Process for purifying sialylated oligosaccharides
Disclosed is a method for purifying sialylated oligosaccharides from a fermentation broth, cell-lysate or biocatalytic reaction mixture for obtaining high amounts of desired sialylated oligosaccharides in high purity. The method is particular suitable for the large-scale economic purification of sialylated human milk oligosaccharides (such as 3′-sialyllactose, 6′-sialyllactose or sialylated lacto-N-tetraose derivatives) from microbial fermentation, using recombinant bacterial cells or yeast cells. The obtained material is of high purity and can be used for food or medical application such like medical nutrition products, infant formula, dietary supplements, general nutrition products (e.g. dairy drinks).
1. A method for purifying one or more desired sialylated oligosaccharides produced by microbial fermentation and secreted into the fermentation broth, the method comprising:
i) separating cells that produced the one or more desired sialylated oligosaccharides during microbial fermentation and which are present in the fermentation broth at the end of the microbial fermentation from the fermentation broth to obtain a substantially cell-free fermentation broth by subjecting the fermentation broth to filtration or centrifugation;
ii) removing one or more cations from the substantially cell-free fermentation broth;
iii) removing one or more anionic impurities from the substantially cell-free fermentation broth;
iv) removing one or more compounds having a molecular weight lower than that of the desired sialylated oligosaccharide to be purified from the fermentation broth; and
v) spray-drying or crystallizing the desired sialylated oligosaccharide,
wherein the method further comprises a step of electrodialysis wherein:
(a) acidic conditions are applied during electrodialysis; or
(b) neutral conditions and bipolar membranes are applied during electrodialysis;
wherein the desired sialylated oligosaccharide is a sialylated human milk oligosaccharide selected from the group consisting of 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c), 3-fucosyl-sialyllactose (3-F-SL), disialyl-lacto-N-tetraose (DS-LNT), and fucosyl-LST-b (F-LST-b), and
wherein the purified desired sialylated oligosaccharide has a purity of ≥80% by dry-weight and possesses less than 10% water by weight.
2. The method according to claim 1 , further comprising one or more of:
i) increasing the concentration of the desired sialylated oligosaccharide;
ii) removing one or more non-desired oligosaccharides;
iii) removing one or more colorants;
iv) removing one or more endotoxins; and/or
v) sterilizing.
3. The method according to claim 1 , wherein said separating of the cells from the fermentation broth is performed by subjecting the fermentation broth to ultrafiltration.
4. The method according to claim 1 , wherein removing cations from the substantially cell-free fermentation broth is performed by cation exchange chromatography.
5. The method according to claim 1 , wherein removing anionic impurities from the substantially cell-free fermentation broth is performed by anion exchange chromatography.
6. The method according to claim 1 , wherein removing compounds having a molecular weight lower than that of the desired sialylated oligosaccharide to be purified is performed by cross-flow filtration.
7. The method according to claim 1 , wherein the concentration of the desired sialylated oligosaccharide to be purified is increased by nanofiltration or evaporation of the solvent.
8. The method according to claim 2 , wherein removing colorants is performed by treating the fermentation broth/solution containing the desired sialylated oligosaccharide with activated carbon.
9. The method according to claim 2 , wherein removing endotoxins is performed by filtration of the solution containing the desired oligosaccharide through a 6 kDa filter or a 3 kDa filter.
10. The method according to claim 2 , wherein sterilizing the solution is performed by filtration of the solution through a 0.2 μm filter.
11. The method according to claim 1 , further comprising simulated moving bed (SMB) chromatography.
12. The method according to claim 3 , wherein the ultrafiltration removes the cells and compounds having a molecular weight of ≥500 kDa from the fermentation broth.
13. The method of claim 3 , wherein the ultrafiltration removes the biomass and compounds having a molecular weight of ≥150 kDa from the fermentation broth.
14. The method of claim 3 , wherein the ultrafiltration removes the biomass and compounds having a molecular weight of ≥100 kDa from the fermentation broth.