EP4651963A1 - Purification of an oligosaccharide or oligosaccharide mixture - Google Patents

Purification of an oligosaccharide or oligosaccharide mixture

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Publication number
EP4651963A1
EP4651963A1 EP24701594.4A EP24701594A EP4651963A1 EP 4651963 A1 EP4651963 A1 EP 4651963A1 EP 24701594 A EP24701594 A EP 24701594A EP 4651963 A1 EP4651963 A1 EP 4651963A1
Authority
EP
European Patent Office
Prior art keywords
gal
lacto
oligosaccharide
glcnac
glc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24701594.4A
Other languages
German (de)
French (fr)
Inventor
Jordy BAUWELINCK
Joeri Beauprez
Gaspard LEQUEUX
Gert PETERS
Dries VAN HERPE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inbiose NV
Original Assignee
Inbiose NV
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Filing date
Publication date
Application filed by Inbiose NV filed Critical Inbiose NV
Publication of EP4651963A1 publication Critical patent/EP4651963A1/en
Pending legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
    • B01D61/422—Electrodialysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/58—Multistep processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/04—Processes using organic exchangers
    • B01J39/05—Processes using organic exchangers in the strongly acidic form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/04—Processes using organic exchangers
    • B01J39/07—Processes using organic exchangers in the weakly acidic form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/16—Organic material
    • B01J39/18—Macromolecular compounds
    • B01J39/20—Macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00—Ion-exchange processes in general; Apparatus therefor
    • B01J47/02—Column or bed processes
    • B01J47/026—Column or bed processes using columns or beds of different ion exchange materials in series
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/04—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F26—DRYING
    • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
    • F26B5/00—Drying solid materials or objects by processes not involving the application of heat
    • F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
    • F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
    • F26B5/065—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing the product to be freeze-dried being sprayed, dispersed or pulverised

Definitions

  • the present invention relates to processes for the purification of an oligosaccharide or oligosaccharide mixture from a solution, a product of such processes, and the use of a product of such processes.
  • 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 and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response.
  • monosaccharides such as e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N- acetylgalactosamine, galacto
  • 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, an N-acetylglucosamine or an N-acetylneuraminic acid residue at their nonreducing end.
  • MOs milk oligosaccharides
  • HMOs human milk oligosaccharides
  • MOs for animal and human infant nutrition are directly linked to their biological activities including protection of the neonate from pathogens, supporting development of the infant's immune system and cognitive abilities.
  • 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.
  • various MOs possess an anti-inflammatory 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.
  • the desired oligosaccharide or oligosaccharide mixture obtained after purification has an ash content of ⁇ 10% on total dry solid, with a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
  • a lead content lower than 0.1 mg/kg dry solid preferably a lead content lower than 0.02 mg/kg dry solid,
  • this and other objects are achieved by providing a process for the purification of an oligosaccharide from a solution.
  • the process comprises passing the solution comprising the desired oligosaccharide through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially in acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH4 + form.
  • said second cationic ion exchange resin is in Na + form.
  • the solution comprising the oligosaccharide 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.
  • the cell cultivation is a fermentation.
  • This invention also provides a purified oligosaccharide by the above-referenced process.
  • 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.
  • the features “synthesize”, “synthesized” and “synthesis” are interchangeably used with the features “produce”, “produced” and “production”, respectively.
  • the expressions “capable of... ⁇ verb>” and “capable to... ⁇ verb>” are preferably replaced with the active voice of said verb and vice versa.
  • the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e., “expresses” is preferably replaced with "capable of expressing”.
  • Said additional compound(s) might be inevitable by-product(s), for example, generated during production of the oligosaccharide or the oligosaccharide mixture of present invention 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.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • 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.
  • sialic acid N-acetylneuraminate
  • N-acylneuraminate N-acetylneuraminic acid
  • Neu(n)Ac molecule refers 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; Neu4, 5,7,8, 9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • monosaccharide 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.
  • phosphorylated monosaccharide refers to a monosaccharide which is phosphorylated.
  • phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate.
  • activated monosaccharide refers to activated forms of monosaccharides.
  • 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
  • glycosyltransferase 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.org).
  • disaccharide refers to a saccharide polymer containing two simple sugars, i.e., monosaccharides.
  • examples of disaccharides comprise lactose (Gal-pi,4-Glc), lactulose (Gal-pi,4-Fru), allolactose (Gal-pi,6-Glc), lacto-N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-oc2,6-Gal, Fuc-al,2-Gal, fucopyranosyl-(l-4)-N-g
  • Oleaccharide refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides.
  • 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.
  • linkage between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein.
  • Gal-bl,4-Glc 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->5, alpha 2->l, 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.
  • polysaccharide refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically.
  • oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), 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 (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide, N-acetylglucos
  • oligosaccharide or “acidic oligosaccharide” are used interchangeably and refer to an oligosaccharide with a negative charge.
  • the negatively charged oligosaccharide is a sialylated oligosaccharide.
  • 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, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal-pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-a2,8-Gal-pi,4-Glc), 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-cc2,8- Neu5Ac-a2,3-Gal-pi
  • 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, galacturonate and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • Neu5Ac N-acetylneuraminic acid
  • Ne5Gc N- glycolylneuraminic acid
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • 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.
  • neutral (non-charged) 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.
  • '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-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAc i-3Gaipi-4Glc), lacto-N-tetraose (LNT, Gaipi- 3GlcNAc i-3Gaipi-4Glc), lacto-N-neotetraose (LNnT, Gaipi-4GlcNAcpi-3Gaipi-4Glc), lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose
  • 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), 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-fucopentaose II (LNFP II), Lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (
  • 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 (Eguus ferus caballus), pigs (Sus scropha), dogs (Canis 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 (Myrmecophag
  • mammalian milk oligosaccharide or “MMO” refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 5-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 II, lacto-N-fucopentaose II,
  • human milk oligosaccharide or "HMO” refer to oligosaccharides found in human breast milk, including preterm human milk, colostrum and term human milk.
  • HMDs 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)).
  • 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-hexao
  • 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.
  • 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.org) (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.
  • 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.
  • CPI cell productivity index
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the term "non- mammary cell(s)" may generally include any mammalian cell of non-mammary lineage.
  • a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component.
  • 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.
  • molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.
  • culture means the culture medium wherein the cell is cultivated, or fermented, or incubated, medium components, the cell itself, and an oligosaccharide that is produced by the cell in whole broth, i.e., inside (intracellularly) as well as outside (extracellularly) of the cell.
  • biomass refers to the suspended, precipitated or insoluble materials originating from fermentation cells, like intact cells, disrupted cells, cell fragments, cell walls, phospholipids, cell membranes, proteins, protein fragments, polysaccharides, polynucleotides and other large organic compounds produced by the cell.
  • the biomass may be in suspension and/or in solution.
  • Biomass as used herein is also to be understood to be non-complex biomass, typically of low or no organisation of cells into peculiar or complex structures. Non-limiting examples are individual cells, cell pairs, cell lumps, oligocellular or multicellular structures, cell layers, biofilms.
  • the non-complex biomass may be of a three- dimensional structure, like e.g.
  • biomass is also to be understood to be complex biomass, wherein said complex biomass is of a complex structure by nature, often a complex three-dimensional structure and comprises many hundreds, thousands, ten-thousands, but more typically hundreds of thousands or millions or more of cellular structures in a complex organisation, often of various cell types with different specialisations.
  • Non-limiting examples are higher plants or animals with a body visible with the naked eye, organs and tissues, including bone and meat or plant parts like fruit, vegetables, straw, sugarcane bagasse, hay, wood, timber.
  • Complex biomass may be the source of non-complex biomass, for example cell lines are typically derived from a tissue or organ but do not maintain the complex structure in cultivation.
  • cell dry weight or “CDW” as used herein refer to the grams of dry weight of biomass per liter of sample after removal of moisture. The procedure for measuring the CDW is disclosed in the working examples below.
  • biocatalysis reaction solution and "enzymatic synthesis reaction” are used interchangeably and refer to a mixture wherein an oligosaccharide is 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, 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 and ii) a buffered solution or the culture or incubation medium wherein said cell was cultivated or incubated.
  • chemical synthesis solution is to be understood as a mixture wherein an oligosaccharide is 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 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.
  • 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.
  • 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.
  • reactor and “incubator” refer to the recipient filled with the cultivation, incubation, chemical synthesis solution or biocatalysis reaction solution.
  • 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.
  • purified refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule.
  • purified refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state.
  • 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.
  • 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 CeliteTM filter aid).
  • protein-free oligosaccharide solution 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.
  • 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.
  • a protein-free oligosaccharide solution is a clarified oligosaccharide solution.
  • purification of an oligosaccharide solution from a cultivation 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.
  • the aqueous medium is pure water.
  • the medium comprises water with a trace amount of one or more organic solvents.
  • the medium comprises less than 1%-wt. (percent by weight) organic solvent.
  • the medium comprises less than 0.1%-wt. organic solvent.
  • the medium comprises less than 0.01%-wt. organic solvent.
  • the medium comprises less than 0.001%-wt. organic solvent.
  • the medium comprises less than 0.0001%-wt. organic solvent.
  • 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.
  • precursor refers to substances which are taken up or synthetized by the cell for the specific production of an oligosaccharide according to the present invention.
  • 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 the biochemical synthesis route of an oligosaccharide.
  • 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.
  • glucose-l-phosphate galactose-l-phosphate, glucose-6- phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-1- 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.
  • the cell used to produce the oligosaccharide is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the list 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.
  • a protein selected from the list 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.
  • acceptor refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase.
  • 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-n
  • 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.
  • the "Brix value” indicates the combined sugar content of the aqueous solution, when the purified oligosaccharide solution comprises two or more different oligosaccharides.
  • 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.
  • dry solid and “dry matter” as used herein are used interchangeably and are further described in Example 1.
  • flow rate 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.
  • 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.
  • bed volume / hour The terms "bed volume / hour”, “bed volume / h", “BV / hour” and "BV/h” are used interchangeably.
  • the ash content is a measure of the total amount of minerals and heavy metals present within a food or ingredients such as oligosaccharides, wherein the mineral content is a measure of the amount of specific inorganic components present within a food, such as e.g., Ca 2+ , Na + , K + , Mg 2+ , phosphate, sulphate and Cl' and wherein the heavy metal content is a measure of the amount of inorganic components present within a food like e.g. lead, arsenic, mercury, cadmium, zinc, manganese, copper and iron.
  • the mineral content is a measure of the amount of specific inorganic components present within a food, such as e.g., Ca 2+ , Na + , K + , Mg 2+ , phosphate, sulphate and Cl'
  • the heavy metal content is a measure of the amount of inorganic components present within a food like e.g. lead, arsenic, mercury, cadmi
  • 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 and heavy metals within a food.
  • Analytical techniques for providing information about the total mineral and heavy metal content are based on the fact that the analyte like minerals and heavy metals 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 and heavy metals 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.
  • Ashing may also be used as the first step in preparing samples for analysis of specific minerals and heavy metals, by atomic spectroscopy or the various traditional methods described below.
  • 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.
  • a dry oligosaccharide sample is generally hygroscopic, and the selected sample should be kept under dry conditions avoiding the absorption of water.
  • samples of 1-10 g 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.
  • 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 and heavy metals in grinders, glassware or crucibles which come into contact with the sample during the analysis.
  • 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 CO2, H2O and N2.
  • Most minerals and heavy metals are converted to oxides, sulphates, phosphates, chlorides or silicates. Although most minerals and heavy metals have fairly low volatility at these high temperatures, some are volatile and may be partially lost, e.g., iron, lead and mercury, for these components ICP-MS analysis of the product is more appropriate for quantification.
  • 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 the process as described herein 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 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 a fermentation as described herein and wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation or said fermentation.
  • the process comprises passing a solution comprising said oligosaccharide through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NHZ form.
  • the cationic ion exchange resin in the second cationic ion exchange is in Na + form.
  • the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H + form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH form.
  • the process further comprises any one or more of the methods selected from the list consisting of homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any
  • the present invention concerns a process for the purification of an oligosaccharide that is provided in a solution comprising said oligosaccharide.
  • the oligosaccharide is selected from the list comprising, consisting of or essentially consisting of fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosamin
  • the oligosaccharide is a fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (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 (
  • the oligosaccharide is a sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-cc2,6)-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
  • the oligosaccharide is an N-acetylglucosamine containing neutral (noncharged) oligosaccharide 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-
  • the oligosaccharide is a milk oligosaccharide.
  • the oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein.
  • the oligosaccharide is a human milk oligosaccharide (HMO) as described herein.
  • the oligosaccharide is an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans.
  • the oligosaccharide is a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans.
  • 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.
  • the 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).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • the oligosaccharide is a sialylated oligosaccharide having one Neu5Ac (neuraminic acid) group. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two sialic acid groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two identical sialic acid groups. In an even more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two Neu5Ac groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having three or more sialic acid groups.
  • the oligosaccharide is a sialylated milk oligosaccharide. In an even more preferred embodiment, the oligosaccharide is a sialylated mammalian milk oligosaccharide (MMO).
  • MMO sialylated mammalian milk oligosaccharide
  • the oligosaccharide is 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
  • the oligosaccharide is a fucosylated oligosaccharide having at least one fucose group. In a more preferred embodiment, the oligosaccharide is fucosylated oligosaccharide having two fucose groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having three or more fucose groups.
  • the oligosaccharide is a fucosylated milk oligosaccharide. In an even more preferred embodiment, the oligosaccharide is a fucosylated mammalian milk oligosaccharide (MMO).
  • MMO mammalian milk oligosaccharide
  • the oligosaccharide is selected from the list comprising, consisting of or essentially consisting of 2'-fucosyllactose (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
  • the oligosaccharide is an oligosaccharide with a degree of polymerization (DP) of at least 3.
  • the oligosaccharide is an oligosaccharide with a DP selected from the list comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.
  • the oligosaccharide is an oligosaccharide with a DP selected from the list comprising 3, 4, 5, 6, 7, 8, 9 and 10.
  • the oligosaccharide is a trisaccharide.
  • the oligosaccharide is a tetrasaccharide.
  • the oligosaccharide is a linear oligosaccharide. In an alternative preferred embodiment, the oligosaccharide is a branched oligosaccharide.
  • the oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide does not contain any protective group.
  • the oligosaccharide is an oligosaccharide that comprises one or more sialic acid groups and one or more monosaccharide building blocks selected from the list comprising fucose, galactose, glucose, xylose, mannose, N-acetylglucosamine, N-acetylgalactosamine, rhamnose, glucuronate, galacturonate, and N-acetylmannosamine.
  • the oligosaccharide is a sialylated oligosaccharide that is also fucosylated.
  • the oligosaccharide is a sialylated oligosaccharide that is not fucosylated.
  • the oligosaccharide is an oligosaccharide comprising one or more fucose groups and one or more sialic acid groups selected from the group 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 KDO.
  • the solution comprising an 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 the process as described herein. More specifically, the solution comprising an 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 a fermentation as described herein. Within the context of the present invention, said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation or by said fermentation.
  • 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).
  • 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.
  • the oligosaccharide is accompanied in said solution by sialic acid as defined herein; ashes, preferably, said ashes comprise sulphates, phosphates, sodium, chloride, potassium, and heavy metals; preferably said heavy metals comprise zinc, manganese, copper, ammonium, lead arsenic, cadmium and/or 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-Gal
  • said solution comprises two or more oligosaccharides.
  • the oligosaccharide 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.
  • the oligosaccharide is accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have the same DP as said oligosaccharide.
  • the oligosaccharide 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.
  • the oligosaccharide is accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have a different DP as said oligosaccharide.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the solution comprises only one or more neutral (non-charged) fucosylated oligosaccharide(s) as defined herein.
  • 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.
  • the solution comprises only one or more neutral (non-charged) oligosaccharide(s).
  • the solution comprises one or more neutral oligosaccharides and one or more charged oligosaccharide(s). In another preferred embodiment, the solution comprises one or more neutral (non-charged) fucosylated oligosaccharide(s) and one or more charged oligosaccharide(s). In another preferred embodiment, the solution comprises one or more neutral (non-charged) fucosylated oligosaccharide(s) and one or more sialylated oligosaccharide(s).
  • the ash content of the solution comprising an oligosaccharide to be purified by a process of present invention is > 10 % on total dry solid before purification by said process.
  • the ash content of a solution can be determined as described herein.
  • the solution comprising an oligosaccharide to be purified by a process of present invention 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.
  • the solution comprising an oligosaccharide to be purified by a process of present invention 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.
  • 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, additives and contaminants like e.g., antifoam.
  • 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.
  • the biomass, when present, that is separated during the process is optionally recycled to the cell cultivation.
  • the oligosaccharide to be purified by a process of present invention is produced by a cell that is cultured in a cell cultivation.
  • the cell cultivation comprises in vitro and/or ex vivo cultivation of cells.
  • the cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.
  • the cell is a metabolically engineered cell.
  • the cell has been metabolically engineered to produce any one or more compound(s) that is/are not (a) oligosaccharide(s).
  • the cell has been metabolically engineered to produce an oligosaccharide.
  • the cell has been metabolically engineered to produce two or more oligosaccharides.
  • the cell produces an oligosaccharide 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.
  • the cell has been metabolically engineered to produce an oligosaccharide 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.
  • the 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.
  • the cell comprises a sialyation pathway.
  • the cell is metabolically engineered to comprise a sialy lation pathway.
  • the cell has been metabolically engineered to comprise a sialylation pathway wherein any one or more of the genes selected 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, phosphoacety
  • the cell comprises a fucosylation pathway.
  • the cell is metabolically engineered to comprise a fucosylation pathway.
  • the cell has been metabolically engineered to comprise a fucosylation pathway wherein any one or more of the genes selected 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.
  • the cell comprises a galactosylation pathway.
  • the cell is metabolically engineered to comprise a galactosylation pathway.
  • the cell has been metabolically engineered to comprise a galactosylation pathway wherein any one or more of the genes selected 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.
  • the cell comprises an 'N-acetylglucosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N- acetylglucosaminylation pathway wherein any one or more of the genes selected from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-1- phosphate acetyltransferase and a glycosyltransferase transferring GIcNAc has/have a modified and/or enhanced expression.
