WO2022072333A1

WO2022072333A1 - Process for purifying a human milk oligosaccharide and related compositions

Info

Publication number: WO2022072333A1
Application number: PCT/US2021/052373
Authority: WO
Inventors: Jyrki Kuusisto; Jari Lewandowski; Antti Koponen; Tero Tuomas MENTUNEN; Wim Soetaert
Original assignee: Dupont Nutrition Biosciences Aps; Inbiose N.V.; Danisco Usa, Inc
Priority date: 2020-09-29
Filing date: 2021-09-28
Publication date: 2022-04-07
Also published as: EP4222156A1; CN116390931A

Abstract

This specification relates to a process for preparing a purified human milk oligosaccharide ("HMO") from an HMO-containing solution (e.g., a fermentation broth) by a process comprising anion ion exchange, cation ion exchange, and mixed bed ion exchange; and a product of such a process.

Description

PROCESS FOR PURIFYING A HUMAN MILK OLIGOSACCHARIDE

AND RELATED COMPOSITIONS

CROSS-REFERENCE TO RELATED PATENT APPLICATION

[1] This specification claims priority to US Provisional Patent Application No. 63/084,834 (filed September 29, 2020) and European Patent Application No. EP20204655.3 (filed October 29, 2020). The entire text of each of the above-referenced patent applications is incorporated by reference into this specification.

FIELD

[2] This specification relates to purifying a human milk oligosaccharide (“HMO”) from an HMO-containing solution (e.g., a fermentation broth) by a process comprising anion ion exchange, cation ion exchange, mixed bed ion exchange; and a product of such a process.

BACKGROUND

[3] Human milk oligosaccharides are important for nutrition and therapeutics. HMOs include, for example, 2’-fucosyllactose (“2’-FL”), 3-fucosyllactose (“3-FL”), lacto-N-tetraose (“LNT”), 6’-sialyllactose (“6’-SL”), 3’-sialyllactose (“3’-SL”), difucosyllactose (“DiFL” or “LDFT”), lacto-N-neotetraose (“LNnT”), lacto-N-fucopentaose, lacto-N-difucohexaose, lacto- N-neodifucohexaose, lacto-N-neooctaose, lacto-N-fucopentaose, lacto-N-neofucopentaose, 3’sialyl-3-fucosyllactose, sialyl-lacto-N-tetraose, LS-tetrasaccharide, lacto-N-triose, lacto-N- neofucopentaose, lacto-N-neofucopentaose, lacto-N-difucohexaose, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose and lacto-N-neohexaose. Many HMOs in human breast milk are fucosylated, unlike oligosaccharides produced by, for example, dairy animals. The most abundant HMO in human breast milk is 2’-FL.

[4] HMO are composed of the five monosaccharide building blocks D-glucose (Glc), D-galactose (Gal), A-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialic acid (N- acetylneuraminic acid). They can be grouped into neutral and charged oligosaccharides, the latter being sialylated. Neutral fucosylated HMOs are neutral and contain fucose at the terminal position (e.g., 2'-fucosyllactose (2'-FL) and lactodifucopentaose). They represent 35% to 50% of the total HMO content. Neutral A-containing (nonfucosylated) HMOs are neutral and contain N- acetylglucosamine at the terminal position (e.g., lacto-A-tetraose), and represent 42% to 55% of the total HMO content. Neutral HMOs account for more than 75% of the total HMOs in human breast milk. [5] Acid (sialylated) HMOs are acidic and contain sialic acid at the terminal position (e.g., 2'-sialyllactose). They represent 12% to 14% of the total HMO content.

[6] Many recent approaches for synthesizing HMOs involve microbial fermentation processes, which produce HMOs (such as 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL) from lactose. In such a process, a given HMO is synthesized by cultured microorganisms, such as recombinant E. coli. The HMO is then isolated from the broth of biomolecules produced by the culture through a series of purification processes. While there has been success with this approach, the fermentation processes generally produce a complex product mixture which includes, besides the desired HMO(s), other ingredients, such as monovalent and divalent salts, lactose, oligosaccharides, monosaccharides, amino acids, polypeptides, proteins, organic acids, nucleic acids, processing aids, etc.

[7] HMOs may be incorporated into a food (e.g., human or pet food), dietary supplement or medicine. HMOs are particularly useful in, for example, infant formula. Thus, there is need for HMOs that are substantially pure.

[8] Accordingly, a need continues to exist for effective, reliable and economically and environmentally feasible processes in industrial scale to provide an HMO product of high quality and purity and good yield.

SUMMARY

[9] Briefly, this specification generally provides, in part, a process for making a purified human milk oligosaccharide (“HMO”) from an HMO solution derived from a fermentation process. The process comprises passing the HMO solution through a first ion exchange material to form a first ion exchange product, passing the first ion exchange product through a second ion exchange material to form a second ion exchange product, and passing the second ion exchange product through mixed bed ion exchange comprising both cation ion exchange material and anion ion exchange material. One of the first and second ion exchange materials comprises cation ion exchange material and no anion ion exchange material. The other of the first and second ion exchange materials comprises anion ion exchange material and no cation ion exchange material. In some embodiments, the composition of the first ion exchange product does not change before it is passed through the second ion exchange material. In some embodiments, the composition of the second ion exchange product does not change before it is passed through the mixed bed ion exchange. In some embodiments, the first ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it is passed through second ion exchange material. In some embodiments, the second ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it is passed through the mixed bed ion exchange.

[10] This specification also provides, in part, a purified HMO (or mixture of HMDs) obtained by the above-referenced process.

[11] This specification also provides, in part, a process for making a food, dietary supplement, infant formula or medicine. The process comprises preparing a purified HMO according to the above-described process, and mixing the purified HMO with an ingredient suitable for the food, dietary supplement, infant formula or medicine.

[12] This specification also provides, in part, a food, dietary supplement, infant formula or medicine prepared by such a process.

[13] Further benefits of the teachings of this specification will be apparent to one skilled in the art from reading this specification.

DETAILED DESCRIPTION

[14] This detailed description is intended to acquaint others skilled in the art with Applicant’s invention, its principles, and its practical application so that others skilled in the art may adapt and apply Applicant’s invention in its numerous forms, as they may be best suited to the requirements of a particular use. This detailed description and its specific examples, while indicating certain embodiments, are intended for purposes of illustration only. This specification, therefore, is not limited to the described embodiments, and may be variously modified.

Definitions

[15] The term “Area%” refers to normalized peak area purity or concentration obtained using HPLC. This is a percentage of peak area relative to the total area of peaks.

[16] The term “2’-FL” or “2’FL” refers to 2’-fucosyllactose (also referred to as “2’-O- fucosyllactose”).

[17] The term “3-FL” or “3FL” refers to 3-fucosyllactose (also referred to as “3-O- fucosyllactose”).

[18] The term “HMO” refers to human milk oligosaccharide. [19] The term “neutral HMO” refers to fiicosylated (contain fucose at the terminal position) and non-fucosylated (N-containing, contain 7V-acetylglucosamine at the terminal position) HMOs.

[20] The term “HPLC” refers to high performance liquid chromatography.

[21] The term “ICUMSA” refers to “International Commission for Uniform Process of Sugar Analysis” sugar color grading system.

[22] The term “MB” refers to a mixed bed.

[23] The term “IEX” refer to ion exchange

[24] The term “SAC” refers to strong acid cation ion exchange material (e.g., resin).

[25] The term “WBA” refers to weak base anion ion exchange material (e.g., resin).

[26] The term “SBA” refers to strong base anion ion exchange material (e.g., resin).

[27] The term “WAC” refers to weak acid cation ion exchange material (e.g., resin).

[28] A “cation ion exchange vessel” is an ion exchange vessel (e.g., a column) that comprises cation ion exchange material (e.g., resin) and no anion ion exchange material (e.g., resin).

[29] An “anion ion exchange vessel” is an ion exchange vessel (e.g., a column) that comprises anion ion exchange material (e.g., resin) and no cation ion exchange material (e.g., resin).

[30] A “mixed bed ion exchange vessel” or “MB ion exchange vessel” is an ion exchange vessel (e.g., a column) that comprises a combination of cation ion exchange material (e.g., resin) with anion ion exchange material (e.g., resin).

