PURIFICATION OF OLIGOSACCHARIDES

Abstract

The invention relates to a method for separating sialylated oligosaccharides, preferably sialylated human milk oligosaccharides (HMOs), from disaccharides, preferably lactose, produced by a fermentation or enzymatic process.

Claims

1. A method for separating a tri- or higher oligosaccharide containing a sialyl moiety from a disaccharide in a feed solution, comprising the steps in the following order: a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the disaccharide to pass, wherein the membrane comprises an active layer of polyamide, and a MgSO.sub.4 rejection of 50-90%, b) conducting a diafiltration process on a retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, wherein the membrane comprises a rejection of said electrolyte is not higher than that of MgSO.sub.4, and c) collecting the retentate enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt from the cation of said electrolyte.

2. A method for separating a tri- or higher oligosaccharide containing a sialyl moiety from mono- and divalent salts which are dissolved in a feed solution, comprising the steps: a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the mono- and divalent salts to pass, wherein the membrane comprises an active layer of polyamide, and a MgSO.sub.4 rejection of 50-90%, and conducting diafiltration of the feed solution with an aqueous solution of an inorganic electrolyte, wherein the membrane comprises a rejection of said electrolyte not higher than that of MgSO.sub.4, and b) collecting a retentate of step a) enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt with the cation of said electrolyte.

3-17. (canceled)

18. The method according to claim 1, wherein the membrane comprises a NaCl rejection lower than the MgSO.sub.4 rejection, and the NaCl rejection of the membrane is not more than 30%.

19. The method according to claim 1, wherein the aqueous solution of an inorganic electrolyte is a neutral solute comprising a single electrolyte, and the single electrolyte is sodium chloride.

20. The method according to claim 1, wherein the polyamide nanofiltration membrane is piperazine-based membrane.

21. The method according to claim 1, wherein the tri- or higher oligosaccharide containing a sialyl moiety comprises said disaccharide in its structure.

22. The method according to claim 21, wherein said disaccharide is lactose.

23. The method according to claim 22, wherein the tri- or higher oligosaccharide containing a sialyl moiety is a sialylated human milk oligosaccharide (HMO).

24. The method according to claim 23, wherein the HMO is produced by fermentation or enzymatically from lactose as precursor.

25. The method according to claim 24, wherein the feed solution is an aqueous medium, and step a) is preceded by at least one of the following steps: i) clarifying the aqueous medium from fermentation or enzymatic reaction to remove suspended particulates and contaminants; and/or ii) removing substantially all proteins from the aqueous medium of the fermentation or enzymatic reaction.

26. The method according to claim 25, wherein at least one of the steps i) and ii) comprises ultrafiltration.

27. The method according to claim 25, wherein step ii) further comprises removing peptides, RNA, DNA, endotoxins and glycolipids from the aqueous medium.

28. The method according to claim 2, wherein the aqueous solution of an inorganic electrolyte is a neutral solute.

29. The method according to claim 28, wherein the neutral solute is a single electrolyte, and the single electrolyte is a uni- or divalent single electrolyte.

30. The method according to claim 29, wherein the univalent single electrolyte is sodium chloride.

31. The method according to claim 30, wherein the membrane comprises a NaCl rejection lower than the MgSO.sub.4 rejection, and the NaCl rejection of the membrane is not more than 30%.

32. The method according to claim 2, wherein the polyamide nanofiltration membrane is piperazine-based membrane.

33. The method according to claim 2, wherein the tri- or higher oligosaccharide containing a sialyl moiety is a sialylated human milk oligosaccharide (HMO).

34. The method according to claim 33, wherein the HMO is produced by fermentation or enzymatically from lactose as precursor.

35. The method according to claim 34, wherein the feed solution is an aqueous medium, and step a) is preceded by at least one of the following steps: i) clarifying the aqueous medium from fermentation or enzymatic reaction to remove suspended particulates and contaminants; and/or ii) removing substantially all proteins from the aqueous medium of the fermentation or enzymatic reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0030] Terms and Definitions

[0031] The term monosaccharide means a sugar of 5-9 carbon atoms that is an aldose (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g. D-fructose, D-sorbose, D-tagatose, etc.), a deoxysugar (e.g. L-rhamnose, L-fucose, etc.), a deoxy-aminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), an uronic acid, a ketoaldonic acid (e.g. sialic acid) or equivalents.

[0032] The term disaccharide means a carbohydrate consisting of two monosaccharide units linked to each other by an interglycosidic linkage.

[0033] The term tri- or higher oligosaccharide containing a sialyl moiety means a sugar polymer consisting of at least three, preferably from three to eight, more preferably from three to six, monosaccharide units (vide supra), wherein at least one monosaccharide unit is sialic acid. The oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.

[0034] The term sialylated human milk oligosaccharide or sialylated HMO means a complex carbohydrate found in human breast milk (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, N.Y., 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). The sialylated HMOs have a core structure being a lactose unit at the reducing end that may be elongated by one or more -N-acetyl-lactosaminyl and/or one or more -lacto-N-biosyl units, and which core structures are substituted by an -N-acetyl-neuraminyl (sialyl) moiety. In this regard, the sialylated HMOs are charged or acidic molecules due to the presence of the sialyl moiety. Examples of acidic HMOs include 3-sialyllactose (3-SL), 6-sialyllactose (6-SL), 3-fucosyl-3-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

[0035] The term sialyl or sialyl moiety means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac), preferably linked with -linkage:

##STR00001##

[0036] The term fucosyl means an L-fucopyranosyl group, preferably linked with -interglycosidic linkage:

##STR00002##

[0037] N-acetyl-glucosaminyl means an N-acetyl-2-amino-2-deoxy-D-glucopyranosyl (GlcNAc) group, preferably linked with -linkage:

##STR00003##

[0038] N-acetyl-lactosaminyl means the glycosyl residue of N-acetyl-lactosamine (LacNAc, Galp1-4G1cNAcp), preferably linked with -linkage:

##STR00004##

[0039] Furthermore, the term lacto-N-biosyl means the glycosyl residue of lacto-N-biose (LNB, Galp1-3G1cNAcp), preferably linked with -linkage:

##STR00005##

[0040] The term aqueous medium [from a fermentation or enzymatic process] preferably means an aqueous suspension resulting from an enzymatic or fermentation process for producing one or more hydrophilic oligosaccharides, preferably a sialylated HMO.