  • the cell comprises an 'N-acetylgalactosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N- acetylgalactosaminylation pathway wherein any one or more of the genes selected 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.
  • the cell comprises a 'mannosylation' pathway.
  • the cell is metabolically engineered to comprise a mannosylation pathway.
  • the cell has been metabolically engineered to comprise a mannosylation pathway wherein any one or more of the genes selected 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.
  • the cell comprises an 'N-acetylmannosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylmannosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N- acetylmannosaminylation pathway wherein any one or more of the genes selected 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-1- phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-GIcNAc 2-epimerase and/or ManNAc kinase and a glycosyltransferase transferring ManNAc has/
  • the cell is metabolically engineered for an enhanced production of an oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of an oligosaccharide, a better efflux of the oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g.
  • ATP 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.
  • the cell produces said oligosaccharide from one or more internalized precursor(s) as defined herein.
  • said precursor is fed to the cell from the culture medium or the incubation.
  • the cell synthesizes one or more precursor(s) that is/are involved in the production of said oligosaccharide.
  • the precursor(s) that is/are used by the cell for the production of said oligosaccharide is/are completely converted into said oligosaccharide.
  • the precursor(s) that is/are used in said solution for the production of said oligosaccharide is/are completely converted into said oligosaccharide.
  • the cell cultivation is a fermentation.
  • 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.
  • 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.
  • 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.
  • the culture medium is a chemically defined medium.
  • 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.
  • the cell is cultivated in a chemically defined medium.
  • the cell is cultivated in a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.
  • the solution comprising an oligosaccharide to 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-morph
  • the solution comprising an oligosaccharide to 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 donor like e.g
  • the solution comprising an oligosaccharide to 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).
  • a polar reaction solvent CH2CI2, CICH2CH2CI
  • toluene an ethereal solvent
  • a nitrile solvent benzene, o-dichlorobenzene
  • urea urea
  • K2CO3 Tris(
  • said first cationic ion exchange comprises a cationic ion exchange resin in H + form.
  • said cationic ion exchange resin in said first cationic ion exchange is provided in H + form by a supplier.
  • said cationic ion exchange resin in said first cationic ion exchange is not provided in H + form but in another form, like e.g., Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form by a supplier and is regenerated upon use in said process into H + form by method(s) known by the person skilled in the art.
  • said first cationic 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.
  • said second cationic ion exchange comprises a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form.
  • said cationic ion exchange resin in said second cationic ion exchange is in Na + form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in Na + form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in Na + form but in another form, like e.g., H + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form 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.
  • said cationic ion exchange resin in said second cationic ion exchange is in K + form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in K + form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in K + form but in another form, like e.g., Na + , H + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form by a supplier and is regenerated upon use in said process into K + form by method(s) known by the person skilled in the art.
  • said cationic ion exchange resin in said second cationic ion exchange is in Ca 2+ form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in Ca 2+ form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in Ca 2+ form but in another form, like e.g., Na + , H + , K + , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form by a supplier and is regenerated upon use in said process into Ca 2+ form by method(s) known by the person skilled in the art.
  • said cationic ion exchange resin in said second cationic ion exchange is in Fe 2+ form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in Fe 2+ form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in Fe 2+ form but in another form, like e.g., Na + , H + , K + , Ca 2+ , Mg 2+ , Fe 3+ , Al 3+ or NH 4 + form by a supplier and is regenerated upon use in said process into Fe 2+ form by method(s) known by the person skilled in the art.
  • said cationic ion exchange resin in said second cationic ion exchange is in Fe 3+ form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in Fe 3+ form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in Fe 3+ form but in another form, like e.g., Na + , H + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Al 3+ or NH 4 + form by a supplier and is regenerated upon use in said process into Fe 3+ form by method(s) known by the person skilled in the art.
  • said cationic ion exchange resin in said second cationic ion exchange is in Al 3+ form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in Al 3+ form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in Al 3+ form but in another form, like e.g., Na + , H + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , or NH 4 + form by a supplier and is regenerated upon use in said process into Al 3+ form by method(s) known by the person skilled in the art.
  • said cationic ion exchange resin in said second cationic ion exchange is in NH + form.
  • said cationic ion exchange resin in said second cationic ion exchange is provided in NH 4 + form by a supplier.
  • said cationic ion exchange resin in said second cationic ion exchange is not provided in NH 4 + form but in another form, like e.g., Na + , H + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , or Al 3+ form by a supplier and is regenerated upon use in said process into NH 4 + form by method(s) known by the person skilled in the art.
  • said second cationic 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.
  • the cationic ion exchange resin present in said first cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin.
  • the cationic ion exchange resin present in said second cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin.
  • WAC weak acid cation
  • SAC strong acid cation
  • 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 alkaline to about neutral pH in the case of a WAC.
  • the first cationic ion exchange comprises a WAC exchange resin in H + form and the second cationic ion exchange comprises a WAC exchange resin in a Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form.
  • the first cationic ion exchange comprises a SAC exchange resin in H + form and the second cationic ion exchange comprises a WAC exchange resin in a Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form.
  • the first cationic ion exchange comprises a WAC exchange resin in H + form and the second cationic ion exchange comprises a SAC exchange resin in a Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form.
  • the first cationic ion exchange comprises a SAC exchange resin in H + form and the second cationic ion exchange comprises a SAC exchange resin in a Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form.
  • 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.
  • 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.
  • 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.
  • the cationic ion exchange resin present in said first cationic ion exchange is in a gel-type version, a porous-type version or in a highly porous-type version.
  • the cationic ion exchange resin present in said second cationic ion exchange is in a gel-type version, a porous-type version or in a highly porous-type version.
  • the cationic ion exchange resin present in said first cationic ion exchange has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix.
  • the cationic ion exchange resin present in said second cationic ion exchange has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix.
  • the matrix used in said cationic ion exchange resin in said first and/or second cationic ion exchange further comprises divinylbenzene (DVB).
  • DVB is cross-linked to a styrene or a polystyrene based matrix used in said cationic ion exchange resin in said first and/or second cationic ion exchange.
  • the cationic exchange resin in said first cationic ion exchange 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 Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form, the cationic ion exchange resin in said second cationic ion exchange needs to be regenerated by means known by the person skilled to obtain a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form, respectively.
  • the flow rate through said first and/or second cationic ion exchange is at least 0.5 bed volume / hour (BV/h). In a more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 1 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 1.5 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 2 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 2.5 BV/h. In a most preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 3 BV/h.
  • said first and/or second cationic ion exchange performed on said solution in the process is/are performed at a temperature ranging from 0°C to 80°C, including 0°C and 80°C in the range.
  • said first and/or second cationic ion exchange is/are performed at a temperature ranging from 4°C to 60°C, including 4°C and 60°C in the range.
  • said first and/or second cationic ion exchange is/are performed at a temperature ranging from 4°C to 40°C, including 4°C and 40°C in the range.
  • said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange is/are performed at a temperature ranging from 20°C to 25°C, including 20°C and 25°C in the range.
  • said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange is/are performed at room temperature.
  • said first and/or second cationic ion exchange is/are 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, 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,
  • 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.
  • performing said first cationic ion exchange step at a specific temperature is to be understood as that the temperature of the cationic ion exchanger used in said first cationic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said first cationic ion exchange is adjusted to said specific temperature.
  • performing said second cationic ion exchange step at a specific temperature is to be understood as that the temperature of the cationic ion exchanger used in said second cationic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said second cationic ion exchange is adjusted to said specific temperature.
  • said first and/or second cationic ion exchange performed on said solution in the process is/are performed at a pH ranging from 2 to 5, including 2 and 5 in the range.
  • said first and/or second cationic ion exchange is/are performed at a pH ranging from 3 to 5, including 3 and 5 in the range.
  • said first and/or second cationic ion exchange is/are performed at a pH ranging from 4 to 5, including 4 and 5 in the range.
  • said first and/or second cationic ion exchange is/are performed at a pH selected from the list comprising about 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 and 5.
  • the process further comprises any one or more of the methods selected from the list comprising, consisting of or essentially consisting of 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, vacuum evaporation, wiped
  • the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H + form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form, preferably in Na + form.
  • said process comprises i) passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially into acids, ii) any one or more of filtration, pH adjustment of the solution to a pH selected from the list comprising about 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 and 5 and/or temperature adjustment before iii) passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH
  • 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 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.
  • 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 0°C to 122°C should be understood as a temperature of 0°C, 1°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,
  • 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
  • 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
  • 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, 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, 25°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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 said first cationic ion exchange comprising a cationic ion exchange resin in H + form.
  • the process further comprises ultrafiltration, nanofiltration and electrodialysis.
  • the process further comprises ultrafiltration, nanofiltration and electrodeionization.
  • the process further comprises ultrafiltration, nanofiltration, electrodialysis and electrodeionization.
  • 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 does not further comprise electrodialysis. In another and/or additional preferred embodiment, the process does not further comprise electrodeionization.
  • 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 H + , Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ , NHZ and wherein said anionic ion exchange resin is in any form selected from the list comprising OH-, Cl' and SO 3 2- .
  • the process comprises two mixed bed ion exchanges.
  • the process comprises two consecutive steps of ultrafiltration and wherein the membrane molecular weight cut-off of the membrane used in the first ultrafiltration step is higher than the membrane molecular weight cutoff of the membrane used in the second ultrafiltration step.
  • said two consecutive steps of ultrafiltration are performed on said solution comprising said oligosaccharide prior to said first cationic ion exchange comprising a cationic ion exchange resin in H + form.
  • the process further comprises clarification, preferably wherein said clarification is performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration.
  • the process further comprises use of a cation exchange adsorbent resin, an anion exchange adsorbent resin and/or use of an adsorbent material.
  • the process further comprises drying, preferably wherein said drying is 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.
  • the process further comprises filtration, preferably wherein said filtration is performed by use of a filtration aid and/or flocculant.
  • a filtration aid is an adsorbing agent, more preferably said filtration aid is active carbon.
  • 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.
  • 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.
  • the process further comprises diafiltration, preferably wherein said diafiltration is performed on said solution 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • the process further comprises nanofiltration, preferably wherein said nanofiltration is performed at a pressure ranging from 5 to 20 bar.
  • 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.
  • 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 list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.
  • one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.
  • 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.
  • the process is a batch process. In an alternative and/or additional preferred embodiment, the process is a continuous process.
  • 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 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: passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially into acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na + form.
  • 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 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: passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially into acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH 4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na
  • the purity of the oligosaccharide 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%.
  • the yield of purification of the oligosaccharide 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 a lead content lower than 0.01 mg/kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with a lead content lower than 0.05 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
  • 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 dry solid, even more preferably lower than 0.05 mg/kg dry solid, even more preferably lower than 0.02 mg/kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with an arsenic content lower than 0.1 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
  • 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.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with a cadmium content lower than 0.05 mg/kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with a cadmium content lower than 0.02 mg/kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of
  • 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 dry solid, even more preferably lower than 0.005 mg/kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the process has an ash content of ⁇ 10% on total dry solid with a mercury content lower than 0.2 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
  • the purified oligosaccharide solution obtained at the end of the process is filter-sterilized.
  • the purified oligosaccharide solution obtained at the end of the process is subjected to endotoxin removal.
  • 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.
  • 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.
  • the purified oligosaccharide solution obtained at the end of the method has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 60 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid.
  • the purified oligosaccharide solution obtained at the end of the method has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA.
  • the purified oligosaccharide solution obtained at the end of the method has an endotoxin content equal to or below 8000 EU per gram dry solid, equal to or below 5000 EU per gram dry solid, equal to or below 3000 EU per gram dry solid, equal to or below 1000 EU per gram dry solid, equal to or below 800 EU per gram dry solid, equal to or below 500 EU per gram dry solid, equal to or below 300 EU per gram dry solid or equal to or below 100 EU per gram dry solid.
  • the purified oligosaccharide solution obtained at the end of the process is free of DNA, proteins, and/or recombinant genetic material.
  • the purified oligosaccharide solution obtained at the end of the method is free of recombinant DNA and/or proteins derived from the recombinant micro-organism.
  • the purified oligosaccharide solution obtained at the end of the method has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters.
  • the purified oligosaccharide solution obtained at the end of the method has less than 0.5 pg/kg epichlorohydrin.
  • the purified oligosaccharide solution obtained at the end of the method has a conductivity of less than 10 mS/cm at a 300 g/L solution.
  • 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, vacuum evaporation, wiped film evaporation, and falling film evaporation.
  • the process further comprises any one or more of nanofiltration, diafiltration, reverse osmosis, evaporation, vacuum evaporation, wiped film evaporation, and falling film evaporation, wherein one or more of said nanofiltration, diafiltration, reverse osmosis, evaporation, vacuum evaporation, wiped film evaporation, and falling film evaporation is performed more than one time during the process.
  • 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.
  • the purified oligosaccharide solution obtained at the end of the process is first subjected to a polishing step prior to concentration.
  • a 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • any of said concentration method is selected from the list comprising using vacuum evaporation or reverse osmosis or nanofiltration.
  • the purified oligosaccharide solution obtained at the end of the process comprises an oligosaccharide 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.
  • a temperature ⁇ 25°C is to be understood as 0°C, l’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 O’C, l’C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C or a temperature below O’C.
  • 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%.
  • 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.
  • the purified oligosaccharide solution obtained at the end of the process is dried by spray-drying, freeze-drying or agitated thin film drying.
  • the pH of the purified oligosaccharide solution is ranging from 2 to 7.
  • 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,
  • the pH of the purified oligosaccharide solution is ranging from 3 to 6; 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,
  • 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.
  • the purified oligosaccharide solution obtained at the end of the process is dried by spray-drying, 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 present invention provides a purified oligosaccharide solution, a purified oligosaccharide or a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein.
  • the purified oligosaccharide solution can comprise one purified oligosaccharide or a purified oligosaccharide mixture.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides an oligosaccharide 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.
  • 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 or less than 0.1% ash on total dry solid.
  • said oligosaccharide is produced through cell cultivation.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that 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.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a lead content ⁇ 0.1 mg/kg dry solid.
  • said purified oligosaccharide has a lead content ⁇ 0.02 mg/kg dry solid.
  • said purified oligosaccharide has a lead content ⁇ 0.01 mg/kg dry solid.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has an arsenic content ⁇ 0.2 mg/kg dry solid.
  • said purified oligosaccharide has an arsenic content ⁇ 0.05 mg/kg dry solid.
  • said purified oligosaccharide has an arsenic content ⁇ 0.02 mg/kg dry solid.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a cadmium content ⁇ 0.1 mg/kg dry solid.
  • said purified oligosaccharide has a cadmium content ⁇ 0.01 mg/kg dry solid.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a mercury content ⁇ 0.5 mg/kg dry solid.
  • said purified oligosaccharide has a mercury content ⁇ 0.1 mg/kg dry solid.
  • said purified oligosaccharide has a mercury content ⁇ 0.005 mg/kg dry solid.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has 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.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant micro-organism and/or recombinant genetic material.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has less than 0.5 pg/kg epichlorohydrin.
  • the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a conductivity of less than 10 mS/cm at a 300 g/L solution.
  • the present invention provides a spray-dried oligosaccharide or a spray-dried oligosaccharide mixture comprising an oligosaccharide, wherein said oligosaccharide or oligosaccharide mixture 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.
  • 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.
  • said oligosaccharide or said oligosaccharide mixture is produced through cell cultivation.
  • 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.
  • 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.
  • said powder contains ⁇ 10%-wt.
  • 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.
  • said powder contains ⁇ 7%-wt.
  • 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.
  • said powder contains ⁇ 5%-wt.
  • 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.
  • 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.
  • said dried powder, preferably spray-dried powder has a mean particle size of 95 to 120 pm as determined by laser diffraction.
  • said dried powder, preferably spray-dried powder has a mean particle size of 110 to 120 pm as determined by laser diffraction.
  • 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.
  • said powder when redissolved in water at a concentration of 10% (mass on volume) provides a solution with 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 or 7.
  • 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.
  • 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, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.
  • 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.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1% or ⁇ 0.5% on total dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder 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.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of ⁇ 10% on total dry solid with a lead content lower than 0.1 mg/kg dry solid, more preferably with a lead content lower than 0.05 mg/kg dry solid, even more preferably with a lead content lower than 0.02 mg/kg dry solid, most preferably with a lead content lower than 0.01 mg/kg dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of ⁇ 10% on total dry solid with an arsenic content lower than 0.2 mg/kg dry solid, more preferably with an arsenic content lower than 0.1 mg/kg dry solid, even more preferably with an arsenic content lower than 0.05 mg/kg dry solid, most preferably with an arsenic content lower than 0.02 mg/kg dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of ⁇ 10% on total dry solid with a cadmium content lower than 0.1 mg/kg dry solid, more preferably with a cadmium content lower than 0.05 mg/kg dry solid, even more preferably with a cadmium content lower than 0.02 mg/kg dry solid, most preferably with a cadmium content below 0.01 mg/kg dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of ⁇ 10% on total dry solid with a mercury content lower than 0.5 mg/kg dry solid, more preferably lower than 0.2 mg/kg dry solid, even more preferably lower than 0.1 mg/kg dry solid, most preferably lower than 0.005 mg/kg dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder 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.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 60 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an endotoxin content equal to or below 8000 EU per gram dry solid, equal to or below 5000 EU per gram dry solid, equal to or below 3000 EU per gram dry solid, equal to or below 1000 EU per gram dry solid, equal to or below 800 EU per gram dry solid, equal to or below 500 EU per gram dry solid, equal to or below 300 EU per gram dry solid or equal to or below 100 EU per gram dry solid.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism and/or recombinant genetic material.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters.
  • the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has less than 0.5 ptg/kg epichlorohydrin. In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder is a spray-dried powder.
  • the present invention provides for a purified oligosaccharide as described herein wherein any one or more of said purified oligosaccharide is a milk oligosaccharide.
  • the present invention provides for a purified oligosaccharide mixture comprising an oligosaccharide as described herein wherein said purified oligosaccharide mixture comprises a milk oligosaccharide.
