Method for the isolation of glycosphingolipids

Abstract

The present invention describes a novel method for the isolation of a glycosphingolipid lacking the amide-linked fatty acyl group, the method comprising the steps of: providing a solution comprising said glycosphingolipid of formula (1) or a salt thereof, and one or more contaminants, diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

Description

[0001] DESCRIPTION

[0002] Method for the Isolation of Glycosphingolipids

[0003] Field of the invention

[0004] The present invention relates to a method for the isolation of glycosphingolipids and to a spray- dried powder comprising one or more glycosphingolipids.

[0005] Background

[0006] Glycosphingolipids (GSLs) are glycoconjugates wherein a glycan moiety is linked to the 1-hydroxyl group of a ceramide or a sphingoid base via a glycosidic linkage.

[0007] GSLs are involved in diverse biological processes and play important structural and functional roles such as cell-cell recognition, communication, and intercellular adhesion. For example, sialylated glycosphingolipids such as gangliosides are found in the brain, and can play roles in neurological diseases especially Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, certain gangliosides are found in the intestinal mucosa and can promote intestinal health, as well as act as anti-infective agents.

[0008] Accordingly, glycosphingolipids hold great potential as therapeutics, cosmetics, and as tools for the study of important biological processes.

[0009] The use of glycosphingolipids as therapeutics and cosmetics requires the production of such compounds at high purity.

[0010] Processes for the isolation of glycosphingolipids were reported.

[0011] Isolation of sialylated glycosphingolipids from extracts of tissues or organs of the nervous system has been described. Particularly, the monosialoganglioside GMl was isolated via ultrafiltration wherein the ultrafiltration was performed in the presence of a-cyclodextrin (EP0469352 Al).

[0012] It is known that gangliosides, such as GMl, may form micellar aggregates in aqueous solutions having molecular weights of several hundred kilodalton (D.B. Gammak, Biochem J 1963, 88, 373), which may not be permeable through ultrafiltration membranes. This property may prevent the direct use of ultrafiltration and / or dialysis for the isolation of glycosphingolipids from raw mixtures.

[0013] As described in EP0469352 Al the a-cyclodextrin is used to complex GMl, thereby forming a GM1- a-cyclodextrin complex which is permeable through the ultrafiltration membrane.

[0014] The main drawback connected to this approach is the use of a large excess of a-cyclodextrin (4 to 6 eq. compared to GMl) in order to form the ganglioside-cyclodextrin complex, and the need of multiple extractions with organic solvents in order to obtain the free ganglioside. A method for the isolation of a sodium salt of the monosialoganglioside GMl from a lipid mixture comprising a step of fractionating said lipid mixture by ion exchange chromatography has been described (US20080312165 Al). The method further comprising several diafiltration steps to remove residual salts, wherein the diafiltration membrane having a molecular weight cut off (MWCO) of 10-100 kDa. Drawbacks connected to this method comprise the use of large amounts of organic solvents, and the need to regenerate and equilibrate the ion exchange resin after every chromatographic separation.

[0015] Furthermore, EP0469352 Al and US20080312165 Al describe methods for the isolation of gangliosides, specifically GMl, but not for the isolation of their lyso forms.

[0016] Accordingly, there is a demand for the development of novel methodologies characterized by high technological feasibility and low costs, which enable the efficient and large-scale isolation of glycosphingolipids.

[0017] Summary of the invention

[0018] In a first aspect the present invention relates to Method for the isolation a glycosphingolipid of formula (1): wherein

[0019] W is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from a sialylated glycosyl moiety, or a neutral glycosyl moiety,

[0020] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl,

[0021] R2is hydrogen or -OR4, wherein R4is selected from hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C2-6 acyl, preferably R4is hydrogen, the bond - may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -

[0022] OR4,

[0023] R3is hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted Ci.g acyl, preferably hydrogen, or a salt thereof, from a solution comprising said glycosphingolipid or said salt, and one or more contaminants, the method comprising the steps of: providing a solution comprising said glycosphingolipid of formula (1) or a salt thereof and one or more contaminants, diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

[0024] Preferably, wherein the diafiltration is performed by using a membrane having a MWCO of 200-300 kDa.

[0025] In a second aspect the present invention relates to a spray-dried powder comprising one or more glycosphingolipids of formula (1), or a salt thereof: wherein

[0026] W is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from a sialylated glycosyl moiety, or a neutral glycosyl moiety,

[0027] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl,

[0028] R2is hydrogen or -OR4, wherein R4is selected from hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C2-6 acyl, preferably R4is hydrogen, the bond - may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -

[0029] OR4,

[0030] R3is hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted Ci.g acyl, preferably hydrogen.

[0031] In a third aspect the present invention relates to the use of the spray-dried powder according to the present invention, for the production of a ceramide or a ganglioside.

