acetylhexosamine pyrophosphorylase UAP1 from H. sapiens (UniProt ID Q16222), the N-acylneuraminate cytidylyltransferases NeuA from M. musculus (UniProt ID Q99KK2), the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N. meningitis (UniProt ID Q9JXQ6) and the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375). Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 1, cells are subjected to UPLC to analyse for production of an oligosaccharide mixture comprising LSTc and 6'SL.

Example 29. Production of an oligosaccharide mixture comprising LSTc and 6'SL using mammalian cell cultures

In another example, an oligosaccharide mixture comprising LSTc and 6'SL is produced by means of mammalian cell cultures. These cell cultures are well known to a person skilled in the art; the cultivation and differentiation of said cell cultures is, e.g., described by Qu et al. (Stem Cell Report 2017, 8, 205-215). The primary cell supernatant of said cell cultures contains lactose or human milk oligosaccharides. To simplify the separation of cells and the produced cell-cultured milk, the cells are immobilized on typical carriers. Microcarriers are for instance Cytodex 3, Cytopore 1 (cyteva), BioNOC II (Cesco), allowing the use of stirred bioreactors for the production of cellular milk.

Example 30. Enzymatic synthesis of one or more oligosaccharide(s)

A cell-free protein expression system (CFPS) can be used to produce functional proteins without the use of living cells. In CFPS, a solution containing all the cellular machinery needed to direct protein synthesis (e.g., ribosomes, tRNAs, enzymes, cofactors, amino acids) is used to transcribe and translate a nucleic acid template (e.g., plasmid DNA, linear DNA or mRNA). There are different types of CFPS systems (Khambhati et al., Front. Bioeng. Biotechnol. 2019, Vol 7, art 248, pag. 1-16). In present example, the PURExpress system (NEB) was used according to the manufacturer's recommendations to produce the alpha-1, 2- fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435), the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375) in separate CFPS reactions. In a next step, different enzymatic synthesis reactions were set up to produce one or more oligosaccharide(s). For each enzymatic synthesis reaction, any one or more of said alpha-1, 2- fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-2, 3-sialyltransferase and/or alpha-2, 6- sialyltransferase was/were added to a reaction mixture together with one or more nucleotide-activated sugars comprising GDP-fucose (GDP-Fuc) and CMP-sialic acid (CMP-Neu5Ac) and with one or more compatible substrate(s) such as e.g., lactose, 2'FL, 3-FL, LNB, LacNAc, LNT, LNnT, and a buffering component such as Tris-HCI or HEPES. Each enzymatic synthesis reaction was then incubated at a certain pH (for example 7), at a certain temperature (for example 37°C) for a certain amount of time (for example

8 hours, 16 hours or 24 hours), during which the substrate(s) was/were converted by the one or more glycosyltransferase(s) added to obtain the desired one or more oligosaccharide(s).

In this way, an enzymatic synthesis reaction was set up comprising said alpha-1, 2-fucosyltransferase produced in a CFPS reaction, 10 mM GDP-Fuc, 10 mM lactose as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain 2'FL. Another enzymatic synthesis reaction was set up comprising said alpha-1, 3-fucosyltransferase produced in a CFPS reaction, 10 mM GDP-Fuc, 10 mM lactose as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain 3-FL. Another enzymatic synthesis reaction was set up comprising said alpha-1, 2-fucosyltransferase produced in a CFPS reaction, 10 mM GDP-Fuc, 10 mM LNT as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain LNFP-I (Fuc-otl,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-1, 3-fucosyltransferase produced in a CFPS reaction, 10 mM GDP-Fuc, 10 mM LNT as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain LNFP-V (Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-(Fuc-al,3)-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-1, 3-fucosyltransferase produced in a CFPS reaction, 10 mM GDP-Fuc, 10 mM lactose and 10 mM LNT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°Cfor 24 hours to obtain an oligosaccharide mixture comprising 3-FL and LNFP- V. Another enzymatic synthesis reaction was set up comprising said alpha-1, 2-fucosyltransferase and said alpha-1, 3-fucosyltransferase both produced in CFPS reactions, 10 mM GDP-Fuc, 10 mM lactose and 10 mM LNT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, LNFP-I and LNFP-V. Another enzymatic synthesis reaction was set up comprising said alpha-1, 2-fucosyltransferase and said alpha-1, 3-fucosyltransferase both produced in CFPS reactions, 10 mM GDP-Fuc, 10 mM lactose, 10 mM LNT and 10 mM LNnT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain an oligosaccharide mixture comprising 2'FL, 3-FL, DiFL, LNFP-I, LNFP-V and LNFP-III (Gal-pi,4-(Fuc-otl,3)- GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-2, 3- sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM lactose as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain 3'SL. Another enzymatic synthesis reaction was set up comprising said alpha-2, 6-sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM lactose as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain 6'SL. Another enzymatic synthesis reaction was set up comprising said alpha- 2,3-sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM LNT as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain LSTa (Neu5Ac-a2,3- Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-2, 6-sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM LNnT as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain LSTc (Neu5Ac-a2,6- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-2, 3-sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM LNnT as compatible substrate and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain LSTd (Neu5Ac-cc2,3- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-2, 3-sialyltransferase and said alpha-2, 6-sialyltransferase both produced in CFPS reactions, 10 mM CMP-Neu5Ac, 10 mM lactose, 10 mM LNnT and 10 mM LNT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain an oligosaccharide mixture comprising 3'SL, 6'SL, LSTa, LSTc and LSTd. Another enzymatic synthesis reaction was set up comprising said alpha-1, 3- fucosyltransferase and said alpha-2, 3-sialyltransferase both produced in CFPS reactions, 10 mM CMP- Neu5Ac, 10 mM GDP-fucose, 10 mM lactose and 10 mM LNnT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain an oligosaccharide mixture comprising 3-FL, 3'SL, LNFP-III and LSTd. Another enzymatic synthesis reaction was set up comprising said alpha-1, 2- fucosyltransferase and said alpha-2, 3-sialyltransferase both produced in CFPS reactions, 10 mM CMP- Neu5Ac, 10 mM GDP-fucose, 10 mM lactose and 10 mM LNT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 24 hours to obtain an oligosaccharide mixture comprising 2'FL, 3'SL, LNFP-I and LSTa. Another enzymatic synthesis reaction was set up comprising said alpha-2, 6- sialyltransferase produced in a CFPS reaction, 10 mM CMP-Neu5Ac, 10 mM lactose and 10 mM LNnT as compatible substrates and a HEPES buffer at pH 7 and incubated at 37°C for 16 hours to obtain an oligosaccharide mixture comprising 6'SL and LSTc.

Example 31. Enzymatic synthesis of 3'SL

In another example, mutant E. coli BL21 strains are used to produce four recombinant proteins: an N- acylglucosamine 2-epimerase originating from B. ovatus (UniProt ID A7LVG6), a sialic acid aldolase NAL form Lactiplantibacillus plantarum WCFS1 (UniProt ID P59407), a CMP-sialic acid synthetase siaC originating from N. meningitidis (UniProt ID Q7DDU0) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). For each protein an expression vector using the pET22b(+) expression system (GenScript Biotech) is constructed with the corresponding gene and transformed into E. coli BL21 cells. The four mutant strains are cultivated in LB medium and recombinant protein expression for each of said N-acylglucosamine 2-epimerase, sialic acid aldolase, CMP-sialic acid synthetase and alpha-2, 3- sialyltransferase is obtained upon induction with 1 mM isopropyl p-D-thiogalactopyranoside (IPTG) for 20 h at 25°C. Purification of each recombinant protein is performed by standard His-tag affinity chromatography. In a next step, 3'sialyllactose is produced in an enzymatic reaction performed at 37°C and 450 rpm in a 1 mL mixture comprising 100 mM Tris/HCI buffer (pH 8), 0.1 pM of each of the four recombinant proteins produced by the mutant E. coli BL21 cells, 20 mM MgCI₂, 0.2 mM L-cysteine, 20mM GIcNAc, 50mM pyruvate, 25 mM CTP, and 20 mM lactose. Example 32. Enzymatic synthesis of LSTc and 6'SL

In another example, mutant E. coli BL21 strains are used to produce four recombinant proteins: an N- acylglucosamine 2-epimerase originating from B. ovatus (UniProt ID A7LVG6), a sialic acid aldolase NAL form Lactiplantibacillus plantarum WCFS1 (UniProt ID P59407), a CMP-sialic acid synthetase siaC originating from N. meningitidis (UniProt ID Q7DDU0) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375). For each protein an expression vector using the pET22b(+) expression system (GenScript Biotech) is constructed with the corresponding gene and transformed into E. coli BL21 cells. The four mutant strains are cultivated in LB medium and recombinant protein expression for each of said N-acylglucosamine 2-epimerase, sialic acid aldolase, CMP-sialic acid synthetase and alpha-2, 6- sialyltransferase is obtained upon induction with 1 mM isopropyl p-D-thiogalactopyranoside (IPTG) for 20 h at 25°C. Purification of each recombinant protein is performed by standard His-tag affinity chromatography. In a next step, 6'SL and LSTc are produced in an enzymatic reaction performed at 37°C and 450 rpm in a mixture comprising 100 mM Tris/HCI buffer (pH 8), 0.1 pM of each of the four recombinant proteins produced by the mutant E. coli BL21 cells, 20 mM MgCI2, 0.2 mM L-cysteine, 20 mM GIcNAc, 50 mM pyruvate, 25 mM CTP, 20 mM lactose and 20 mM LNnT.

Example 33. Composition determination of the fermentation or cultivation broth or of the oligosaccharide solutions obtained after enzymatic synthesis reactions

For the fermentation or cultivation broths obtained in Examples 2-29 the composition was determined by measuring the cell dry mass of the broth, the ash content of the supernatant and the broth, the oligosaccharide content of the supernatant and the broth and the total dry solids in the broth in accordance with the methods described in Example 1.

Also, the composition of the solutions obtained in Examples 30-32 was determined by measuring the ash content of the supernatant and the oligosaccharide content of the supernatant and the total dry solids in the supernatant in accordance with the methods described in Example 1. For all samples the total oligosaccharide content was below 80% on total dry solids. The oligosaccharide mixture purity in the broth ranged from 30% to 77%.