[0041] The term protein-free aqueous medium preferably means an aqueous medium or broth from a fermentation or enzymatic process, which has been treated to remove substantially all the proteins, as well as peptides, peptide fragments, RNA and DNA, as well as endotoxins and glycolipids that could interfere with the eventual purification of the one or more hydrophilic oligosaccharides, especially a sialylated HMO, from the fermentation or enzymatic process. Such removal of proteins, peptides, peptide fragments, RNA and DNA can be accomplished in a conventional manner by ion exchange chromatography, affinity chromatography, ultrafiltration, and size exclusion chromatography.

[0042] The term clarified aqueous medium preferably means an aqueous medium or broth from a fermentation or enzymatic process, which has been treated to remove suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris from a fermentation process, that could interfere with the eventual purification of the one or more hydrophilic oligosaccharides, especially a sialylated HMO, from the fermentation or enzymatic process. Such a clarification treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which may include a Celite filter aid).

[0043] Rejection factor of a salt or an electrolyte (in percent) is calculated as (1.sub.p/.sub.r).Math.100, wherein .sub.p is the conductivity of the salt in the permeate and .sub.r is the conductivity of the salt in the retentate. The retentate concentration is practically equal to the feed concentration concerning the salt. The procedure for measuring rejection of salts is disclosed in the examples.

[0044] Rejection factor of a carbohydrate (in percent) is calculated as (1C.sub.p/C.sub.r).Math.100, wherein C.sub.p is the concentration of the carbohydrate in the permeate and C.sub.r is the concentration of the carbohydrate in the retentate. The retentate concentration is practically equal to the feed concentration concerning the carbohydrate. The procedure for measuring rejection of a carbohydrate is disclosed in the examples.

[0045] Separation factor concerning two carbohydrates is calculated as (C.sub.p1/C.sub.r1)/(C.sub.p2/C.sub.r2), wherein C.sub.p1 and C.sub.p2 are the concentrations of the first and the second carbohydrate, respectively, in the permeate, and C.sub.r1 and C.sub.r2 are the concentrations of the first and the second carbohydrate, respectively, in the retentate.

[0046] Pure water flux is defined as the volume of purified water (e.g. distilled water, RO water) that passes through a membrane per unit time, per unit area and per unit of transmembrane pressure under specified conditions (at 23-25 C., 10 bar and constant cross-flow of 300 l/h). The procedure for measuring the pure water flux is disclosed in the working examples below.

[0047] Separating a Tri- or Higher Oligosaccharide Containing a Sialyl Moiety from a Disaccharide and/or Mono- and Divalent Salts

[0048] The separation and therefore the purification of valuable oligosaccharides such as human milk oligosaccharides from the medium in which they have been produced, e.g. a fermentation broth or enzymatic reaction mixture, has always been a complicated multistep process due to the presence of numerous contaminants and by-products of different physical and chemical characters. One of the most difficult problem is to separate compounds of similar nature from each other, like to separate a desired carbohydrate from other, non-desired carbohydrates or separate charged compounds like tri- or higher oligosaccharide containing a sialyl moiety from ions (salts).

[0049] The present inventors have surprisingly found that a nanofiltration step significantly facilitates the separation and purification of charged oligosaccharides, advantageously sialylated human milk oligosaccharides, from a complex broth.

[0050] Accordingly, in a first aspect, a method is provided for separating a tri- or higher oligosaccharide containing a sialyl moiety from a disaccharide which are dissolved in a feed solution, particularly in an aqueous medium from a fermentation or enzymatic process, comprising the steps in the following order: [0051] a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the disaccharide to pass, wherein the active (top) layer of the membrane is composed of polyamide, and wherein the MgSO.sub.4 rejection of the membrane is 50-90%, [0052] b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, wherein the rejection of said electrolyte is not higher than that of MgSO.sub.4, followed by optional diafiltration with pure water to remove excess of the electrolyte, [0053] c) and collecting the retentate enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt with the cation of said electrolyte.

[0054] In a second aspect of the invention, a method is provided for separating a tri- or higher oligosaccharide containing a sialyl moiety from mono- and divalent salts which are dissolved in a feed solution, particularly in an aqueous medium from a fermentation or enzymatic process, comprising the steps: [0055] a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the mono- and divalent salts to pass, wherein the active (top) layer of the membrane is composed of polyamide, and wherein the MgSO.sub.4 rejection of the membrane is 50-90%, by way of diafiltration with an aqueous solution of an inorganic electrolyte, wherein the rejection of said electrolyte is not higher than that of MgSO.sub.4, followed by optional diafiltration with pure water to remove excess of the electrolyte, [0056] b) and collecting the retentate of step a) enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt with the cation of said electrolyte.