Human milk oligosaccharide

[31] There are over 150 known human milk oligosaccharides generally present in human breast milk. A process described in this specification may be used to prepare a single purified HMO or a purified mixture of two or more HMOs.

[32] In some embodiments, a process of this specification comprises preparing a purified HMO selected from fucosyllactoses (such 2 ’-FL, 3 -FL or DiFL) , LNT, LNnT, lacto-N- fucopentaose, lacto-N-difucohexaose, lacto-N-neodifucohexaose, lacto-N-neooctaose, lacto-N- fucopentaose, lacto-N-neofucopentaose, LS-tetrasaccharide, lacto-N -triose, lacto-N-neo fucopentaose, lacto-N -neofucopentaose, lacto-N- difucohexaose, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose or lacto-N-neohexaose. In some embodiments, a process of this specification comprises preparing a purified HMO mixture comprising one or more of the above-listed HMOs. In some embodiments, a process of this specification comprises preparing a purified HMO mixture comprising at least two of the above-listed HMOs.

[33] In some embodiments, a process of this specification is used to prepare a purified neutral HMO.

[34] In some embodiments, a process of this specification is used to prepare a purified HMO selected from a fucosyllactose (e.g, 2’-FL, 3-FL or DiFL) or 7V-containing (nonfucosylated) HMO (e.g., LNT or LNnT).

[35] In some embodiments, a process of this specification is used to prepare a purified fucosyllactose (also referred to as “FL”). At room temperature and pressure, a fucosyllactose is typically a white to ivory colored solid and soluble in water. In some embodiments, the purified fucosyllactose is 2’-FL. In some embodiments, the purified fucosyllactose is 3-FL. In some embodiments, a process of this specification is used to prepare a purified HMO mixture comprising a fucosyllactose. In some embodiments, a process of this specification is used to prepare a purified HMO mixture comprising 2’-FL, 3-FL or DiFL. In some embodiments, a process of this specification is used to prepare a purified HMO mixture comprising at least two fucosyllactoses. In some embodiments, a process of this specification is used to prepare a purified HMO mixture comprising 2 ’-FL and DiFL.

[36] In some embodiments, a process of this specification comprises preparing purified LNT. In some embodiments, the process of this specification is used to make a purified HMO mixture comprising LNT.

[37] In some embodiments, a process of this specification comprises preparing purified LNnT. In some embodiments, the process of this specification is used to make a purified HMO mixture comprising LNnT.

HMO Solution

[38] An “HMO solution” from which an HMO is purified in accordance with this specification generally comprises an aqueous medium. The aqueous medium comprises both the HMO and other ingredients, for example, monovalent and divalent salts, lactose, oligosaccharides (other than HMO), monosaccharides, amino acids, polypeptides, proteins, organic acids and nucleic acids.

[39] In some embodiments, the aqueous medium is water.

[40] In some embodiments, the HMO is selected from 2’-FL, 3-FL, LNT, DiFL, LNnT, lacto-N-fucopentaose, lacto-N-difucohexaose, lacto-N-neodifucohexaose, lacto-N- neooctaose, lacto-N-fucopentaose, lacto-N-neofucopentaose, LS-tetrasaccharide, lacto-N-triose, lacto-N-neo fucopentaose, lacto-N-neofucopentaose, lacto-N-difiicohexaose, 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, and lacto-N-neohexaose.

[41] In some embodiments, the HMO is a fucosyllactose.

[42] In some embodiments, the HMO is 2 ’-FL.

[43] In some embodiments, the HMO is 3 -FL.

[44] In some embodiments, the HMO is DiFL.

[45] In some embodiments, the HMO is LNnT.

[46] In some embodiments, the HMO is LNT.

[47] In some embodiments, the HMO solution comprises at least two HMOs. In some embodiments, the HMO solution comprises at least three HMOs. In some embodiments, the HMO solution comprises at least four HMOs. In some embodiments, the HMO solution comprises at least five HMOs.

[48] In some embodiments, the HMO solution comprises two or more HMOs selected from fucosyllactoses, LNnT and LNT. In some such embodiments, the fucosyllactoses are selected from 2 ’-FL, DiFL and 3 -FL.

[49] In some embodiments, the HMO solution comprises 2’-FL and 3-FL.

[50] In some embodiments, the HMO solution comprises 2’-FL and DiFL.

[51] Typically, the HMO solution further comprises one or more ingredients in addition to the HMO(s) to be purified. Such other ingredients may include, for example, monovalent and divalent salts, lactose, oligosaccharides, monosaccharides, amino acids, polypeptides, proteins, organic acids, nucleic acids, etc.

[52] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more additional HMOs and/or one or more other types of carbohydrates.

[53] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more oligosaccharides.

[54] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more additional HMOs.

[55] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more additional HMOs selected from 2 ’-FL, 3-FL, LNT, DiFL, LNnT, lacto-N-fucopentaose, lacto-N-difiicohexaose, lacto-N-neodifucohexaose, lacto-N-neooctaose, lacto-N-fucopentaose, lacto-N-neofucopentaose, LS-tetrasaccharide, lacto-N -triose, lacto-N- neo fucopentaose, lacto-N -neofucopentaose, lacto-N- difucohexaose, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N -hexaose, and lacto-N -neohexaose. [56] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) 2’-O-fucosyl lactulose.

[57] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) DiFL.

[58] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) lactose.

[59] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) lactulose.

[60] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more monosaccharides.

[61] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) fucose.

[62] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified and the second carbohydrate) glucose.

[63] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) galactose.

[64] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more monovalent salts.

[65] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more divalent salts.

[66] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more amino acids.

[67] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more proteins.

[68] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more organic acids.

[69] In some embodiments, the HMO solution comprises (in addition to the HMO(s) to be purified) one or more nucleic acids.

[70] In some embodiments, the HMO solution comprises (or is derived in whole or in part from) a product of a fermentation. In some such embodiments, the HMO solution is (or derived in whole or in part from) the product of a fermentation used to make the HMO(s) to be purified. In some such embodiments, the other carbohydrate(s) in the solution is/are from the culture medium used in the fermentation and/or formed during and/or after the fermentation. In some embodiments, the fermentation comprises culturing, in an aqueous culture medium comprising a carbohydrate (such as lactose and/or fucose), a recombinant microorganism comprising at least one recombinant polynucleotide sequence encoding an enzyme capable of producing an HMO. The product of the fermentation process may be referred to as a fermentation “product” or “broth.”

[71] The fermentation product typically comprises many ingredients in addition to the HMO(s) to be purified. Such ingredients may include, for example, monovalent and divalent salts, lactose, oligosaccharides, monosaccharides, amino acids, polypeptides, proteins, organic acids, nucleic acids, etc.

[72] In some embodiments, the fermentation product comprises one or more ingredients selected from divalent salts, lactose, oligosaccharides besides the HMO(s) to be purified, monosaccharides, amino acids, polypeptides, proteins, organic acids and nucleic acids. In some embodiments, the fermentation product comprises a divalent salt, lactose, an oligosaccharide besides the HMO(s) to be purified, a monosaccharide, an amino acid, a polypeptide, a protein, an organic acid and a nucleic acid.

[73] In some embodiments, the fermentation product comprises one or more ingredients selected from salts, acids, human milk oligosaccharides besides the HMO(s) to be purified, lactose and monomeric sugars. In some embodiments, the fermentation product comprises a salt, an acid, a human milk oligosaccharide besides the HMO(s) to be purified, lactose and a monomeric sugar.

[74] In some embodiments, an HMO to be purified is a fucosyllactose, and the HMO solution comprises (or is derived in whole or in part from) a product of a fermentation process wherein the fermentation process comprises culturing, in an aqueous culture medium comprising a carbohydrate (such as lactose and/or fucose), a recombinant microorganism comprising a recombinant polynucleotide sequence encoding an a-l,2-fucosyl transferase (EC 2.4.1.69) or a- 1,3-fucosyl transferase (EC 2.4.1.214).

[75] In general, when the HMO solution comprises (or is derived in whole or in part from) a product of a fermentation process, the process of this specification generally comprises one or more process steps wherein the cell biomass of the microorganisms used in the fermentation is separated from the fermentation product. In general, at least a portion of (or all) the cell mass is removed before the ion exchange disclosed in this specification.

[76] Cell biomass may be separated from a fermentation product using, for example, filtration, centrifugation, sedimentation and/or other process suitable for removing cell biomass.