Example 34. Processing a sialylated oligosaccharide syrup

The broth originating from a 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested and further concentrated to Brix 21 using vacuum distillation whereafter the pH of the solution was adjusted to 2.95 using H₂SO₄. A 500 m L glass column (ACE) was filled with 250 mL of Amberlite FPC22 (Dupont), i.e., a strong acid cation exchanger, and 250 mL of Amberlite FPA51 (Dupont), i.e., a weak base anion exchanger, and wetted with demineralized water (10- 30 tS/cm). Hereafter the column was shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH was pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the 6'SL syrup, having a pH of 2.95, was pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1. The ash content of the influent was measured to be 10% of the total dry weight whereas the ash content of the effluent was 7%. As it is desired to produce a 6'SL sodium salt, the sodium content is preferably as high as possible. In the influent, only 3% of the total cation concentration was Na+ while this was 29% after the mixed bed process. 95% of the 6'SL in the influent was recovered in the effluent after being treated with the mixed bed resin.

Example 35. Processing a sialylated oligosaccharide syrup

The broth originating from a 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested and further concentrated to Brix 21 using vacuum distillation whereafter the pH of the solution was adjusted to 3.99 using H2SO4. A 500 mL glass column (ACE) was filled with 250 mL of Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and 250 mL of Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, and wetted with demineralized water (10-30 pS/cm). Hereafter the column was shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH was pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the 6'SL syrup, having a pH of 3.99, was pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1. The ash content of the influent was measured to be 10% of the total dry weight whereas the ash content of the effluent was 9%. As it is desired to produce a 6'SL sodium salt, the sodium content is preferably as high as possible. In the influent, only 3% of the total cation concentration was Na+ while this was 43% after the mixed bed process. More than 90% of the 6'SL in the influent was recovered in the effluent after being treated with the mixed bed resin.

Example 36. Removing heavy metal traces from a 2'FL syrup

The broth originating from a 2'FL fermentation as described in Example 3 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested and further concentrated to Brix 20 using vacuum distillation whereafter the pH of the solution was adjusted to 4.4 using H₃PO₄. A 500 m L glass column (ACE) was filled with 250 mLof Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and 250 mLof Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, and wetted with demineralized water (10-30 pS/cm). Hereafter the column was shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH was pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the 2'FL syrup, having a pH of 4.4, was pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1. The ash content of the influent was measured to be 0.38% of the total dry weight whereas the ash content of the effluent was 0.19%. Of all 2'FL that was present in the influent, more than 85% was recovered in the effluent after being treated with the mixed bed resin. Furthermore, the cadmium, mercury, arsenic and lead concentrations were below the detection limits of 0.1 pg/L. Afterwards the pH of the effluent was corrected to 6.5 using 4% H2SO4.

Example 37. Removing heavy metal traces from a LNT syrup

The broth originating from a LNT fermentation as described in Example 5 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested and further concentrated to Brix 20 using vacuum distillation whereafter the pH of the solution was adjusted to 3.99 using H3PO4. A 500 mL glass column (ACE) was filled with 250 mL of Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and 250 mL of Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, and wetted with demineralized water (10-30 pS/cm). Hereafter the column was shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH was pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the LNT syrup, having a pH of 3.99, was pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1. The ash content of the influent was measured to be 0.2% of the total dry weight whereas the ash content of the effluent was 0.12%. Of all LNT that was present in the influent, more than 85% was recovered in the effluent after being treated with the mixed bed resin. Furthermore, the cadmium, mercury, arsenic and lead concentrations were below the detection limits of 0.1 pg/L. Afterwards, the pH of the effluent was corrected to 6.5 using 4% H₂SO₄. Example 38. Separating sialic acid from an oligosaccharide mixture

Broth originating from a fermentation comprising an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharides as obtained in Examples 12-13 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested and further concentrated to a syrup of Brix 20 using vacuum distillation whereafter the pH of this syrup was adjusted to 4 using H₃PO₄. A 500 mL glass column (ACE) was filled with 250 mL of Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and 250 mL of Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, and wetted with demineralized water (10- 30 pS/cm). Hereafter the column was shaken to obtain a uniform mixed bed. Then, 5 bed volumes of 4% NaOH was pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the syrup was pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC, Dionex, and ash content measurements as described in Experiment 1. The ash content of the influent was measured to be 8% of the total dry weight whereas the ash content of the effluent was 5%. Of all fucosylated oligosaccharides that were present in the influent, more than 90% was recovered in the effluent after being treated with the mixed bed resin. Similarly, of all sialylated oligosaccharides that were present in the influent, more than 85% was recovered in the effluent after being treated with the mixed bed resin. However, of all sialic acid that was present in the influent, more than 95% was retained in the mixed bed resin and thus removed from the effluent.

Example 39. Removing heavy metal traces from a syrup comprising neutral oligosaccharides

The broth originating from a fermentation as described in Examples 4 and 7 or an aqueous solution from an enzymatic synthesis reaction for the production of one or more neutral oligosaccharide(s) as described in Example 30 is subjected to ultrafiltration of a 15 kDa molecular weight cut-off ceramic membrane (Tami) whereafter the resulting ultrafiltrate is subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate is harvested. The retentate is further concentrated using vacuum distillation whereafter the pH of the solution is adjusted to 4 using H₃PO₄. A glass column (ACE) is filled with equal volumes of Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and of Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, wetted with demineralized water (10-30 pS/cm) and shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH is pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the syrup comprising one or more neutral oligosaccharide(s) and having a pH of 4, is pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step was performed at room temperature. Both the influent and the effluent of the column is collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1 for determination of the ash content and the one or more neutral oligosaccharide(s) before and after the mixed bed passage. After the mixed bed treatment, > 85 % of the neutral oligosaccharide(s) are present in the effluent.

Example 40. Removing heavy metal traces from a syrup comprising one or more sialylated oligosaccharide(s)

The broth originating from a fermentation or cell cultivation comprising one or more sialylated oligosaccharide(s) as described in Examples 8, 14, 17, 19, 23, 26 and 27 or the aqueous solution from an enzymatic synthesis reaction for the production of one or more sialylated oligosaccharide(s) as described in Examples 30 to 32 is subjected to a 15 kDa molecular weight cut-off ceramic membrane (Tami) whereafter the resulting ultrafiltrate is subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate is harvested and further concentrated using vacuum distillation whereafter the pH of the solution is adjusted to 4 using H3PO4. A glass column (ACE) is filled with equal volumes of Diaion PK228 (Mitsubishi), i.e., a strong acid cation exchanger, and of Diaion HPA25 (Mitsubishi), i.e., a strong base anion exchanger, wetted with demineralized water (10-30 pS/cm) and shaken to obtain a uniform mixed bed. Then, 6 bed volumes of 4% NaOH is pumped over the column at a flow rate of 6 BV/h to condition the cation and anion resin in the Na+ and OH- form, respectively. In a next step, the syrup comprising one or more sialylated oligosaccharide(s) and having a pH of 4, is pumped over the resin at a flow rate of 3 BV/h. The mixed bed ion exchange step is performed at room temperature. Both the influent and the effluent of the column is collected and subjected to UPLC, Dionex, and ash content measurements as described in Example 1 for determination of the ash content, sialic acid and the one or more sialylated oligosaccharide(s) before and after the mixed bed passage. After the mixed bed treatment, > 85 % of the sialylated oligosaccharide(s) are present in the effluent.

Example 41. Testing anion exchange on a 6'sialyllactose solution

The broth originating from the 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested. A 5L aliquot of this retentate was further concentrated to Brix 10 using vacuum distillation and divided into two fractions. The pH of the first fraction was adjusted to 1 using H2SO4, whereas the pH of the second fraction was adjusted to 7 using NaOH. In a next step, 40 mL of each fraction was mixed with 3 mL of an anionic ion exchange resin that was provided in either OH- form or Cl- form. Table 2 summarizes the different anionic ion exchange resins tested. Each mixture was incubated for lh on a shaking platform at 200 rpm set at room temperature. Before and after incubation with each anionic ion exchange resin at different ionic form, the syup was analyzed for 6'SL using UPLC, and for salts (i.e., sulphate and phosphate) using Dionex as described in Example 1. The results are summarized in Table 3. The most desirable result was obtained with the Amberlite FPA90 resin in OH' form at pH 1 as only 5% of 6'SL was lost, and a large majority of the unwanted anions were removed. At most resins there is no major difference between performing the experiment at pH 1 or 7 except for Amberlite FPA51 which is the only weak base anion exchanger. It is known that these types of resins lose their effectiveness at a higher pH value. A 6'SL loss of 63% was obtained when performing ion exchange with the Diaion SA20A resin in OH" form at pH 1 which is the least desirable result obtained in the experiment. The other resins show mainly a 6'SL loss between 10-25 % and good sulphate and phosphate removal.

Table 2. Overview of anionic ion exchange resins tested

Table 3. Loss (%) of 6'SL, sulphate or phosphate in a 6'SL syrup originating from a fermentation broth using different anionic ion exchange resins in either OH- or Cl-form

Example 42. Deashing a 6'SL syrup using a sequential cation/anion exchange setup

The broth originating from the 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested. A 5L aliquot of this retentate was further concentrated to Brix 10 using vacuum distillation, whereafter the pH of the solution was adjusted to 2.9 using H₂SO₄. A 200 mL glass column (YMC) was filled with 105 mL of Amberlite FPC22Na cation exchange resin (Dupont) and wetted with demineralized water (10-30 pS/cm). A second glass column (YMC) was filled with 105 mL of Amberlite FPA51 (OH) anion exchange resin (Dupont). In a next step, IL of the aforementioned 6'SL syrup, having a pH of 2.9, was pumped first over the cation exchange resin and then over the anion exchange resin at a flow rate of 3 BV/h. Both ion exchange steps were performed at room temperature. Before and after the sequential cation/anion exchange, the syrup was subjected to UPLC, Dionex, color and ash content measurements. The ash content of the syrup was measured to be 6% of the total dry weight before passage on the sequential cation/anion exchange whereas the ash content of the syrup was decreased to 4% after said passage on the sequential cation/anion exchange resin. The experiment also showed that 88% of the 6'SL was recovered in the effluent obtained after said passage on the sequential cation/anion exchange resin. Example 43. Separation of 6'SL from LSTc

The broth originating from the fermentation as described in Example 10 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested. After nanofiltration, the retentate was harvested. A 5L aliquot of this retentate was further concentrated to Brix 10 using vacuum distillation, whereafter the pH of the solution was adjusted to 2.9 using H2SO4. A 200 mL glass column (YMC) was filled with 105 mt of Diaion SA20A (OH) anion exchange resin (Mitsubishi) and wetted with demineralized water (10-30 piS/cm). Then, IL of the syrup was pumped over the resin at a flow rate of 3 BV/h. The anionic ion exchange step was performed at room temperature. The effluent of the column was collected and subjected to UPLC analysis to determine the sugar composition. The experiment demonstrated that the influent contained 76% of LSTc, 3.3% of 6'SL and 20.7% of other carbohydrates comprising LN3 and LNnT, whereas the effluent contained 80% of LSTc and 20% of other carbohydrate comprising LN3 and LNnT and 0% 6'SL, indicating that said experimental set-up resulted in a good removal of 6'SL from the syrup whilst maintaining LSTc in the syrup.