  • the milk oligosaccharide is a mammalian milk oligosaccharide (MMO).
  • MMO mammalian milk oligosaccharide
  • HMO human milk oligosaccharide
  • the milk oligosaccharide a neutral (noncharged) milk oligosaccharide, preferably a neutral (non-charged) human milk oligosaccharide (HMO), selected from the list comprising 2'-fucosyllactose, 3-fucosyllactose, 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 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-ne
  • 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-he
  • the present invention provides for a purified oligosaccharide or purified oligosaccharide mixture as described herein, 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.
  • the present invention provides for a purified oligosaccharide comprising 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.
  • a heavy metal selected from the list comprising lead, arsenic, cadmium, mercury, zinc, manganese, copper, iron, magnesium and calcium.
  • the purified oligosaccharide comprises an ash content of ⁇ 10% on total dry solid wherein said ash content of ⁇ 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
  • the purified oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosaminoglycan
  • the fucosylated oligosaccharide is selected from the list comprising 2'- fucosyllactose (2'FL), 3-fucosyl lactose (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
  • the sialylated 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-a2,6)- Glc), 8,3-disialyllactose (Neu5Ac-cc2,8-Neu5Ac-cc2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, 3'- sialyllactosamine, sialyllacto-N-tetraose a (
  • the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
  • the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
  • the monomeric building blocks e.g. the monosaccharide or glycan unit composition
  • the anomeric configuration of side chains e.g. the anomeric configuration of side chains
  • the presence and location of substituent groups e.g. the degree of polymerization/molecular weight and the linkage pattern
  • 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 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.
  • GC-MS gas chromatography-mass spectrometry
  • MALDI-MS Microx-assisted laser desorption/ionization-mass spectrometry
  • ESI-MS Electropray ionization-mass spectrometry
  • 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).
  • SEC-HPLC high performance size-exclusion chromatography
  • To identify the monomeric components of the oligosaccharide 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.
  • said oligosaccharide 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 (gasliquid chromatography coupled with mass spectrometry).
  • GLC/MS gasliquid chromatography coupled with mass spectrometry
  • said oligosaccharide 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.
  • an enzyme that is specific for a particular type of linkage e.g., beta-galactosidase, or alpha-glucosidase, etc.
  • NMR may be used to analyse the products.
  • the present invention provides for a purified oligosaccharide or purified oligosaccharide mixture as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
  • the present invention provides use of a purified oligosaccharide 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.
  • said oligosaccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • Said purified oligosaccharide 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.
  • 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.
  • 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.
  • a preparation is provided that further comprises at least one probiotic microorganism.
  • said preparation is a nutritional composition.
  • 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.
  • 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.
  • 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 B. infantis (e.g., Bi-26)), and Saccharomyces boulardii.
  • said oligosaccharide produced and/or purified by a process of this specification is orally administered in combination with such microorganism.
  • 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.
  • oligosaccharides such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose
  • said oligosaccharide 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.
  • 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.
  • said oligosaccharide 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.
  • said purified oligosaccharide is mixed with one or more ingredients of the infant formula.
  • 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 (HMDs).
  • 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,
  • 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 in the infant formula is approximately the same concentration as the concentration of the oligosaccharide generally present in human breast milk. In some embodiments, an oligosaccharide 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.
  • the present invention provides for a saccharide composition
  • a saccharide composition comprising an ash content of ⁇ 10% on total dry solid and an oligosaccharide content of > 90 % 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.
  • said ash content of ⁇ 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
  • the saccharide composition comprises more than one oligosaccharide.
  • the oligosaccharide(s) present in the saccharide composition is/are selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N- acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate
  • the fucosylated oligosaccharide is selected from the list comprising 2'-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 I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-neofucop
  • the sialylated oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(Neu5Ac-oc2,6)-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
  • the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
  • the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
  • the saccharide composition further comprises one or more disaccharide(s).
  • the saccharide composition further comprises a protein source, a vitamin, an oil, a mineral, a fatty acid, an enzyme, and/or a probiotic strain.
  • the saccharide composition is a composition selected from the list comprising a food composition, a pharmaceutical composition, a cosmetic composition, a nutritional composition, a liquid food composition, a solid food composition, an infant food formula, a toddler food formula, a medical nutrition product, a medical food composition, a dietary supplement, a sachet product, a liquid ready-to-use infant nutrition product, a solid infant nutrition product, a liquid ready-to-use toddler nutrition product, a granulated product, a spray-dried infant formula product, a premix, a dairy drink, an infant formula and combinations thereof.
  • 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, the process comprising passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion
  • said cationic ion exchange resin in said first and/or second cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin.
  • WAC weak acid cation
  • SAC strong acid cation
  • the flow rate through said first and/or second cationic 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.
  • 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, 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
  • process further comprises any one or more of the methods selected from the list comprising homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and
  • 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.
  • the temperature of said solution is adjusted to a temperature of 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.
  • process comprises enzymatic treatment comprising incubation of said solution with one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.
  • one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase.
  • Process according to any one of embodiments 8 to 22, wherein said process comprises enzymatic treatment converting lactose, sucrose, malto-oligosaccharides, maltotriose, sorbitol, trehalose, starch, cellulose, hemi-cellulose, lignocellulose, molasses, corn-steep liquor and/or high-fructose syrup to monosaccharides.
  • said process is a batch or continuous process.
  • oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosaminoglycan
  • said oligosaccharide is a sia lylated 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).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • oligosaccharide is 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
  • ash content of said solution comprising said oligosaccharide is > 10 % on total dry solid before purification by said process.
  • said solution comprising said oligosaccharide 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.
  • said solution comprising said oligosaccharide 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.
  • said solution is a cell cultivation using a cell, preferably a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein the purity of said oligosaccharide in said cell cultivation is ⁇ 70 %, ⁇ 50 %, ⁇ 50 %, ⁇ 40 %, ⁇ 30 %, ⁇ 20 %, ⁇ 10 % on total dry solid before purification by said process.
  • said solution is a cell cultivation using a cell, preferably a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein, when present, biomass separated during said process is optionally recycled to said cell cultivation.
  • said solution is a cell cultivation using at least one cell that has been metabolically engineered to produce said oligosaccharide.
  • 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.
  • said cell produces said oligosaccharide from one or more internalized precursor(s). 37.
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.
  • 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, maltooligosaccharides, 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 minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.
  • oligosaccharide 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, preferably wherein all of said other oligosaccharides have the same DP as said oligosaccharide.
  • DP degree of polymerization
  • oligosaccharide 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, preferably wherein all of said other oligosaccharides have a different DP as said oligosaccharide.
  • DP degree of polymerization
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the purified oligosaccharide solution obtained at the end of said process comprises an oligosaccharide 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 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 or the purified oligosaccharide mixture 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 57, 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 oligosaccharide mixture obtainable, preferably obtained, by a process according to any one of embodiments 1 to 57, 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.
  • Oligosaccharide purified according to the process according to any one of embodiments 1 to 57 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.
  • 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 57 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.
  • oligosaccharide according to any one of embodiments 58, 59, 61 to 65, 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).
  • MMO mammalian milk oligosaccharide
  • HMO human milk oligosaccharide
  • Purified oligosaccharide mixture comprising an oligosaccharide according to any one of embodiments 58, 60 to 65, wherein said purified oligosaccharide mixture comprises a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO).
  • MMO mammalian milk oligosaccharide
  • HMO human milk oligosaccharide
  • Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 58 to 68, 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.
  • Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 58 to 69 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
  • the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement
  • the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • a milk oligosaccharide according to any one of embodiment 67 or 68 as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food.
  • a purified oligosaccharide comprising 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 according to embodiment 75 wherein said ash content of ⁇ 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
  • oligosaccharide according to any one of embodiment 75 or 76, wherein said purified oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (noncharged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, N-acetylglucosamine containing neutral (non-charged) oligosaccharide, N-acetyllactosamine containing oligosaccharide, lacto-N-biose containing oligosaccharide, a galactose containing oligosaccharide, non-fucosylated neutral (noncharged) oligosaccharide, chitosan, chitosan comprising oligosaccharide, heparosan, chondroitin
  • a saccharide composition comprising an ash content of ⁇ 10% on total dry solid and an oligosaccharide content of > 90 % 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.
  • Composition according to embodiment 78 wherein said ash content of ⁇ 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
  • said oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N- acetylglucosamine containing oligosaccharide, 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
  • the present invention relates to the following preferred specific embodiments:
  • 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 a fermentation, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation or said fermentation, the process comprising passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H + form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NHZ form, and wherein: the eluate obtained from said first
  • said cationic ion exchange resin in said first and/or second cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin, has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix, and/or has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix wherein said matrix further comprises divinylbenzene (DVB).
  • WAC weak acid cation
  • SAC strong acid cation
  • the flow rate through said first and/or second cationic ion exchange is at least 0.5, at least 1, at least 2, at least 2.5 and/or at least 3 bed volume / hour (BV/h)
  • said first and/or second cationic ion exchange is/are performed at a temperature ranging from 0°C to 80°C, from 4°C to 60°C, from 4°C to 40°C, from 10°C to 37°C, from 20°C to 30°C, from 20°C to 25°C, from 22°C to 24°C and/or from 23°C to 24°C
  • said first and/or second cationic ion exchange is/are performed at a pH ranging from 2 to 5, from 3 to 5, and/or from 4 to 5.
  • process further comprises any one or more of the methods selected from the list consisting of 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, vacuum evaporation, wiped film evapor
  • any one or more of said method(s) succeed(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ or NH form, and/or
  • the temperature of said solution is adjusted to a temperature of: from 36°C to 65°C, from 36°C to 60°C, from 40°C to 55°C, and/or from 40°C to 45°C wherein said temperature is within 5°C of a temperature at which the solution exhibits maximum turbidity, and/or - from 0°C to 122°C, from 2°C to 80°C, from 4°C to 50°C, from 10°C to 55°C, from 20°C to 45°C, from 21°C to 40°C, from 22°C to 37°C and/or from 25°C to 30°C.
  • oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing neutral (noncharged) 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; chondroitin sulphate; glycosaminogly
  • the purity of said oligosaccharide in said solution is ⁇ 70 %, ⁇ 60 %, ⁇ 50 %, ⁇ 40 %, ⁇ 30 %, ⁇ 20 %, ⁇ 10 % on total dry solid
  • the ash content of said solution comprising said oligosaccharide is > 10 % on total dry solid
  • said solution comprising said oligosaccharide has an ash content of > 10% on total dry solid
  • 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
  • said solution comprising said oligosaccharide 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 pur
  • oligosaccharide is accompanied in said solution by sialic acid, ashes, ashes selected from the list comprising 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); O-antigen; enterobacterial common antigen (ECA); the oligosaccharide is accompanied in said solution by si
  • said solution is a cell cultivation: using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein the purity of said oligosaccharide in said cell cultivation is ⁇ 70 %, ⁇ 50 %, ⁇ 50 %, ⁇ 40 %, ⁇ 30 %, ⁇ 20 %, ⁇ 10 % on total dry solid before purification by said process, using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein, when present, biomass separated during said process is optionally recycled to said cell cultivation, using at least one cell that has been metabolically engineered to produce said oligosaccharide, and/or using at least one cell that has been metabolically engineered to produce said oli
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.
  • 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, culture medium comprising a carbon source wherein 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, treha
  • a carbon source comprising a monosaccharide, disaccharide, oli
  • said oligosaccharide 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), at least one of said other oligosaccharides has a different DP, all of said other oligosaccharides have the same DP, or all of said other oligosaccharides have a different DP as said oligosaccharide.
  • DP degree of polymerization
  • 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, and/or 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%.
  • the purified oligosaccharide solution obtained at the end of said process has an ash content of ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1% and/or ⁇ 0.5% on total dry solid, 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, has an ash content of ⁇ 10% on total dry solid with a lead content lower than 0.1 mg/kg dry solid, lower than 0.02 mg/kg dry solid and/or lower than 0.01 mg/kg dry solid, has an ash content of ⁇ 10% on total dry solid with an arsenic content lower than 0.2 mg/kg dry solid, lower than 0.05 mg/kg dry solid, and/or lower
  • the purified oligosaccharide solution, the purified oligosaccharide or the purified oligosaccharide mixture obtainable or obtained by a process according to any one of previous preferred embodiments.
  • Purified oligosaccharide obtainable or obtained by a process according to any one of preferred embodiments 1 to 16, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is i) dried, 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, at least 30% dry matter or at least 40% dry matter.
  • a purified oligosaccharide comprising an ash content of ⁇ 10% on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid.
  • Purified oligosaccharide according to preferred embodiment 21, wherein said purified oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing neutral (noncharged) 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; chondroitin sulphate; glycos
  • a saccharide composition comprising an ash content of ⁇ 10% on total dry solid and an oligosaccharide content of > 90 % on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid.
  • 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 cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH4CI, 5.00 g/L (NF hSC , 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H 2 O, 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.
  • Vitamin solution consisted of 3.6 g/L FeCI 2.4 H 2 O, 5.0 g/LCaCI 2 .2H 2 0, 1.3 g/L MnCI 2 .2H 2 O, 0.38 g/L CuCI 2 .2H 2 O, 0.5 g/L CoCI 2 .6H 2 O, 0.94 g/L ZnCI 2 , 0.0311 g/L H3BO4, 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine. HCI.
  • the molybdate solution contained 0.967 g/L NaMoO 4 .2H 2 O.
  • the selenium solution contained 42 g/L Seo2.
  • the minimal medium for fermentations contained 6.75 g/L NH 4 CI, 1.25 g/L (NH 4 ) 2 SO 4 , 2.93 g/L KH 2 PO 4 and 7.31 g/L KH 2 PO 4 , 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H 2 O, 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.
  • 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), carbenicill in (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • a preculture for the bioreactor was started from an entire 1 m L 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, Melsoder, 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 H 2 S0 4 and 20% NH 4 OH.
  • the exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
  • 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.
  • 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 mL 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. Strains, plasmids and mutations
  • 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 (NH ⁇ SC , 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSC>4.7H2O, 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 CaCl2.2H 2 O, 0.1 g/L MnCI 2 .2H 2 O, 0.033 g/L CuCI 2 .2H 2 O, 0.06 g/L CoCI 2 .6H 2 O, 0.17 g/L ZnCL, 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 FeCI 3 .6H 2 O, 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.
  • 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.
  • 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.
  • 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 CaCI 2 , 10 g/L FeSO 4 .7H 2 O, 10 g/L MnSO 4 .H 2 O, 1 g/L ZnSO 4 .7H 2 O, 0.2 g/L CuSO 4 , 0.02 g/L NiCI 2 .6H 2 O, 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 (NH 4 ) 2 SO 4 , 5 g/L urea, 1 g/L KH 2 PO 4 , 1 g/L K 2 HPO 4 , 0.25 g/L MgSO 4 .7H 2 O, 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.
  • 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.
  • 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.
  • Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7).
  • 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.
  • 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).
  • TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm2).
  • 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).
  • 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).
  • 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 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.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q.61420), and a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.
  • the mutant strain was derived from C.
  • 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 D3Q.Y14) from E.
  • LgtB Uniprot ID Q.51116, sequence version 02, 01 Dec 2000
  • 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).
  • 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).
  • 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).
  • 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.
  • FBS foetal bovine serum
  • 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.
  • Hassiotou et al. 2012, Stem Cells. 30(10): 2164-2174
  • 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 - C356; each of which is incorporated herein by reference in their entireties for all purposes.
  • 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.
  • 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.
  • penicillin-streptomycin 100 U/mL penicillin, 100 ug/mL streptomycin
  • 5 pg/mL insulin for 48h.
  • 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.
  • serum is removed from the complete induction medium.
  • 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.
  • growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • 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.
  • 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.
  • 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.
  • Mammocult media available from Stem Cell Technologies
  • DMEM mammary cell enrichment media
  • 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.
  • remodelling systems such as CRISPR/Cas9
  • select genes of interest such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins
  • 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.
  • isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W0220
  • 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.
  • the cells Upon exposure to the lactation media, the cells start to differentiate and stop growing.
  • 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.
  • 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.
  • a cell-free protein expression system can be used to produce functional proteins without the use of living cells.
  • 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).
  • a nucleic acid template e.g., plasmid DNA, linear DNA or mRNA.
  • 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-l,3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375) in separate CFPS reactions.
  • NEB PURExpress system
  • enzymatic synthesis reactions were set up to produce one or more oligosaccharide(s).
  • any one or more of said alpha-1, 2- fucosyltransferase, alpha-l,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).
  • a certain pH for example 7
  • a certain temperature for example 37°C
  • time for example 8 hours, 16 hours or 24 hours
  • 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.
  • ELSD Evaporative Light Scattering Detector
  • Rl Refractive Index
  • 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
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection.
  • Rl Refractive Index
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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. Ill
  • oligosaccharide e.g., HMO
  • a Bunsen burner 5 g
  • 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.
  • ICP-MS inductively coupled plasma-mass spectrometry
  • Nitric acid > 65%, Sigma-Aldrich was used for microwave digestion and standard/sample preparation. All dilutions were done using 18.2 MfTcm (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.
  • CEM microwave digestion
  • 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.
  • HEHe High Energy He
  • 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.
  • 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:
  • CDW Cell dry weight
  • An alternative method for determination of cell dry mass or cell dry weight uses an Infrared Moisture Analyzer (Sartorius MA150).
  • the equipment was allowed to warm-up for 30 min, and the standby temperature was set to 60°C.
  • the balance was automatically tared with the weighing aluminium pan containing a glass microfibre filter pad (0.2pm) after drying (105°C in moisture analyser to achieve stable weight, lasting about 1 min) and equilibration.
  • the drying temperature was set to 105°C. Samples were evenly added to the glass microfibre pad, and the drying programme was set to end when the weight change was less than 0-1 mg min -1 .
  • the dried glass fibre filter pad was used to filter off the biomass from 10 mL of broth and the filtered broth was washed 2 times with physiological solution.
  • the filter was then dried again by means of the method described above, drying the already tared aluminium pan and the filter pad at 105°C until stable weight.
  • a method is used that is compatible with reducing agents, such as reducing sugars or oligosaccharides with a reducing end.
