Process for purification of neutral human milk oligosaccharide using simulated moving bed chromatography
The present application discloses a process for the purification of a neutral human milk oligosaccharide (neutral HMO). The process uses simulated moving bed (SMB) chromatography which allows the continuous purification of large quantities of HMOs with high purity. Contrary to chemical synthesis routes of neutral HMOs, and their subsequent purification, the presented process allows the provision of HMOs free of noxious chemicals, such as e.g. trace amounts of heavy metals or organic solvents. The individual neutral HMO product may be obtained in solid form by spray drying or as a concentrated syrup. The provided neutral HMO is very well-suited for use in food applications.
1. A process for purification of a neutral human milk oligosaccharide from a crude solution, comprising
i. obtaining a crude solution from microbial fermentation, chemical synthesis, enzymatic biocatalysis and/or combinations thereof, wherein the crude solution comprises the-neutral human milk oligosaccharide and contaminants, and wherein the contaminants in the crude solution comprise oligosaccharides generated by a glycosyltransferase used in the synthesis of the neutral human milk oligosaccharide,
ii. applying the crude solution to simulated moving bed chromatography, wherein each column in the simulated moving bed chromatography comprises a cation exchange resin, and
iii. obtaining a purified solution comprising the neutral human milk oligosaccharide in a raffinate stream,
wherein the neutral human milk oligosaccharide has more than three monosaccharide units.
2. The process of claim 1 , the neutral human milk oligosaccharide in the purified solution has a purity of ≥80%.
3. The process of claim 1 , wherein the eluent comprises water and wherein the simulated moving bed chromatography is performed at an operating temperature of 15° to 60° C.
4. The process of claim 1 , wherein the simulated moving bed chromatography is operated continuously.
5. The process of claim 1 , wherein the crude solution is obtained by microbial fermentation by a method comprising
a) filtering the microbial fermentation solution to separate soluble material from insoluble material;
b) subjecting the filtered solution from step a) to at least one of cation exchange chromatography or anion exchange chromatography;
c) concentrating the solution obtained in step b); and
d) dialysing the solution obtained in step c) to obtain the crude solution.
6. The process of claim 1 , wherein before applying the crude solution to the simulated moving bed chromatography system, the crude solution is
i) filtered to remove any insoluble material, and/or
ii) subjected to at least one of cation or anion exchange chromatography.
7. The process of claim 1 , wherein before applying the crude solution to the simulated moving bed chromatography system, the crude solution is
i) electrodialysed and/or
ii) diafiltered.
8. The process of claim 1 , wherein before applying the crude solution to the simulated moving bed chromatography system, the crude solution is dialysed and then concentrated to a concentration of ≥100 g/L.
9. The process of claim 1 , further comprising sterile filtering and/or subjecting the purified solution to endotoxin removal.
10. The process of claim 1 , wherein the purified solution is concentrated to obtain a concentrated solution comprising 20-60% (w/v) of the purified neutral human milk oligosaccharide, and wherein the concentrated solution is further spray dried to obtain the purified neutral human milk oligosaccharide in solid granule or power form.
11. The process of claim 1 , wherein the neutral human milk oligosaccharide is selected from the group consisting of 2′3-difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose, lacto-N-neofucopentaose I, lacto-N fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6′-galactosyllactose, 3′-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, and difocosyl-lacto-N-neohexaose.
12. The process of claim 11 , wherein the neutral human milk oligosaccharide is lacto-N-tetraose.
13. The process of claim 8 , wherein the crude solution is dialysed to a conductivity of ≤15 mS/cm.
14. The process of claim 1 , wherein the purified solution is further
i) dialysed and/or
ii) diafiltered.
15. The process of claim 14 , wherein the purified solution is electrodialysed to a conductivity of ≤15 mS/cm.
16. The process of claim 3 , wherein the eluent comprises water and ethanol.
17. The process of claim 3 , wherein the operating temperature of the simulated moving bed chromatography is 25° to 50° C.
18. The process of claim 1 , wherein the simulated moving bed chromatography system comprises four zones, wherein each zone has at least two columns.
19. The process of claim 18 , wherein the columns in each of the four zones have flow rates as follows:
a. 28-32 ml/min in zone I;
b. 19-23 ml/min in zone II;
c. 21-25 ml/min in zone III; and
d. 16-20 ml/min in zone IV.
20. The process of claim 19 , wherein the feed rate of the crude solution is 2-4 ml/min and flow rate of the eluent is 10-13 ml/min.
21. The process of claim 19 , wherein all the columns in the four zones comprise a cation exchange resin and wherein the raffinate is removed between zone III and zone IV.
22. The process of claim 1 , wherein the simulated moving bed chromatography system has eight zones.
23. The process of claim 1 , wherein the cation exchange resin comprises hydrogen or calcium cations.
24. The process of claim 1 , wherein the cation exchange resin comprises sulfonic acid groups.
25. The process of claim 1 , wherein the neutral human milk oligosaccharide is a tetrasaccharide, pentasaccharide, or hexasaccharide.
26. The process of claim 21 , wherein the cation exchange resin comprises hydrogen or calcium cations.
27. The process of claim 21 , wherein the cation exchange resin comprises sulfonic acid groups.
28. The process of claim 5 , wherein all the columns in the simulated moving bed chromatography comprises a cation exchange resin.