Example 44. Removing buffer components from an enzymatically produced 3'SL syrup

The aqueous solutions from the enzymatic 3'SL production process as described in Examples 30 and 31 are filtered using a 15 kDa molecular weight cut-off ceramic membrane (Tami). Afterwards, the ultrafiltrates are concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surface, Mann+Hummel). The pH of the NF retentates are corrected to 4 using H2SO4 and then pumped over 2 glass columns (YMC) wherein the first column contains a packed bed of Amberlite FPC22Na cation exchange resin (Dupont) and the second column contains a packed bed of Amberlite FPA51 OH- anion exchange resin (Dupont). Both ion exchange steps were performed at room temperature. After said passage, less than 15% of 3'SL is lost in the effluent, whereas the remaining concentration of buffer in the effluent is less than 2%.

Example 45. Purification of a chemically synthesized sialylated oligosaccharide

A sialylated oligosaccharide structure is synthesized via chemical synthesis as described in Example 1. In a next step, the chemical synthesis solution is subjected to ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami) and the resulting ultrafiltrate is then subjected to nanofiltration over a Puramem S MWCO 600 Da (single element 2540 format 1.8 m², Evonik). After nanofiltration, the retentate is evaporated. In a next step, the powder is dissolved in water and passed through a column filled with Dowex Optipore L493 (Lenntech). The effluent is further passed over a column containing cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange is performed at room temperature. After cation exchange, the pH is adjusted to 4 by using 50% NaOH. The resulting solution is spray-dried to powder, which is redissolved in demineralized water (10-30 pS/cm) and pumped over a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent is 4. The mixed bed ion exchange step is performed at room temperature. After said mixed bed step, the effluent is analysed for the presence of the sialylated oligosaccharide and for ash content.

Example 46. Deashing a 6'SL syrup with a mixed bed resin in batch mode

The broth originating from the 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate at Brix 6.5 was harvested. Said retentate still contained some traces of sialic acid. A IL aliquot of this retentate was further concentrated to Brix 10 using vacuum distillation. In a next step, five different 50 mL Falcon tubes were each filled with 30 mL of the syrup at Brix 10 thus obtained. Depending on the Falcon tube, the pH of the syrup was adjusted to pH 2, 3, 4, 5 or 6 using H3PO4 and NaOH. The volume changes due to the pH adjustment were minimal. After pH adjustment, 3 mL of an Amberlite MB20 resin (Dupont) was added to each Falcon tube. Then, the Falcon tubes were incubated on a shaking incubator for 1 hour at 200 rpm at 37°C. The sugar and ash concentrations were determined as described in Example 1, before and after the one-hour incubation with the MB20 resin. The results are shown in Table 4.

Due to the pH correction with H3PO4, the ash contents of the syrup at pH 2 and 3 were rather high. However, a significant reduction of more than 50% of ashes was observed in the syrup tested at all pH values (Table 4).

The amount of sialic acid, 6'SL and ash retained on the mixed bed (MB) and thus removed from the syrup is summarized in Table 5. Table 5 shows that increasing the pH of the syrup, i.e., the influent, prior to MB increased the retention of 6'SL on the resin. When the pH of the syrup was adjusted to 3 prior to MB, only 5% of the starting concentration of 6'SL was retained by the resin leaving 95% of 6'SL in the syrup. When the pH of the syrup was adjusted to 5 prior to MB, 27% of the 6'SL was retained by the resin. In contrast, sialic acid was much better retained on the resin at low pH. When the pH of the syrup was adjusted to 3 prior to MB, already 85% of the sialic acid was retained by the resin and thus removed from the syrup and from the 6'SL present in the syrup. Also, the majority of ashes was retained by the resin when the pH of the syrup was adjusted to 3 prior to MB. Based on Table 5, a process comprising adjustment of the pH of an oligosaccharide syrup to 3 prior to MB can be used for efficiently deashing and removing sialic acid from said oligosaccharide syrup. Table 4. Ash and sugar concentrations (g/L) in an oligosaccharide syrup set at different pH before and after mixed bed (MB) treatment

Table 5. Retainment (%) of sialic acid, 6'SL and ash on a MB20 resin using an oligosaccharide syrup influent with a different pH prior to MB treatment

Example 47. Deashing a 6'SL syrup using a packed mixed bed ion exchange resin

The broth originating from the 6'SL fermentation as described in Example 2 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate at Brix 7.2 was harvested. A 5L aliquot of this retentate was further concentrated to a syrup of Brix 10 using vacuum distillation, whereafter the pH of this syrup was adjusted to 2.9 using H2SO4. A 200 mL glass column (YMC) was filled with 105 mL of Amberlite MB20 mixed bed ion exchange resin (Dupont) and wetted with demineralized water (10-30 pS/cm). The mixed bed ion exchange step was performed at room temperature. The syrup, having a pH of 2.9, was pumped over said resin at a flow rate of 3 bed volumes / hour (BV/h). The effuent of the column was collected in fractions of 500 mL and subjected to UPLC, Dionex, color and ash content measurements as described in Example 1. The ash content of the influent was measured to be 6% of the total dry weight whereas the ash content in the effluent was decreased to 4%. However, 89% of the 6'SL in the influent was recovered in the effluent after being treated with the mixed bed resin.

Example 48. Deashing and removing sialic acid from an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide structures

The broth originating from a fermentation as described in Example 12, comprising an oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide structures, was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate was harvested. The pH of this retentate, i.e., syrup, was adjusted to 3 using H2SO4. A 500 mL glass column (ACE-glass) was filled with 250 ml of Amberlite FPC22H strong acid cation exchange resin (Dupont) and 250 mL of Amberlite FPA90 (OH-) strong base anion exchange resin (Dupont) and wetted with demineralized water (10-30 pS/cm). The column was shaken to obtain a uniform mixed bed. The mixed bed ion exchange step was performed at room temperature. The syrup, having a pH of 3, was pumped over the mixed bed resin at a flow rate of 3 BV/h. The effluent of the column was collected and subjected to UPLC measurement as described in Example 1. The experiment demonstrated that more than 95% of the fucosylated oligosaccharide structures and more than 90% of the sialylated oligosaccharide structures (e.g., 5'SL) were recovered in the effluent whereas more than 99% of sialic acid was removed from the effluent. The ash content in the effluent was less than 5% of the total oligosaccharide concentration.

Example 49. Deashing a syrup comprising 3'SL or 6'SL using a packed mixed bed ion exchange resin

The broth from the cultivations of Examples 14, 17, 18, 20, 24 and 26 is used in a cell lysis experiment. A soft release of the product (i.e., 3'SL, 6'SL or an oligosaccharide mixture comprising 3'SL or 6'SL) is established by heating for 1 hour the broth to a temperature between 60°C and 80°C. Other methods encompass freeze thawing, and/or shear stress through sonication, mixing, use of a homogenizer, French press or enzymatic cell lysis. Afterwards, the solutions obtained are cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrates are subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentates are harvested and aliquots of all retentates are further concentrated to different syrups of Brix 10 using vacuum distillation, whereafter the pH of each of said syrups is adjusted to 2.9 using H2SO4. Then, for each of said syrups, a glass column (YMC) is filled 50% with an Amberlite MB20 mixed bed ion exchange resin (Dupont) and wetted with demineralized water (10-30 pS/cm). Every mixed bed ion exchange step is performed at room temperature. Each syrup is separately pumped over such a MB20 resin at a flow rate of 3 BV/h. For every MB run, the effluent of the column is collected and subjected to UPLC, Dionex, color and ash content measurements as described in Example 1. After MB, the ash content in each effluent was lower than the ash content in the respective influent. However, > 85 % of 3'SLor 6'SL, respectively, that was present in the influent is recovered in the effluent after being treated with the mixed bed resin whereas sialic acid and LSTc, when present, are retained in the mixed bed resin.

Example 50. Deashing and removing sialic acid from an oligosaccharide mixture comprising fucosylated and sialylated HMOs

The broth originating from a cell cultivation as described in Example 27 , comprising an oligosaccharide mixture comprising fucosylated and sialylated HMOs, is used in a cell lysis experiment. A soft release of the oligosaccharide mixture comprising fucosylated and sialylated HMOs is established by heating for 1 hour the broth to a temperature between 60°C and 80°C. Other methods encompass freeze thawing, and/or shear stress through sonication, mixing, use of a homogenizer, French press or enzymatic cell lysis. Afterwards, the solution obtained is cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate is subjected to nanofiltration over a Trisep XN45 membrane (1 single element of 4040 format, 7.8 m² membrane surface, Mann+Hummel). After nanofiltration, the retentate is harvested. The pH of this retentate, i.e. syrup, is adjusted to 3 using H2SO4. A 500 mL glass column (ACE-glass) is filled with 250 mL of Amberlite FPC22H strong acid cation exchange resin (Dupont) and 250 mL of Amberlite FPA90 (OH-) strong base anion exchange resin (Dupont) and wetted with demineralized water (10-30 pS/cm). The column is shaken to obtain a uniform mixed bed. The mixed bed ion exchange step is performed at room temperature. The syrup, having a pH of 3, is pumped over the mixed bed resin at a flow rate of 3 BV/h. The effluent of the column is collected and subjected to UPLC measurement as described in Example 1 for measurement of the fucosylated and the sialylated oligosaccharide structures, sialic acid and the ash content.