[77] In some embodiments, separation of microorganisms from a fermentation product comprises ultrafiltration (also referred to as “UF”). Ultrafiltration can also be particularly beneficial to, for example, remove large biomolecules, such as endotoxins, proteins, nucleic acids and lipopolysaccharides.

[78] In some embodiments, the ultrafiltration is carried out using a cross-flow filtration. The polymeric membrane configuration used can be, for example, a spiral wound, hollow fiber or plate and frame unit. The ultrafiltration can also be carried out with tubular or ceramic disc membranes. Typically, the ultrafiltration membrane pore size can be chosen from about 0.1 to about 0.001 pm, or from about 200 kD to about 1 kD.

[79] In some embodiments, separation of microorganisms from a fermentation product comprises cross-flow microfiltration (also referred to as “MF”). Typically, the MF membrane pore size is from about 0.1 pm to about 3 pm. The polymeric membrane configuration used can be, for example, a spiral wound, hollow fiber or plate and frame unit. The cross-flow microfiltration can also be carried out with ceramic tubular or ceramic disc membranes. Furthermore, MF membranes made of steel can be used.

[80] In some embodiments, separation of microorganisms from a fermentation product comprises centrifugation. Typically, such a centrifugation may be carried out using disc stack separator reaching from about 3000 to about 20000 G-force. The clarified solution can be further purified with, for example, filtration technologies to obtain liquid essentially free of microbes.

[81] In some embodiments, the cell biomass removal is carried out at a temperature from about 5 °C to about 20°C.

[82] In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 18°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 16°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 16°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 15°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 15 °C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 10°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 10°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 9°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 9°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 8°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 8°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 7°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 7°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 6°C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 6°C. In some embodiments, the cell biomass removal is carried out at a temperature of no greater than about 5 °C. In some embodiments, the cell biomass removal is carried out at a temperature of less than about 5°C.

Ion exchange

[83] The process of this specification generally comprises passing the HMO solution through two ion exchange materials in series to form an initial ion exchange product, which, in turn, is passed through a mixed bed ion exchange comprising a combination of cation ion exchange material (e.g., resin) and anion ion exchange material (e.g., resin). One of the two initial ion exchange materials comprises cation ion exchange material (e.g., resin) and no anion ion exchange material. The other of the two initial ion exchange materials comprises anion ion exchange material (e.g., resin) and no cation ion exchange material.

[84] In some embodiments, the first of the two initial ion exchange materials comprises cation ion exchange material and no anion ion exchange material; and the second comprises anion exchange material and no cation exchange material.

[85] In some embodiments, the first of the two initial ion exchange materials comprises anion ion exchange material and no cation ion exchange material; and the second comprises cation exchange material and no anion exchange material.

[86] In some embodiments, the composition of the initial ion exchange product does not change before it enters the mixed bed ion exchange material.

[87] In some embodiments, the initial ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it enters the mixed bed ion exchange material. In some embodiments, no base or acid is added to the initial ion exchange product before it enters the mixed bed ion exchange material.

[88] Ion exchange is generally a reversible interchange of ions between a solid ion exchange material (or “ion exchanger”) and a liquid such as water. The ion exchange reaction typically occurs in an ion exchange vessel (often an ion exchange column), where a process solution is passed through the solid that facilitates the exchange of ions. There is generally no permanent change in the structure of the solid. Ion exchange is used in water treatment and also provides a method of separation in many non-water processes. It is widely used in chemical synthesis, medical research, food processing, mining, agriculture and a variety of other areas. [89] The ion exchange material is generally an insoluble solid material (often a specialized resin) which carries exchangeable cations or anions. The ions can be exchanged for a stoichiometrically equivalent number of other ions of the same electrical charge when the ion exchange material is in contact with an electrolyte solution. Carriers of exchangeable cations are called cation ion exchangers, and carriers of exchangeable anions are called anion ion exchangers. Ion exchange resins are polymers that are capable of exchanging ions with ions in a solution that is passed through them. Mixed bed ion exchange resin is a mixture of cation ion exchange resin and anion ion exchange resin.

[90] Strong acid cation (SAC) exchange resins may be, for example, polystyrene based resins with sulfonic acid as functional group.

[91] Weak acid cation (WAC) exchange resins may be, for example, polyacrylic based resins with formic acid as functional group.

[92] Strong base anion (SBA) exchange resins may be, for example, polystyrene or polyacrylic based resins. SBA resins are often categorized as Type 1 and Type 2, based on the functional group used. Type 1 resins generally have trimethylamine as functional group. Type 2 resins generally have dimethyl ethanolamine as a functional group.

[93] Weak base anion (WBA) exchange resins may be, for example, polystyrene or polyacrylic based resins with tertiary amine as a functional group.

[94] In some embodiments, the ion exchange is conducted after removal of cell biomass.

[95] In some embodiments, the ion exchange is conducted after an ultrafiltration step.

[96] In some embodiments, the ion exchange is conducted after a nanofiltration step.

[97] In some embodiments, the ion exchange is conducted before a nanofiltration step.

[98] In some embodiments, the ion exchange is conducted after an active carbon treatment step.

[99] In some embodiments, the ion exchange is conducted before an active carbon treatment step.

[100] In some embodiments, the ion exchange is conducted after an evaporation step.

[101] In some embodiments, the ion exchange is conducted before an evaporation step.

[102] In some embodiments, the ion exchange is conducted after an electrodialysis step.

[103] In some embodiments, the ion exchange is conducted before an electrodialysis step.

[104] In some embodiments, the ion exchange is conducted after an antifoam removal step. [105] In some embodiments, the ion exchange is conducted before an antifoam removal step.

[106] In some embodiments, the ion exchange is conducted after dissolving the HMO to be purified. In some such embodiments, the HMO to be purified with ion exchange comprises a previously crystalized or spray-dried HMO. Typically, when the ion exchange is conducted on a crystalline or spray dried HMO, the HMO is first dissolved, and then the resulting solution is passed through the ion exchange.

[107] In some embodiments, the ion exchange is conducted for reprocessing dissolved crystalline product.

[108] In some embodiments, the ion exchange is conducted for reprocessing dissolved spray dried product.

[109] The HMO solution fed into the the ion exchange may be selected from, for example, the fermentation broth after cell removal, a permeate from ultrafiltration, concentrate from nanofiltration, or the product of an active carbon treatment.

[HO] A final HMO product of the process disclosed herein may be, for example, a syrup, spray dried powder or crystalline product.

[111] In some embodiments, the ion exchange system disclosed herein is used to make a final HMO product.

[112] In general, the HMO purification process of this specification comprises passing the HMO solution through three ion exchange steps in series:

• a cation ion exchange, wherein the solution passes through cation ion exchange material in the absence of anion ion exchange material;

• an anion ion exchange, wherein the solution passes through anion ion exchange material in the absence of cation ion exchange material, and

• a mixed bed ion exchange, wherein the solution passes through a mixture of cation ion exchange material with anion ion exchange material.

In general, these steps are directly connected to each other in series with no other purification steps (e.g., enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying) in between. In some such embodiments, the order of the steps is: (1) the cation ion exchange, (2) the anion ion exchange, and (3) the mixed bed ion exchange. In other embodiments, the order of the steps is: (1) the anion ion exchange, (2) the cation ion exchange, and (3) the mixed bed ion exchange. Each ion exchange material typically comprises an ion exchange material (e.g., resin). In some embodiments, the above ion exchange steps are the only ion exchange steps in the HMO purification process. In other embodiments, the purification process comprises additional ion exchange steps.

[113] In some embodiments, the HMO purification process of this specification comprises passing the HMO solution through an ion exchange system comprising a series of ion exchange steps as follows:

• one or more cation ion exchange vessels comprising cation ion exchange material and no anion ion exchange material;

• one or more anion ion exchange vessels comprising anion ion exchange material an no cation ion exchange material, and

• one or more mixed bed ion exchange vessels comprising both cation ion exchange material and anion ion exchange material.