  • reducing agents such as reducing sugars or oligosaccharides with a reducing 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.
  • DIW de-ionized water
  • 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® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • Threshold assay 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® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • 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.
  • LAL limulus amebocyte lysate
  • 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.
  • the system 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 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.
  • 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.
  • 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. Typically, the obtained product concentration was above 50 g/L.
  • 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. Typically, the obtained product concentration was above 50 g/L.
  • An E. coli K12 MG1655 strain engineered for production of 2' FL (Fuc-al,2-Gal-pi,4-Glc) as described e.g., in WO22129470 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.
  • 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 was measured using UPLC as described in Example 1. Typically, the obtained product concentration was above 50 g/L.
  • An E. coli K12 MG1655 strain engineered for production of 3-FL (Gal- i,4-[Fuc-al,3]-Glc) as described e.g., in WO20127417 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.
  • 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 was measured using UPLC as described in Example 1. Typically, the obtained product concentration was above 50 g/L.
  • An E. coli K12 MG1655 strain engineered for production of an oligosaccharide mixture comprising 2'FL (Fuc-al,2-Gal-pi,4-Glc), 3-FL (Gal-pi,4-[Fuc-al,3]-Glc) and DiFL (Fuc-al,2-Gal-pi,4-[Fuc-al,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.
  • 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 glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), 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.
  • a compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)),
  • 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 Q.9CLP3).
  • 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.
  • 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 glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), 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.
  • a first compatible yeast expression plasmid comprising constitutive transcriptional units for the lactose permease LAC12 from K. lactis (UniProt ID P07921), the glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), the phosphata
  • 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 Q.9JXQ.6) and the N- acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (Uniprot ID 0.51116, 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. 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.
  • 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).
  • 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.
  • the mutant strain is modified with a transcriptional unit encoding the alpha-l,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.
  • 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.
  • 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 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.
  • 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, Sequence version 03 (10 Feb 2009)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from H. sapiens (UniProt ID Q.96EK6), the phosphoacetylglucosamine mutase PGM3 from H. sapiens (UniProt ID 095394), the UDP-N-acetylhexosamine pyrophosphorylase UAP1 from H.
  • 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 3'SL.
  • 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.
  • 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.
  • An enzymatic synthesis reaction was set up comprising the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) produced in a CFPS reaction as described in Example 1, 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 the alpha-1, 2-fucosyltransferase HpFutC from H.
  • pylori (UniProt ID Q9X435) produced in a CFPS reaction as described in Example 1, 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-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc).
  • Another enzymatic synthesis reaction was set up comprising said alpha-1, 3-fucosyltransferase HpFucT from H.
  • Another enzymatic synthesis reaction was set up comprising said alpha-1, 2-fucosyltransferase HpFutC and said alpha-1, 3-fucosyltransferase HpFucT both produced in CFPS reactions as described in Example 1, 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 the alpha-2, 3-sialyltransferase PmultST3 from P.
  • multocida (UniProt ID Q9CLP3) 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, 3-sialyltransferase PmultST3 from P.
  • multocida (UniProt ID Q9CLP3) 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 the alpha-2,3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3) and the alpha-2, 6-sialyltransferase PdSt6 from P.
  • damselae 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 (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) and LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc).
  • Another enzymatic synthesis reaction was set up comprising the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) and the alpha-2, 3-sialyltransferase PmultST3 from P.
  • multocida (UniProt ID Q9CLP3) 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 the human ST6GalNAc5 (UniProt ID Q.9BVH7, purchased from Glyco Expression Technologies Inc), 10 mM CMP-Neu5Ac, 10 mM MgCI 2 and 25 mM LSTa as compatible substrate and a Tris buffer at pH 8.8 and incubated at 37°C for 24 hours to obtain DSLNT (disialyllacto-N- tetraose or Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-pi,4-Glc).
  • DSLNT disialyllacto-N- tetraose or Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-pi,4-Glc).
  • mutant E. coli BL21 strains are used to produce four recombinant proteins: an N- acylglucosamine 2-epimerase AGE 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 Q.7DDU0) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3).
  • N- acylglucosamine 2-epimerase AGE originating from B. ovatus
  • sialic acid aldolase NAL form Lactiplantibacillus plantarum WCFS1 UniProt ID P59407
  • 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.
  • Example 3 Composition determination of the fermentation or cultivation broth or of the solutions obtained after enzymatic synthesis reactions
  • Example 2 For the fermentation or cultivation broths obtained in Example 2 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. 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%.
  • Broth comprising 3'SL was produced via fermentation as described in Example 2.
  • Said 3'SL was produced in the salt form with the medium cations as counter ion, wherein potassium and ammonium ions were most dominant.
  • the biomass was removed by well-known methods in the art such as e.g., ultrafiltration, removing proteins, DNA and other macro molecules from the solution.
  • the resulting solution was directly passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H + form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na + form.
  • the solution was first treated in a nanofiltration step using, e.g., a Trisep XN45 membrane, which removes water, salts and carbohydrates, after which the retentate is passed over the cation exchange columns as described above.
  • a part of the solution was further treated in an activated charcoal step.
  • the pH of the effluent of the second cation exchange column was 2.3 which was corrected by addition of NaOH to pH 6.5.
  • the cationic counter ions were all exchanged for Na + ions after the second cation exchange step.
  • the solution was further concentrated or was directly spray dried to form a white stable powder which was analyzed according to the methods in Example 1.
  • the ash content in the powder was between 5 and 8 % and the Na + concentration was between 3.5 and 5%.
  • Broth comprising 2'FL was produced via fermentation as described in Example 2.
  • the biomass was removed by well-known methods in the art, such as e.g., microfiltration and ultrafiltration, removing proteins, DNA and other macro molecules from the solution.
  • the resulting solution was directly passed over a cation exchange column containing Amberlite FPC88 H (Dupont, USA) in H + form followed by a cation exchange column containing Amberlite FPC88 H (Dupont, USA) in Na + form.
  • a nanofiltration step using, e.g., a Trisep XN45 membrane, which removes water, salts and carbohydrates, after which the retentate is passed over the cation exchange columns as described above.
  • Broth comprising 6'SL was produced via fermentation as described in Example 2.
  • Said 6'SL was produced in the salt form with the medium cations as counter ion, wherein potassium and ammonium ions were most dominant.
  • the biomass was removed by well-known methods in the art such as e.g., ultrafiltration, removing proteins, DNA and other macro molecules from the solution.
  • the resulting solution was directly passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H + form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na + form.
  • AC activated charcoal
  • Broth comprising 3'-sialyl-3-fucosyllactose is produced via fermentation as described in Example 2.
  • the biomass is removed by microfiltration.
  • the filtrate is then further purified by ultrafiltration removing proteins and other macro molecules.
  • the resulting filtrate is treated with nanofiltration to remove water, salts and unwanted carbohydrates.
  • the nanofiltration retentate is then further treated with activated charcoal (AC) (Chemviron, France) to remove color components.
  • AC activated charcoal
  • the syrup is passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H + form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na + form.
  • the pH of the effluent is ranging between 1.8 and 3.0, which is corrected by addition of NaOH to pH 6.5.
  • traces of heavy metals are mostly removed resulting in a lead content lower than 0.02 mg/kg dry solid, an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid and a mercury content lower than 0.1 mg/kg dry solid.
  • the resulting syrup is then spray-dried to form a white stable powder.
  • Example 9 Processing a solution comprising DSLNT
  • the heavy metals present comprise a lead content lower than 0.02 mg/kg dry solid, an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid and a mercury content lower than 0.1 mg/kg dry solid.
  • the resulting solution is then spray-dried to form a white stable powder.

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Abstract

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

Description

Purification of an oligosaccharide or oligosaccharide mixture
Field of the invention
The present invention relates to processes for the purification of an oligosaccharide or oligosaccharide mixture from a solution, a product of such processes, and the use of a product of such processes.
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 and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response. 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, an N-acetylglucosamine or an N-acetylneuraminic acid residue at their nonreducing end. 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 anti-inflammatory 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 needs to be purified.
Description
Summary of the invention
It is an object of the present invention to provide for methods by means of which an oligosaccharide or an oligosaccharide mixture 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 or oligosaccharide mixture. Preferably, the desired oligosaccharide or oligosaccharide mixture obtained after purification has an ash content of < 10% on total dry solid, with a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
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 passing the solution comprising the desired oligosaccharide through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially in acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4+ form. Preferably, said second cationic ion exchange resin is in Na+ form. The solution comprising the oligosaccharide 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 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 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 or the oligosaccharide mixture of present invention 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; Neu4, 5,7,8, 9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
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.
The term "phosphorylated monosaccharide" as used herein refers to a monosaccharide which is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate.
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, UDP-xylose and CMP-KDO. Nucleotide-sugars 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.org). 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. Examples of disaccharides comprise lactose (Gal-pi,4-Glc), lactulose (Gal-pi,4-Fru), allolactose (Gal-pi,6-Glc), lacto-N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-oc2,6-Gal, Fuc-al,2-Gal, fucopyranosyl-(l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-al,2-Fru), maltose (Glc-al,4-Glc) and melibiose (Gal-al,6-Glc).
"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. 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->5, alpha 2->l, 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 (ECA), 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 (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide, N-acetylglucosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, N-acetyllactosamine-containing oligosaccharides, N-acetylglucosamine containing sialylated oligosaccharides, N-acetylglucosamine containing neutral (non-charged) oligosaccharides, N- acetylglucosamine containing negatively charged oligosaccharides, N-acetylglucosamine containing fucosylated oligosaccharides, N-acetylglucosamine containing non-fucosylated oligosaccharides, lacto-N- biose containing sialylated oligosaccharides, lacto-N-biose containing neutral (non-charged) oligosaccharides, lacto-N-biose containing negatively charged oligosaccharides, lacto-N-biose containing fucosylated oligosaccharides, lacto-N-biose containing non-fucosylated oligosaccharides, N- acetyllactosamine containing sialylated oligosaccharides, N-acetyllactosamine containing neutral (noncharged) oligosaccharides, N-acetyllactosamine containing negatively charged oligosaccharides, N- acetyllactosamine containing fucosylated oligosaccharides, N-acetyllactosamine containing non- fucosylated oligosaccharides, chitosan, chitosan comprising oligosaccharide, heparosan, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, dermatan sulphate, hyaluronan, hyaluronic acid, keratan sulphate, erlose (Glc-al,4-Glc-al,2-Fru), lactul-N-triose II (GlcNAc-pi,3-Gal-pi,4- Fru), lactul-N-tetraose, lactul-N-neotetraose and globotriose.
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, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal-pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-a2,8-Gal-pi,4-Glc), 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-cc2,8- Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, 3'-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 (Neu5Ac-oc2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), LSTd (Neu5Ac-cc2,3- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), 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, Neu5Ac-oc2,3-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal-pi,3-(Neu5Ac- oc2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), 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, 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,3GlcNacP-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'- fucosyl lactose, 6'-Sialyl-3-fucosyl lactose, 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 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, GMl Gaip-l,3GalNAc -l,4(Neu5Aca-2,3)Gaip- l,4Glc, GDla Neu5Aca-2,3Gai -l,3GalNAc -l,4(Neu5Aca-2,3)Gai -l,4Glc, GTla Neu5Aca-2,8Neu5Aca- 2,3Gai -l,3GalNAc -l,4(Neu5Aca-2,3)Gaip-l,4Glc, GD2 GalNAc -l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip- l,4Glc, GT2 GalNAcP-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,3GalNAc -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 -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.
"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, galacturonate and 2-keto-3-deoxymanno-octulonic acid (KDO). 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 (non-charged) 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-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAc i-3Gaipi-4Glc), lacto-N-tetraose (LNT, Gaipi- 3GlcNAc i-3Gaipi-4Glc), lacto-N-neotetraose (LNnT, Gaipi-4GlcNAcpi-3Gaipi-4Glc), 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, 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), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, 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, GIcNAc- bl,5-(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, GIcNAc- 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 '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), 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-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 (a), Difucosyllacto-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, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'-fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-sialyl-3- fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.
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 (Eguus ferus caballus), pigs (Sus scropha), dogs (Canis 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, 5-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 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, lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-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. HMDs 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.org) (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 "non- mammary 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 that is produced by the cell in whole broth, i.e., inside (intracellularly) as well as outside (extracellularly) of the cell.
The term "biomass" as used herein refers to the suspended, precipitated or insoluble materials originating from fermentation cells, like intact cells, disrupted cells, cell fragments, cell walls, phospholipids, cell membranes, proteins, protein fragments, polysaccharides, polynucleotides and other large organic compounds produced by the cell. The biomass may be in suspension and/or in solution. Biomass as used herein is also to be understood to be non-complex biomass, typically of low or no organisation of cells into peculiar or complex structures. Non-limiting examples are individual cells, cell pairs, cell lumps, oligocellular or multicellular structures, cell layers, biofilms. The non-complex biomass may be of a three- dimensional structure, like e.g. cells forming a layer or a biofilm or attached to a surface of a reactor or incubator. As used herein, biomass is also to be understood to be complex biomass, wherein said complex biomass is of a complex structure by nature, often a complex three-dimensional structure and comprises many hundreds, thousands, ten-thousands, but more typically hundreds of thousands or millions or more of cellular structures in a complex organisation, often of various cell types with different specialisations. Non-limiting examples are higher plants or animals with a body visible with the naked eye, organs and tissues, including bone and meat or plant parts like fruit, vegetables, straw, sugarcane bagasse, hay, wood, timber. Complex biomass may be the source of non-complex biomass, for example cell lines are typically derived from a tissue or organ but do not maintain the complex structure in cultivation.
The terms "cell dry weight" or "CDW" as used herein refer to the grams of dry weight of biomass per liter of sample after removal of moisture. The procedure for measuring the CDW is disclosed in the working examples below.
The terms "biocatalysis reaction solution" and "enzymatic synthesis reaction" are used interchangeably and refer to a mixture wherein an oligosaccharide is 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, 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 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 is 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 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.
In some embodiments, the purity of an oligosaccharide in a solution is measured on total dry solid. Herein, the total dry solid comprises particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids, lipids, endotoxins, salts, vitamins, heavy metals, and carbohydrates.
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 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 the biochemical synthesis route of an oligosaccharide. 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 the metabolic pathway of an oligosaccharide. 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 the metabolic pathway of an oligosaccharide. 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-l-phosphate, galactose-l-phosphate, glucose-6- phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-1- 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 is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the list 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 ash content is a measure of the total amount of minerals and heavy metals present within a food or ingredients such as oligosaccharides, wherein the mineral content is a measure of the amount of specific inorganic components present within a food, such as e.g., Ca2+, Na+, K+, Mg2+, phosphate, sulphate and Cl' and wherein the heavy metal content is a measure of the amount of inorganic components present within a food like e.g. lead, arsenic, mercury, cadmium, zinc, manganese, copper and iron. 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 and heavy metals within a food. Analytical techniques for providing information about the total mineral and heavy metal content are based on the fact that the analyte like minerals and heavy metals 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 and heavy metals 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 and heavy metals, 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 g 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 and heavy metals 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 CO2, H2O and N2. Most minerals and heavy metals are converted to oxides, sulphates, phosphates, chlorides or silicates. Although most minerals and heavy metals have fairly low volatility at these high temperatures, some are volatile and may be partially lost, e.g., iron, lead and mercury, for these components 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 the process as described herein and wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation. More specifically, 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 a fermentation as described herein and wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation or said fermentation. The process comprises passing a solution comprising said oligosaccharide through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NHZ form. In a preferred embodiment, the cationic ion exchange resin in the second cationic ion exchange is in Na+ form. Herein, the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H+ form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH form. Alternatively, the process further comprises any one or more of the methods selected from the list consisting of homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any one or more of said method(s) succeed(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and precede(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form.
The present invention concerns a process for the purification of an oligosaccharide that is provided in a solution comprising said oligosaccharide.
In a preferred embodiment, the oligosaccharide is selected from the list comprising, consisting of or essentially consisting of fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan 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, 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 and globotriose.
In a more preferred embodiment, the oligosaccharide is a fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (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 GIcNAc- bl,3-Gal-bl,4-(Fuc-al,3-)Glc. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-cc2,6)-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, sialyl lacto-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,3GlcNacP-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-a2,6-(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 any combination thereof. In another more preferred embodiment, the oligosaccharide is an N-acetylglucosamine containing neutral (noncharged) oligosaccharide 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 another more preferred embodiment, the oligosaccharide is a milk oligosaccharide. In an even more preferred embodiment, the oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another even more preferred embodiment, the oligosaccharide is a human milk oligosaccharide (HMO) as described herein. In another more preferred embodiment, the oligosaccharide is an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans. In another more preferred embodiment, the oligosaccharide is a plant oligosaccharide 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 another more preferred embodiment, the 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 oligosaccharide is a sialylated oligosaccharide having one Neu5Ac (neuraminic acid) group. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two sialic acid groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two identical sialic acid groups. In an even more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having two Neu5Ac groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having three or more sialic acid groups.
In another more preferred embodiment, the oligosaccharide is a sialylated milk oligosaccharide. In an even more preferred embodiment, the oligosaccharide is a sialylated mammalian milk oligosaccharide (MMO). In a most preferred embodiment, the oligosaccharide is 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 combinations thereof.
In another more preferred embodiment, the oligosaccharide is a fucosylated oligosaccharide having at least one fucose group. In a more preferred embodiment, the oligosaccharide is fucosylated oligosaccharide having two fucose groups. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide having three or more fucose groups.
In another more preferred embodiment, the oligosaccharide is a fucosylated milk oligosaccharide. In an even more preferred embodiment, the oligosaccharide is a fucosylated mammalian milk oligosaccharide (MMO). In a most preferred embodiment, the oligosaccharide is a fucosylated human milk oligosaccharide (HMO), selected from the list comprising 2'-fucosyllactose (2' FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyl lactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I (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-I I), difucosyl-lacto-N-hexaose and difucosyl- lacto-N-neohexaose.