Example 51. Deashing an enzymatically produced oligosaccharide mixture

The aqueous solution from an enzymatic synthesis reaction set up for the production of an oligosaccharide mixture comprising e.g., 3'SL, 6'SL, LSTa, LSTc and LSTd as described in Example 30 is filtered using a 15 kDa molecular weight cut-off ceramic membrane (Tami). In a next step, the ultrafiltrate is concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surface, Mann+Hummel). The pH of the NF retentate is corrected to 4 using H2SO4 and then pumped over a glass column (YMC) containing a mixed bed of equal volumes of an Amberlite FPC22H cation exchange resin (Dupont) and an Amberlite FPA51 anion exchange resin (Dupont) to remove any remaining Tris-HCI or HEPES buffer. The mixed bed ion exchange step is performed at room temperature. The effluent of the column is collected and subjected to UPLC measurement as described in Example 1 for measurement of the oligosaccharide structures, and the ash content. Example 52. Deashing an enzymatically produced oligosaccharide

The aqueous solution from an enzymatic synthesis reaction set up for the production of an oligosaccharide like e.g., 3'SL as described in Example 31 is filtered using a 15 kDa molecular weight cut-off ceramic membrane (Tami). Afterwards, the ultrafiltrate is concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surface, Mann+Hummel). The pH of the NF retentate is corrected to 4 using H2SO4 and then pumped over a glass column (YMC) containing a mixed bed of equal volumes of an Amberlite FPC22H cation exchange resin (Dupont) and an Amberlite FPA51 anion exchange resin (Dupont) to remove any remaining Tris-HCI buffer. The mixed bed ion exchange step is performed at room temperature. The effluent of the column is collected and subjected to UPLC measurement as described in Example 1 for measurement of 3'SL and the ash content.

Example 53. Cell lysis

In many of the above-described mutant strains the product is readily excreted from the cell. Larger molecules however tend to be released more difficult during the fermentation or cultivation process. Therefore, an additional step is optionally introduced to release the product from the cell.

The broth from the fermentation processes of Examples 2 to 8, 10 and 12 is used in a cell lysis experiment. A soft release of the product was established by heating for 1 h the broth to a temperature between 60°C and 80°C. The higher the temperature, the more release was obtained, but color formation increased. The product release was most optimal at a pH below 6.5 and above 3. The least monosaccharide formation was found at a pH of above 3.9. The release of the product was quantified by measuring the total oligosaccharide pool as described in Example 1 in the broth before and after treatment. When observing an increase in oligosaccharide concentration, the product was released from the cells.

A similar cell lysis process may be used on the broth originating from the cultivation processes of Examples 14 to 19, 22, 23, 26 and 27.

To disrupt the cell integrity, even more other methods are also commonly used comprising e.g., freeze thawing and/or shear stress through sonication, mixing, use of a homogenizer, French press.

Example 54. Broth clarification

The broth originating from the fermentations as described in Examples 2 to 8, 10 and 12 and, as the case may be, subjected to a lysis step as described in Example 53 are further clarified through microfiltration. For filtration several types of microfiltration membranes have been used to clarify the fermentation broth with a pore size ranging between 0.1 to 10 pm (ceramic, PES, PVDF membranes). The membrane types were first used as dead-end filtration and further optimization was performed in cross-flow filtration. The cross-flow microfiltration was followed by diafiltration to increase product yield after this purification step. The membranes are capable of separating large, suspended solids such as colloids, particulates, fat, bacteria, yeasts, fungi, cells, while allowing sugars, proteins, salts, and low molecular weight molecules pass through the membrane. The particle concentration in the filtrate was measured with a spectrophotometer at light adsorption at 600 nm. This method allows the validation of particle removal and filtration optimization.

Alternative to microfiltration membranes, ultrafiltration membranes are used. Ultrafiltration membranes with a cut-off between 1 kDa and 10 kDa were tested (microdyne Nadir (3 kDa PES), Synder (3 kDa, PES), Synder Filtration MT (5 kDa, PES) and Synder Filtration ST (10 kDa, PES)) according to the method described in Example 26. Alternative membranes with larger cut-offs will also work for broth clarification. The membranes were used in cross-flow mode, and diafiltrations were applied similar to the microfiltration operation described above to increase product yield. The filtration efficiency is evaluated based on the particle concentration of the filtrate. Apart from cells and cell debris, membranes below 10 kDa efficiently remove DNA, protein and endotoxin, which were measured with the methods described in Example 1. Higher cut-off membranes between 10 and 500 kDa remove cell mass efficiently, but do not retain smaller molecular weight products as efficiently, therefore requiring an additional ultrafiltration step with a molecular weight cut-off below 10 kDa. A final recovery through ultrafiltration for broth clarification of above 95% was obtained.

To enhance broth clarification through centrifugation, flocculants/coagulants have been used. Generally, Gypsum, Alum, calcium hydroxide, polyaluminium chloride and Aluminium chlorohydrate are used as good flocculation agents. These flocculants were applied at a pH > 7 and at temperatures between 4°C and 20°C, more preferably between 4°C and 10°C. A pH < 7 released toxic cations which are removed further through cation exchange. Alternative flocculants tested are based on polyacrylamide or biopolymer (chitosan), Floquant (SNF inc), Superfloc (Kemira) or hyperfloc (Hychem inc), Tramfloc. These flocculants were used in different concentrations: 0.05, 0.1 and 0.2 v/v%. After diluting the broth 1:1 with RO-water, they were directly added to the broth and gently mixed for 10 minutes at room temperature. The pH was kept at neutral conditions, between pH 6 and 7. At higher pH some degradation of the flocculant occurs, leading to compounds that are removed by means of ion exchange.

To test flocculation efficiency centrifugation was performed at 4000 g and the pellet strength and supernatant turbidity was evaluated after different centrifugation times. The oligosaccharide yield was measured by measuring the oligosaccharide supernatant concentration and the total supernatant volume. The pellet was washed several times to increase the release of oligosaccharides. A final oligosaccharide recovery between 90 and 98% was obtained.

Example 55. Ultrafiltration

Ultrafiltration was performed on a Colossus apparatus (Convergence Industry, The Netherlands) controlled by a PC running Convergence Inspector software. Temperature, pressures and conductivity of both retentate and filtrate were measured inline, pH was measured offline with a calibrated pH probe (Hanna Instruments). The membrane to further remove DNA, protein and endotoxin was a 10 kDa membrane based on PES (Synder), used in crossflow. After filtration, the DNA, protein and endotoxin content was measured in the filtrate. The protein content was below 100 mg per kg dry solid, the DNA content below 10 ng per gram dry solid and the endotoxin was below 10000 EU per gram dry solid. No DNA from the production hosts could be detected in the filtrate.

Although in this example a polysulfon based membrane was used, other membrane materials will perform equally, these membrane materials can be a ceramic or made of a synthetic or natural polymer, e.g., polypropylene, poly(piperazine-amide), cellulose acetate or polylactic acid from suppliers such as Synder, Tami, TriSep, Microdyn Nadir, GE.

Example 56. Ion and mono- and disaccharide removal through nanofiltration

Tangential flow nanofiltration was performed on a Colossus apparatus (Convergence Industry, The Netherlands) controlled by a PC running Convergence Inspector software. Temperature, pressures and conductivity of both retentate and filtrate were measured inline, pH was measured offline with a calibrated pH probe (Hanna Instruments). Clarified liquid treated with ultrafiltration from Example 55 was further subjected to nanofiltration and sequential diafiltrations. To this end, a polyamide base membrane with a cut-off between 300 and 500 Da was used (TriSep XN-45 (TriSep Corporation, USA)) at 40°C. The diafiltrations were performed with deionized water with a total volume of 5 times the volume of the oligosaccharide concentrate. This step reduced the disaccharide fraction on dry solid below 5% and reduced the total ash content of the liquid with 50%. The oligosaccharide concentration was increased to about 200 g/L. Examples of the ash content, the conductivity and ion composition of the retentate for different oligosaccharide solutions obtained from 3'SL, 6'SL, LNnT or LNT fermentations after said steps of ultrafiltration, nanofiltration and sequential diafiltrations are given in Table 6 below.

Table 6- Conductivity, ion composition, pH and Brix value of the retentate for different oligosaccharide solutions obtained from 3'SL, 6'SL, LNT or LNnT fermentations after ultrafiltration, nanofiltration and sequential diafiltrations.

Example 57. Ion removal through electrodialysis

The ED equipment used is a PCCell ED 64004 lab-scale ED stack (PCCell GmbH, Germany), fitted with 5 cell pairs of the PC SA and PC SK standard ion-exchange membranes. The initial diluate and concentrate both consisted of 1.5L of the feed stream obtained after the clarification and ultrafiltration of broth samples from different fermentations as described in Examples 54 and 55. The liquids obtained in these examples contained oligosaccharides and cations and anions with an ash content above 10% on dry solid. The desalination was performed against a concentration gradient. Both streams were recirculated while a constant voltage of 7.5 V was applied and the current and conductivity are monitored. Samples were taken at the beginning and end and periodically during the experiment. Water transport across the membranes was monitored by measuring the volume of all streams at the end of the experiment. To ensure efficient transfer of the current to the stack, an electrolyte solution of 60 g/L NaNO3 was recirculated at the electrodes.

The ED experiment was maintained until a stabilization of the current and conductivity was noticed. This indicates the point where desalination becomes slow and more inefficient. The conductivity decreased from 3.79 mS/cm in the feed to 0.88 mS/cm at the end of the experiment, indicating an overall desalination of 77%. The multivalent anions were removed up to 90%. The final oligosaccharide recovery was between 90 and 99%. The ash content on dry solid after electrodialysis was about 3% on dry solid.

Example 58. Ion removal through ion exchange

To remove ions from the broth to an ash content lower than 1%, first a cation exchange and second an anion exchange step was performed. Depending on the mixture of oligosaccharides different anion exchange resins were selected to enhance the yield of the purification step.

For clarified broths originating from Examples 3, 4, 5, 6 and 7, containing non-charged oligosaccharides, were first passed through a strong acid cation exchange resin containing column (IL of Amberlite IR120) in the proton form at a temperature of 10°C, resulting in exchange of all cations with a proton in the liquid. The liquid resulting from the cation exchange step was subjected to a weak base anion exchange resin containing column (IL of Amberlite I R400) in the hydroxide form at a temperature of 10°C, exchanging the anions in the liquid for hydroxide ions. After both cation and anion exchange, the pH was set to a pH between 6 and 7. The oligosaccharide recovery was between 95 and 98%. Alternative cation and anion exchange resins are Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet 1200 and Amberjet 4200, Amberjet 4600, Amberlite IR400, Amberlite IR410, Amberlite IR458, Diaion SA, Diaion UBA120, Lewatit MonoPlus M, Lewatit S7468. The cation and anion exchange treated liquids were then tested on ash, oligosaccharide content and heavy metal content. The ash content after treatment was below 0.5% (on total dry solid), the lead content was lower than 0.1 mg/kg dry solid, the arsenic content was lower than 0.2 mg/kg dry solid, the cadmium content was lower than 0.1 mg/kg dry solid and the mercury content was lower than 0.5 mg/kg dry solid.