In general, no other types of purification steps (e.g., enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying) occur between the above-referenced ion exchange vessels. In some such embodiments, the HMO solution passes through the ion exchange system in the following order: (1) the cation ion exchange vessel(s), (2) the anion ion exchange vessel(s), and (3) the mixed bed ion exchange vessel(s). In other embodiments, the HMO solution passes through the ion exchange system in the following order: (1) the anion ion exchange vessel(s), (2) the cation ion exchange vessel(s), and (3) the mixed bed ion exchange vessel(s). When more than one cation ion exchange vessel is used, the cation ion exchange vessels may be in series and/or in parallel. When more than one anion ion exchange vessel is used, the anion ion exchange vessels may be in series and/or in parallel. When more than one mixed bed ion exchange vessel is used, the mixed bed ion exchange vessels may be in series and/or in parallel. Each ion exchange material typically comprises an ion exchange resin. In some embodiments, the above-referenced ion exchange vessels are the only ion exchange vessels used in the HMO purification process. In other embodiments, the purification process comprises additional ion exchange vessels.

[114] In some embodiments, one or more ion exchange vessel(s) is/are used in addition to the above-descussed ion exchange system. In some such embodiments, one or more additional ion exchange vessel(s) is/are located downstream of the above-discussed ion exchange system. In some such embodiments, an additional ion exchange vessel is connected directly to a mixed bed ion exchange vessel of the ion exchange system. In other embodiments, an additional downstream ion exchange vessel(s) is/are separated from the ion exchange system by other purification steps (e.g., nanofiltration, electrodialysis, chromatography, antifoam removal, activated carbon, sterile filtration, crystallization, spray-drying and evaporation). When more than one additional ion exchange vessels are used, they may be in parallel with each other, connected in series to each other, and/or in series but separated from each other by one or more other purification steps (e.g., nanofiltration, electrodialysis, chromatography, antifoam removal, activated carbon, sterile filtration, crystallization, spray-drying and evaporation).

[115] In some embodiments, the process comprises the use of an additional cation ion exchange vessel comprising cation ion exchange resin and no anion ion exchange resin.

[116] In some embodiments, the process comprises the use of an additional anion ion exchange vessel comprising anion ion exchange resin and no cation ion exchange resin.

[117] In some embodiments, the process comprises the use of both (1) an additional cation ion exchange vessel comprising cation ion exchange resin and no anion ion exchange resin, and (2) an additional cation ion exchange vessel comprising cation ion exchange resin and no anion ion exchange resin. In such embodiments, the additional cation ion exchange vessel and additional anion ion exchange vessel are typically in series. In some such embodiments, the additional cation ion exchange vessel is first in the series. In other such embodiments, the additional anion ion exchange vessel is first in the series.

[118] In some embodiments, the process comprises pretreatment ion exchange, which occurs before (in some embodiments, immediately before) the above-referenced ion exchange system. In some embodiments, the pretreatment ion exchange comprises passing the HMO solution through (i) cation ion exchange material in the absence of anion ion exchange material, and (ii) anion ion exchange material in the absence of cation ion exchange material. In some such embodiments, the pretreatment ion exchange comprises passing the HMO solution through cation ion exchange material and then through anion ion exchange material. In other such embodiments, the pretreatment ion exchange comprises passing the HMO solution through anion ion exchange material and then through cation ion exchange material. In some embodiments, the composition of the product from the first pretreatment ion exchange material is the same as the composition fed into the second pretreatment ion exchange material. In some embodiments, the composition of the product from the first pretreatment ion exchange material is not subjected to other purification steps (e.g., nanofiltration, electrodialysis, chromatography, antifoam removal, activated carbon, sterile filtration, crystallization, spray-drying and evaporation) before being fed into the second pretreatment ion exchange material. In some embodiments, the product of the pretreatment ion exchange is fed directly into the abovediscussed ion exchange system. [119] In some embodiments, at least one additional mixed bed ion exchange vessel is used. In some embodiments, two additional mixed bed ion exchange vessels are used. In some embodiments, three or more mixed bed ion exchange vessels are used.

[120] In some embodiments, the cation ion exchange resin and anion ion exchange resin to be used in a mixed bed ion exchange vessel are mixed before packing into the mixed bed ion exchange vessel (e.g., column). When the resin is packed together as a mixture, it may be mixed before packing into the mixed bed ion exchange column from a selected cation ion exchange resin and selected anion ion exchange resin in a selected volume ratio. In some embodiments, the mixture that is packed into the mixed bed ion exchange vessel is a uniform mixture. Mixed bed ion exchange resins are also available as a ready-mixed resin, for example, AMBERTEC™ UP6040 by DuPont.

[121] In some embodiments, the cation ion exchange resin and anion ion exchange resin to be used in a mixed bed ion exchange vessel are packed into the mixed bed ion exchange vessel (e.g., column) in alternating layers. In some embodiments, the alternating layers have the same volume. In other embodiments, the alternating layers have different volumes. In some embodiments, the cation ion exchange resin and anion ion exchange resin are packed into a mixed bed ion exchange column in 6 or more alternating layers. In some embodiments, the cation ion exchange resin and anion ion exchange resin are packed into a mixed bed ion exchange column in 30 or more alternating layers. In some embodiments, the cation ion exchange resin and anion ion exchange resin are packed into a mixed bed ion exchange column in 100 or more alternating layers.

[122] In some embodiments, the volume ratio of cation ion exchange material (e.g., resin) to anion ion exchange material (e.g., resin) used in a mixed bed ion exchange is from about 10:90 to about 90:10. In some embodiments, the ratio is from about 30:70 to about 70:30. In some embodiments, the ratio is from about 20:80 to about 80:20. In some embodiments, the ratio is from about 40:60 to about 60:40. In some embodiments, the ratio is about 50:50. In some embodiments, the ratio is selected based on the properties of the feed liquor fed to the ion exchange system.

[123] In some embodiments, the mixed bed ion exchange vessel is packed with strong acid cation (SAC) and strong base anion (SBA) ion exchange resins. In some embodiments, SAC:SBA resin volume ratio is from about 10:90 to about 90:10. . In some embodiments, SAC:SBA resin volume ratio is from about 30:70 to about 70:30. In some embodiments, SAC:SBA resin volume ratio is from about 20:80 to about 80:20. In some embodiments, SAC:SBA resin volume ratio is from about 40:60 to about 60:40. In some embodiments, SAC:SBA resin volume ratio is about 50:50. In some embodiments, resin volume ratio is selected based on the properties of the feed liquor fed to the ion exchange system.

[124] In some embodiments, mixed ion exchange vessel is packed with strong acid cation (SAC) and weak base anion (WBA) ion exchange resins. In some embodiments, SAC:WBA resin volume ratio is from about 10:90 to about 90:10. In some embodiments, SAC:WBA resin volume ratio is from about 30:70 to about 70:30. In some embodiments, SAC:WBA resin volume ratio is from about 20:80 to about 80:20. In some embodiments, SAC:WBA resin volume ratio is from about 40:60 to about 60:40. In some embodiments, SAC:WBA resin volume ratio is about 50:50. In some embodiments, resin volume ratio is selected based on the properties of the feed liquor fed to the ion exchange system.

[125] In some embodiments, the mixed bed ion exchange vessel is packed with strong acid cation (WAC) and strong base anion (WBA) ion exchange resins. In some embodiments, WAC:WBA resin volume ratio is from about 10:90 to about 90:10. . In some embodiments, WAC:WBA resin volume ratio is from about 30:70 to about 70:30. In some embodiments, WAC:WBA resin volume ratio is from about 20:80 to about 80:20. In some embodiments, WAC:WBA resin volume ratio is from about 40:60 to about 60:40. In some embodiments, WAC:WBA resin volume ratio is about 50:50. In some embodiments, resin volume ratio is selected based on the properties of the feed liquor fed to the ion exchange system.

[126] In some embodiments, the mixed bed ion exchange vessel is packed with strong acid cation (WAC) and strong base anion (SBA) ion exchange resins. In some embodiments, WAC:SBA resin volume ratio is from about 10:90 to about 90:10. . In some embodiments, WAC:SBA resin volume ratio is from about 30:70 to about 70:30. In some embodiments, WAC:SBA resin volume ratio is from about 20:80 to about 80:20. In some embodiments, WAC:SBA resin volume ratio is from about 40:60 to about 60:40. In some embodiments, WAC:SBA resin volume ratio is about 50:50. In some embodiments, resin volume ratio is selected based on the properties of the feed liquor fed to the ion exchange system.