[0057] The term ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety preferably means that, during the nanofiltration step, the tri- or higher oligosaccharides containing a sialyl moiety do not pass, or at least significantly do not pass, through the membrane and thus will be present in the retentate. The term allowing at least a part of the disaccharide to pass through the membrane preferably means, that the disaccharide, at least partially can penetrate the membrane and be collected in the permeate. In case of high rejection (about 90%) of the disaccharide, a subsequent diafiltration with pure water may be necessary to bring all or at least the majority of the disaccharide in the permeate. The higher the disaccharide rejection the more diafiltration water is necessary for efficient separation. The term allowing at least a part of the mono- and divalent salts to pass through the membrane preferably means, that the mono- and divalent salt, at least partially, but preferably their majority, can penetrate the membrane and be collected in the permeate. The mono- and divalent salts comprised in the feed solution are inorganic salts of mono- or divalent cations with mono- or divalent anions from strong inorganic acids and bases, and that are used e.g. as minerals in the fermentation process for producing tri- or higher oligosaccharide containing a sialyl moiety, or as a part of a buffer solution in an enzymatic reaction for producing tri- or higher oligosaccharide containing a sialyl moiety, or any salts formed in or after a fermentation or enzymatic process but prior to step a) in the method according to the second aspect of the invention, as a result of pH adjustment.

[0058] The applied nanofiltration membrane shall be tight for tri- and higher oligosaccharides in order that they are efficiently retained. Preferably, the rejection of the tri- or higher oligosaccharides containing a sialyl moiety is more than 95%, more preferably 97%, even more preferably 99%. Membranes with MWCO of more than 3500 Da are expected to allow more or significant amount of tri-or higher oligosaccharide pass through the membrane thus show a reduced retention of tri- or higher oligosaccharide containing a sialyl moiety and therefore are not suitable for the purposes of the invention, and can be excluded. In the same time, membranes with MWCO of less than 600 Da can also be excluded, becausetogether with the retention of tri- and higher oligosaccharidesthat of the mono- and disaccharides is also expected, meaning that the overall separation of the compounds would likely be poor. In this regard, it is preferred that the rejection of the disaccharide is not more than 80-90%. If the disaccharide rejection turns to be 9012%, the tri- or tetrasaccharide rejection shall preferably be around 99% or higher in order to achieve a practically satisfying separation.

[0059] It has been found that the above requirements are simultaneously fulfilled when the membrane is relatively loose for MgSO.sub.4, that is its rejection is about 50-90%. In this regard the above specified membrane is tight for tri- and higher oligosaccharides, and loose for mono- and disaccharides, and as well as for MgSO.sub.4. Therefore, it is possible to separate e.g. lactose, which is a precursor in making human milk oligosaccharides enzymatically or by fermentation, from the human milk oligosaccharides product by nanofiltration with a good efficacy, and additionally a substantial part of divalent ions also passes to the permeate. In some embodiments, the MgSO.sub.4 rejection factor is 60-90%, 70-90%, 50-80%, 50-70%, 60-70% or 70-80%. Preferably, the MgSO.sub.4 rejection factor on said membrane is 80-90%.Also preferably, the membrane has a rejection factor for NaCl that is lower than that for MgSO.sub.4. In one embodiment, the rejection factor for NaCl is not more than 50%. In other embodiment, the rejection factor for NaCl is not more than 40%. In other embodiment, the rejection factor for NaCl is not more than 30%. In this latter embodiment a substantial reduction of all monovalent salts in the retentate is also achievable.

[0060] Also preferably, in some embodiments, the pure water flux of the membrane is at least 50 l/m.sup.2h. Preferably, the pure water flux of the membrane is at least 60 l/m.sup.2h, at least 70 l/m.sup.2h, at least 80 l/m.sup.2h or at least 90 l/m.sup.2h.

[0061] The active or the top layer of nanofiltration membrane suitable for the purpose of the invention is preferably made of polyamide. Although membranes of different type seem to have promising separation efficacy, for example NTR-7450 having sulphonated PES as active layer for separating lactose and 3-SL (Luo et al. (Biores. Technol. 166, 9 (2014); Nordvang et al. (Separ. Purif. Technol. 138, 77 (2014)), the above specified membrane used in the invention shows always better separation of lactose from an HMO. In addition, the above mentioned NTR-7450 membrane is subject to fouling, which typically results in a drop in flux, increasing the lactose rejection and therefore a reduced separation factor (see examples). Also surprisingly, NTR-7450 membrane showed undesired lower retention of sialylated oligosaccharides at higher flux due to higher pressure applied.

[0062] Yet preferably, the polyamide membrane is a polyamide with phenylene diamine or piperazine building blocks as amine, more preferably piperazine (referred to as piperazine-based polyamide, too).

[0063] Yet preferably, the membrane suitable for the purpose of the present invention is a thin-film composite (TFC) membrane.

[0064] An example of suitable piperazine based polyamide TFC membranes is TriSep UA60.

[0065] The claimed method applies a nanofiltration membrane characterized by some or all of the above features and thus one or more of the following benefits are provided: selectively and efficiently removes disaccharide, preferably lactose, from tri- or higher oligosaccharides containing a sialyl moiety, preferably sialylated HMOs, yielding an enriched tri- or higher oligosaccharide containing a sialyl moiety, preferably sialylated HMO, fraction; removes efficiently monovalent as well as divalent salts therefore no ion exchange step is necessary to obtain a tri- or higher oligosaccharide containing a sialyl moiety, preferably sialylated HMO, in high purity; higher flux during the nanofiltration/diafiltration can be maintained compared to other membranes used for the same or similar purpose in the prior art, which reduces the operation time; the membrane applied in the claimed method is less prone to getting clogged compared to the prior art solutions; the membrane applied in the claimed can be cleaned and regenerated completely therefore can be recycled without substantial reduction of its performance.