For clarified broths originating from Examples 2, 8, 10 and 12, specific anion exchange resins were used that do not retain the negatively charged oligosaccharides (containing a sialyl group). These resins are characterized to have a moisture content of 30-48% and preferably a gel type anion exchanger. Examples of such resins are DIAION SA20A, Diaion WA20A (Mitsubishi), XA4023 (Applexion), Dowex 1-X8 (Dow). In a first step the liquid was first passed through a strong acid cation exchange resin containing column (IL of Amberlite IR120) in the proton form at a temperature of 10°C, resulting in exchange of all cations with a proton in the liquid. This was then passed immediately through an anion exchange resin column (IL of XA4023), exchanging salts like phosphates and sulphates for hydroxide ions. The resulting liquid was set to a pH between 5 and 7. The ash content corrected for the sodium counter ions for the sialylated oligosaccharides was below 1% (on total dry solid) after ion exchange treatment, the lead content was lower than 0.1 mg/kg dry solid, the arsenic content was lower than 0.2 mg/kg dry solid, the cadmium content was lower than 0.1 mg/kg dry solid and the mercury content was lower than 0.5 mg/kg dry solid. An alternative to sequential cation and anion exchange steps is mixed bed ion exchange. The resins are mixed in a ratio typically within the range of 35:65 and 65:35 volume percentage. Typically, a mixed bed ion exchange step is introduced in the process after a first de-ionization step such as a nanofiltration step, an electrodialysis step or ion exchange step but is also used as sole ion exchange step. The broth from the fermentation runs described in Examples 4, 6 and 7, each containing an oligosaccharide mixture of noncharged oligosaccharides, were subjected to clarification and ultrafiltration before they were subjected to a mixed bed column of Amberlite FPC 22H and Amberlite FPA51 mixed in a ratio 1:1,3 on a IL column. The mixed bed step was performed at a temperature between 4°C and 10°C. Finally, the liquids were set to a pH between 5 and 7 and the ash content of the solutions was measured to be below 1%. The oligosaccharide recovery in each oligosaccharide solution was between 95 and 98%. The broth from the fermentation runs described in Examples 8, 10 and 12, each containing an oligosaccharide mixture comprising negatively charged (sialylated) oligosaccharides, were subjected to clarification and ultrafiltration before they were subjected to a mixed bed column of Diaion SA20A and Amberlite FPC 22H mixed in a ratio 1,3:1 on a IL column. The mixed bed step was performed at a temperature between 4°C and 10°C. Finally, the liquids were set to a pH between 5 and 7 and the ash content of the solutions was measured to be below 1%. None of the sialylated oligosaccharides were retained in this step, retaining the mixture composition, the oligosaccharide recovery was between 95 and 98%.

Example 59. Concentration through nanofiltration

Nanofiltration was carried out with an NF-2540 membrane (DOW) with a cut-off of 200 Da to concentrate the de-ionized solutions after ion exchange, electrodialysis or nanofiltration, as obtained in Examples 56 to 58, up to 25 Brix. During the filtration process a pressure across the membrane in the range of 20-25 bar was used and a process temperature of 45°C. The solution was continuously recirculated over the membrane for concentration, leading to a dry matter content of the concentrate up to 25% Brix.

To remove some of the monosaccharides formed during the cation and/or anion exchange step, typically a nanofiltration step of 300 to 500 Da is used at a membrane pressure of 20 to 25 bar and at a temperature above 30°C. The membrane allows concentrating the oligosaccharide solution to about 15 to 20% Brix.

Example 60. Further ion removal by electrodeionization

Partially deionized solutions originating from Examples 56 to 59 were further treated by electrodeionization. The EDI stack comprised lonac MC-3470 and MA-3475 membranes. The initial conductivity was 4 mS/cm for broth comprising neutral (non-charge) oligosaccharides and 20 mS/cm for broth comprising negatively charged (sialylated) oligosaccharides, at 4 to 18% Brix. Final conductivity of the processed syrup was 0.004 - 25 mS/cm for broth comprising neutral (non-charge) oligosaccharides and 5 - 12 mS/cm for broth comprising negatively charged (sialylated) oligosaccharides, at 4 to 18% Brix.

Example 61. Further ion removal by electrodeionization at low pH

To minimize scaling of the EDI membranes, the pH of the solutions obtained in Examples 56 to 59 was reduced to a pH of about 3 to 6, preferably lower than 6, more preferably lower than 5, more preferably lower than 4.5, more preferably lower than 4, more preferably between 3 and 4.

Example 62. Further ion removal by electrodeionization after reduction of magnesium and calcium ions To minimize scaling of the EDI membrane due to magnesium and calcium ions, these ions are removed by means of nanofiltration as described in Example 56 wherein the diafiltrations are performed to such an extent that the magnesium concentration is lower than 1000 ppm, preferably lower than 500 ppm, more preferably lower than 400 ppm, more preferably lower than 300 ppm, more preferably lower than 200 ppm, more preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 10 ppm and/or the calcium concentration is lower than 200 ppm, preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 20 ppm, more preferably lower than 10 ppm, more preferably lower than 5 ppm, more preferably lower than 2 ppm, more preferably lower than 1 ppm, more preferably lower than 0.5 ppm, more preferably lower than 0.1 ppm. After diafiltration, the EDI treatment may also be performed at low pH to further avoid membrane scaling as described in Example 61.

Example 63. Further ion removal by electrodeionization after reduction of magnesium and calcium ions To minimize scaling of the EDI membrane due to magnesium and calcium ions, these ions are removed by means of electrodialysis described in Example 57 wherein the current and the applied flow rate ensures that the magnesium concentration is lower than 1000 ppm, preferably lower than 500 ppm, more preferably lower than 400 ppm, more preferably lower than 300 ppm, more preferably lower than 200 ppm, more preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 10 ppm and/or the calcium concentration is lower than 200 ppm, preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 20 ppm, more preferably lower than 10 ppm, more preferably lower than 5 ppm, more preferably lower than 2 ppm, more preferably lower than 1 ppm, more preferably lower than 0.5 ppm, more preferably lower than 0.1 ppm.

After electrodialysis, the EDI treatment may also be performed at low pH to further avoid membrane scaling as described in Example 61.

Example 64. Further ion removal by electrodeionization after reduction of magnesium and calcium ions To minimize scaling of the EDI membrane due to magnesium and calcium ions, these ions are removed by means of cation exchange described in Example 58 wherein the magnesium concentration is lower than 1000 ppm, preferably lower than 500 ppm, more preferably lower than 400 ppm, more preferably lower than 300 ppm, more preferably lower than 200 ppm, more preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 10 ppm and/or the calcium concentration is lower than 200 ppm, preferably lower than 100 ppm, more preferably lower than 50 ppm, more preferably lower than 20 ppm, more preferably lower than 10 ppm, more preferably lower than 5 ppm, more preferably lower than 2 ppm, more preferably lower than 1 ppm, more preferably lower than 0.5 ppm, more preferably lower than 0.1 ppm.

After cation exchange, the EDI treatment may also be performed at low pH to further avoid membrane scaling as described in Example 61.

Example 65. Further ion removal by electrodeionization

To enable stability of the oligosaccharides at low pH, minimizing the scaling of the EDI membranes as described in Examples 60-64 the solution is kept preferably at a temperature below 20°C, more preferably below 15°C, even more preferably below 10°C during the EDI process step. Example 66. Color removal

To achieve decolourization, several samples throughout the process were subjected to activated charcoal treatment with Norit SX PLUS activated charcoal (0.5% m/v). Color removal was measured with a spectrophotometer at 420 nm. In all samples the color intensity at 420 nm was reduced 50 to 100-fold.

Example 67. Spray drying of oligosaccharides

A single oligosaccharide or mixture of oligosaccharides at different concentrations was spray-dried with pilot spray-dry equipment. The equipment had an evaporation capacity of 25 kg/h.

For spray-drying the liquid was heated to a temperature between 50 and 100°C, to lower the viscosity. The pH of the liquid was set to a pH of 4 to 6. More preferably, the pH is set to a pH of 4 to 5 and temperatures are kept between 50 and 70°C.

The total oligosaccharide concentration in the feed is between 20% and 80% Brix. These concentrations were obtained by rotary evaporation or wiped film evaporation. The concentrated liquids were fed to the spray dryer at a rate between 50 and 90%. The higher the percentage Brix, the faster the feed rate.

The used inlet temperature ranged between 120 and 280°C. The outlet temperature ranged between 100°C and 180°C. The atomizer wheel rotation speed was set between 10000 and 28000 rpm. In one specific test the inlet temperature was set at 184°C, outlet temperature was set at 110°C and atomizer rate was set at 21500 rpm.

The obtained powder had a white to off white color and the pH after redissolving water at a concentration of 10%, was between 4 and 6. The purity of the oligosaccharide or oligosaccharide mixture was above 80% of oligosaccharides on dry solid. The spray-dried oligosaccharide or oligosaccharide mixture had about 3 to 10% of water content, the protein content was below 100 mg per kg dry solid, the DNA content below 10 ng per gram dry solid and the endotoxin was below 10000 EU per gram dry solid. No DNA from the production hosts could be detected in the filtrate. The ash content after treatment was below 5% (on total dry solid), the lead content was typically lower than 0.1 mg/kg dry solid, more typically lower than 0.05 mg/kg even more typically below 0.02 mg/kg; the arsenic content was typically lower than 0.2 mg/kg dry solid, more typically lower dan 0.1 mg/kg, even more typically lower than 0.05 mg/kg; the cadmium content was typically lower than 0.1 mg/kg dry solid, more typically lower than 0.05 mg/kg, even more typically below 0.02 mg/kg; and the mercury content was typically lower than 0.5 mg/kg dry solid, more typically lower than 0.2 mg/kg even more typically below 0.1 mg/kg.