[127] In some embodiments, the resin in the cation ion exchange vessel comprises an SAC resin.

[128] In some embodiments, the resin in the cation ion exchange vessel comprises an SAC resin in the H⁺-ion form.

[129] In some embodiments, the resin in the cation ion exchange vessel comprises an SAC resin in the Na⁺-ion form.

[130] In some embodiments, the resin in the cation ion exchange vessel comprises an

WAC resin. [131] In some embodiments, the resin in the cation ion exchange vessel comprises an WAC resin in the H⁺-ion form.

[132] In some embodiments, the resin in the cation ion exchange vessel comprises an WAC resin in the Na⁺-ion form.

[133] In some embodiments, the resin in the anion ion exchange vessel comprises a WBA resin.

[134] In some embodiments, the resin in the anion ion exchange vessel comprises a WBA resin in the OH" -ion form (also referred to as free base form).

[135] In some embodiments, the resin in the anion ion exchange vessel comprises a WBA resin in the Cl'-ion form.

[136] In some embodiments, the resin in the anion ion exchange vessel comprises an SBA resin.

[137] In some embodiments, the resin in the anion ion exchange vessel comprises an SBA resin in the OH" -ion form (also referred to as free base form).

[138] In some embodiments, the resin in the anion ion exchange vessel comprises an SBA resin in the Cl'-ion form.

[139] In some embodiments, the cation ion exchange resin in the mixed bed ion exchange vessel comprises an SAC resin.

[140] In some embodiments, the cation ion exchange resin in the mixed bed ion exchange vessel comprises an SAC resin in the H⁺-ion form.

[141] In some embodiments, the cation ion exchange resin in the mixed bed ion exchange vessel comprises an SAC resin in the Na⁺-ion form.

[142] In some embodiments, the cation ion exchange resin in the mixed bed exchange vessel comprises an WAC resin.

[143] In some embodiments, the cation ion exchange resin in the mixed bed exchange vessel comprises an WAC resin in the H⁺-ion form.

[144] In some embodiments, the cation ion exchange resin in the mixed bed exchange vessel comprises an WAC resin in the Na⁺-ion form.

[145] In some embodiments, the anion ion exchange resin in the mixed bed exchange vessel comprises a WBA resin.

[146] In some embodiments, the anion ion exchange resin in the mixed bed exchange vessel comprises a WBA resin in the OH" -ion form (also referred to as free base form).

[147] In some embodiments, the anion ion exchange resin in the mixed bed exchange vessel comprises a WBA resin in the Cl'-ion form. [148] In some embodiments, the anion ion exchange resin in the mixed bed exchange vessel comprises an SBA resin.

[149] In some embodiments, the anion ion exchange resin in the mixed bed exchange vessel comprises an SBA resin in the OH" -ion form (also referred to as free base form).

[150] In some embodiments, the anion ion exchange resin in the mixed bed ion exchange vessel comprises an SBA resin in the Cl'-ion form.

[151] In some embodiments, the cation ion exchange material used in the cation ion exchange step is the same as the cation ion exchange material used in the mixed bed ion exchange step. In some embodiments, the cation ion exchange material used in the cation ion exchange step is different from the cation ion exchange material used in the mixed bed ion exchange step.

[152] In some embodiments, the anion ion exchange material used in the anion ion exchange step is the same as the anion ion exchange material used in the mixed bed ion exchange step. In some embodiments, the anion ion exchange material used in the anion ion exchange step is different from the anion ion exchange material used in the mixed bed ion exchange step.

[153] In some embodiments, the cation ion exchange material used in the cation ion exchange step comprises SAC, the anion ion exchange material used in the anion ion exchange step comprises WBA, the cation ion exchange material used in the mixed bed ion exchange step comprises SAC, and the anion ion exchange material used in the mixed bed ion exchange step comprises SBA.

[154] In some embodiments, the cation ion exchange material used in the cation ion exchange step comprises SAC, the anion ion exchange material used in the anion ion exchange step comprises WBA, the cation ion exchange material used in the mixed bed ion exchange step comprises SAC, and the anion ion exchange material used in the mixed bed ion exchange step comprises WBA.

[155] HMOs have varying stabilities. In general, HMO stability is dependent on pH. Typically, stability of an HMO solution is better in a slightly acidic (at a pH of from about 4.5 to about 6) or neutral (at about pH 7) pH range.

[156] In some embodiments, a mixed bed ion exchange is used for adjusting pH of the HMO solution. In some embodiments, pH adjustment of the process stream with acid or alkali addition is avoided by using the mixed bed ion exchanger to adjust the pH. In some embodiments, mixed bed ion exchange is used to neutralize the HMO solution. [157] In some embodiments, the pH of the HMO stream at the exit of the mixed bed ion exchange vessel is from about 4.5 to about 7. In some embodiments, the pH of the HMO stream at the exit of the mixed bed ion exchange vessel is from about 4.5 to about 6. In some embodiments, the pH of the HMO stream at the exit of the mixed bed ion exchange vessel is from about 6 to about 7.

[158] HMO stability is also generally dependent on temperature. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 0°C to about 60°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 5 °C to about room temperature. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 5 °C to about 25°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 5°C to about 20°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 0°C to about 10°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is from about 5°C to about 10°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is about 10°C. In some embodiments, the temperature during at least one ion exchange step (and, in some embodiments, all ion exchange steps) is about 5°C.

[159] In some embodiments, the dry substance concentration in the HMO solution is from about 3 to about 65 g/100 g when the solution is fed into the ion exchange system described herein. In some embodiments, the dry substance concentration in the HMO solution is from about 3 to about 60 g/100 g when the solution is fed into the ion exchange system described herein. In some embodiments, the dry substance concentration in the HMO solution is from about 3 to about 50 g/100 g when the solution is fed into the ion exchange system described herein. In some embodiments, the dry substance concentration in the HMO solution is from about 12 to about 20 g/100 g when the solution is fed into the ion exchange system described herein. In some embodiments, the dry substance concentration in the HMO solution is from about 3 to about 30 g/100 g when the solution is fed into the ion exchange system described herein. In some embodiments, the dry substance concentration in the HMO solution is from about 5 to about 50 g/100 g when the solution is fed into the ion exchange system described herein. [160] In some embodiments, the flowrate through the mixed bed ion exchange vessel (e.g., column) is from about 0.5 BV7h to about 10 BV/h or greater. In some embodiments, the flowrate through the mixed bed ion exchange vessel is from about 2 BV/h to about 5 BV/h. In some embodiments, the flowrate through the mixed bed ion exchange vessel is from about 2 BV/h to about 3 BV/h. In some embodiments, the flowrate through the mixed bed ion exchange vessel is about 2 BV/h. In some embodiments, the flowrate through the mixed bed ion exchange vessel is about 2.5 BV/h. In some embodiments, the flowrate through the mixed bed ion exchange vessel is about 3 BV/h.

[161] In some embodiments, the HMO yield using a process of this specification is greater than 80%. In some embodiments, the HMO yield is greater than 85%. In some embodiments, the HMO yield is greater than 90%. In some embodiments, the HMO yield is greater than 95%. In some embodiments, the HMO yield is greater than 97%.

[162] Cationic compounds, anionic compounds and color and conductivity can generally be efficiently removed (or at least diminished) by using an ion exchange system disclosed in this specification. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that falls outside the desired product specification. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that has too high pH. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that has too low pH. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that has too much color. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that contains microbial contaminants. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that has too high conductivity. In some embodiments, the ion exchange system disclosed in this specification is used to reprocess an HMO product that has too high salt concentration.

Additional treatments

[163] In some embodiments, the HMO purification process comprises subjecting the HMO solution to one or more of the following treatments: an enzymatic treatment (e.g., enzymatic hydrolysis of lactose), ultrafiltration, nanofiltration, electrodialysis, chromatography, antifoam removal, activated carbon, sterile filtration, crystallization, evaporation and/or spraydrying. [164] The additional treatments may typically be carried out in various orders, as well as being repeated at different points in the process. In some embodiments, the process comprises a combination of at least three of the above additional treatments. In some embodiments, the process comprises a combination of at least four of the above additional treatments.

[165] In some embodiments, the HMO solution is subjected to nanofiltration. In some embodiments, the nanofiltration is carried out under conditions discussed in W02020/154565 (incorporated by reference into this specification).

