Process for the purification of lacto-N-neotetraose
Disclosed is a process for the purification of LNnT (lacto-N-neotetraose) from a fermentation broth, the process comprises subjecting a fermentation broth to a first step of membrane filtration, thereby providing a filtrated solution, such filtrated solution is subjecting to a second step of simulated moving bed chromatography, obtaining a purified solution thereof, then subjecting this purified solution to a third step of crystallization, obtaining crystals containing the LNnT of interest, and subjecting the crystals to a fourth and final step of drying, thereby providing a highly purified powder of LNnT.
1 . A process for purification of LNnT (lacto-N-neotetraose) from a fermentation broth, wherein the process comprises:
providing a fermentation broth which contains LNnT, biomass, medium components, contaminants and carbohydrates other than LNnT;
subjecting the fermentation broth to at least one membrane filtration using nanofiltration membranes, thereby providing a filtrated solution containing LNnT;
subjecting the filtrated solution to at least one simulated moving bed chromatography, thereby providing a purified solution which contains LNnT;
subjecting the purified solution to at least one crystallization from water, thereby providing a crystal mass which contains LNnT
wherein the crystallization comprises
obtaining a mixture of at least two oligosaccharides, containing at least LNnT in a concentration of at least about 50% DSC and such LNnT purity of this mixture is of at least about 60%;
crystallizing the purified solution obtained from the simulated moving bed chromatography from a starting temperature of at least about 20-30° C. and letting the saturated mass crystallize by cooling down to at least 10-20° C. or at least 0-10° C., or from a starting temperature of at least about 30-40° C. and letting the saturated mass crystallize by cooling down to at least about 20-30° C., at least 10-20° C. or at least 0-10° C., or from a starting temperature of at least about 40-50° C. and letting the saturated mass crystallize by cooling down to at least about 30-40° C. to at least about 20-30° C., at least 10-20° C. or at least 0-10° C., or from a starting temperature of at least about 50-60° C. and letting the saturated mass crystallize by cooling down to at least 40-50° C., at least about 30-40° C., at least about 20-30° C., or at least about 10-20° C., or at least about 0-10° C. to obtain a homogeneous crystal mass;
treating the crystal mass with a mixture of an alcohol, solvent, or water-miscible alcohol or solvent and water, wherein the alcohol or solvent content of the solution is between 10 and 90 vol. % to obtain a mixture of the alcohol or solvent-containing solution and the crystal mass; and
at least one washing of the alcohol or solvent-containing or sugar-containing fraction of the crystal mass in order to reduce content of lower saccharides from the crystal mass; and
subjecting such crystal mass to at least one homogenization, wherein the homogenization step is selected from freeze-drying, spray-drying, roller or drum drying and band drying, thereby providing a homogenized and dried purified LNnT;
wherein the purified preparation contains the LNnT with a purity of ≥80%.
2 . The process according to claim 1 , wherein the membrane filtration is nanofiltration, using a membrane which has a molecular weight cut-off of between 0.2 and 3.5 kDa.
3 . The process according to claim 1 , wherein the membrane has a molecular weight cut-off of between 0.2 and 2.0 kDa.
4 . The process according to claim 1 , wherein the membrane has a molecular weight cut-off of between 0.2 and 1.0 kDa.
5 . The process according to claim 1 , wherein the filtrated solution obtained from the membrane filtration contains LNnT with a purity of more than 60%, more than 65%, or more than 70%.
6 . The process according to claim 1 , wherein the simulated moving bed chromatography comprises
i) at least 4 columns, optionally at least 8 columns, optionally at least 12 columns, wherein at least one column comprises a weak or strong cation exchange resin, optionally a cation exchange resin in the H+-form, Na+-form, K+-form or Ca2+-form; and/or
ii) four zones I, II, Ill and IV with different flow rates; and/or
iii) an eluent comprising or consisting of water, optionally ethanol and water, optionally 5-15 vol. % ethanol and 85-95 vol. % water, optionally 9-11 vol. % ethanol and 89-91 vol. % water,
iv) an operating temperature of 15 to 60° C., optionally 20 to 55° C., optionally 25 to 50° C.
7 . The process according to claim 1 , wherein the simulated moving bed chromatography comprises
i) four zones I, II, Ill and IV with different flow rates, wherein the flow rates are optionally 22-32 ml/min in zone 1, 17-23 ml/min in zone II, 18-25 ml/min in zone Ill and/or 14-20 ml/min in zone IV; and/or
ii) a feed rate of 0.5-4 ml/min, optionally 2 ml/min; and/or
iii) an eluent flow rate of 6-12 ml/min, optionally 8 ml/min; and/or
iv) a switching time of 14-20 min, optionally 16-18 min, optionally 17 min, optionally, at least one of the columns comprises 0.1 to 5000 kg of cation exchange resin, optionally 0.2 to 500 kg of cationic exchange resin, optionally 0.5 to 50 kg of cation exchange resin, optionally 1.0 to 20 kg of cation exchange resin.
8 . The process according to claim 1 , wherein the purified solution obtained from the simulated moving bed chromatography contains LNnT with a purity of more than 75% or more than 80%.
9 . The process according to claim 1 , wherein the purified solution obtained from at least one simulated moving bed chromatography can be subjected to at least one other further purification using simulating moving bed chromatography, wherein a purified solution comprising LNnT with a purity of >85%, optionally >90%; optionally >93% is provided.
10 . The process according to claim 1 , wherein the crystallization step comprises
treating the crystal mass with a mixture of an alcohol, solvent, or water-miscible alcohol or solvent and water, wherein the alcohol or solvent content of the solution is between 30 and 80 vol. % and optionally between 50 and 70 vol. %, to obtain a mixture of the alcohol or solvent-containing solution and the crystal mass.
11 . The process according to claim 1 , wherein washing of the alcohol or solvent-containing or sugar-containing fraction of the crystal can be repeated at least two times in order to reduce content of lower saccharides.
12 . The process according to claim 1 , wherein content of lower saccharides is less than 10%, optionally less than 5% and the optionally less than 3%.
13 . The process according to claim 1 , wherein the crystals obtained from the crystallization step contain LNnT with a purity of more than 85%, more than 90% or more than 95%.
14 . The process according to claim 1 , wherein the purity of the LNnT in the purified preparation is ≥85% with respect to the dry matter of the purified preparation.
15 . The process according to claim 14 , wherein the purity of the LNnT in the purified preparation is ≥90% with respect to the dry matter of the purified preparation.