Example 68. Stepwise purification of neutral oligosaccharides

The broth of Example 6 was clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 50°C and a pH of 4 to 5. The filtrate of the microfiltration step was in a second step subjected to ultrafiltration in which a PES membrane of 10 kDa was used, removing protein, endotoxin and DNA. The resulting filtrate was further concentrated by nanofiltration, partially removing salts and disaccharides from the liquid with a polyamide membrane of 300 to 500 Da at 40°C. In the nanofiltration step the oligosaccharide mixture was concentrated to a concentration of about 200 g/L or 20 Brix. The resulting concentrate was further decolored by means of activated charcoal and cations and anions were removed by means of EDI preferably at a pH of 3 to 4. This liquid was set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solution was spray dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The obtained powder had a white to off white color and the pH after redissolving water at a concentration of 10% was between 4 and 6. The purity of the oligosaccharide mixture was above 80% of oligosaccharides on dry solid. The spray-dried oligosaccharide mixtures had about 3 to 10% of water content, the protein content was below 100 mg per kg dry solid, the DNA content below 10 ng per gram dry solid and the endotoxin was below 10000 EU per gram dry solid. No DNA from the production hosts could be detected in the filtrate. The ash content after treatment was below 5% (on total dry solid), the lead content was lower than 0.1 mg/kg dry solid, the arsenic content was lower than 0.2 mg/kg dry solid, the cadmium content was lower than 0.1 mg/kg dry solid and the mercury content was lower than 0.5 mg/kg dry solid. The oligosaccharides present in the obtained powder are Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para-Lacto-N-neohexaose, para-Lacto-N-hexaose, beta-(l,3)galactosyl-para-Lacto-N-neopentaose and beta-(l,4)galactosyl-para-Lacto-N-pentaose.

Example 69. Stepwise purification of a fucosylated oligosaccharide mixture

The broth of Example 7 was clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 60°C and a pH of 4 to 5. The filtrate of the microfiltration step was in a second step subjected to ultrafiltration in which a PES membrane of 10 kDa was used, removing protein, endotoxin and DNA. The resulting filtrate was further concentrated by nanofiltration, partially removing salts and disaccharides from the liquid with a polyamide membrane of 300 to 500 Da at 40°C. In the nanofiltration step the oligosaccharide mixture was concentrated to a concentration of about 200 g/L or 20 Brix. The resulting concentrate was further decolored by means of activated charcoal and cations and anions were removed by means of EDI preferably at a pH of 3 to 4. This liquid was set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solution was spray dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The obtained powder had a white to off white color and the pH after redissolving water at a concentration of 10% was between 4 and 6. The purity of the oligosaccharide mixture was above 80% of oligosaccharides on dry solid. The spray dried oligosaccharide mixtures had about 3 to 10% of water content, the protein content was below 100 mg per kg dry solid, the DNA content below 10 ng per gram dry solid and the endotoxin was below 10000 EU per gram dry solid. No DNA from the production hosts could be detected in the filtrate. The ash content after treatment is below 5% (on total dry solid), the lead content is lower than 0.1 mg/kg dry solid, the arsenic content is lower than 0.2 mg/kg dry solid, the cadmium content is lower than 0.1 mg/kg dry solid and the mercury content is lower than 0.5 mg/kg dry solid. The oligosaccharides present in the obtained powder are 2'FL, 3-FL, DiFL, LN3, LNT and LNFP-I.

Example 70. Stepwise purification of a sialylated oligosaccharide mixture

The broth of Example 12 was clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 60°C and a pH of 4 to 5. The filtrate of the microfiltration step was in a second step subjected to ultrafiltration (UF) in which a PES membrane of 10 kDa was used, removing protein, endotoxin and DNA. The UF filtrate was further treated in a nanofiltration step concentrating the oligosaccharide mixture to a concentration of about 200 g/L or 20 Brix. The resulting concentrate was further decolored by means of activated charcoal and cations and anions were further removed by means of EDI preferably at a pH of 3 to 4. This liquid was set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solution was spray-dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The obtained powder had a white to off white color and the pH after redissolving water at a concentration of 10% was between 4 and 6. The purity of the oligosaccharide mixture was above 80% of oligosaccharides on dry solid. The spray dried oligosaccharide mixtures had about 3 to 10% of water content, the protein content was below 100 mg per kg dry solid, the DNA content below 10 ng per gram dry solid and the endotoxin was below 10000 EU per gram dry solid. No DNA from the production hosts could be detected in the filtrate. The ash content after treatment is below 10% (on total dry solid), the lead content is lower than 0.1 mg/kg dry solid, the arsenic content is lower than 0.2 mg/kg dry solid, the cadmium content is lower than 0.1 mg/kg dry solid and the mercury content is lower than 0.5 mg/kg dry solid.

Example 71. Purification of one or more oligosaccharide(s)

Each of the enzymatic reaction mixtures comprising one or more oligosaccharide(s) produced as described in Example 30 is purified by ultrafiltration on a Colossus apparatus (Convergence Industry, The Netherlands) controlled by a PC running Convergence Inspector software as described in Example 55. Temperature, pressures and conductivity of both retentate and filtrate are measured inline, the pH is measured offline with a calibrated pH probe (Hanna Instruments). The membrane to further remove DNA and protein is a 10 kDa membrane based on PES (Synder), used in crossflow. After filtration, the DNA and protein content are measured in the filtrate. This filtrate is further treated in a tangential flow nanofiltration as described in Example 56. Temperature, pressures and conductivity of both retentate and filtrate are measured inline, the pH is measured offline with a calibrated pH probe (Hanna Instruments). Clarified liquid is further subjected to nanofiltration and sequential diafiltrations. To this end a polyamide base membrane with a cut-off between 300 and 500 Da can be used (TriSep XN-45 (TriSep Corporation, USA)) at 40°C. The diafiltrations are done with deionized water with a total volume of 5 times the volume of the oligosaccharide concentrate. The retentate of the nanofiltration unit operation is then treated by means of EDI as described in Examples 60 to 64. The pH of said solution is set lower than 5, more preferably lower than 4.5, even more preferably lower than 4 more preferably between 3 and 4, so that the mixed bed resin in the electrodeionization system does not retain the oligosaccharide(s) but selectively removes inorganic salts such as e.g., sulphates, phosphates, nitrates, chlorides, chlorates, and/or organic acids such as e.g., sialic acid, CMP-sialic acid, Tris buffer, CTP, pyruvic acid, PEP. At the end of the enzymatic synthesis and/or after purification, the production of the oligosaccharide(s) is measured via analytical methods as described in Example 1 and known by the person skilled in the art.

Example 72. Purification of 3'sialyllactose

The 3'sialyllactose produced in an enzymatic reaction as described in Example 31 is purified from the enzymatic reaction mixture by ultrafiltration on a Colossus apparatus (Convergence Industry, The Netherlands) controlled by a PC running Convergence Inspector software as described in Example 55. Temperature, pressures and conductivity of both retentate and filtrate are measured inline, the pH is measured offline with a calibrated pH probe (Hanna Instruments). The membrane to further remove DNA, protein and endotoxin is a 10 kDa membrane based on PES (Synder), used in crossflow. After filtration, the DNA, protein and endotoxin content is measured in the filtrate. This filtrate is further treated in a tangential flow nanofiltration as described in Example 56. Temperature, pressures and conductivity of both retentate and filtrate are measured inline, the pH is measured offline with a calibrated pH probe (Hanna Instruments). Clarified liquid is further subjected to nanofiltration and sequential diafiltrations. To this end a polyamide base membrane with a cut off between 300 and 500 Da is used (TriSep XN-45 (TriSep Corporation, USA)) at 40°C. The diafiltrations are done with deionized water with a total volume of 5 times the volume of the 3'sialyllactose concentrate. This step reduces the monosaccharide (N- acetylglucosamine, N-acetylmannosamine, sialic acid, CMP-sialic acid) and disaccharide (lactose) fraction on dry solid and reduces the total ash content of the liquid, while the 3'sialyllactose concentration increases. The retentate of the nanofiltration unit operation is then treated by means of EDI as described in Examples 60 to 64. The pH of said solution is set lower than 5, more preferably lower than 4.5, even more preferably lower than 4 more preferably between 3 and 4, so that the mixed bed resin in the electrodeionization system does not retain the 3'sialyllactose but selectively removes inorganic salts such as e.g., sulphates, phosphates, nitrates, chlorides, chlorates, and/or organic acids such as e.g., sialic acid, CMP-sialic acid, tris buffer, CTP, pyruvic acid, PEP, cysteine, acetic acid, formic acid, lactic acid, citric acid, butyric acid, propionic acid, succinic acid, fumaric acid, fumaric acid, malic acid, maleic acid.

Example 73. Purification of one or more oligosaccharide(s)

In another example, the milk obtained by means of mammalian cell cultures as described in Example 27 may be sterilized (e.g., UHT sterilization) or pasteurized after cultivation. The milk is fractionated based on component sizes by microfiltration, ultrafiltration, nanofiltration and reverse osmosis. Microfiltration separates the possible presence of cells, bacteria, yeasts, fungi and fat globules from the liquid solution. The ultrafiltration (U F) step separates the feed (e.g., skim milk) into two streams, allowing water, dissolved salts, lactose, oligosaccharides, and acids to pass through it in either direction, while retaining (and thereby concentrating) proteins and fat. Nanofiltration separates a range of minerals and monosaccharides from a liquid, retaining the oligosaccharides. Through diafiltrations the ionic content of the milk is minimized. The retentate of the nanofiltration step is subjected to a further ion removal step, either an anion and cation ion exchange step, a mixed bed ion exchange step, a single cation exchange step or an electrodeionization step, as described within the present disclosure. A variant purification process replaces the nanofiltration step by electrodialysis, leaving all lactose in the solution followed by a further ion removal step, either an anion and cation ion exchange step, a mixed bed ion exchange step, a single cation exchange step or an electrodeionization step, as described within the present disclosure. The de-ionized liquid is further concentrated by means of reversed osmosis, falling film evaporation and/or wiped film evaporation before being dried, e.g., by means of agitated thin film drying.