[166] In some embodiments, the HMO solution is subjected to an antifoam removal step. In some embodiments, the antifoam removal is carried out under conditions discussed in PCT/US20/48379 (incorporated by reference into this specification).

[167] In some embodiments, the HMO solution is subjected to evaporation. This can be helpful, for example, to concentrate the HMO by removing a solvent (e.g., water). In some embodiments, evaporation is the final purification step of the desired HMO.

[168] In some embodiments, the HMO solution is subjected to spray drying. In some embodiments, the spray-drying is carried out under conditions discussed in WO2019/160922 (incorporated by reference into this specification). In some embodiments, spray-drying is the final purification step for the desired HMO.

[169] In some embodiments, the process comprises crystallization. In some embodiments, no organic solvent is used during the crystallization. In some embodiments, the crystallization comprises a crystallization process disclosed in WO2018/164937 (incorporated by reference into this specification). In some embodiments, crystallization is the final purification step of the desired HMO. In some embodiments, the process comprises both crystallization and evaporation. In some embodiments, the process comprises both crystallization and spray-drying.

[170] In some embodiments, no base or acid is added to the HMO solution downstream of the ion exchange system before the HMO solution is passed through an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying. In some embodiments, no base or acid is added to the human milk oligosaccharide downstream of the ion exchange system.

EXAMPLES

[171] The following examples are merely illustrative, and not limiting to the remainder of this specification in any way. [172] Color of the HMO solutions was measured at room temperature.

Example 1

2’FL ion exchange with SAC + WBA + MB

[173] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars was fed into a 2-step ion exchange process including SAC, WBA and SBA resins. In the first step, the solution was treated with SAC and WBA resins in separate ion exchange columns connected in series. In the second step, the solution was treated with SAC and SBA resins in one mixed bed ion exchange column. The SAC resin was Dowex88, the WBA resin was Dowex66, and the SBA resin was Dowex22.

[174] Before the ion exchange process, the Dowex88 strong acid cation ion exchange resin (SAC) was regenerated with 5% sulfuric acid solution to the H⁺ form, the Dowex66 weak base anion resin (WBA) was regenerated with 4% NaOH solution to the free base form, and the Dowex22 strong base anion resin (SBA) was regenerated with 4% NaOH solution to the OH" form. After each resin regeneration step, the resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. The SAC and SBA resins for the mixed bed ion exchange column were also properly mixed after resin regeneration and flushed with water to ensure good performance.

[175] The first step of the ion exchange process included two ion exchange columns in series: one ion exchange column containing about 1000 liters of SAC resin and one ion exchange column containing about 1000 liters of WBA resin. The second step of the ion exchange process included about 200 liters of SAC resin and about 200 liters of SBA resin in one mixed bed ion exchange column.

[176] In the first step of the ion exchange process, about 2,419 kg of feed solution containing about 278 kg of 2’FL was fed at 2,500 1/h to the ion exchange column series at a temperature of about 10°C. For the second ion exchange step, products from three first step ion exchange cycles were combined, and about 10,813 kg of feed solution containing about 769 kg 2’FL was fed at 2,500 1/h to the mixed bed ion exchange column at a temperature of about 10°C.

[177] Properties of the first step ion exchange feed are shown in Table 1-1. TABLE 1-1: Properties of the first step ion exchange feed

[178] Properties of the first step ion exchange product (the second ion exchange step feed) are shown in Table 1-2.

TABLE 1-2: Properties of the first step ion exchange product

[179] Properties of the final product after both steps of the ion exchange process are shown in Table 1-3, wherein the HPLC analyses are given on peak area-% basis.

TABLE 1-3: Properties of the product after two step ion exchange process

[180] In the 2-step ion exchange process, about 11,000 kg of mixed bed ion exchange column product containing about 750 kg of 2’FL was produced. Overall, 2’FL yield over the ion exchange process, including both steps, was about 90%.

Example 2 2’FL ion exchange with SAC + WBA + MB

[181] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars was fed into an exchange process including SAC, WBA and SBA resins. The solution was treated with an ion exchange system including SAC, WBA and mixed bed ion exchange columns connected in series. The SAC resin was Dowex88, the WBA resin was Dowex66, and the SBA resin was Dowex22.

[182] Before the ion exchange process, the Dowex88 strong acid cation resin in the SAC ion exchange column was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex66 resin in the WBA ion exchange column was regenerated with 4% NaOH solution to the free base form. Resins in the mixed bed ion exchange column were already used for 7 similar ion exchange cycles after regeneration of the SAC resin with 5% sulfuric acid to the H⁺ form and regeneration of SBA resin with 4% NaOH to the OH" form. After each resin regeneration step, all resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. The SAC and SBA resins in the mixed bed ion exchange column were also properly mixed after resin regeneration and flushed with water to ensure good performance.

[183] The ion exchange process included one ion exchange column containing about 1000 liters of SAC resin, one ion exchange column containing about 1000 liters of WBA resin, and a mixed bed ion exchange column containing about 200 liters of SAC resin and about 200 liters of SBA resin.

[184] In the ion exchange process, about 5,960 kg of feed solution containing about 888 kg of 2’FL was fed at 2,500 1/h to the ion exchange column series at temperature of about 10°C.

[185] Properties of the ion exchange feed are shown in Table 2-1. TABLE 2-1: Properties of the ion exchange feed

[186] Properties of the product after ion exchange are shown in Table 2-2, whereby the HPLC analyses are given on peak area-% basis.

TABLE 2-2: Properties of the product after ion exchange

[187] In the ion exchange process, about 7,175 kg of mixed bed product containing about 862 kg of 2’FL was produced. Overall, the 2’FL yield over the ion exchange process was about 97%.

Example 3

2’FL purification using SAC + WBA + MB (with alternating resin layers)

[188] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars was fed to a 2-step ion exchange process including SAC, WBA and SBA resins. In the first step, the solution was treated with SAC and WBA resins in separate ion exchange columns connected in series. In the second step, the solution was treated with SAC and SBA resins in one mixed bed ion exchange column. The SAC resin was Dowex88, the WBA resin was Dowex66, and the SBA resin was Dowex22. [189] Before the ion exchange process, the Dowex88 resin in the SAC ion exchange column was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex66 resin in the WBA ion exchange column was regenerated with 4% NaOH solution to the free base form. The Dowex 88 resin for the MB ion exchange column was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex 22 resin for the MB ion exchange column was regenerated with 4% NaOH solution to the OH" form. After each resin regeneration step, the resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. The Dowex 88 and Dowex 22 resins were packed into the MB ion exchange column in alternating layers of the same volume.

[190] The first step of the ion exchange process included one ion exchange column containing 1,000 mL of SAC resin, and one ion exchange column containing 1,000 mL of WBA resin. The flow rate in the first step was 2,500 mL/h (2.5 BV7h). The first step was conducted twice to produce enough material for the second ion exchange step. The SAC and WBA resins were regenerated in between the two cycles. The second step of the ion exchange process included one mixed bed ion exchange column containing 50 mL of SAC resin and 50 mL of SBA resin. The flow rate in the second step was 1,000 mL/h (10 BV/h). A temperature of 10°C was used in both ion exchange steps.

[191] The properties of the feed solution for the SAC and WBA ion exchange column series are shown in Table 3-1. The properties of the outlet solution for the first cycle of the SAC and WBA ion exchange column series are shown in Table 3-2, and the properties of the outlet solution from the second cycle of the SAC and WBA ion exchange column series are shown in Table 3-3.

TABLE 3-1: Properties of the SAC + WBA ion exchange system feed

TABLE 3-2: Properties of SAC + WBA ion exchange system cycle 1 outlet.

TABLE 3-3: Properties of SAC + WBA ion exchange system cycle 2 outlet.

[192] The maximum cycle length in both SAC + WBA cycles was approximately 11 BV. In the first cycle, 99.9% of the conductivity was removed, while in the second cycle, 97.0% of the conductivity was removed. The products from SAC + WBA cycle 1 and cycle 2 were fed into the MB ion exchange column one after the other. The MB ion exchange column was flushed with IEX water in between. The properties of the outlet solution from MB ion exchange column are shown in Table 3-4. Maximum cycle length of approximately 175 BV was reached in MB ion exchange column. At this point, a clear conductivity breakthrough from the ion exchange column was observed. The pH of the combined product within 0-175 BV was 6.05. TABLE 3-4: Properties of mixed bed ion exchange column outlet

Example 4

2’FL purification using MB column compared to granular carbon column

[193] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars, which was first treated with SAC and WBA resins in separate ion exchange columns connected in series, was treated with two different purification systems. The first purification system included a single mixed bed ion exchange column with SAC and SBA resins. The second purification system included a single column with activated carbon granule. The SAC resin was Dowex88, and the SBA resin was Dowex22. The activated carbon was CHEMVIRON CPG.