In another more preferred embodiment, the oligosaccharide is selected from the list comprising, consisting of or essentially consisting of 2'-fucosyllactose (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, GlcNAc-bl,3- Gal-bl,4-(Fuc-al,3-)Glc, 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-2,3Gaip-l,3GalNac -l,3Gala-l,4Gai -l,4Gal, KDO-2,3Gaip-l,3GlcNac - l,3Gaip-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, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), 5'-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 another preferred embodiment, the oligosaccharide is an oligosaccharide with a degree of polymerization (DP) of at least 3. In another preferred embodiment, the oligosaccharide is an oligosaccharide with a DP selected 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 is an oligosaccharide with a DP selected from the list comprising 3, 4, 5, 6, 7, 8, 9 and 10. In another preferred embodiment, the oligosaccharide is a trisaccharide. In another preferred embodiment, the oligosaccharide is a tetrasaccharide. In another preferred embodiment, the oligosaccharide is a pentasaccharide. In another preferred embodiment, the oligosaccharide is a hexasaccharide. In another preferred embodiment, the oligosaccharide is a heptasaccharide.
In another preferred embodiment, the oligosaccharide is a linear oligosaccharide. In an alternative preferred embodiment, the oligosaccharide is a branched oligosaccharide. The oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide does not contain any protective group.
In another preferred embodiment, the oligosaccharide is an oligosaccharide that comprises one or more sialic acid groups and one or more monosaccharide building blocks selected 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 is a sialylated oligosaccharide that is also fucosylated. In another more preferred embodiment, the oligosaccharide is a sialylated oligosaccharide that is not fucosylated. In another more preferred embodiment, the oligosaccharide is an oligosaccharide comprising one or more fucose groups and one or more sialic acid groups selected from the group 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 KDO.
Within the context of present invention, the solution comprising an 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 the process as described herein. More specifically, the solution comprising an 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 a fermentation as described herein. Within the context of the present invention, said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution, or by a cell cultivated in said cell cultivation or by said fermentation. 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, 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.
In another and/or additional preferred embodiment, the oligosaccharide is accompanied in said solution by sialic acid as defined herein; ashes, preferably, said ashes comprise sulphates, phosphates, sodium, chloride, potassium, and heavy metals; preferably said heavy metals comprise zinc, manganese, copper, ammonium, lead arsenic, cadmium and/or 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-pi,4-Glc), lacto-N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,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 (ECA); 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 2'-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 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), 5'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(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-2,3Gaip-l,3GalNac -l,3Gala-l,4Gaip-l,4Gal, KDO- 2,3Gai -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-a2,6-(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 any combination thereof; N- acetylglucosamine containing 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-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; 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 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. In a more preferred embodiment, the oligosaccharide is accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have the same DP as said oligosaccharide. In an alternative preferred embodiment, the oligosaccharide 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. In a more preferred alternative embodiment, the oligosaccharide is accompanied in said solution by one or more other oligosaccharide(s) wherein all of said other oligosaccharides have a different DP as said oligosaccharide.
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 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 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 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 another preferred embodiment, the solution comprises one or more neutral (non-charged) fucosylated oligosaccharide(s) and one or more charged oligosaccharide(s). In another preferred embodiment, the solution comprises one or more neutral (non-charged) fucosylated oligosaccharide(s) and one or more sialylated oligosaccharide(s).
In another and/or additional preferred embodiment of the process of present invention, the ash content of the solution comprising an oligosaccharide to be purified by a process of present invention is > 10 % on total dry solid before purification by said process. The ash content of a solution can be determined as described herein.
In another and/or additional preferred embodiment of the process of present invention, the solution comprising an oligosaccharide to be purified by a process of present invention 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.
In another and/or additional preferred embodiment of the process of present invention, the solution comprising an oligosaccharide to be purified by a process of present invention 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.
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, additives and contaminants like e.g., antifoam. 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 more preferred embodiment, the biomass, when present, that is separated during the process, is optionally recycled to the cell cultivation.
In a preferred embodiment, the oligosaccharide to be purified by a process of present invention is 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.
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. In an even more preferred embodiment, the cell has been metabolically engineered to produce two or more oligosaccharides.
In another and/or additional preferred embodiment, the cell produces an oligosaccharide 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 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 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. In an even more preferred embodiment, the cell is metabolically engineered to comprise a sialy lation 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 selected 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-l-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9- phosphatase, d-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate 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. 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 selected 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. 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 selected 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. 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 selected from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-1- 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. 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 selected 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. 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 selected 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. 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 selected 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-1- 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, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of an oligosaccharide, a better efflux of the oligosaccharide, 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 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. In another preferred embodiment, the precursor(s) that is/are used by the cell for the production of said oligosaccharide is/are completely converted into said oligosaccharide. In another preferred embodiment, the precursor(s) that is/are used in said solution for the production of said oligosaccharide is/are completely converted into said oligosaccharide.
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 cell is cultivated in a chemically defined medium. In another and/or additional preferred embodiment, the cell is cultivated in 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 to 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 to 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 to 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 said invention, said solution is passed through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, and passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form. In a preferred embodiment, the cationic ion exchange resin in the second cationic ion exchange is in Na+ form.
In another specific embodiment, said first cationic ion exchange comprises a cationic ion exchange resin in H+ form. In a preferred embodiment, said cationic ion exchange resin in said first cationic ion exchange is provided in H+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said first cationic ion exchange is not provided in H+ form but in another form, like e.g., Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form by a supplier and is regenerated upon use in 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 first cationic 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 specific embodiment, said second cationic ion exchange comprises a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form.
In a preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Na+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Na+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Na+ form but in another form, like e.g., H+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 +form 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 second cationic ion exchange is in K+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in K+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in K+ form but in another form, like e.g., Na+, H+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form by a supplier and is regenerated upon use in said process into K+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Ca2+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Ca2+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Ca2+ form but in another form, like e.g., Na+, H+, K+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form by a supplier and is regenerated upon use in said process into Ca2+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Mg2+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Mg2+form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Mg2+ form but in another form, like e.g., Na+, H+, K+, Ca2+, Fe2+, Fe3+, Al3+ or NH4 + form by a supplier and is regenerated upon use in said process into Mg2+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Fe2+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Fe2+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Fe2+ form but in another form, like e.g., Na+, H+, K+, Ca2+, Mg2+, Fe3+, Al3+ or NH4 + form by a supplier and is regenerated upon use in said process into Fe2+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Fe3+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Fe3+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Fe3+ form but in another form, like e.g., Na+, H+, K+, Ca2+, Mg2+, Fe2+, Al3+ or NH4 + form by a supplier and is regenerated upon use in said process into Fe3+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in Al3+ form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in Al3+ form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in Al3+ form but in another form, like e.g., Na+, H+, K+, Ca2+, Mg2+, Fe2+, Fe3+, or NH4 + form by a supplier and is regenerated upon use in said process into Al3+ form by method(s) known by the person skilled in the art.
In another preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is in NH + form. In a more preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is provided in NH4 + form by a supplier. In an alternative preferred embodiment, said cationic ion exchange resin in said second cationic ion exchange is not provided in NH4 + form but in another form, like e.g., Na+, H+, K+, Ca2+, Mg2+, Fe2+, Fe3+, or Al3+ form by a supplier and is regenerated upon use in said process into NH4 + form by method(s) known by the person skilled in the art.
In an alternative and/or additional preferred embodiment, said second cationic 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 cationic ion exchange resin present in said first cationic 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 cationic ion exchange resin present in said second cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin. The terms "WAC" and "SAC" 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 alkaline to about neutral pH in the case of a WAC.
In another and/or additional preferred embodiment, the first cationic ion exchange comprises a WAC exchange resin in H+ form and the second cationic ion exchange comprises a WAC exchange resin in a Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form.
In another and/or additional preferred embodiment, the first cationic ion exchange comprises a SAC exchange resin in H+ form and the second cationic ion exchange comprises a WAC exchange resin in a Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form.
In another and/or additional preferred embodiment, the first cationic ion exchange comprises a WAC exchange resin in H+ form and the second cationic ion exchange comprises a SAC exchange resin in a Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form.
In another and/or additional preferred embodiment, the first cationic ion exchange comprises a SAC exchange resin in H+ form and the second cationic ion exchange comprises a SAC exchange resin in a Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form.
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 first cationic 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 second cationic 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 first cationic 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 cationic ion exchange resin present in said second cationic 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 matrix used in said cationic ion exchange resin in said first and/or second cationic 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 first and/or second cationic ion exchange.
Examples of cationic ion exchange resins that can be used in said first and/or second cationic ion exchange comprise Amberlite FPC22 H (Dupont), Amberlite FPC88 H (Dupont), Amberlite FPC23 UPS (Dupont), Amberlite FPC11 Na (Dupont), Amberlite FPC23 UPS H (Dupont), Diaion PK208 (Mitsubishi chemical), Diaion PK212 (Mitsubishi chemical), Diaion PK216 (Mitsubishi chemical), Diaion PK220 (Mitsubishi chemical), Diaion PK228 (Mitsubishi chemical), Lewatit K 2431 (Lanxess), Lewatit K 2620 (Lanxess), Lewatit MDS 1368 Ca 320 (Lanxess), Lewatit C 249 NS (Lanxess), Dowex 88 (Dow chemical) and Dowex monosphere C-600 B (Dow chemical). If not already present in H+ form, the cationic exchange resin in said first cationic ion exchange 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 Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, the cationic ion exchange resin in said second cationic 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+, Fe2+, Fe3+, Al3+ or NH4 + form, respectively.
In another and/or additional preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 0.5 bed volume / hour (BV/h). In a more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 1 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 1.5 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 2 BV/h. In an even more preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 2.5 BV/h. In a most preferred embodiment, the flow rate through said first and/or second cationic ion exchange is at least 3 BV/h.
In another and/or additional preferred embodiment, said first and/or second cationic ion exchange performed on said solution in the process 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange is/are 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange 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, said first and/or second cationic ion exchange is/are performed at room temperature. In another more preferred embodiment, said first and/or second cationic ion exchange is/are 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, 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 and 80°C.
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. Within the context of present invention, performing said first cationic ion exchange step at a specific temperature is to be understood as that the temperature of the cationic ion exchanger used in said first cationic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said first cationic ion exchange is adjusted to said specific temperature. Further within the context of present invention, performing said second cationic ion exchange step at a specific temperature is to be understood as that the temperature of the cationic ion exchanger used in said second cationic ion exchange step is adjusted to said specific temperature and/or the temperature of the solution that is added as influent to said second cationic ion exchange is adjusted to said specific temperature.
In another and/or additional preferred embodiment, said first and/or second cationic ion exchange performed on said solution in the process is/are performed at a pH ranging from 2 to 5, including 2 and 5 in the range. In a more preferred embodiment, said first and/or second cationic ion exchange is/are performed at a pH ranging from 3 to 5, including 3 and 5 in the range. In an even more preferred embodiment, said first and/or second cationic ion exchange is/are performed at a pH ranging from 4 to 5, including 4 and 5 in the range. In another more preferred embodiment, said first and/or second cationic ion exchange is/are performed at a pH selected from the list comprising about 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 and 5.
In another and/or additional preferred embodiment, the process further comprises any one or more of the methods selected from the list comprising, consisting of or essentially consisting of 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, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying and wherein any one or more of said method(s) (a) precede(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and/or (b) any one or more of said method(s) succeed(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably in Na+ form. It is to be understood herein that no one of said method(s) is performed succeeding said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and preceding said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably in Na+ form. In other words, the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H+ form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 +form, preferably in Na+ form. In a more preferred embodiment, any or more of said methods is performed more than one time during said process preceding said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and/or succeeding said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form, preferably in Na+ form.
In another and/or additional preferred embodiment, the process further comprises any one or more of the methods selected from the list comprising, consisting of or essentially consisting of homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any one or more of said method(s) succeed(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and precede(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form, preferably in Na+ form.
Herein, pH adjustment ranging from 2 to 5 is to be understood as an adjustment of the pH of the solution to a pH ranging from 2 to 5 including 2 and 5 in the range before said solution is passed through said second cationic ion exchange as described herein. Preferably, the pH of the solution is adjusted to a pH selected from the list comprising about 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 and 5 before said solution is passed through said second cationic ion exchange as described herein. Use of solvents with a pH ranging from 2 to 5 is to be understood as use of solvents that have a pH selected from the list comprising about 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 and 5. Drying and dissolving in a liquid with a pH ranging from 2 to 5 is to be understood that said solution is dried and dissolved in a liquid with a pH selected from the list comprising about 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 and 5. In a more preferred embodiment, any or more of said methods is performed more than one time during said process succeeding said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and preceding said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably in Na+ form.
In another more preferred embodiment, said process comprises i) passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, immediately followed by ii) passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form and iii) further purification of said solution to obtain purified oligosaccharide.
In another more preferred embodiment, said process comprises i) any one or more of ultrafiltration, microfiltration and/or nanofiltration of said solution comprising said oligosaccharide, ii) passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, iii) passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form and iv) further purification of said solution to obtain purified oligosaccharide.
In another more preferred embodiment, said process comprises i) passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, ii) any one or more of filtration, pH adjustment of the solution to a pH selected from the list comprising about 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 and 5 and/or temperature adjustment before iii) passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form and iv) further purification of said solution to obtain purified oligosaccharide.
In another more preferred embodiment, said process comprises i) passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, ii) any one or more of filtration, pH adjustment of the solution to a pH selected from the list comprising about 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 and 5, concentration, dilution and/or temperature adjustment, before iii) passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form and iv) further purification of said solution to obtain purified oligosaccharide. 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 0°C to 122°C should be understood as a temperature of 0°C, 1°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, 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, 25°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 said first cationic ion exchange comprising a cationic ion exchange resin in H+ form. 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 does not further comprise electrodialysis. In another and/or additional preferred embodiment, the process does not further comprise electrodeionization. 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 selected from the list comprising H+, Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+, NHZ and wherein said anionic ion exchange resin is in any form selected from the list comprising OH-, Cl' and SO3 2-. In another and/or additional preferred embodiment, the process comprises two mixed bed ion exchanges. In another and/or additional preferred embodiment, the process comprises two consecutive steps of ultrafiltration and wherein the membrane molecular weight cut-off of the membrane used in the first ultrafiltration step is higher than the membrane molecular weight cutoff of the membrane used in the second ultrafiltration step. In a more preferred embodiment, said two consecutive steps of ultrafiltration are performed on said solution comprising said oligosaccharide prior to said first cationic ion exchange comprising a cationic ion exchange resin in H+ form.
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 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.
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 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 on said solution 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 list 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 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 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: passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ 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: passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids and passing said solution comprising said salts fully or partially converted into acids through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH4 + form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form.
In another and/or additional preferred embodiment of the present invention, the purity of the oligosaccharide 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 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, 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 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.
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 a lead content lower than 0.01 mg/kg dry solid. Preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a lead content lower than 0.05 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
< 10% on total dry solid with a lead content 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 an arsenic content lower than 0.2 mg/kg dry solid, more preferably lower than 0.1 mg/kg dry solid, even more preferably lower than 0.05 mg/kg dry solid, even more preferably lower than 0.02 mg/kg dry solid. Preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with an arsenic content lower than 0.1 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
< 10% on total dry solid with an arsenic content 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. Preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a cadmium content lower than 0.05 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a cadmium content lower than 0.02 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
< 10% on total dry solid with a cadmium content 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 dry solid, even more preferably lower than 0.005 mg/kg dry solid. Preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a mercury content lower than 0.2 mg/kg dry solid. More preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of
< 10% on total dry solid with a mercury content lower than 0.1 mg/kg dry solid. Even more preferably, the purified oligosaccharide solution obtained at the end of the process has an ash content of < 10% on total dry solid with a mercury content 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 embodiment, the purified oligosaccharide solution obtained at the end of the method has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 60 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid. In another and/or additional more preferred embodiment, the purified oligosaccharide solution obtained at the end of the method has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA. In another and/or additional more preferred embodiment, the purified oligosaccharide solution obtained at the end of the method has an endotoxin content equal to or below 8000 EU per gram dry solid, equal to or below 5000 EU per gram dry solid, equal to or below 3000 EU per gram dry solid, equal to or below 1000 EU per gram dry solid, equal to or below 800 EU per gram dry solid, equal to or below 500 EU per gram dry solid, equal to or below 300 EU per gram dry solid or equal to or below 100 EU per gram dry solid. In an even more preferred embodiment, the purified oligosaccharide solution obtained at the end of the process is free of DNA, proteins, and/or recombinant genetic material. In another more preferred embodiment, the purified oligosaccharide solution obtained at the end of the method is free of recombinant DNA and/or proteins derived from the recombinant micro-organism.
In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the method has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters. In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the method has less than 0.5 pg/kg epichlorohydrin.
In another and/or additional preferred embodiment, the purified oligosaccharide solution obtained at the end of the method has a conductivity of less than 10 mS/cm at a 300 g/L solution.
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, vacuum 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, vacuum evaporation, wiped film evaporation, and falling film evaporation, wherein one or more of said nanofiltration, diafiltration, reverse osmosis, evaporation, vacuum 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 selected 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 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. A temperature < 25°C is to be understood as 0°C, l’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 O’C, l’C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C or a temperature below O’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 7. 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, 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 or 7. In an additional even more preferred embodiment, the pH of the purified oligosaccharide solution is ranging from 3 to 6; 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,
5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6. 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 spray-drying, 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 a second aspect, the present invention provides a purified oligosaccharide solution, a purified oligosaccharide or a purified oligosaccharide mixture comprising an oligosaccharide obtainable, preferably obtained, by a process as described herein. The purified oligosaccharide solution can comprise one purified oligosaccharide or a purified oligosaccharide mixture.
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 and/or additional preferred embodiment, the present invention provides an oligosaccharide 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. 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 or less than 0.1% ash on total dry solid.