Example 74. Stepwise purification of 3-FL or of an oligosaccharide mixture comprising fucosylated oligosaccharides

In another example, the cultivations from 1) an engineered yeast strain producing 3-FL or an oligosaccharide mixture A comprising 2' FL, DiFL, LN3, LNT, LNFP-I and 2'FLacNAc as obtained in Example 15 and 2) a modified C. glutamicum strain producing an oligosaccharide mixture B comprising 3-FL, LNFP- III, LN3 and LNnT as obtained in Example 23 are each clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 60°C and a pH of 4 to 5. The filtrate of the microfiltration step is in a second step subjected to ultrafiltration in which a PES membrane of 10 kDa is used. The resulting filtrates are further concentrated by nanofiltration, partially removing salts and disaccharides from the liquids with a polyamide membrane of 300 to 500 Da at 40°C. In the nanofiltration step the 3-FL obtained from the yeast cultivation and the oligosaccharide mixtures A and B are each concentrated to a concentration of about 200 g/L or 20 Brix. The resulting concentrates are further decolored by means of activated charcoal and cations and anions are removed by means of EDI preferably at a pH of 3 to 4. These liquids are set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solutions are spray-dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The powders obtained have a white to off white color and the pH after redissolving water at a concentration of 10% is between 4 and 6. The purity of the purified 3-FL and of the purified oligosaccharide mixtures obtained from oligosaccharide mixtures A and B is above 80% of oligosaccharides on dry solid. The spray dried 3-FL and the spray dried oligosaccharide mixtures obtained from oligosaccharide mixtures A and B each have about 3 to 10% of water content, the protein content is below 100 mg per kg dry solid and the DNA content is below 10 ng per gram dry solid. No DNA from the production hosts is detected in the filtrates. The ash content after treatment is below 5% (on total dry solid), the lead content is lower than 0.1 mg/kg dry solid, the arsenic content is lower than 0.2 mg/kg dry solid, the cadmium content is lower than 0.1 mg/kg dry solid and the mercury content is lower than 0.5 mg/kg dry solid.

Example 75. Stepwise purification of a sialylated oligosaccharide mixture

The cultivations from 1) an engineered yeast strain producing 3'SL or an oligosaccharide mixture C comprising 6'SL, LN3, LNnT and LSTc as obtained in Example 14, 2) a modified B. subtilis strain producing 5'SL as obtained in Example 17 or an oligosaccharide mixture D comprising LN3, LNnT, 6'SL and LSTc as obtained in Example 18, 3) modified C. reinhardtii cells producing LSTc as obtained in Example 26 and 4) mammary-like cells producing 3'SL as obtained in Example 30 are each clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 60°C and a pH of 4 to 5. The filtrate of the microfiltration step is in a second step subjected to ultrafiltration (UF) in which a PES membrane of 10 kDa is used. The UF filtrate is further treated in a nanofiltration step concentrating each oligosaccharide (3'SL, 6'SL or LSTc, respectively) or oligosaccharide mixtures C and D each to a concentration of about 200 g/L or 20 Brix. The resulting concentrates are further decolored by means of activated charcoal and cations and anions were further removed by means of EDI preferably at a pH of 3 to 4. These liquids are set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solutions are spray-dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The powders obtained have a white to off white color and the pH after redissolving water at a concentration of 10% was between 4 and 6. The purity of each purified oligosaccharide (3'SL, 6'SL or LSTc, respectively) or purified oligosaccharide mixtures obtained from oligosaccharide mixtures C and D is above 80% of oligosaccharides on dry solid. Each spray-dried oligosaccharide (3'SL, 6'SL or LSTc, respectively) or spray-dried oligosaccharide mixture obtained from oligosaccharide mixtures C and D have about 3 to 10% of water content, the protein content is below 100 mg per kg dry solid and the DNA content is below 10 ng per gram dry solid. No DNA from the production hosts is detected in the filtrate. The ash content after treatment is below 5% (on total dry solid), the lead content is lower than 0.02 mg/kg dry solid, the arsenic content is lower than 0.05 mg/kg dry solid, the cadmium content is lower than 0.05 mg/kg dry solid and the mercury content is lower than 0.1 mg/kg dry solid.

Example 76. Purification of an oligosaccharide mixture comprising fucosylated and sialylated oligsoaccharides

The cultivations from 1) an engineered yeast strain producing an oligosaccharide mixture E comprising 2'FL, 3-FL, Di FL, 3'SL and 6'SL as obtained in Example 16 and 2) a modified C. glutamicum strain producing an oligosaccharide mixture F comprising 2'FL, 3-FL, DiFL, 3'SL and 6'SL as obtained in Example 19 are each clarified by first applying microfiltration with a 0.45 pm pore sized membrane, removing biomass at 60°C and a pH of 4 to 5. The filtrate of the microfiltration step is in a second step subjected to ultrafiltration ( UF) in which a PES membrane of 10 kDa is used. The UF filtrate is further treated in a nanofiltration step concentrating oligosaccharide mixtures E and F each to a concentration of about 200 g/L or 20 Brix. The resulting concentrates are further decolored by means of activated charcoal. In a next step the fucosylated oligosaccharides are separated from the sialylated oligosaccharides by means of EDI preferably at a pH between 3 and 5, forming an oligosaccharide mixture G containing 2'FL, 3-FL and DiFL, as well as an oligosaccharide mixture H, containing 3'SL and 6'SL. The oligosaccharide mixtures G and H are set to a pH between 4 and 7 and concentrated by means of evaporation to about 50 Brix. The final solutions obtained of oligosaccharide mixtures G and H are spray-dried with an inlet temperature of 160°C, outlet temperature of 75°C, an airflow of 600 L/h and a feed rate of 8 mL/min on a Procept spray dryer. The powders obtained have a white to off white color and the pH after redissolving water at a concentration of 10% is between 4 and 6. The purity of the purified oligosaccharide mixtures G and H is above 80% of oligosaccharides on dry solid. Each spray-dried oligosaccharide mixture has about 3 to 10% of water content, the protein content is below 100 mg per kg dry solid and the DNA content is below 10 ng per gram dry solid. No DNA from the production hosts is detected in the filtrate. The ash content after treatment is below 5% (on total dry solid), the lead content is lower than 0.1 mg/kg dry solid, the arsenic content is lower than 0.2 mg/kg dry solid, the cadmium content is lower than 0.1 mg/kg dry solid and the mercury content is lower than 0.5 mg/kg dry solid.

Example 77. Separation of LSTc from 6'SL using mixed bed

The broth originating from a fermentation as described in Example 3 was cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrate was subjected to nanofiltration over a Synder NFG membrane (1 single element of 2540 format, 2.7 m² membrane surface, Synder filtration). After nanofiltration, the retentate was harvested and further concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surfacen Mann+Hummel). This resulted in 1.5L of retentate, i.e. syrup, at 9 Brix. This syrup was treated with 3% w/w powdered Filtercarb activated carbon (Carbonitalia) for 1 hour. The mixture was then centrifugated and the remaining activated carbon was filtered off using a 0.45 pm filter which resulted in 3L of decolored syrup at Brix 3.4. The syrup was then passed over a column containing 170 mL of cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange was performed at room temperature. After cation exchange, the pH was adjusted to 6.5 by using 50% NaOH. The resulting syrup was spray-dried to powder comprising about 70% LSTc. Ten grams of this powder were redissolved in 90 mL demineralized water (10-30 pS/cm) and pumped over 15 mL of a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent was 6.5 and the conductivity 4.3 mS/cm. The mixed bed ion exchange step was performed at room temperature. After said mixed bed step, 84% of the LSTc could be recovered in the effluent whereas all the 6'SL present in the influent was retained in the MB resin. No concentration changes were observed for the neutral molecules (like e.g., LN3 and LNnT). The influent of the mixed bed ion exchange contained a total amount of 0.33 g of inorganic salts while the treated effluent of the mixed bed ion exchange contained 0.12 g inorganic salts equalling a removal of 64%.

Example 78. Purification of LSTc via mixed bed

The broth from the cultivations of Examples 9, 11, 13, 14, 18, 21, 25, 28 and 29 is used in a cell lysis experiment. A soft release of the product (i.e., an oligosaccharide mixture comprising LN3, LNnT, 6'SL and LSTc) is established by heating for 1 hour the broth to a temperature between 60°C and 80°C. Other methods encompass freeze thawing, and/or shear stress through sonication, mixing, use of a homogenizer, French press or enzymatic cell lysis. Afterwards, the solutions obtained from both cultivations are cleared from cells, DNA, peptides and other macromolecular products by means of ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrates are subjected to nanofiltration over a Synder NFG membrane (1 single element of 2540 format, 2.7 m² membrane surface, Synder filtration). After nanofiltration, the retentates are harvested and further concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surface, Mann+Hummel). These syrups are treated with 3% w/w powdered Filtercarb activated carbon (Carbonitalia) for 1 hour and then centrifugated and the remaining activated carbon is filtered off using a 0.45 pm filter which resulted in decolored syrups. The syrups are then passed over a column containing cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange step is performed at room temperature. After cation exchange, the pH is adjusted to 6.5 by using 50% NaOH. The resulting syrups are spray-dried to powder. Ten grams of this powder are redissolved in 90 mL demineralized water (10-30 pS/cm) and pumped over 15 mL of a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent is 6.5 and the conductivity 4.3 mS/cm. The mixed bed step is performed at room temperature. After said mixed bed step, the majority of LSTc is recovered in the effluent whereas all the 6'SL present in the influent is retained in the MB resin. No concentration changes are observed for the neutral molecules (like e.g., LN3 and LNnT). The influent of the mixed bed ion exchange contains more inorganic salts compared to the treated effluent of the mixed bed ion exchange.

Example 79. Purification of LSTc via mixed bed

The enzymatic reaction mixtures of Example 30 are subjected to ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami). The resulting ultrafiltrates are subjected to nanofiltration over a Synder NFG membrane (1 single element of 2540 format, 2.7 m² membrane surface, Synder filtration). After nanofiltration, the retentates are harvested and further concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surface, Mann+Hummel). These syrups are treated with 3% w/w powdered Filtercarb activated carbon (Carbonitalia) for 1 hour and then centrifugated and the remaining activated carbon is filtered off using a 0.45 pm filter which resulted in decolored syrups. The syrups are then passed over a column containing cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange step is performed at room temperature. After cation exchange, the pH is adjusted to 6.5 by using 50% NaOH. The resulting syrups are spray-dried to powder. Ten grams of this powder are redissolved in 90 mL demineralized water (10-30 piS/cm) and pumped over 15 mL of a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent is 6.5 and the conductivity 4.3 mS/cm. The mixed bed step is performed at room temperature. After said mixed bed step, the majority of LSTc is recovered in the effluent whereas all the 6'SL present in the influent is retained in the MB resin. No concentration changes are observed for the neutral molecules (like e.g., LN3 and LNnT). The influent of the mixed bed ion exchange contains more inorganic salts compared to the treated effluent of the mixed bed ion exchange.