[0066] The nanofiltration membrane defined in the method of the invention is more beneficial compared to the prior art membranes used for the same or similar purpose as that of the present invention. Specifically, sulphonated PES membrane of Luo et al. or Nordvang et al. (NTR-7450, MWCO: 600-800, Nitto-Denko), besides showing lower separation factor of tri- to hexasaccharides over lactose, gets easily clogged; GE GE (polyamide, MWCO: 1000 Da) and GE GH (polyamide, MWCO: 2500 Da) membranes of WO 98/15581, besides showing lower separation factor of tri- to hexasaccharides over lactose, operate at lower flux and retain higher amount of salts in the permeate due to high NaCl rejection factor.

[0067] Accordingly, in one embodiment of the first aspect, a method is provided for separating a tri- or higher oligosaccharide from a disaccharide which are dissolved in a feed solution, particularly in an aqueous medium from a fermentation or enzymatic process, comprising: [0068] a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 1000-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the disaccharide to pass, wherein the active (top) layer of the membrane is composed of polyamide, and wherein the MgSO.sub.4 rejection of the membrane is 80-90%, and wherein [0069] the NaCl rejection factor on said membrane is lower than that for MgSO.sub.4, and/or [0070] the pure water flux value of said membrane is at least 50 l/m.sup.2h, [0071] b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, wherein the rejection of said electrolyte is not higher than that of MgSO.sub.4, followed by optional diafiltration with pure water to remove excess of the electrolyte, [0072] c) and collecting the retentate enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt with the cation of said electrolyte.

[0073] In one embodiment of the second aspect, a method is provided for separating a tri- or higher oligosaccharide containing a sialyl moiety from mono- and divalent salts which are dissolved in a feed solution, particularly in an aqueous medium from a fermentation or enzymatic process, comprising the steps: [0074] a) contacting the feed solution with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 1000-3500 Da ensuring the retention of the tri- or higher oligosaccharide containing a sialyl moiety and allowing at least a part of the mono- and divalent salts to pass, wherein the active (top) layer of the membrane is composed of polyamide, and wherein the MgSO.sub.4 rejection of the membrane is 80-90%, by way of diafiltration with an aqueous solution of an inorganic electrolyte, wherein the rejection of said electrolyte is not higher than that of MgSO.sub.4, followed by optional diafiltration with pure water to remove excess of the electrolyte, [0075] b) and collecting the retentate of step a) enriched in the tri- or higher oligosaccharide containing a sialyl moiety in the form of a salt with the cation of said electrolyte.

[0076] Preferably, in both embodiments disclosed above, the NaCl rejection factor of the membrane is at most the half of the MgSO.sub.4 rejection factor.

[0077] More preferably, the nanofiltration membrane to be applied in the claimed invention: [0078] is a piperazine-based polyamide membrane with a MWCO of 1000-3500 Da, [0079] has a MgSO.sub.4 rejection of 50-90%, preferably 80-90%, [0080] has a NaCl rejection of not more than 30%, and [0081] has a pure water flux value of at least 50 l/m.sup.2h, preferably 90 l/m.sup.2h.

[0082] Also in a preferred embodiment, the structure of the tri- or higher oligosaccharide containing a sialyl moiety comprises the structure of the disaccharide, which disaccharide is to be separated from the tri- or higher oligosaccharide containing a sialyl moiety by the method of invention. Accordingly, the tri- or higher oligosaccharides containing a sialyl moiety are derivatives of the disaccharide, namely they are glycosylated variants of the particular disaccharide, wherein at least one glycosyl moiety is sialyl. If the disaccharide is glycosylated by adding one monosaccharide unit, it results in a trisaccharide comprising the disaccharide moiety; if the disaccharide is glycosylated by adding two monosaccharide units, it results in a tetrasaccharide comprising the disaccharide moiety; etc. The matching combinations of the disaccharide and the tri- or higher oligosaccharide containing a sialyl moiety are due to the practical reason that the tri- or higher oligosaccharides containing a sialyl moiety are produced from the disaccharide as a precursor by chemical, enzymatic or fermentative ways, especially enzymatically or by fermentation, and the disaccharide left in the reaction mixture as unreacted or because it was added in excess.

[0083] According to a more preferred embodiment, the disaccharide is lactose and the tri- or higher oligosaccharide containing a sialyl moiety is a glycosylated lactose, wherein at least one glycosyl moiety is sialyl. In this regard, concerning the above more preferred embodiment, the trisaccharide containing a sialyl moiety is a sialylated lactose; a tetrasaccharide containing a sialyl moiety is a double glycosylated lactose wherein at least one of the two glycosyl groups is sialyl, which sialyl group may be directly attached to the lactose or the other glycosyl group provided that the other glycosyl group is not sialyl, and preferably the other glycosyl group is a fucosyl or N-acetylglucosaminyl; a pentasaccharide containing a sialyl moiety is a triple glycosylated lactose wherein at least one of the three glycosyl groups is sialyl, which sialyl group may be directly attached to the lactose or one of the other two glycosyl groups provided that that other glycosyl group is not sialyl, and preferably the other two glycosyl groups consists of a N-acetylglucosaminyl and a galactosyl moiety attached to each other to form a lacto-N-biosyl or a N-acetyllactosaminyl moiety. Particularly, tri- or higher oligosaccharide containing a sialyl moiety is a sialylated (charged) HMO, preferably selected from the list consisting of 3-SL, 6-SL, FSL, LST a, LST b, LST c, FLST a, FLST b, FLST c and DS-LNT. Particularly preferred sialylated HMOs are 3-SL and 6-SL.