Preferably, said oligosaccharide is produced through cell cultivation.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that 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, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a lead content < 0.1 mg/kg dry solid. In a more preferred embodiment, said purified oligosaccharide has a lead content < 0.02 mg/kg dry solid. In an even more preferred embodiment, said purified oligosaccharide has a lead content < 0.01 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has an arsenic content < 0.2 mg/kg dry solid. In a more preferred embodiment, said purified oligosaccharide has an arsenic content < 0.05 mg/kg dry solid. In an even more preferred embodiment, said purified oligosaccharide has an arsenic content < 0.02 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a cadmium content < 0.1 mg/kg dry solid. In a more preferred embodiment, said purified oligosaccharide has a cadmium content < 0.01 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a mercury content < 0.5 mg/kg dry solid. In a more preferred embodiment, said purified oligosaccharide has a mercury content < 0.1 mg/kg dry solid. In an even more preferred embodiment, said purified oligosaccharide has a mercury content < 0.005 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has 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 embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid. In another and/or additional more preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA. In another and/or additional more preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has an endotoxin content equal to or below 8000 EU per gram dry solid, equal to or below 5000 EU per gram dry solid, equal to or below 3000 EU per gram dry solid, equal to or below 1000 EU per gram dry solid, equal to or below 800 EU per gram dry solid, equal to or below 500 EU per gram dry solid, equal to or below 300 EU per gram dry solid or equal to or below 100 EU per gram dry solid. In an even more preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant micro-organism and/or recombinant genetic material.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3-monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has less than 0.5 pg/kg epichlorohydrin.
In another and/or additional preferred embodiment, the present invention provides an oligosaccharide that is purified according to a process as described herein and that has a conductivity of less than 10 mS/cm at a 300 g/L solution.
According to a third aspect, the present invention provides a spray-dried oligosaccharide or a spray-dried oligosaccharide mixture comprising an oligosaccharide, wherein said oligosaccharide or oligosaccharide mixture 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. 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. Preferably, said oligosaccharide or said oligosaccharide mixture is produced through cell cultivation. 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 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, 4.1, 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 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, 4.1, 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 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, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 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, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6. In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of < 10%, < 9%, < 8%, < 7%, < 6%, < 5%, < 4%, < 3%, < 2%, < 1% or < 0.5% on total dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder 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 present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of < 10% on total dry solid with a lead content lower than 0.1 mg/kg dry solid, more preferably with a lead content lower than 0.05 mg/kg dry solid, even more preferably with a lead content lower than 0.02 mg/kg dry solid, most preferably with a lead content lower than 0.01 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of < 10% on total dry solid with an arsenic content lower than 0.2 mg/kg dry solid, more preferably with an arsenic content lower than 0.1 mg/kg dry solid, even more preferably with an arsenic content lower than 0.05 mg/kg dry solid, most preferably with an arsenic content lower than 0.02 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of < 10% on total dry solid with a cadmium content lower than 0.1 mg/kg dry solid, more preferably with a cadmium content lower than 0.05 mg/kg dry solid, even more preferably with a cadmium content lower than 0.02 mg/kg dry solid, most preferably with a cadmium content below 0.01 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an ash content of < 10% on total dry solid with a mercury content lower than 0.5 mg/kg dry solid, more preferably lower than 0.2 mg/kg dry solid, even more preferably lower than 0.1 mg/kg dry solid, most preferably lower than 0.005 mg/kg dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder 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 embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has a protein content equal to or below 99 mg per kg dry solid, equal to or below 95 mg per kg dry solid, equal to or below 90 mg per kg dry solid, equal to or below 80 mg per kg dry solid, equal to or below 70 mg per kg dry solid, equal to or below 60 mg per kg dry solid, equal to or below 50 mg per kg dry solid, equal to or below 40 mg per kg dry solid, equal to or below 30 mg per kg dry solid, equal to or below 20 mg per kg dry solid or equal to or below 10 mg per kg dry solid. In another and/or additional more preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has a DNA content equal to or below 9 ng per gram dry solid, equal to or below 8 ng per gram dry solid, equal to or below 7 ng per gram dry solid, equal to or below 6 ng per gram dry solid, equal to or below 5 ng per gram dry solid, equal to or below 4 ng per gram dry solid, equal to or below 3 ng per gram dry solid, equal to or below 2 ng per gram dry solid, equal to or below 1 ng per gram dry solid, or no DNA. In another and/or additional more preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has an endotoxin content equal to or below 8000 EU per gram dry solid, equal to or below 5000 EU per gram dry solid, equal to or below 3000 EU per gram dry solid, equal to or below 1000 EU per gram dry solid, equal to or below 800 EU per gram dry solid, equal to or below 500 EU per gram dry solid, equal to or below 300 EU per gram dry solid or equal to or below 100 EU per gram dry solid.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism and/or recombinant genetic material.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters.
In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder has less than 0.5 ptg/kg epichlorohydrin. In another and/or additional preferred embodiment, the present invention provides dried powder of a purified oligosaccharide or of a purified oligosaccharide mixture comprising an oligosaccharide wherein said powder is a spray-dried powder.
In another and/or additional preferred embodiment, the present invention provides for a purified oligosaccharide as described herein wherein any one or more of said purified oligosaccharide is a milk oligosaccharide. In another embodiment, the present invention provides for a purified oligosaccharide mixture comprising an oligosaccharide 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 (noncharged) milk oligosaccharide, preferably a neutral (non-charged) human milk oligosaccharide (HMO), selected from the list comprising 2'-fucosyllactose, 3-fucosyllactose, 4-fucosyllactose (4FL), 6- fucosyllactose (6FL), 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-fucopentaose VI, lacto-N-difucohexaose I, lacto-N- difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, 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 preferred embodiment, the present invention provides for a purified oligosaccharide or purified oligosaccharide mixture as described herein, 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.
According to another aspect, the present invention provides for a purified oligosaccharide comprising 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 a preferred embodiment, the purified oligosaccharide comprises an ash content of < 10% on total dry solid wherein said ash content of < 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
In another and/or additional preferred embodiment, the purified oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan 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, 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 and globotriose. In a more preferred embodiment, the fucosylated oligosaccharide is selected from the list comprising 2'- fucosyllactose (2'FL), 3-fucosyl lactose (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-fucosyl lactulose, 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 more preferred embodiment, the sialylated 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-a2,6)- Glc), 8,3-disialyllactose (Neu5Ac-cc2,8-Neu5Ac-cc2,3-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-2,3Gaip-l,3GalNac -l,3Gala-l,4Gaip-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-a2,6-(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 any combination thereof In another and/or additional more preferred embodiment, 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, 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 another and/or additional more preferred embodiment, the milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO). In another and/or additional more preferred embodiment, the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans. In another and/or additional more preferred embodiment, the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
For identification of said oligosaccharide 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 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 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 (gasliquid 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 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 or purified oligosaccharide mixture 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 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 is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. Said purified oligosaccharide 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 B. 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 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 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 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 (HMDs). 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 in the infant formula is approximately the same concentration as the concentration of the oligosaccharide generally present in human breast milk. In some embodiments, an oligosaccharide 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.
In another aspect, the present invention provides for a saccharide composition comprising an ash content of < 10% on total dry solid and an oligosaccharide content of > 90 % 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. Preferably, said ash content of < 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid. In a more preferred embodiment, the saccharide composition comprises an oligosaccharide content of > 90 % on total dry solid and a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid.
In another and/or additional preferred embodiment, the saccharide composition comprises more than one oligosaccharide.
In another and/or additional preferred embodiment, the oligosaccharide(s) present in the saccharide composition is/are selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N- acetylglucosamine containing oligosaccharide, 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, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan 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, 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 and globotriose. In a more preferred embodiment, the fucosylated oligosaccharide is selected from the list comprising 2'-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 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 GIcNAc- bl,3-Gal-bl,4-(Fuc-al,3-)Glc. In another and/or additional more preferred embodiment, the sialylated oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(Neu5Ac-oc2,6)-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-2,3Gaip-l,3GalNacP-l,3Gala-l,4Gaip-l,4Gal, KDO- 2,3Gaip-l,3GlcNacP-l,3Gaip-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-a2,5-(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-a2,5-(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 any combination thereof In another and/or additional more 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 another and/or additional more preferred embodiment, the milk oligosaccharide is a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO). In another and/or additional more preferred embodiment, the animal oligosaccharide is selected from the list consisting of N-glycans and O-glycans. In another and/or additional more preferred embodiment, the plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans. In another and/or additional preferred embodiment, the saccharide composition further comprises one or more disaccharide(s).
In an additional and/or alternative preferred embodiment, the saccharide composition further comprises a protein source, a vitamin, an oil, a mineral, a fatty acid, an enzyme, and/or a probiotic strain.
In another and/or additional preferred embodiment, the saccharide composition is a composition selected from the list comprising a food composition, a pharmaceutical composition, a cosmetic composition, a nutritional composition, a liquid food composition, a solid food composition, an infant food formula, a toddler food formula, a medical nutrition product, a medical food composition, a dietary supplement, a sachet product, a liquid ready-to-use infant nutrition product, a solid infant nutrition product, a liquid ready-to-use toddler nutrition product, a granulated product, a spray-dried infant formula product, a premix, a dairy drink, an infant formula and combinations thereof.
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 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, the process comprising passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4+ form, preferably said cationic ion exchange resin in said second cationic ion exchange is in Na+ form. 2. Process according to of embodiment 1, wherein said cationic ion exchange resin in said first and/or second cationic ion exchange is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin.
3. Process according to any one of embodiment 1 or 2, wherein said cationic ion exchange resin in said first and/or second cationic ion exchange has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix.
4. Process according to embodiment 3, wherein said matrix further comprises divinylbenzene (DVB).
5. Process according to any one of previous embodiments, wherein the flow rate through said first and/or second cationic 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 embodiments, wherein said first and/or second cationic 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 embodiments, wherein said first and/or second cationic ion exchange is/are performed at a pH ranging from 2 to 5, preferably from 3 to 5, more preferably from 4 to 5.
8. 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, 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, and wherein any one or more of said method(s) (a) precede(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and/or (b) any one or more of said method(s) succeed(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4+ form, preferably in Na+ form, preferably wherein any one or more of said methods is performed more than one time during said process. 9. 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 ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any one or more of said method(s) succeed(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and precede(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH form, preferably in Na+ form.
10. 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.
11. Process according to any one of previous embodiments, wherein the temperature of said solution is adjusted to a temperature of 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.
12. Process according to any one of embodiments 8 to 11, wherein said process comprises two consecutive steps of nanofiltration.
13. Process according to any one of embodiments 8 to 12, wherein said process comprises two consecutive steps of electrodialysis.
14. Process according to any one of embodiments 8 to 12, wherein said process does not comprise electrodialysis.
15. Process according to any one of embodiments 8 to 14, wherein said 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.
16. Process according to any one of embodiments 8 to 15, wherein said process comprises clarification performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration.
17. Process according to any one of embodiments 8 to 16, wherein said process comprises 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. Process according to any one of embodiments 8 to 17, wherein said process comprises filtration performed by use of a filtration aid and/or flocculant, preferably said filtration aid is an adsorbing agent, more preferably active carbon. Process according to any one of embodiments 8 to 18, wherein said process comprises 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. Process according to any one of embodiments 8 to 19, wherein said process comprises nanofiltration wherein the nanofiltration membrane used in said nanofiltration has a size exclusion limit of < 20 A. Process according to any one of embodiments 8 to 20, wherein said process comprises diafiltration performed on said solution 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 8 to 21, wherein said process comprises enzymatic treatment comprising incubation of said solution with one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase. Process according to any one of embodiments 8 to 22, wherein said process comprises enzymatic treatment 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 previous embodiments, wherein said process is a batch or continuous process. 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, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, 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, 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 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 and globotriose; preferably wherein said fucosylated oligosaccharide is selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (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 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 sialylated oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(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, sialyl lacto-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,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-a2,6-(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 any combination 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, 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,5-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc; 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 embodiments, wherein said oligosaccharide is a sia lylated 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 previous embodiments, 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 embodiments, wherein said oligosaccharide is 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 and 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-cc2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-cc2,6- 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-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNacP-l,3Gaip-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 any combination thereof; N-acetylglucosamine containing 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- 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. Process according to any one of previous embodiments, wherein the ash content of said solution comprising said oligosaccharide is > 10 % on total dry solid before purification by said process. Process according to any one of previous embodiments, wherein said solution comprising said oligosaccharide 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. Process according to any one of previous embodiments, wherein said solution comprising said oligosaccharide 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. 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 is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein the purity of said oligosaccharide in said cell cultivation is < 70 %, < 50 %, < 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 is produced by said cell, the cell cultivation comprising said oligosaccharide, 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. 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 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. Process according to any one of previous embodiments, wherein said cell produces said oligosaccharide from one or more internalized precursor(s). 37. 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.
38. Process according to any one of previous embodiments, 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, maltooligosaccharides, 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.
39. Process according to embodiment 38, 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.
40. Process according to any one of previous embodiments, wherein said cell cultivation is a fermentation.
41. Process according to any one of previous embodiments, wherein said oligosaccharide 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, preferably wherein all of said other oligosaccharides have the same DP as said oligosaccharide.
42. Process according to any one of embodiments 1 to 40, wherein said oligosaccharide 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, preferably wherein all of said other oligosaccharides have a different DP as said oligosaccharide.
43. Process according to any one of embodiments 28 to 42, wherein said oligosaccharide is purified from said sialic acid and/or said ashes by said process.
44. Process according to any one of embodiments 28 to 42, wherein said oligosaccharide and said one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by said process.
45. Process according to any one of previous embodiments, 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.
46. Process according to any one of previous embodiments, 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%. 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. 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. 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. 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. 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. 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. 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. Process according to any one of previous embodiments, wherein the purified oligosaccharide solution obtained at the end of said process comprises an oligosaccharide 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. Process according to any one of embodiments 1 to 53, 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 55, 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 7, preferably from 3 to 6, more preferably from 4 to 5. Process according to any one of embodiment 55 or 56, 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 or the purified oligosaccharide mixture 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 57, 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 oligosaccharide mixture obtainable, preferably obtained, by a process according to any one of embodiments 1 to 57, 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. Oligosaccharide purified according to the process according to any one of embodiments 1 to 57 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. 62. Oligosaccharide purified according to the process according to any one of embodiments 1 to 57 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.
63. Oligosaccharide purified according to the process according to any one of embodiments 1 to 57 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.
64. 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 57 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.
65. Dried powder of purified oligosaccharide solution obtained from a process according to any one of embodiments 55 to 57, 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.
66. Dried powder according to any one of embodiments 59, 60, 64, 65, 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.
67. Purified oligosaccharide according to any one of embodiments 58, 59, 61 to 65, 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).
68. Purified oligosaccharide mixture comprising an oligosaccharide according to any one of embodiments 58, 60 to 65, wherein said purified oligosaccharide mixture comprises a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO).
69. Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 58 to 68, 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.
70. Purified oligosaccharide or purified oligosaccharide mixture according to any one of embodiments 58 to 69 for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
71. Use of a purified oligosaccharide obtained by a process of any one of embodiments 1 to 57 in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.
72. Use of a purified oligosaccharide according to any one of embodiments 58 to 69 in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.
73. Use according to any one of embodiment 71 or 72, wherein: the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement, and/or the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
74. Use of a milk oligosaccharide according to any one of embodiment 67 or 68 as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food.
75. A purified oligosaccharide comprising 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.
76. Purified oligosaccharide according to embodiment 75, wherein said ash content of < 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid.
77. Purified oligosaccharide according to any one of embodiment 75 or 76, wherein said purified oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (noncharged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N-acetylglucosamine containing oligosaccharide, N-acetylglucosamine containing neutral (non-charged) oligosaccharide, N-acetyllactosamine containing oligosaccharide, lacto-N-biose containing oligosaccharide, a galactose containing oligosaccharide, non-fucosylated neutral (noncharged) 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, 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 and globotriose; preferably wherein said fucosylated oligosaccharide is selected from the list comprising 2'-fucosyllactose (2' FL), 3-fucosyllactose (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; preferably wherein said sialylated 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-oc2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-oc2,6-Gal- pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-oc2,8-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,4Gaip-l,4Gal, KDO-2,3Gai -l,3GlcNac -l,3Gai -l,4Glc, KDO-2,3Gai -l,4GlcNac -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 any combination 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, 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; 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. A saccharide composition comprising an ash content of < 10% on total dry solid and an oligosaccharide content of > 90 % 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. Composition according to embodiment 78, wherein said ash content of < 10% on total dry solid comprises a lead content lower than 0.1 mg/kg dry solid, preferably a lead content lower than 0.02 mg/kg dry solid, more preferably a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.2 mg/kg dry solid, preferably an arsenic content lower than 0.05 mg/kg dry solid, more preferably an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.1 mg/kg dry solid, 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, preferably a mercury content lower than 0.1 mg/kg dry solid, more preferably a mercury content lower than 0.005 mg/kg dry solid. Composition according to any one of embodiment 78 or 79, wherein said composition comprises more than one oligosaccharide. Composition according to any one of embodiments 78 to 80, wherein said oligosaccharide is selected from the list comprising fucosylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, sialylated oligosaccharide, Lewis type antigen, N- acetylglucosamine containing oligosaccharide, 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, 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, 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 and globotriose; preferably wherein said fucosylated oligosaccharide is selected from the list comprising 2'-fucosyllactose (2' FL), 3-fucosyllactose (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; preferably wherein said sialylated 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-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-2,3Gaip-l,3GalNacP-l,3Gala- l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNac -l,3Gai -l,4Glc, KDO-2,3Gai -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 any combination 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, 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; 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.
More specifically, the present invention relates to the following preferred 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 a fermentation, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation or said fermentation, the process comprising passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NHZ form, and wherein: the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H+ form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH form, or said process further comprises any one or more of the methods selected from the list consisting of homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any one or more of said method(s) succeed(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and precede(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NHZ form.
2. Process according to preferred embodiment 1, wherein said cationic ion exchange resin in said first and/or second cationic ion exchange: is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin, has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix, and/or has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix wherein said matrix further comprises divinylbenzene (DVB).
3. Process according to any one of preferred embodiment 1 or 2, wherein: the flow rate through said first and/or second cationic ion exchange is at least 0.5, at least 1, at least 2, at least 2.5 and/or at least 3 bed volume / hour (BV/h), said first and/or second cationic ion exchange is/are performed at a temperature ranging from 0°C to 80°C, from 4°C to 60°C, from 4°C to 40°C, from 10°C to 37°C, from 20°C to 30°C, from 20°C to 25°C, from 22°C to 24°C and/or from 23°C to 24°C, and/or said first and/or second cationic ion exchange is/are performed at a pH ranging from 2 to 5, from 3 to 5, and/or from 4 to 5.