Example 80. Separation of LSTc from 3'SL using mixed bed

A solution comprising LSTc and 3'SL is first subjected to ultrafiltration over a 15 kDa molecular weight cutoff ceramic membrane (Tami) and the resulting ultrafiltrate is then subjected to nanofiltration over a Synder NFG membrane (1 single element of 2540 format, 2.7 m² membrane surface, Synder filtration). After nanofiltration, the retentate is harvested and further concentrated using nanofiltration over a Trisep XN45 membrane (1 single element of 1812 format, 0.35 m² membrane surfacen Mann+Hummel). This resulted in a retentate, i.e. syrup, that is further treated with 3% w/w powdered Filtercarb activated carbon (Carbonitalia) for 1 hour. The mixture is then centrifugated and the remaining activated carbon is filtered off using a 0.45 pm filter which results in a decolored syrup. The latter syrup is then passed over a column containing cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange is performed at room temperature. After cation exchange, the pH is adjusted to 6.5 by using 50% NaOH. The resulting syrup is spray-dried to powder. Ten grams of this powder are redissolved in 90 mL demineralized water (10-30 pS/cm) and pumped over 15 mL of a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent is 6.5 and the conductivity 4.3 mS/cm. The mixed bed ion exchange step is performed at room temperature. After said mixed bed step, the effluent is analysed for the presence of LSTc and for ash content.

Example 81. Purification of LSTc

A sialylated oligosaccharide structure like LSTc can be synthesized via chemical synthesis as described in Example 1. In a next step, the chemical synthesis solution is subjected to ultrafiltration over a 15 kDa molecular weight cut-off ceramic membrane (Tami) and the resulting ultrafiltrate is then subjected to nanofiltration over a Puramem S MWCO 600 Da (single element 2540 format 1.8 m², Evonik). After nanofiltration, the retentate is evaporated. In a next step, the powder is dissolved in water and passed through a column filled with Dowex Optipore L493 (Lenntech). The effluent is further passed over a column containing cation exchange resin (Amberlite FPC22H, Dupont) in H+ form. The cation exchange is performed at room temperature. After cation exchange, the pH is adjusted to 6.5 by using 50% NaOH. The resulting solution is spray-dried to powder, which is redissolved in demineralized water (10-30 pS/cm) and pumped over a mixed bed Amberlite MB20 resin (Dupont) at a flow rate of 3 BV/h. The pH of the influent is 6.5. The mixed bed ion exchange step is performed at room temperature. After said mixed bed step, the effluent is analysed for the presence of the sialylated oligosaccharide and for ash content.

Claims

1. A process for purification of an oligosaccharide from a solution, wherein said solution comprising said oligosaccharide is a solution chosen from the list comprising a biocatalysis reaction solution, a chemical synthesis solution, a cell cultivation and any process stream of said process, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation, the process comprising: i) pH adjustment of said solution to a pH ranging from 2 to 7 , preferably from 3 to 7, more preferably from 3 to 6, even more preferably from 3 to 5, most preferably from 3 to 4, and ii) passing said pH adjusted solution through: an anionic ion exchange using an anionic ion exchange resin in OH- form, optionally preceded by a cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺ form, preferably said cationic ion exchange resin is in Na⁺ form, and/or a mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form and an anionic ion exchange resin in OH- form, preferably said cationic ion exchange resin in said mixed bed ion exchange is in Na⁺ form.

2. Process according to claim 1, wherein said pH adjustment is obtained by any one or more of addition of an acidic agent, an alkaline agent and/or a buffered solution; filtration; nanofiltration; dialysis; electrodialysis; electrodeionization; ion exchange; mixed bed ion exchange; ion exchange chromatography; reverse osmosis; use of activated carbon or charcoal.

3. Process according to any one of previous claims, wherein said mixed bed ion exchange comprises an ion exchange column packed with a mixture of said cationic ion exchange resin and said anionic ion exchange resin in any volume ratio.

4. Process according to any one of previous claims, wherein in said mixed bed ion exchange the total ion exchange capacity of said anionic ion exchange resin is equal to the total ion exchange capacity of said cationic ion exchange resin.

5. Process according to any one of previous claims, wherein the flow rate through said anionic ion exchange, said cationic ion exchange, when present, and/or said mixed bed ion exchange is at least 0.5 bed volume / hour (BV/h), preferably at least 1 BV/h, more preferably at least 2 BV/h, more preferably at least 2.5 BV/h, most preferably at least 3 BV/h.

6. Process according to any one of previous claims, wherein said anionic ion exchange, said cationic ion exchange, when present, and /or said mixed bed ion exchange is/are performed at a temperature ranging from 0°C to 80°C, preferably from 4°C to 60°C, more preferably from 4°C to 40°C, even more preferably from 10°C to 37°C, even more preferably from 20°C to 30°C, even more preferably from 20°C to 25°C, even more preferably from 22°C to 24°C, most preferably from 23°C to 24°C.

7. Process according to any one of previous claims, wherein said process further comprises any one or more of the methods chosen from the list comprising homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, electrodialysis, electrodeionization, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange, ion exchange chromatography, mixed bed ion exchange, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, anion exchange adsorbent resin, use of an adsorbent material, use of ion exchange resin, evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying in any order, preferably wherein any one or more of said methods is performed more than one time during said process. Process according to claim 7, wherein (a) any one or more of said method(s) precede(s) said i) pH adjustment, ii) anionic ion exchange using an anionic ion exchange resin in OH- form, iii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na+ form, and/or iv) mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺, preferably in Na+ form, and an anionic ion exchange resin in OH- form and/or (b) any one or more of said method(s) succeed(s) said i) pH adjustment, ii) anionic ion exchange using an anionic ion exchange resin in OH- form, iii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na+ form, and/or iv) mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NH₄ ⁺, preferably in Na+ form, and an anionic ion exchange resin in OH- form. Process according to any one of claim 7 or 8, wherein any one or more of said method(s) succeed(s) said pH adjustment and precede(s) said i) anionic ion exchange using an anionic ion exchange resin in OH- form, ii) when present, cationic ion exchange using a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ form, preferably in Na+ form, and/or iii) mixed bed ion exchange comprising a cationic ion exchange resin in Na⁺, K⁺, Ca²⁺, Mg²⁺, Al³⁺ or NHZ, preferably in Na+ form, and an anionic ion exchange resin in OH- form. Process according to any one of previous claims, wherein said oligosaccharide is selected from the group comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing neutral (non-charged) oligosaccharide, N-acetyllactosamine containing oligosaccharide, lacto-N-biose containing oligosaccharide, non-fucosylated neutral (non-charged) oligosaccharide, chitosan, chitosan comprising oligosaccharide, heparosan, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, chondroitin sulphate, dermatan sulphate, hyaluronan or hyaluronic acid, keratan sulphate, a milk oligosaccharide, O-antigen, enterobacterial common antigen (ECA), the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan, an amino- sugar, an antigen of the human ABO blood group system, an animal oligosaccharide and a plant oligosaccharide; preferably wherein said milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); preferably wherein said animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans; preferably wherein said plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans. Process according to any one of previous claims, wherein the purity of said oligosaccharide in said solution is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process. Process according to any one of previous claims, wherein said solution is a cell cultivation using at least one cell that has been metabolically engineered to produce said oligosaccharide. Process according to any one of previous claims, wherein said oligosaccharide is accompanied in said solution by sialic acid, ashes, preferably, said ashes comprise sulphates and phosphates, one or more monosaccharide(s), one or more activated monosaccharide(s), one or more phosphorylated monosaccharide(s), one or more disaccharide(s), and/or one or more other oligosaccharide(s) selected from the group comprising a neutral (noncharged) oligosaccharide, a negatively charged oligosaccharide, a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the group consisting of N-glycans and O- glycans; a plant oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharide; sialylated oligosaccharide preferably selected from the group comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose and combinations thereof; N-acetylglucosamine containing neutral (non-charged) oligosaccharide; N-acetyllactosamine containing oligosaccharide; lacto-N-biose containing oligosaccharide; non-fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; chondroitin sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.

14. Process according to any one of previous claims, wherein said solution is a cell cultivation using at least one cell that has been metabolically engineered to produce said oligosaccharide and one or more of i) sialic acid, ii) one or more monosaccharide(s), iii) one or more activated monosaccharide(s), iv) one or more phosphorylated monosaccharide(s), v) one or more disaccharide(s) and/or vi) one or more other oligosaccharides.

15. Process according to any one of previous claims, wherein said cell produces said oligosaccharide from one or more internalized precursor(s).

16. Process according to any one of previous claims, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.

17. Process according to any one of claims 13 to 16, wherein said oligosaccharide is purified from said sialic acid and/or said ashes by said process.

18. Process according to any one of previous claims, wherein the purity of the oligosaccharide obtained in the purified oligosaccharide solution at the end of said process is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% on total dry solid.

19. Process according to any one of previous claims, wherein the yield of purification of the oligosaccharide obtained in the purified oligosaccharide solution at the end of said process is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%.

20. Process according to any one of previous claims, wherein the purified oligosaccharide solution obtained at the end of said process has an ash content of < 10% on total dry solid, preferably < 9% on total dry solid, more preferably < 8% on total dry solid, even more preferably < 7% on total dry solid, even more preferably < 6% on total dry solid, even more preferably < 5% on total dry solid, even more preferably < 4% on total dry solid, even more preferably < 3% on total dry solid, even more preferably < 2% on total dry solid, even more preferably < 1% on total dry solid, most preferably < 0.5% on total dry solid.

21. The purified oligosaccharide solution, the purified oligosaccharide or the purified oligosaccharide mixture obtainable, preferably obtained, by a process according to any one of previous claims.

22. Purified oligosaccharide or purified oligosaccharide mixture according to claim 21 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.

23. Use of a purified oligosaccharide obtained by a process of any one of claims 1 to 20 in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.

24. Use of a purified oligosaccharide according to any one of claim 21 or 22 in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.