# US 12,760,053 B2 · Process for the purification of an acidic human milk oligosaccharide from fermentation broth

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- Number: US 12,760,053 B2
- Document ID: US12760053B2
- Status: Granted
- Kind: B2
- Application number: 18/032,933
- Filed: 2021-10-29
- Published: 2023-12-07
- Granted: 2026-10-13
- Earliest claimed: 2020-10-30
- Assignees: FrieslandCampina Nederland B.V.
- Applicants: FrieslandCampina Nederland B.V.
- Inventors: Alfred Willy Bonte; Franciscus Johannes Hubertus Jeurissen; Martina Henrica Maria Van Genuchten
- Web page: https://patentgrep.com/patent/US12760053B2

## CPC

- C07H 1/08: from natural products
- B01D 15/362: {Cation-exchange}
- B01D 15/363: {Anion-exchange}
- B01D 61/029: {Multistep processes comprising different kinds of membrane processes selected from reverse osmosis, hyperfiltration or nanofiltration}
- B01J 39/05: in the strongly acidic form
- B01J 41/07: in the weakly basic form
- B01J 41/13: obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention relates to a process for the purification of an acidic human milk oligosaccharide (HMO) from a fermentation broth using ion exchange methods. This process allows for a reduction of the number and/or extent of desalting operations, such as electrodialysis. It is even possible to refrain from such operations.

## Claims

### Claim 1

A process for the purification of an acidic human milk oligosaccharide (HMO) from a fermentation broth, the process comprising:
(i) separating biomass from the fermentation broth to provide a crude solution;
(ii) subjecting the crude solution to cation exchange using a cation exchange material in the H+ form, thereby obtaining a solution with a pH in the range 1-3;
(iii) subjecting the solution with pH in the range 1-3 to anion exchange using a weakly basic anion exchange material in the Cl− form, thereby obtaining a solution with a pH in the range 1.5-5.5;
(iv) subjecting the solution with pH in the range 1.5-5.5 to adsorption using an adsorbent material in order to remove neutral organic compounds;
(v) optionally adjusting the pH of the solution having been subjected to adsorption to a value in the range 5-6;
(vi) optionally subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution, this optional step being conducted between steps (iii) and (iv) or steps (iv) and (v);
thereby obtaining a purified solution comprising said acidic human milk oligosaccharide.

### Claim 2 (depends on 1)

The process according to claim 1, wherein the acidic human milk oligosaccharide is selected from the group consisting of 6′-sialyllactose (6′-SL), 3′-sialyllactose (3′-SL), disialyllacto-N-tetraose, 3′-sialyl-3-fucosyllactose, and sialyllacto-N-tetraose.

### Claim 3 (depends on 1)

The process according to claim 1, wherein the cation exchange material in step (ii) is a strong acid cation exchange material.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the weakly basic anion exchange material in step (iii) is selected from the group consisting of acrylic gel-type and styrene/divinylbenzene gel-type matrices with tertiary amine.

### Claim 5 (depends on 1)

The process according to claim 1, wherein the adsorbent material in step (iv) is a weak cation exchange material.

### Claim 6 (depends on 1)

The process according to claim 1, comprising subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution, this step being conducted between steps (iii) and (iv) or steps (iv) and (v), and wherein the anion exchange resin is a weakly basic anion exchange resin.

### Claim 7 (depends on 1)

The process according to claim 1, wherein step (i) includes the use of a membrane having a molecular weight cut-off of 5 kDa or less.

### Claim 8 (depends on 1)

The process according to claim 1, wherein step (i) includes a microfiltration step which is performed at a temperature in the range of 20-75° C.

### Claim 9 (depends on 8)

The process according to claim 8, wherein the microfiltration step forms a microfiltration (MF) permeate and the MF permeate is subjected to a cooling step before step (ii).

### Claim 10 (depends on 1)

The process according to claim 1, comprising subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution, this step being conducted between steps (iii) and (iv) or steps (iv) and (v), and wherein the anion exchange resin is in the OH− form.

### Claim 11 (depends on 1)

The process according to claim 1, wherein the process further comprises subjecting the purified solution to nanofiltration and/or reverse osmosis.

### Claim 12 (depends on 1)

The process according to claim 1, wherein the purified solution is, optionally after one or more further treatment steps, subjected to a drying or crystallisation step.

### Claim 13 (depends on 12)

The process according to claim 12, wherein the human milk oligosaccharide is obtained in the form of a powder having a water content of less than 10 wt %.

### Claim 14 (depends on 1)

The process according to claim 1, wherein the acidic human milk oligosaccharide is obtained in the form of a syrup.

### Claim 15 (depends on 1)

A method for producing an acidic human milk oligosaccharide, the method comprising: producing an acidic human milk oligosaccharide by microbial fermentation in a fermentation broth and purifying the produced acidic human milk oligosaccharide using the process according to claim 1.

### Claim 16 (depends on 1)

The process according to claim 1, comprising adjusting the pH of the solution having been subjected to adsorption to a value in the range 5-6.

### Claim 17 (depends on 16)

The process according to claim 16, subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution this step being conducted between steps (iii) and (iv).

### Claim 18 (depends on 16)

The process according to claim 16, subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution this step being conducted between steps (iv) and (v).

### Claim 19 (depends on 1)

The process according to claim 1, subjecting the solution to anion exchange using an anion exchange resin, thereby binding the acidic human milk oligosaccharide to the resin followed by eluting the acidic human milk oligosaccharide from the resin using a salt solution.