4. Process according to any one of previous preferred embodiments, wherein said process further comprises any one or more of the methods selected from the list consisting of 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, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying, and wherein any one or more of said method(s):
(a) precede(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+form,
(b) any one or more of said method(s) succeed(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH form, and/or
(c) is performed more than one time during said process.
5. Process according to any one of previous preferred embodiments, wherein the temperature of said solution is adjusted to a temperature of: from 36°C to 65°C, from 36°C to 60°C, from 40°C to 55°C, and/or from 40°C to 45°C wherein said temperature is within 5°C of a temperature at which the solution exhibits maximum turbidity, and/or - from 0°C to 122°C, from 2°C to 80°C, from 4°C to 50°C, from 10°C to 55°C, from 20°C to 45°C, from 21°C to 40°C, from 22°C to 37°C and/or from 25°C to 30°C.
5. Process according to any one of preferred embodiment 4 or 5, wherein said process: comprises two consecutive steps of nanofiltration, comprises two consecutive steps of electrodialysis or no electrodialysis, comprises two consecutive ultrafiltration steps wherein the membrane molecular weight cutoff used in the first ultrafiltration step is higher than that used in the second ultrafiltration step, comprises clarification performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration, comprises 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, comprises filtration performed by use of a filtration aid and/or flocculant, comprises filtration performed by use of a filtration aid wherein said filtration aid is an adsorbing agent, comprises filtration performed by use of a filtration aid wherein said filtration aid is active carbon, comprises 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, comprises nanofiltration wherein the nanofiltration membrane used in said nanofiltration has a size exclusion limit of < 20 A, comprises diafiltration performed on said solution until a conductivity is reached of < 15 mS/cm, < 10 mS/cm, < 5 mS/cm, < 1 mS/cm, < 0.1 mS/cm, < 0.01 mS/cm and/or < 0.001 mS/cm, comprises enzymatic treatment comprising incubation of said solution with one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase, comprises enzymatic treatment converting lactose, sucrose, malto-oligosaccharides, maltotriose, sorbitol, trehalose, starch, cellulose, hemi-cellulose, lignocellulose, molasses, corn-steep liquor and/or high-fructose syrup to monosaccharides, and/or is a batch or continuous process.
7. Process according to any one of previous preferred embodiments, wherein said oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing neutral (noncharged) 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; a fucosylated oligosaccharide 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; a sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-cc2,6)-Gal-pi,4-Glc), 6,6'- disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-oc2,6)-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-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNacp-l,3Gaip-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 any combination thereof; an N- acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans; 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).
8. Process according to any one of previous preferred embodiments, wherein: the purity of said oligosaccharide in said solution is < 70 %, < 60 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid, the ash content of said solution comprising said oligosaccharide is > 10 % on total dry solid, said solution comprising said oligosaccharide 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, and/or said solution comprising said oligosaccharide 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. Process according to any one of previous preferred embodiments, wherein said oligosaccharide is accompanied in said solution by sialic acid, ashes, ashes selected from the list comprising 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; a mammalian milk oligosaccharide (MMO); 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; an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide; a plant oligosaccharide 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; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (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; sialylated oligosaccharide; sialylated oligosaccharide 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-2,3Gaip-l,3GalNacP- l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNac -l,3Gaip-l,4Glc, KDO-2,3Gaip-l,4GlcNac - 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-a2,5-(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 any combination thereof; N- acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing oligosaccharide 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- 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; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.
10. Process according to any one of previous preferred embodiments, wherein said solution is a cell cultivation: using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein the purity of said oligosaccharide in said cell cultivation is < 70 %, < 50 %, < 50 %, < 40 %, < 30 %, < 20 %, < 10 % on total dry solid before purification by said process, using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein, when present, biomass separated during said process is optionally recycled to said cell cultivation, using at least one cell that has been metabolically engineered to produce said oligosaccharide, and/or 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. Process according to any one of previous preferred embodiments, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. Process according to any one of previous preferred embodiments, 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, culture medium comprising a carbon source wherein 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, a chemically defined medium, and/or a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium. Process according to any one of previous preferred embodiments, wherein said oligosaccharide 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), at least one of said other oligosaccharides has a different DP, all of said other oligosaccharides have the same DP, or all of said other oligosaccharides have a different DP as said oligosaccharide. Process according to any one of preferred embodiments 9 to 13, wherein: said oligosaccharide is purified from said sialic acid and/or said ashes by said process, or said oligosaccharide and said one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by said process. Process according to any one of previous preferred embodiments, 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, and/or 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%. Process according to any one of previous preferred embodiments, wherein the purified oligosaccharide solution obtained at the end of said process: has an ash content of < 10%, < 9%, < 8%, < 7%, < 6%, < 5%, < 4%, < 3%, < 2%, < 1% and/or < 0.5% on total dry solid, 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, has an ash content of < 10% on total dry solid with a lead content lower than 0.1 mg/kg dry solid, lower than 0.02 mg/kg dry solid and/or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.2 mg/kg dry solid, lower than 0.05 mg/kg dry solid, and/or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.1 mg/kg dry solid and/or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.5 mg/kg dry solid, lower than 0.1 mg/kg dry solid and/or lower than 0.005 mg/kg dry solid, is filter-sterilized and/or subjected to endotoxin removal, and/or subjected to endotoxin removal by filtration through a 3 kDa filter, 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, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism, and/or recombinant genetic material, has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, has less than 0.5 pg/kg epichlorohydrin, is further i) concentrated to a syrup of at least 20% dry matter, at least 30% dry matter, at least 40% dry matter; ii) crystallised; iii) dried to a powder or iv) granulated, is further concentrated to an oligosaccharide concentration of > 100 g/L, > 200 g/L, > 300 g/L, > 400 g/L, > 500 g/L, > 600 g/L and/or between 300 g/L and 650 g/L, and/or at a temperature of < 80°C, < 60°C, < 50°C, between 20°C to 50°C, and/or 30°C to 45°C, is further concentrated by a method comprising using vacuum evaporation or reverse osmosis or nanofiltration, comprises an oligosaccharide which is concentrated to a concentration of > 1.5 M and cooled to a temperature < 25 °C, and/or < 8 °C, to obtain crystalline material of the oligosaccharide, 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, is dried by spray-drying, freeze-drying or agitated thin film drying and wherein the pH of said purified oligosaccharide solution is ranging from 2 to 7, from 3 to 6 and/or from 4 to 5, and/or is spray-dried at an oligosaccharide solution concentration of 20-60 (w/v), 30-50 (w/v) and/or 35-45 (w/v), with a nozzle temperature of 110-150°C, 120-140°C and/or 125-135°C and/or an outlet temperature of 60-80°C and/or 65-70°C. The purified oligosaccharide solution, the purified oligosaccharide or the purified oligosaccharide mixture obtainable or obtained by a process according to any one of previous preferred embodiments. Purified oligosaccharide obtainable or obtained by a process according to any one of preferred embodiments 1 to 16, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is i) dried, 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, at least 30% dry matter or at least 40% dry matter. Oligosaccharide purified according to the process according to any one of preferred embodiments 1 to 16 and wherein the purified oligosaccharide obtained after said process: has an ash content of < 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism, and/or recombinant genetic material, has less than 125 tg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, and/or has less than 0.5 pig/kg epichlorohydrin. Dried powder of purified oligosaccharide solution obtained from a process according to preferred embodiment 16, wherein said dried powder: contains < 15%-wt., < 10%-wt., < 7%-wt. and/or < 5%-wt. of water, has a mean particle size of 50 to 250 pm, 95 to 120 pm or 110 to 120 pm, wherein said particle size is determined by laser diffraction, has an ash content of < 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism and/or recombinant genetic material, has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3-monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, and/or has less than 0.5 pg/kg epichlorohydrin. A purified oligosaccharide comprising an ash content of < 10% on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid. Purified oligosaccharide according to preferred embodiment 21, wherein said purified oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing neutral (noncharged) 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (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; sialylated oligosaccharide 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- oc2,3-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,3Gaip-l,4Glc, KDO- 2,3Gaip-l,4GlcNac -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 any combination thereof; N- acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
23. A saccharide composition comprising an ash content of < 10% on total dry solid and an oligosaccharide content of > 90 % on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid.
24. Composition according to preferred embodiment 23, wherein said composition comprises more than one oligosaccharide and/or wherein said oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; 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; fucosylated oligosaccharide selected from the list comprising 2'- fucosyllactose (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; sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-cc2,6)-Gal-pi,4-Glc), 6,6'- disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac- oc2,3-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-a2,6-(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 any combination thereof; N- acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
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 and cultivation
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 cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH4CI, 5.00 g/L (NF hSC , 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. Depending on the experiment, 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/LCaCI2.2H20, 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. HCI. The molybdate solution contained 0.967 g/L NaMoO4.2H2O. 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), carbenicill in (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
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 96well 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 m L 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.
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. All strains were stored in cryovials at -80°C (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).
8. Saccharomyces cerevisiae
Media and cultivation
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).
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 mL 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. Strains, plasmids and mutations
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). 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 and cultivation
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 (NH^SC , 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSC>4.7H2O, 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 CaCl2.2H2O, 0.1 g/L MnCI2.2H2O, 0.033 g/L CuCI2.2H2O, 0.06 g/L CoCI2.6H2O, 0.17 g/L ZnCL, 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.
8. 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 50 min at 50°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).
Strains, plasmids and mutations
8. 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.
D. Corynebacterium glutamicum
Media and cultivation
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.
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).
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.
E. Chlamydomonas reinhardtii
Media and cultivation
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/cm2).
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).
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 Q.61420), and a Golgi-localised sialyltransferase selected 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 and/or an alpha-l,3-fucosyltransferase. 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 Q.9JXQ.6). 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 D3Q.Y14) from E. coli 055:1-17 or an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q.51116, 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).
F. Animal cells
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 - C356; 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. Enzymatic synthesis
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-l,3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511), the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3) 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-l,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).
/. 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.
J. 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 (sequence version 01, UniProt Database 2021_03 of 09 June 2021) as described in present disclosure
* Sequence version 02 (01 Feb 2005) as present in the UniProt Database 2021 03 of 09 June 2021
** Sequence version 03 (10 Feb 2009) as present in the UniProt Database 2021_03 of 09 June 2021
*** Sequence version 04 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021 **** Sequence version 02 (01 Dec 2000) as present in the UniProt Database 2021_03 of 09 June 2021 ° Sequence version 02 (13 Oct 2009) as present in the UniProt Database 2021_03 of 09 June 2021
Sequence version 02 (01 Feb 2005) as present in the UniProt Database 2021_03 of 09 June 2021 Sequence version 03 (05 Jul 2005) as present in the UniProt Database 2021_03 of 09 June 2021
K. 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.
L. 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. Ill
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 MfTcm (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.
M. Biomass dry mass content (cell dry mass)
Cell dry weight (CDW) 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.
An alternative method for determination of cell dry mass or cell dry weight, also well known to a person skilled in the art, uses an Infrared Moisture Analyzer (Sartorius MA150). The equipment was allowed to warm-up for 30 min, and the standby temperature was set to 60°C. The balance was automatically tared with the weighing aluminium pan containing a glass microfibre filter pad (0.2pm) after drying (105°C in moisture analyser to achieve stable weight, lasting about 1 min) and equilibration. The drying temperature was set to 105°C. Samples were evenly added to the glass microfibre pad, and the drying programme was set to end when the weight change was less than 0-1 mg min-1. The dried glass fibre filter pad was used to filter off the biomass from 10 mL of broth and the filtered broth was washed 2 times with physiological solution. The filter was then dried again by means of the method described above, drying the already tared aluminium pan and the filter pad at 105°C until stable weight.
N. 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. O. 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.
P. 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.
Q. 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.
R. 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 one or more oligosaccharide(s)
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. Typically, the obtained product concentration was above 50 g/L. 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 D3Q.Y14) 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-l,4-galactosyltransferase LgtB (Uniprot ID 0.51116, sequence version 02, 01 Dec 2000) from Neisseria 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. Typically, the obtained product concentration was above 50 g/L.
An E. coli K12 MG1655 strain engineered for production of 2' FL (Fuc-al,2-Gal-pi,4-Glc) as described e.g., in WO22129470 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 was measured using UPLC as described in Example 1. Typically, the obtained product concentration was above 50 g/L.
An E. coli K12 MG1655 strain engineered for production of 3-FL (Gal- i,4-[Fuc-al,3]-Glc) as described e.g., in WO20127417 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 was measured using UPLC as described in Example 1. Typically, the obtained product concentration was above 50 g/L.
An E. coli K12 MG1655 strain engineered for production of an oligosaccharide mixture comprising 2'FL (Fuc-al,2-Gal-pi,4-Glc), 3-FL (Gal-pi,4-[Fuc-al,3]-Glc) and DiFL (Fuc-al,2-Gal-pi,4-[Fuc-al,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. Typically, the obtained product concentration was above 50 g/L.
E. coli K12 MG1655 strains modified for production of i) an oligosaccharide mixture comprising 2'FL, DiFL, LN3, LNT, LNFP-I and LNFP-II or ii) 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, LN3, 3'SLN3, 6'S-LN3, LNT, LNFP-I and LSTa as described e.g., in WO22034067 were 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 of each fermentation were taken and evaluated via UPLC for production of said oligosaccharide mixtures. Typically, the obtained product concentration was above 50 g/L.
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 glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), 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 Q.9CLP3). 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 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 glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), 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 Q.9JXQ.6) and the N- acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N. meningitidis (Uniprot ID 0.51116, 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. 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.
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 0.03417) 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-l,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.
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 Q.9NR45, Sequence version 03 (13 Oct 2009)), the N-acylneuraminate cytidylyltransferase CMAS from H. sapiens (UniProt ID Q8NFW8, Sequence version 02 (01 Feb 2005)) 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. 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, Sequence version 03 (10 Feb 2009)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from H. sapiens (UniProt ID Q.96EK6), the phosphoacetylglucosamine mutase PGM3 from H. sapiens (UniProt ID 095394), the UDP-N-acetylhexosamine pyrophosphorylase UAP1 from H. sapiens (UniProt ID Q.16222, Sequence version 03 (05 Jul 2005)), the N-acylneuraminate cytidylyltransferases NeuA from M. musculus (UniProt ID Q99KK2, Sequence version 02 (01 Feb 2005)) and the CMP-N-acetylneuraminate-beta-1,4- galactoside alpha-2, 3-sialyltransferase ST3GAL3 from H. sapiens (UniProt ID Q11203). 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 3'SL.
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. 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.
An enzymatic synthesis reaction was set up comprising the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) produced in a CFPS reaction as described in Example 1, 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 the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) produced in a CFPS reaction as described in Example 1, 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-al,2-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc). Another enzymatic synthesis reaction was set up comprising said alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) produced in a CFPS reaction as described in Example 1, 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, 2-fucosyltransferase HpFutC and said alpha-1, 3-fucosyltransferase HpFucT both produced in CFPS reactions as described in Example 1, 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 the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) 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, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) 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 the alpha-2,3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3) and the alpha-2, 6-sialyltransferase PdSt6 from P. damselae (UniProt ID 066375) 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 (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) and LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc). Another enzymatic synthesis reaction was set up comprising the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) 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 the human ST6GalNAc5 (UniProt ID Q.9BVH7, purchased from Glyco Expression Technologies Inc), 10 mM CMP-Neu5Ac, 10 mM MgCI2 and 25 mM LSTa as compatible substrate and a Tris buffer at pH 8.8 and incubated at 37°C for 24 hours to obtain DSLNT (disialyllacto-N- tetraose or Neu5Ac-a2,3-Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-pi,4-Glc).
In another example, mutant E. coli BL21 strains are used to produce four recombinant proteins: an N- acylglucosamine 2-epimerase AGE 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 Q.7DDU0) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q.9CLP3). 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 3. Composition determination of the fermentation or cultivation broth or of the solutions obtained after enzymatic synthesis reactions
For the fermentation or cultivation broths obtained in Example 2 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. 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 4. Processing a sialylated oligosaccharide syrup
Broth comprising 3'SL was produced via fermentation as described in Example 2. Said 3'SL was produced in the salt form with the medium cations as counter ion, wherein potassium and ammonium ions were most dominant. The biomass was removed by well-known methods in the art such as e.g., ultrafiltration, removing proteins, DNA and other macro molecules from the solution. The resulting solution was directly passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H+ form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na+form. In an alternative process, after biomass removal the solution was first treated in a nanofiltration step using, e.g., a Trisep XN45 membrane, which removes water, salts and carbohydrates, after which the retentate is passed over the cation exchange columns as described above. To remove color components, a part of the solution was further treated in an activated charcoal step. The pH of the effluent of the second cation exchange column was 2.3 which was corrected by addition of NaOH to pH 6.5. The cationic counter ions were all exchanged for Na+ ions after the second cation exchange step. The solution was further concentrated or was directly spray dried to form a white stable powder which was analyzed according to the methods in Example 1. The ash content in the powder was between 5 and 8 % and the Na+ concentration was between 3.5 and 5%. Heavy metal analysis indicated that cadmium, mercury, lead and arsenic concentrations were below 20 pg/kg powder.