[194] Before the ion exchange process, the Dowex88 resin was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex22 resin was regenerated with 4% NaOH solution to the OH" form. After each resin regeneration step, both resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. In the first purification system, 250 mL of Dowex88 resin and 250 mL of Dowex22 resin were packed into a single MB ion exchange column in alternating layers of the same volume. In the second purification system, 500 mL of CHEMVIRON CPG activated carbon was packed into a single column. A temperature of less than 25°C was used in both systems.

[195] The properties of the purification feed solution are shown in Table 4-1. The same feed solution was used in both purification systems. The properties of the outlet solution from the 250 mL SAC + 250 mL SBA mixed bed ion exchange system are shown in Table 4-2, and the properties of the outlet solution from the activated carbon purification system are shown in Table 4-3.

TABLE 4-1: Properties of the purification feed solution

TABLE 4-2: Properties of SAC + SBA mixed bed ion exchange system outlet

TABLE 4-3: Properties of activated carbon granule purification system outlet

[196] Both purification methods were shown to adjust pH to between 6 and 7, and to remove 96-98% of the color. In addition, the mixed bed ion exchange system was also shown to remove conductivity. Conductivity removal was not shown with the activated carbon system. Instead, a slight increase in conductivity was shown.

Example 5 2’FL purification with a single MB column with different SAC + SBA resin ratios

[197] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars, which was first treated with SAC and WBA resins in separate ion exchange columns connected in series, was treated with two different ion exchange process systems including SAC and SBA resins. The ion exchange systems included a single mixed bed ion exchange column, but with different ratios of SAC and SBA resins. The SAC resin was Dowex88, and the SBA resin was Dowex22.

[198] Before the ion exchange process, the Dowex88 resin was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex22 resin was regenerated with 4% NaOH solution to the OH" form. After each resin regeneration step, the resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. In the first ion exchange system, 25 mL of Dowex88 resin and 25 mL of Dowex22 resin were packed into the MB ion exchange column in alternating layers of the same volume. In the second ion exchange system, 15 mL of Dowex88 and 35 mL of Dowex22 were packed into the MB ion exchange column in alternating layers of different volume. The flow rate in both ion exchange systems was 300 mL/h (6 BV/h). A temperature of less than 25°C was used in both ion exchange systems.

[199] The properties of the ion exchange feed solution are shown in Table 5-1. The same feed solution was used in both ion exchange systems. The properties of the outlet solution from the 25 mL SAC + 25 mL SB A mixed bed ion exchange system are shown in Table 5-2, and the properties of the outlet solution from 15 mL SAC + 35 mL SB A mixed bed ion exchange system are shown in Table 5-3.

TABLE 5-1: Properties of the ion exchange feed solution

TABLE 5-2: Properties of SAC + SBA 50/50 volume ratio mixed bed ion exchange outlet

TABLE 5-3: Properties of SAC + SBA 30/70 volume ratio mixed bed ion exchange outlet

[200] The mixed bed ion exchange column with the SAC and SBA resins in a volume ratio of 30/70 showed greater capacity for the ion exchange of the feed solution compared to the mixed bed ion exchange column with the SAC and SBA resins in a volume ratio of 50/50. Approximately 4 BV cycle length was reached with the SAC + SBA 50/50 volume ratio mixed bed ion exchange system, while approximately 8 BV cycle length was reached with the SAC + SBA 30/70 volume ratio mixed bed ion exchange system. After these points, a clear conductivity and color breakthrough from the ion exchange column was observed.

Example 6

2’FL purification with a single MB column with SAC + SBA or SAC + WBA

[201] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars, which was first treated with SAC and WBA resins in separate ion exchange columns connected in series, was treated with two different ion exchange process systems including SAC, WBA and SBA resins. Both ion exchange systems included a single mixed bed ion exchange column with different resins. The SAC resin was Dowex88, the WBA resin was Dowex66, and the SBA resin was Dowex22.

[202] Before the ion exchange process, the Dowex88 resin was regenerated with 5% sulfuric acid solution to the H⁺ form, the Dowex66 resin was regenerated with 4% NaOH solution to the free base form, and the Dowex22 resin was regenerated with 4% NaOH solution to the OH" form. After each resin regeneration step, the resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. In the first ion exchange system, 25 mL of the Dowex88 resin and 25 mL of the Dowex22 resin were packed into the MB ion exchange column in alternating layers. In the second ion exchange system, 25 mL of the Dowex88 and 25 mL of the Dowex66 were packed into the MB ion exchange column in alternating layers. The flow rate in both ion exchange systems was 150 mL/h (3 BV/h). A temperature of less than 25°C was used in both ion exchange systems.

[203] The properties of the ion exchange feed solution are shown in Table 6-1. The same feed solution was used in both ion exchange systems. The properties of the outlet solution from the 25 mL SAC + 25 mL SB A mixed bed ion exchange system are shown in Table 6-2, and the properties of the outlet solution from the 25 mL SAC + 25 mL WBA mixed bed ion exchange system are shown in Table 6-3. TABLE 6-1: Properties of the ion exchange feed solution

TABLE 6-2: Properties of SAC + SBA mixed bed ion exchange system outlet

TABLE 6-3: Properties of SAC + WBA mixed bed ion exchange system outlet

[204] Both ion exchange systems were shown to adjust pH closer to neutral and lower the conductivity within their capacity range. Approximately 8 BV cycle length was reached with the SAC + SBA mixed bed ion exchange system, while approximately 4 BV cycle length was reached with the SAC + WBA mixed bed ion exchange system. After these points, a clear conductivity and color breakthrough from the ion exchange column was observed. Example ?

3FL ion exchange with SAC + WBA + MB

[205] A fermentation-based solution containing various salts, acids, color, human milk oligosaccharides, lactose and monomeric sugars was treated with an ion exchange process system including SAC, WBA and SBA resins. The ion exchange system included SAC, WBA and mixed bed ion exchange columns connected in series. The SAC resin was Dowex88, the WBA resin was Dowex66, and the SBA resin was Dowex22.

[206] Before the ion exchange process, the Dowex88 strong acid cation resin in the SAC ion exchange column was regenerated with 5% sulfuric acid solution to the H⁺ form, and the Dowex66 resin in the WBA ion exchange column was regenerated with 4% NaOH solution to the OH" form. The resins in the mixed bed ion exchange column were regenerated separately. The SAC resin was regenerated with 5% sulfuric acid to the H⁺ form, and the SBA resin was regenerated with 4% NaOH to the OH" form. After each resin regeneration step, the resins were flushed with water to remove excess regeneration chemicals before the ion exchange process. The regenerated resins for the mixed bed were packed into a single ion exchange column.

[207] The ion exchange process included one ion exchange column containing 220 mL of SAC resin, one ion exchange column containing 200 mL of WBA resin, and one mixed bed ion exchange column containing 40 mL of SAC resin and 40 mL of SBA resin.

[208] In the ion exchange process, the feed solution containing 3-FL was fed at 500 mL/h flow to the ion exchange column series in varying temperatures. The temperatures were 5 and 10°C. The properties of the ion exchange feed are shown in Table 7-1.

TABLE 7-1: Properties of the ion exchange feed

[209] The products were collected in 200 mL fractions. Properties of the product fractions after ion exchange at 5°C are shown in Table 7-2.

TABLE 7-2: Properties of the product after ion exchange at 5°C

[210] The product fractions after ion exchange at 10°C are shown in Table 7-3.