[0084] Also in a preferred embodiment, the separation factor of a disaccharide over a tri- or higher oligosaccharide containing a sialyl moiety is more than 5, preferably more than 10, more preferably more than 25, even more preferably more than 100. Especially, the separation factor of lactose over a sialylated human milk oligosaccharide is more than 10, preferably more than 25, more preferably more than 50, even more preferably more than 100.

[0085] Yet preferably, the separation factor of a disaccharide over a trisaccharide containing a sialyl moiety is more than 5, preferably more than 10, more preferably more than 25. Especially, the separation factor of lactose over 3-SL or 6-SL is more than 20, preferably more than 50.

[0086] Yet preferably, the separation factor of a disaccharide over a tetra- or pentasaccharide containing a sialyl moiety is more than 25, preferably more than 50, more preferably more than 100.

[0087] The method of the invention can be conducted under conditions used for conventional nanofiltration or nanofiltration/diafiltration with tangential flow or cross-flow filtration with positive pressure compared to permeate side where both operations could be performed in a batch mode or preferably in continuous mode. The diafiltration is conducted, in the first aspect of the invention, by adding pure water or an electrolyte to the retentate after the nanofiltration according to step a) disclosed above and continuing the filtration process. The diafiltration is conducted, in the second aspect of the invention, by adding pure water or an electrolyte directly to the feed solution.

[0088] In both aspects, the diafiltration is performed with an aqueous solution of an inorganic electrolyte. The inorganic electrolyte implies an inorganic salt that is at least partially soluble, preferably well soluble in water. The rejection of the inorganic salt on the membrane used in the method of invention shall not be higher than that of MgSO.sub.4. In one embodiment, the aqueous solution of the inorganic electrolyte (salt) is neutral, that is a salt of a strong inorganic acid and a strong inorganic base. Exemplary embodiments of strong inorganic acids are HCl, HBr, sulfuric acid, nitric acid, phosphoric acid and perchloric acid, and those of strong inorganic bases are NaOH, KOH, LiOH, Mg(OH).sub.2 and Ca(OH).sub.2; the salts formed from these acid and bases are comprised in the group of neutral inorganic electrolytes applicable for the purpose of the invention. In other embodiment, the aqueous solution of the inorganic electrolyte is slightly basic, this is the case when a strong inorganic base (see above) forms a salt with a weak inorganic acid, e.g. carbonic acid (making carbonates) or acetic acid (making acetates).

[0089] The inorganic electrolyte, usually, is an univalent or a bivalent anion electrolyte. Univalent anion electrolytes are e.g. the salts of HCl, HBr, nitric acid or perchloric acid, particularly chlorides such as LiCl, NaCl, KCl, MgCl.sub.2, CaCl.sub.2, or bicarbonates such as NaHCO.sub.3. Divalent anion electrolytes are e.g. the salts of sulfuric acid such as Na.sub.2SO.sub.4, K.sub.2SO.sub.4, MgSO.sub.4, Na.sub.2SO.sub.4, or carbonates such as Na.sub.2CO.sub.3, K.sub.2CO.sub.3. Especially preferred inorganic electrolyte is NaCl.

[0090] Preferably, the aqueous solution of an inorganic electrolyte used in the diafiltration is a single electrolyte, that is the aqueous solution contains one and only electrolyte (salt).

[0091] The diafiltration membrane shall ensure the high retention of the tri- or higher oligosaccharide containing a sialyl moiety and allow the electrolyte to pass. In this regard the membrane used in the diafiltration step is preferably the one defined in both methods of the invention.

[0092] Diafiltration step b) can be conducted with feeding an electrolyte or pure water to the retentate or the feed, as the case may be, with constant removal of permeate. The preferred mode of an electrolyte or water addition is continuous, i.e. the addition flow rate is matching approximately the permeate flow rate. The addition of electrolyte is terminated when the desired degree of replacement, e.g. 99% or more, of unwanted salts is achieved. Then the excess of electrolyte can be removed completely or to the acceptable low level by diafiltration with pure water, which could be monitored for example by substantial reduction in conductivity of a permeate stream to a constant low level.

[0093] During diafiltration step, a tri- or higher oligosaccharide containing a sialyl moiety, preferably a sialylated HMO, particularly 3-SL or 6-SL, obtained in the retentate after step a) (in the first aspect), or containing in the feed (in the second aspect), is subjected to salt exchange with the inorganic electrolyte. The tri- or higher oligosaccharide containing a sialyl moiety as dissolved in the retentate or the feed may be in non-uniform form, meaning thatdepending on how and under which conditions it has been previously synthetizedit may occur in partially acid and salt form, moreover the cations of the salt can be mixed, e.g. those employed in the fermentation medium as nutrients for the producing cell or in the enzymatic reaction as buffer. The diafiltration is conducted for sufficient time that the cations of the tri- or higher oligosaccharide containing a sialyl moiety are exchanged to the cation of the electrolyte employed; e.g. if the tri- or higher oligosaccharide containing a sialyl moiety is 3-SL and the electrolyte is NaCl, substantially all amount of 3-SL is converted to 3-SL sodium salt. In this regard, the tri- or higher oligosaccharide containing a sialyl moiety is brought to a single and uniform salt form at the end of the diafiltration step.

[0094] Optionally, the diafiltration may comprise addition of pure water after diafiltration with the aqueous solution with an inorganic electrolyte. This optional step is advisable when the rejection of the inorganic electrolyte on the membrane specified in the method is close to that of MgSO.sub.4.

[0095] By the pure water diafiltration substantially all uni- and divalent salts can be removed to the filtrate.