Example 5. Processing a fucosylated oligosaccharide syrup
Broth comprising 2'FL was produced via fermentation as described in Example 2. The biomass was removed by well-known methods in the art, such as e.g., microfiltration and ultrafiltration, removing proteins, DNA and other macro molecules from the solution. The resulting solution was directly passed over a cation exchange column containing Amberlite FPC88 H (Dupont, USA) in H+ form followed by a cation exchange column containing Amberlite FPC88 H (Dupont, USA) in Na+ form. In an alternative process, after biomass removal the solution was first treated in a nanofiltration step using, e.g., a Trisep XN45 membrane, which removes water, salts and carbohydrates, after which the retentate is passed over the cation exchange columns as described above. To remove color components, a part of the solution was further treated in an activated charcoal step. The pH of the effluent of the second cation exchange column was 3.1 which was corrected by addition of NaOH to pH 6.5. The unwanted cations, such as K+ and NH4 + ions, were all exchanged for the desired Na+ ions after the second cation exchange step. The solution was further concentrated or was directly spray dried to form a white stable powder which was analyzed according to the methods in Example 1. The ash content in the powder was between 0.3 and 1.5 %. Heavy metal analysis indicated that cadmium, mercury, lead and arsenic concentrations were below 20 pg/kg powder. Example 6. Processing a sialylated oligosaccharide syrup
Broth comprising 6'SL was produced via fermentation as described in Example 2. Said 6'SL was produced in the salt form with the medium cations as counter ion, wherein potassium and ammonium ions were most dominant. The biomass was removed by well-known methods in the art such as e.g., ultrafiltration, removing proteins, DNA and other macro molecules from the solution. The resulting solution was directly passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H+ form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na+ form. A 0.2 pm Sartrobran P Filter cartridge (Sartorius, Germany) was placed in between the two cation exchange columns to prevent fines leaching from the resin in the H+ column entering the Na+ column. In an alternative process, after biomass removal the solution was first treated in a nanofiltration step using, e.g., a Trisep XN45 membrane, which removes water, salts and carbohydrates, after which the retentate was passed over the cation exchange columns as described above. To remove color components, a part of the solution was further treated in an activated charcoal step. The pH of the effluent of the second cation exchange column was 2.3 which was corrected by addition of NaOH to pH 5.5. The cationic counter ions were all exchanged for Na+ ions after the second cation exchange step. The solution was further concentrated or was directly spray dried to form a white stable powder which was analyzed according to the methods in Example 1. The ash content in the powder was between 5 and 8 % and the Na+ concentration was between 3.5 and 5%. Heavy metal analysis indicated that cadmium, mercury, lead and arsenic concentrations were below 20 pg/kg powder.
Example 7. Processing a 3'SL syrup
Broth comprising 3'Sialyllactose (3'SL), wherein said 3'SL had a purity of 58 % on total dry solid, was produced via fermentation as described in Example 2. After fermentation, the biomass was removed by microfiltration. The filtrate was then further purified via ultrafiltration removing proteins and other macro molecules. Next, the resulting filtrate was treated with nanofiltration using e.g., a Trisep XN45 (Mann+Hummel, Germany) membrane, which removes water, salts and unwanted carbohydrates. The nanofiltration retentate was then further treated with activated charcoal (AC) (Chemviron, France) to remove color components. After this step, the 3'SL formed a salt mainly with potassium and ammonium. Furthermore, some traces of heavy metals were still present (Table 2, part "after AC"). The ash content was measured to be 2.81 % of the total dissolved solids. In a next step the syrup was passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H+ form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na+ form. The pH of the effluent was 2.3 which was corrected by addition of NaOH to pH 6.5 which caused a slight increase in ash content to 6.6 % of the total dissolved solids. After said both cation exchange steps the K+ and NH4 + ions were well exchanged for Na+ whereas the traces of heavy metals were mostly removed resulting in a lead content lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.01 mg/kg dry solid, a cadmium content lower than 0.005 mg/kg dry solid and a mercury content lower than 0.01 mg/kg dry solid (Table 2, part "after IEX").
Table 2. Concentration of cations and heavy metals in process streams obtained in a purification process of a solution comprising 3'SL obtained by fermentation and clarified by microfiltration, ultrafiltration and nanofiltration, when measured after activated charcoal (AC) treatment of the nanofiltration retentate and after a first cation ion exchange in H+ form and a second cation ion exchange in Na+ form of the activated charcoal treated solution.
Example 8. Processing a 3*-sialyl-3-fucosyllactose syrup
Broth comprising 3'-sialyl-3-fucosyllactose is produced via fermentation as described in Example 2. After fermentation, the biomass is removed by microfiltration. The filtrate is then further purified by ultrafiltration removing proteins and other macro molecules. Next, the resulting filtrate is treated with nanofiltration to remove water, salts and unwanted carbohydrates. The nanofiltration retentate is then further treated with activated charcoal (AC) (Chemviron, France) to remove color components. In a next step the syrup is passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H+ form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na+ form. The pH of the effluent is ranging between 1.8 and 3.0, which is corrected by addition of NaOH to pH 6.5. After said both cation exchange steps traces of heavy metals are mostly removed resulting in a lead content lower than 0.02 mg/kg dry solid, an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid and a mercury content lower than 0.1 mg/kg dry solid. The resulting syrup is then spray-dried to form a white stable powder.
Example 9. Processing a solution comprising DSLNT
A solution comprising DSLNT is produced enzymatically as described in Example 2. After enzymatic catalysis, the solution is treated by microfiltration and ultrafiltration removing proteins and other macro molecules. Next, the resulting filtrate is treated with nanofiltration. The nanofiltration retentate is then further treated with activated charcoal (AC) (Chemviron, France). In a next step the solution is passed over a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in H+form followed by a cation exchange column containing Diaion PK216 resin (Mitsubishi chemical, Japan) in Na+ form. The pH of the effluent is ranging between 1.8 and 3.0 which is corrected by addition of NaOH to pH 6.5. After said both cation exchange steps the heavy metals present comprise a lead content lower than 0.02 mg/kg dry solid, an arsenic content lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid and a mercury content lower than 0.1 mg/kg dry solid. The resulting solution is then spray-dried to form a white stable powder.

Claims

Claims
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 a fermentation, wherein said oligosaccharide is produced by said biocatalysis reaction solution, said chemical synthesis solution or by a cell cultivated in said cell cultivation or said fermentation, the process comprising passing said solution through a first cationic ion exchange comprising a cationic ion exchange resin in H+ form, hereby converting the salts in said solution fully or partially into acids, characterized in that said solution comprising said salts fully or partially converted into acids is passed through a second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NHZ form, and wherein: the eluate obtained from said first cationic ion exchange comprising a cationic ion exchange resin in H+ form is immediately passed through said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+ or NH form, or said process further comprises any one or more of the methods selected from the list consisting of homogenization, clarification, clearing, concentration, centrifugation, decantation, dilution, pH adjustment ranging from 2 to 5, temperature adjustment, filtration, ultrafiltration, microfiltration, diafiltration, reverse osmosis, nanofiltration, dialysis, use of activated charcoal or carbon, use of solvents with a pH ranging from 2 to 5, use of alcohols, use of aqueous alcohol mixtures, use of charcoal, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, hydrophobic interaction chromatography, gel filtration, ligand exchange chromatography, column chromatography, cation exchange adsorbent resin, use of an adsorbent material, evaporation, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization, drying and dissolving in a liquid with a pH ranging from 2 to 5, and wherein any one or more of said method(s) succeed(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+ form and precede(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH form.
2. Process according to claim 1, wherein said cationic ion exchange resin in said first and/or second cationic ion exchange: is selected from the list comprising a weak acid cation (WAC) exchange resin and a strong acid cation (SAC) exchange resin, has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix, and/or has an acrylic based, a methacrylic based, a styrene based or a polystyrene based matrix wherein said matrix further comprises divinylbenzene (DVB).
3. Process according to any one of claim 1 or 2, wherein: the flow rate through said first and/or second cationic ion exchange is at least 0.5, at least 1, at least 2, at least 2.5 and/or at least 3 bed volume / hour (BV/h), said first and/or second cationic ion exchange is/are performed at a temperature ranging from 0°C to 80°C, from 4°C to 60°C, from 4°C to 40°C, from 10°C to 37°C, from 20°C to 30°C, from 20°C to 25°C, from 22°C to 24°C and/or from 23°C to 24°C, and/or said first and/or second cationic ion exchange is/are performed at a pH ranging from 2 to 5, from 3 to 5, and/or from 4 to 5.
4. Process according to any one of previous claims, wherein said process further comprises any one or more of the methods selected from the list consisting of 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, vacuum evaporation, wiped film evaporation, falling film evaporation, pasteurization, enzymatic treatment, decolorization and drying, and wherein any one or more of said method(s):
(a) precede(s) said first cationic ion exchange comprising a cationic ion exchange resin in H+form,
(b) any one or more of said method(s) succeed(s) said second cationic ion exchange comprising a cationic ion exchange resin in Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Al3+or NH4 + form, and/or
(c) is performed more than one time during said process.
5. Process according to any one of previous claims, wherein the temperature of said solution is adjusted to a temperature of: from 36°C to 65°C, from 36°C to 60°C, from 40°C to 55°C, and/or from 40°C to 45°C wherein said temperature is within 5°C of a temperature at which the solution exhibits maximum turbidity, and/or
- from 0°C to 122°C, from 2°C to 80°C, from 4°C to 60°C, from 10°C to 55°C, from 20°C to 45°C, from 21°C to 40°C, from 22°C to 37°C and/or from 25°C to 30°C.
6. Process according to any one of claim 4 or 5, wherein said process: comprises two consecutive steps of nanofiltration, comprises two consecutive steps of electrodialysis or no electrodialysis, comprises two consecutive ultrafiltration steps wherein the membrane molecular weight cutoff used in the first ultrafiltration step is higher than that used in the second ultrafiltration step, comprises clarification performed by any one or more of microfiltration, centrifugation, flocculation or ultrafiltration, comprises 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, comprises filtration performed by use of a filtration aid and/or flocculant, comprises filtration performed by use of a filtration aid wherein said filtration aid is an adsorbing agent, comprises filtration performed by use of a filtration aid wherein said filtration aid is active carbon, comprises 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, comprises nanofiltration wherein the nanofiltration membrane used in said nanofiltration has a size exclusion limit of < 20 A, comprises diafiltration performed on said solution until a conductivity is reached of < 15 mS/cm, < 10 mS/cm, < 5 mS/cm, < 1 mS/cm, < 0.1 mS/cm, < 0.01 mS/cm and/or < 0.001 mS/cm, comprises enzymatic treatment comprising incubation of said solution with one or more enzymes selected from the list comprising glycosidase, lactase, p-galactosidase, fucosidase, sialidase, maltase, amylase, hexaminidase, glucuronidase, trehalase, and invertase, comprises enzymatic treatment converting lactose, sucrose, malto-oligosaccharides, maltotriose, sorbitol, trehalose, starch, cellulose, hemi-cellulose, lignocellulose, molasses, corn-steep liquor and/or high-fructose syrup to monosaccharides, and/or is a batch or continuous process.
7. Process according to any one of previous claims, wherein said oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; 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; a fucosylated oligosaccharide selected from the list comprising 2'- fucosyllactose (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; a sialylated oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 8'sialyllactose (8'SL), 3,6-disialyllactose (Neu5Ac-oc2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'- disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-oc2,6)-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,3Gaip-l,4Glc, KDO- 2,3Gaip-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-a2,6-(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 any combination thereof; an N- acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans; 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).
8. 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, the ash content of said solution comprising said oligosaccharide is > 10 % on total dry solid, said solution comprising said oligosaccharide 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, and/or said solution comprising said oligosaccharide 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.
9. Process according to any one of previous claims, wherein said oligosaccharide is accompanied in said solution by sialic acid, ashes, ashes selected from the list comprising 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; a mammalian milk oligosaccharide (MMO); 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; an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide; a plant oligosaccharide 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; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (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; sialylated oligosaccharide; sialylated oligosaccharide 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-2,3Gaip-l,3GalNacp- l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNac -l,3Gaip-l,4Glc, KDO-2,3Gaip-l,4GlcNac - 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 any combination thereof; N- acetylglucosamine containing oligosaccharide; N-acetylglucosamine containing oligosaccharide 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- 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; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.
10. Process according to any one of previous claims, wherein said solution is a cell cultivation: using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein 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, using a cell or a metabolically engineered cell, wherein said oligosaccharide is produced by said cell, the cell cultivation comprising said oligosaccharide, biomass, medium components and contaminants, wherein, when present, biomass separated during said process is optionally recycled to said cell cultivation, using at least one cell that has been metabolically engineered to produce said oligosaccharide, and/or 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.
11. 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.
12. Process according to any one of previous claims, 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, culture medium comprising a carbon source wherein 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, a chemically defined medium, and/or a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.
13. Process according to any one of previous claims, wherein said oligosaccharide 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), at least one of said other oligosaccharides has a different DP, all of said other oligosaccharides have the same DP, or all of said other oligosaccharides have a different DP as said oligosaccharide.
14. Process according to any one of claims 9 to 13, wherein: said oligosaccharide is purified from said sialic acid and/or said ashes by said process, or said oligosaccharide and said one or more other oligosaccharide(s) are purified from said sialic acid and/or said ashes by said process.
15. 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, and/or 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%.
16. 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%, < 9%, < 8%, < 7%, < 6%, < 5%, < 4%, < 3%, < 2%, < 1% and/or <
0.5% on total dry solid, 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, has an ash content of < 10% on total dry solid with a lead content lower than 0.1 mg/kg dry solid, lower than 0.02 mg/kg dry solid and/or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.2 mg/kg dry solid, lower than 0.05 mg/kg dry solid, and/or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.1 mg/kg dry solid and/or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.5 mg/kg dry solid, lower than 0.1 mg/kg dry solid and/or lower than 0.005 mg/kg dry solid, is filter-sterilized and/or subjected to endotoxin removal, and/or subjected to endotoxin removal by filtration through a 3 kDa filter, 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, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism, and/or recombinant genetic material, has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, has less than 0.5 pg/kg epichlorohydrin, is further i) concentrated to a syrup of at least 20% dry matter, at least 30% dry matter, at least 40% dry matter; ii) crystallised; iii) dried to a powder or iv) granulated, is further concentrated to an oligosaccharide concentration of > 100 g/L, > 200 g/L, > 300 g/L, > 400 g/L, > 500 g/L, > 600 g/L and/or between 300 g/L and 650 g/L, and/or at a temperature of < 80°C, < 60°C, < 50°C, between 20°C to 50°C, and/or 30°C to 45°C, is further concentrated by a method comprising using vacuum evaporation or reverse osmosis or nanofiltration, comprises an oligosaccharide which is concentrated to a concentration of > 1.5 M and cooled to a temperature < 25 °C, and/or < 8 °C, to obtain crystalline material of the oligosaccharide, 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, is dried by spray-drying, freeze-drying or agitated thin film drying and wherein the pH of said purified oligosaccharide solution is ranging from 2 to 7, from 3 to 6 and/or from 4 to 5, and/or is spray-dried at an oligosaccharide solution concentration of 20-60 (w/v), 30-50 (w/v) and/or 35-45 (w/v), with a nozzle temperature of 110-150°C, 120-140°C and/or 125-135°C and/or an outlet temperature of 60-80°C and/or 65-70°C.
17. The purified oligosaccharide solution, the purified oligosaccharide or the purified oligosaccharide mixture obtainable or obtained by a process according to any one of previous claims.
18. Purified oligosaccharide obtainable or obtained by a process according to any one of claims 1 to 16, wherein the purified oligosaccharide solution comprising said purified oligosaccharide is i) dried, 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, at least 30% dry matter or at least 40% dry matter.
19. Oligosaccharide purified according to the process according to any one of claims 1 to 16 and wherein the purified oligosaccharide obtained after said process: has an ash content of < 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism, and/or recombinant genetic material, has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3- monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, and/or has less than 0.5 pg/kg epichlorohydrin.
20. Dried powder of purified oligosaccharide solution obtained from a process according to claim 16, wherein said dried powder: contains < 15%-wt., < 10%-wt., < 7%-wt. and/or < 5%-wt. of water, has a mean particle size of 50 to 250 pm, 95 to 120 pm or 110 to 120 pm, wherein said particle size is determined by laser diffraction, has an ash content of < 10% on total dry solid with a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, has an ash content of < 10% on total dry solid with a cadmium content lower than 0.01 mg/kg dry solid, has an ash content of < 10% on total dry solid with a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid, is free of DNA, recombinant DNA, proteins, proteins derived from the recombinant microorganism and/or recombinant genetic material, has less than 125 pg/kg of 3-MCPD, wherein said 3-MCPD is the sum of 3-monochloropropanediol (3-MCPD) and 3-MCPD fatty acid esters, and/or has less than 0.5 pig/kg epichlorohydrin.
21. A purified oligosaccharide comprising an ash content of < 10% on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid.
22. Purified oligosaccharide according to claim 21, wherein said purified oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; 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; fucosylated oligosaccharide selected from the list comprising 2'- fucosyllactose (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; sialylated oligosaccharide 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), 6,5'- disialyllactose (Neu5Ac-oc2,6-Gal-pi,4-(Neu5Ac-oc2,6)-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-2,3Gaip-l,3GalNac -l,3Gala-l,4Gai -l,4Gal, KDO-2,3Gai -l,3GlcNacP-l,3Gaip-l,4Glc, KDO- 2,3Gai -l,4GlcNac -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 any combination thereof; N- acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
23. A saccharide composition comprising an ash content of < 10% on total dry solid and an oligosaccharide content of > 90 % on total dry solid, wherein said ash comprises: a lead content lower than 0.02 mg/kg dry solid or lower than 0.01 mg/kg dry solid, an arsenic content lower than 0.05 mg/kg dry solid or lower than 0.02 mg/kg dry solid, a cadmium content lower than 0.01 mg/kg dry solid, and/or a mercury content lower than 0.1 mg/kg dry solid or lower than 0.005 mg/kg dry solid.
24. Composition according to claim 23, wherein said composition comprises more than one oligosaccharide and/or wherein said oligosaccharide is selected from the list consisting of fucosylated oligosaccharide; neutral (non-charged) oligosaccharide; negatively charged oligosaccharide; sialylated oligosaccharide; Lewis type antigen; N-acetylglucosamine containing oligosaccharide; 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; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan 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; 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; fucosylated oligosaccharide selected from the list comprising 2'-fucosyllactose (2' FL), 3- fucosyllactose (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 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 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-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal, KDO- 2,3Gaip-l,3GlcNac -l,3Gaip-l,4Glc, KDO-2,3Gaip-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-a2,6-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 any combination thereof; N-acetylglucosamine containing neutral (non-charged) oligosaccharide 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; a mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMD); an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans; a plant oligosaccharide is selected from the list consisting of N-glycans and O-glycans.
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