TABLE 7-3: Properties of the product after ion exchange at 10°C

Claims

We claim:

1. A process for preparing a purified human milk oligosaccharide (HMO) from an HMO solution derived from a fermentation process, wherein: the process comprises:

(a) passing the HMO solution through a first ion exchange material to form a first ion exchange product,

(b) passing the first ion exchange product through a second ion exchange material to form a second ion exchange product, and

(c) passing the second ion exchange product through a mixed bed ion exchange comprising both cation ion exchange material and anion ion exchange material; one of the first and second ion exchange materials comprises cation ion exchange material and no anion ion exchange material; the other of the first and second ion exchange materials comprises anion ion exchange material and no cation ion exchange material; the composition of the first ion exchange product does not change before it is passed through the second ion exchange material; and the composition of the second ion exchange product does not change before it is passed through the mixed bed ion exchange.

2. A process for preparing a purified human milk oligosaccharide (HMO) from an HMO solution derived from a fermentation process, wherein: the process comprises:

(c) passing the second ion exchange product through a mixed bed ion exchange comprising both cation ion exchange material and anion ion exchange material; one of the first and second ion exchange materials comprises cation ion exchange material and no anion ion exchange material; the other of the first and second ion exchange materials comprises anion ion exchange material and no cation ion exchange material;

37 the first ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it is passed through second ion exchange material; and the second ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it is passed through the mixed bed ion exchange.

3. A process according to claim 2, wherein no base or acid is added to the second ion exchange product before it is passed through the mixed bed ion exchange.

4. A process according to any one of the preceding claims, wherein the first ion exchange material comprises cation ion exchange material and no anion ion exchange material.

5. A process according to any one of claims 1-3, wherein the first ion exchange material comprises anion ion exchange material and no cation ion exchange material.

6. A process according to any one of the preceding claims, wherein the mixed bed ion exchange comprises an ion exchange column packed with a uniform mixture of cation ion exchange resin and anion ion exchange resin.

7. The process according to any one of claims 1-5, wherein the mixed bed ion exchange comprises an ion exchange column packed with alternating layers of anion ion exchange resin and cation ion exchange resin.

8. The process according to claim 7, wherein the alternating layers each have the same volume.

9. The process according to any one of the preceding claims, wherein the anion ion exchange material used in Step (a) or Step (b) comprises a strong base anion (SBA) exchange resin.

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10. The process according to any one of claims 1-8, wherein the anion ion exchange material used in Step (a) or Step (b) comprises a weak base anion (WBA) exchange resin.

11. The process according to any one of the preceding claims, wherein the cation ion exchange material used in Step (a) or Step (b) comprises a strong acid cation (SAC) exchange resin.

12. The process according to any one of claims 1-10, wherein the cation ion exchange material used in Step (a) or Step (b) comprises a weak acid cation (WAC) exchange resin.

13. The process according to any one of the preceding claims, wherein: the cation ion exchange material in the mixed bed ion exchange comprises a strong acid cation (SAC) exchange resin, and the anion ion exchange material in the mixed bed ion exchange comprises a strong base anion (SBA) exchange resin.

14. The process according to any one of claims 1-12, wherein: the cation ion exchange material in the mixed bed ion exchange comprises a strong acid cation (SAC) exchange resin, and the anion ion exchange material in the mixed bed ion exchange comprises a weak base anion (WBA) exchange resin.

15. The process according to any one of claims 1-12, wherein: the cation ion exchange material in the mixed bed ion exchange comprises a weak acid cation (WAC) exchange resin, and the anion ion exchange material in the mixed bed ion exchange comprises a weak base anion (WBA) exchange resin.

16. The process according to any one of claims 1-12, wherein: the cation ion exchange material in the mixed bed ion exchange comprises a weak acid cation (WAC) exchange resin, and the anion ion exchange material in the mixed bed ion exchange comprises a strong base anion (SBA) exchange resin.

17. The process according to any one of the preceding claims, wherein: the mixed bed ion exchange is carried out in at least one ion exchange column comprising cation ion exchange resin and anion ion exchange resin, and the cation ion exchange resin and anion ion exchange resin are present in the at least one column at a volume ratio of from about 50:50 to about 30:70.

18. The process according to any one of the preceding claims, wherein no base is added to the HMO solution downstream of the mixed bed ion exchange before the HMO solution is passed through an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying.

19. The process according to any one of the preceding claims, wherein no base is added to the human milk oligosaccharide downstream of the mixed bed ion exchange.

20. The process according to any one of the preceding claims, wherein no acid is added to the HMO solution downstream of the mixed bed ion exchange before the HMO solution is passed through an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying.

21. The process according to any one of the preceding claims, wherein no acid is added to the human milk oligosaccharide downstream of the mixed bed ion exchange.

22. The process according to any one of the preceding claims, wherein the process comprises no more than one mixed bed ion exchange vessel.

23. The process according to any one of the preceding claims, wherein the process comprises passing the HMO solution through at least two mixed bed ion exchange vessels.

24. The process according to any one of the preceding claims, wherein the process comprises passing the HMO solution through at least two mixed bed ion exchange vessels in series.

25. The process according to any one of the preceding claims, wherein the HMO solution comprises: the HMO being purified; and an ingredient selected from monovalent and divalent salts, lactose, oligosaccharides besides the HMO being purified, monosaccharides, amino acids, polypeptides, proteins, organic acids and nucleic acids.

26. The process according to any one of the preceding claims, wherein the HMO solution comprises: the HMO being purified; and an ingredient selected from a salt, an acid, human milk oligosaccharides besides the HMO being purified, lactose and monomeric sugars.

27. The process according to any one of the preceding claims, wherein: the process further comprises subjecting the HMO solution to an ion exchange pretreatment before passing it through the first ion exchange material; the ion exchange pretreatment comprises:

(i) passing the HMO solution through a first pretreatment ion exchange material to form a first pretreatment ion exchange product, and

(ii) passing the first pretreatment ion exchange product through a second pretreatment ion exchange material to form a final pretreatment ion exchange product; one of the first and second pretreatment ion exchange materials comprises cation ion exchange material and no anion ion exchange material; the other of the first and second pretreatment ion exchange materials comprises anion ion exchange material and no cation ion exchange material; the composition of the first pretreatment ion exchange product does not change before it is passed through the second pretreatment ion exchange material; and the composition of the final pretreatment ion exchange product is the same as the composition of the HMO solution that is passed through the first ion exchange material.

28. The process according to any one of claims 1-26, wherein: the process further comprises subjecting the HMO solution to an ion exchange pretreatment before passing it through the first ion exchange material; the ion exchange pretreatment comprises:

(ii) passing the first pretreatment ion exchange product through a second pretreatment ion exchange material to form a final pretreatment ion exchange product; one of the first and second pretreatment ion exchange materials comprises cation ion exchange material and no anion ion exchange material; the other of the first and second pretreatment ion exchange materials comprises anion ion exchange material and no cation ion exchange material; the first pretreatment ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before it is passed through the second pretreatment ion exchange material; and the final pretreatment ion exchange product is not subjected to an enzymatic treatment, ultrafiltration, nanofiltration, sterile filtration, electrodialysis, chromatography, an antifoam removal step, activated carbon, crystallization or spray-drying before being passed through the first ion exchange material.

29. A process according to claim 27 or claim 28, wherein the first pretreatment ion exchange material comprises cation ion exchange material and no anion ion exchange material.

30. A process according to claim 27 or claim 28, wherein the first pretreatment ion exchange material comprises anion ion exchange material and no cation ion exchange material.

31. The process according to any one of the preceding claims, wherein the HMO is 2’- fucosyllactose.

32. The process according to any one of claims 1-30, wherein the HMO is 3- fiicosyllactose.

33. A purified HMO obtained by a process of any one of the preceding claims.

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34. A process for making a food, dietary supplement, infant formula or medicine, wherein the process comprises: preparing a purified HMO according to the process of any one of the preceding claims, and mixing the purified HMO with an ingredient suitable for the food, dietary supplement, infant formula or medicine.

35. The process according to claim 34, wherein the HMO is a dried HMO.

36. The process according to claim 34 or claim 35, wherein an infant formula is made.

37. The process according to claim 36, wherein the process comprises mixing the HMO with one or more infant formula ingredients selected from nonfat milk, a carbohydrate source, a protein source, a fat source, a vitamin, a mineral and other human milk oligosaccharides.

38. The process according to claim 36, wherein the process comprises mixing the HMO with one or more infant formula ingredients selected from lactose, whey protein concentrate and high oleic safflower oil.

39. A food, dietary supplement, or medicine prepared by the process of any one of claims 34-38.

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