[0096] Both methods comprising diafiltration described above provides a retentate solution that contains a particular salt of the tri- or higher oligosaccharide containing a sialyl moiety and practically lacks any uni- and divalent salts. Therefore, a salt of a tri- or higher oligosaccharide containing a sialyl moiety is available in uniform salt form and high purity or essay in a simple way, and no further purification steps such ion exchange treatment, as suggested by the prior art, is necessary.

[0097] The pH of the feed solution applied for the NF separation or the NF/DF step according to the present invention is not higher than 7, preferably between 3 and 7, more preferably around 4 and 5. Too low pH may influence the membrane's and the solute's properties adversely.

[0098] The convenient temperature range applied is between 10 and 80 C. Higher temperature provides a higher flux and thus accelerates the process. The membrane is expected to be more open for flow-through at higher temperatures, however this doesn't change the separation factors significantly. A preferred temperature range for conducting the nanofiltration separation according to the invention is 20-45 C.

[0099] The applied pressure in the nanofiltration and nanofiltration/diafiltration separation is about 2-50 bars, the higher the pressure the higher the flux.

[0100] In step c) of the method according to the first aspect or in step b) according to the second aspect of the invention, the salt of a tri- or higher oligosaccharide containing a sialyl moiety can then be isolated from the aqueous solution obtained as retentate after diafiltration in solid form in a conventional manner, e.g. by evaporation, spray-drying drying (see e.g. WO 2013/185780), crystallization (see e.g. WO 2010/116317, WO 2017/086443) or lyophilization.

[0101] The sialylated HMOs can be produced from lactose in a conventional manner enzymatically and/or by fermentation of genetically transformed bacteria in an aqueous medium or broth. In this regard, for fermentation methods see for example WO 01/04341, WO 2006/034225, US 2007/0020736, WO 2007/101862, WO 2014/153253, Priem at al. Glycobiology 12, 235 (2002), Antoine et al. Angew. Chem. Int. Ed. 44, 1350 (2005), Fierfort et al. J. Biotechnol. 134, 261 (2008), Drouillard et al. Carbohydr. Res. 345, 1394 (2010). For enzymatic syntheses, see for example WO 96/32492, WO 99/31224, Maru et al. Biosci. Biotech. Biochem. 56, 1557 (1992), Gilbert et al. Nature Biotechnol. 16, 769 (1998), Masuda et al. J. Biosci. Bioeng. 89, 119 (2000), Mine et al. J. Carbohydr. Chem. 29, 51 (2010), Luo et al. Biores. Technol. 166, 9 (2014), Nordvang et al. Separ. Purif. Technol. 138, 77 (2014).

[0102] In carrying out this invention, an aqueous medium, which can come directly from an enzymatic or preferably a fermentation process, particularly from E. coli or yeast fermentation, and which contains the tri- or higher oligosaccharide containing a sialyl moiety accompanied by different contaminants, is optionally treated by, preferably prior step a) according to both aspects, the following steps: [0103] i) clarifying the aqueous medium to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris from a fermentation process; and/or [0104] ii) removing substantially all the proteins, as well as peptides, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent purification step, from the aqueous medium, preferably after clarifying it.

[0105] In step i), the aqueous medium, which contains the tri- or higher oligosaccharide containing a sialyl moiety, is clarified in a conventional manner, e.g. by centrifugation or ultrafiltration. Preferably the aqueous medium is first flocculated and then centrifuged or filtered to remove any remaining insoluble particulates and contaminants, as well as cells and cell components and insoluble metabolites and debris.

[0106] In step ii), proteins and related impurities are removed from the aqueous medium in a conventional manner, e.g. by a second ultrafiltration step or tangential flow ultrafiltration.

[0107] According to steps i) or ii), the broth obtained from fermentation is subjected to ultrafiltration. The fermentation broth typically contains, besides the N-acetylglucosamine containing neutral oligosaccharides produced, the biomass of the cells of the used microorganism together with proteins, protein fragments, DNA, endotoxins, biogenic amines, inorganic salts, unreacted carbohydrate acceptor such as lactose, sugar-like by-products, monosaccharides, colorizing bodies, etc. The ultrafiltration step is to separate the biomass and, preferably, also high molecular weight suspended solids from the soluble components of the broth which pass through the ultrafiltration membrane in the permeate. This UF permeate (UFP) is an aqueous solution containing the produced tri- or higher oligosaccharide containing a sialyl moiety.

[0108] Any conventional ultrafiltration membrane can be used having a molecular weight cut-off (MWCO) range between about 1 and about 500 kDa, such as 10-250, 50-100, 200-500, 100-250, 5-100, 5-50, 10-25, or any other suitable sub-ranges. The membrane material can be a ceramic or made of a synthetic or natural polymer, e.g. polysulfone, polypropylene, cellulose acetate or polylactic acid. The ultrafiltration step can be applied in dead-end or cross-flow mode. These steps may comprise more than one ultrafiltration step using membranes with different MWCO, e.g. using two ultrafiltration separations wherein the first membrane has a higher MWCO than that of the second membrane. This arrangement may provide a better separation efficacy of the higher molecular weight components of the broth. After this separation step the permeate contains materials that have a molecular weight lower than the MWCO of the second membrane, including the oligosaccharide of interest.

[0109] The one or more sialylated HMOs separated from lactose and/or mono- and divalent salts and therefore purified by the method of invention can then be isolated from the aqueous retentate and from the optional the aqueous wash in a conventional manner, e.g. by evaporation, freeze drying, crystallization or lyophilisation.

EXAMPLES

Example 1

Determination of a Substance Rejection Factor on a Membrane

[0110] The NaCl and MgSO.sub.4 rejection on a membrane is determined as follows: in a membrane filtration system, a NaCl (0.1%) or a MgSO.sub.4 (0.2%) solution is circulated across the selected membrane sheet with (for Tami: tubular module) while the permeate stream is circulated back into the feed tank. The system is equilibrated at 10 bars and 25 C. for 10 minutes before taking samples from the permeate and retentate. The rejection factor is calculated from the measured conductivity of the samples: (1.sub.p/.sub.r).Math.100, wherein .sub.p is the conductivity of NaCl or MgSO.sub.4 in the permeate and .sub.r is the conductivity of NaCl or MgSO.sub.4 in the retentate.

TABLE-US-00001 NaCl rej. factor MgSO.sub.4 rej. factor supplier lab. supplier lab. membrane active layer MWCO spec. measurement spec. measurement Trisep UA60 piperazine-PA 1000-3500 10% 80% 81-89%.sup. GE GH PA 2500 81% 76% NTR-7450 sulph. PES 600-800 50% 56% 32% Tami ceramic 1000 0%

[0111] A carbohydrate rejection factor is determined in a similar way with the difference that the rejection factor is calculated from the concentration of the samples (determined by HPLC): (1C.sub.p/C.sub.r).Math.100, wherein C.sub.p is the concentration of the carbohydrate in the permeate and C.sub.r is the concentration of the carbohydrate in the retentate.

Example 2

[0112] 3-SL was made by fermentation using a genetically modified E. coli cell of LacZ.sup., LacY.sup.+ phenotype carrying heterologous neuBCA, wherein said cell comprises a recombinant gene encoding an -2,3-sialyl transferase which is able to transfer the sialic acid of GMP-sialic acid to the internalized lactose, and deleted or inactivated nanKETA. The fermentation was performed by culturing said cell in the presence of exogenously added lactose and a suitable carbon source, thereby producing 3-SL. The obtained fermentation broth containing 3-SL was divided into two parts and they were subjected to cell removal by centrifugation with additional washing of separated cell debris. The obtained clarified supernatants (feed #1: 5.2 kg; feed #2: 4.5 kg) were combined and passed through a UF membrane (Synder XT2B-1812F, 1 kDa) installed into MMS SW18 membrane filtration system at p=8 bar, T=40 C. and a cross-flow of approximately 300 l/h. After most of the solution was passed through, the remaining retentate (ca. 2 l) was diafiltrated with 5 l of water to give 13 kg of combined permeate. It was concentrated by nanofiltration using a tight membrane (Synder NFG-2B-1812F, MWCO 600-800 Da) with subsequent diafiltration applying 10 l of water to give cca. 2000 g of retentate (NF1 retentate). A sample from NF1 was taken and freeze-dried (100 ml=103.5 g gave 10.5 g of a solid) and analysed for ion composition and carbohydrate content. NF1 was de-colorized with 25 g of activated charcoal then concentrated under reduced pressure and freeze-dried to give 173.7 g of white solid of crude 3-SLmixed salt containing K.sup.+, Mg.sup.2+ and Na.sup.+ as major counter-ions.

[0113] Part of the obtained freeze-dried solid (90 g) was re-dissolved in water (1.5 l) and diafiltrated continuously through Trisep TurboClean 1812-UA60-31 membrane (piperazine PA, MWCO1000-3500 Da, measured MgSO.sub.4 rejection is 89%) using 10 l of 1% NaCl solution at a cross-flow of 300 l/h, p=20 bar, T=20-25 C. and initial pH=5.2. Then excess of NaCl was removed by additional diafiltration with 10 l of deionized water until constant and low conductivity of the permeate (0.068 mS/cm vs 5.89 mS/cm before diafiltration). The so-obtained retentate was pumped out from the system to give 794 g of 3-SL solution (an estimated dead volume of 119 g remained in the system). A sample from this retentate (300 g) was freeze-dried to give 3-SL Na salt as a white solid (27 g, NF2-retentate). The remaining solution plus washes from the system gave additional 42 g of the final product after freeze-drying. Estimated yield of the diafiltration step is 88%. The analytical data are summarized in the table below.

TABLE-US-00002 feed#1 feed#2 NF2 retentate (after cell (after cell NF1 NF1 retentate after after DF with removal) removal) retentate activated charcoal NaCl 3-SL assay NMR 88.5% (acid form) 3-SL assay HPAEC 69.74% 49.82% 88.54% 89.53% 84.1% Na (theoretical: 3.27 0.53% 0.45% 0.55% 0.57% 3.06% %) H.sub.2O (KF) 6.97 Impurities lactose n.a. 0.43% 0 0 0 sialic acid 6.0% 6.42% 0.17% 0.23% 0 potassium 1.53% 5.04% 2.44% 2.51% 0.0060% magnesium 0.22% 0.31% 0.55% 0.53% 0.0150% calcium 0.005% 0.0046% 0.0046% 0.0083% <0.0010% ammonia n.a. n.a. n.a. 1.71% n.d. (0) orthophosphate 0.52% 7.06% 0.46% 0.46% n.d. (0) sulphate 0.42% 0.16% 0.0015% 0.0012% <0.05% proteins (Bradfort 1.52% 2.98% 879 ppm 82 ppm 0.0074% assay)

Example 3

[0114] 6-SL was made by fermentation using a genetically modified E. coli cell of LacZ.sup., LacY.sup.+ phenotype carrying heterologous neuBCA, wherein said cell comprises a recombinant gene encoding an -2,6-sialyl transferase which is able to transfer the sialic acid of GMP-sialic acid to the internalized lactose, and deleted or inactivated nanKETA. The fermentation was performed by culturing said cell in the presence of exogenously added lactose and a suitable carbon source, thereby producing 6-SL. The obtained fermentation broth containing 6-SL (ca. 600 1) was subjected to cell removal by ultrafiltration/diafiltration (ceramic membrane, MWCO 15 kDa), followed by charcoal treatment and partial concentration by nanofiltration (MWCO 150-300 Da). Part of the obtained solution (36.4 kg) was divided into 8 portions (cca. 4.5 kg each, 6-SL content: 25.4 g/l, total 6-SL content: 924 g). Each portion was acidified with acetic acid to adjust the pH to 5.0 and subjected to further enrichment by additional cross-flow ultrafiltration (UF) in a first membrane filtration equipment (1 kDa, Synder XT-1812-2B spiral-wound membrane, pressure: 20-30 bar, T=20-30 C.; 6-SL rejection 31%) and the permeate stream (that contains 6-SL) was immediately processed by nanofiltration (NF) (Trisep 1812-UA60-31, piperazine PA, MWCO1000-3500 Da, measured MgSO.sub.4 rejection is 89%; 6-SL rejection 99.8%, separation factor over lactose: 170) in a second membrane filtration equipment. When most of the feed was passed through the UF membrane, the remaining part was diafiltered and the permeate was processed by the second (NF) membrane filtration equipment. At the end of UF/DF in the first equipment, the UF retentate contained 0.8% of the original amount of 6-SL in the feed, showing that practically all 6-SL passed through the UF membrane to the feed of the NF and processed by NF. Them, diafiltration (DF) was conducted in the second (NF) unit at p=30-35 bar and T=30-35 C. with 20 l of 2% NaCl solution followed by 20 l of deionized water at approximately 8.9 l/h feed rate matching the permeate flow rate. After completion of the addition of the DF water, the retentate was further concentrated at p=40 bar until permeate flux dropped to 14 l/m.sup.2h. In the end of the NF/DF process in the second equipment, the retentates of all 8 batches were combined to give 4.3 kg of 6-SL Na-salt solution (6-SL amount is 886 g, 92%). A small sample was freeze-dried (50 ml=54.2 g gave 10.9 g of a solid) and analysed. The obtained remaining retentate was treated with activated charcoal, filtered and freeze-dried to give 845 g of the final 6-SL Na-salt as a white solid (assay by HPAEC: 95.25%, Na.sup.+: 3.1% [theoretical: 3.27%], K.sup.+: 0.026%, Mg.sup.2+: 0.009%, PO.sub.4.sup.3: 0.0137%, SO.sub.4.sup.2: 0.076%).

Example 4

[0115] The nanofiltration membranes are, in general, subject to fouling due to the presence of larger molecules in the feed solution like peptide fragments, lipids, anti-foam, etc., which causes a drop in flux and/or decrease the separation factor. The purpose of this investigation is how the membranes can be cleaned and regenerated.

[0116] Flat sheet membranes (d=20 cm, active membrane area 280 cm.sup.2 for each sheet) were installed into a cross-flow flat sheet cell of the MMS SW18 membrane filtration system. Pure water was equilibrated at 10 bars and 23-25 C. with constant cross-flow (300 l/h) for at least 10 min. Then small portion (5-30 ml) of permeate fractions were collected and exact mass or volume was measured. Flux was calculated according to the following formula: F=V/(t.Math.A) where V is the collected permeate volume in litres, t is the time required to collect the measured volume in hours and A is the membrane area in m.sup.2.

[0117] The following pure water flux values were measured:

TABLE-US-00003 membrane active layer MWCO flux (l/m.sup.2h) Trisep UA60 piperazine-PA 1000-3500 100.8 GE GH PA 2500 17 NTR-7450 sulph. PES 600-800 99.6

[0118] Then, for the Trisep UA60 and Nitto-Denko NTR-7450 membranes, water was replaced by a feed solution which was prepared as follows: crude LNnT solid sample was obtained from fermentation broth after cell removal by UF (15 kDa), NF (150-300 Da) with diafiltration, decolouration with activated charcoal and freeze-drying. The obtained solid contained LNnT (54.6%), lactose (9.86%), lacto-N-triose II (7.32%) and pLNnH (8.67%, all by weight), from which 41 g was dissolved in 2050 g of water, obtaining a solution having a pH of 5.71 and conductivity of 0.825 mS/cm. The flux of the feed solution was measured under the same conditions.

[0119] Then the membranes were washed with pure water (cleaning in place, CIP1), and water flux was re-measured.

[0120] Following this, the membranes were washed with an aqueous cleaning solution containing 0.1% sodium dodecyl sulphate, 0.5% EDTA and 0.5% sodium tripolyphosphate (cleaning in place, CIP2, 30 min, 5 bar, 20-25 C.), and water flux was remeasured.

[0121] The data show that the NTR-7450 membrane is more prone to being fouled than Trisep UA60 when a pre-treated oligosaccharide solution obtained after fermentation is applied. Furthermore, while pure water washing regenerated the Trisep UA60 membrane to reach 85% of the original water flux, it was inefficient to do so for the NTR-7450 membrane. In addition, whereas a detergent containing cleaning solution completely cleaned the Trisep UA60 membrane, the NTR-4750 membrane was regenerated only partially.

TABLE-US-00004 flux (l/m2h) Trisep UA60 NTR-7450 initial water flux 100.8 99.6 flux with feed solution 55.1 30.3 water flux after CIP1 85.4 23.9 after CIP1 relative to initial 85% 24% water flux after CIP2 119 71.3 after CIP2 relative to initial 118% 72%