[0032] Detailed description of the invention The present invention describes, for the first time, a method for the isolation a of glycosphingolipid lacking the amide-linked fatty acyl group. Said glycosphingolipids are preferably isolated from a raw solution comprising said glycosphingolipid ad one or more other compounds, e.g. proteins, sugars, salts ("contaminants") via direct diafiltration, and wherein the diafiltration is performed with a membrane having a molecular weight cut-off (MWCO) of 100-300 kDa, preferably of 200-300 kDa. Surprisingly, it has been found that glycosphingolipids lacking the amide-linked fatty acyl group are not permeable through the diafiltration membranes used in the present invention. This effect can advantageously be utilized for the efficient removal of organic molecules, biological material, and monovalent as well as divalent salts and therefore no ion exchange step is necessary. Furthermore, when using membranes with a high MWCO, such as those used in the present invention, a high flux can be maintained during the diafiltration, which reduces the operation time and costs.

[0033] The diafiltration retentate comprising the glycosphingolipid can be spray dried to afford a highly homogenous and easy-to-handle spray-dried glycosphingolipid powder which can be utilized for the production of ceramides and gangliosides.

[0034] The method of the present invention comprising the steps of:

[0035] - providing a solution comprising said glycosphingolipid of formula (1) or a salt thereof and one or more contaminants,

[0036] - diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

[0037] Non-limiting embodiments of different aspects of the invention are described below and illustrated by non-limiting examples.

[0038] The terms, definitions and embodiments described throughout the specification of the invention relate to all aspects and embodiments of the invention.

[0039] The term "a" grammatically is a singular, but it may as well mean the plural of e.g., the intended compound. For example, a skilled person would understand that in the expression "a glycosphingolipid", the provision of not only one single glycosphingolipid, but of a variety of glycosphingolipids of the same type or of different types is meant.

[0040] The term "at least" means an unlimited range of values starting from the indicated value or, in case of wt.% range, a range starting from the indicated value and up to 100 wt.%.

[0041] Throughout the specification "wt.% " is meant the weight of the named substance contained in the 100 g of the named composition e.g., a spray-dried powder comprising at least about 70 wt.% of the glycosphingolipid of formula (1) means that 100 g of the spray-dried powder typically contains at least about 70 g of the glycosphingolipid of formula (1).

[0042] As used herein, the various functional groups or substituents represented will be understood to have a point of attachment at the functional group or atom having the dash (-). For example, in the case of -OR4it will be understood that the point of attachment is the oxygen atom. If a group is listed without a dash, then the attachment point is indicated by the plain and ordinary meaning of the recited group.

[0043] As used herein, the term "alkyl" refers to an acyclic straight or branched hydrocarbyl group having 1-50 carbon atoms which may be saturated or contain one or more double and / or triple bonds (so, forming for example an alkenyl or an alkynyl), and / or which may be substituted or unsubstituted, as herein further described. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neo-pentyl, n-hexyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl,l-pentenyl, 2-pentenyl, 2-methyl-l-butenyl, 3- methyl-l-butenyl, 2-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, methylpentenyl, dimethylbutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, pentynyl, and hexynyl, each of which may be substituted or unsubstituted. Typically, the term alkyl refers to a straight saturated acyclic hydrocarbyl group having 1-31 carbons, which may be substituted or unsubstituted.

[0044] As used herein, the term "aryl" refers to an aromatic cyclic hydrocarbyl group having 5-14 ring carbon atoms, which may be mono- or polycyclic, which may contain fused rings, preferably 1 to 3 fused or unfused rings, and which may contain one or more heteroatoms, and / or which may be substituted or unsubstituted, as herein further described. Examples of "aryl" include, but are not limited to, phenyl, naphtyl, anthracyl, phenantryl, pyrrolyl, imidazolyl, thiophenyl, furanyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzofuranyl, each of which may be substitute or unsubstituted. Typically, the term "aryl" refers to a substituted or unsubstituted phenyl.

[0045] As used herein, the term "acyl" refers to a group derived by the removal of one or more hydroxyl groups from an oxoacid, preferably from a carboxylic acid. The acyl group according to the present invention is typically a saturated or unsaturated C2-32acyl, which may be substituted or unsubstituted.

[0046] As used herein, the term "substituted" means that the group in question is substituted with a group which typically modifies the general chemical characteristics of the group in question. The substituents can be used to modify characteristics of the molecule, such as molecule stability, molecule solubility and the ability of the molecule to form crystals. The person skilled in the art will be aware of other suitable substituents of a similar size and charge characteristics, which could be used as alternatives in a given situation.

[0047] In connection with the terms "alkyl", "aryl", and "acyl" the term substituted means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from hydroxy (which when bound to an unsaturated carbon atom may be present in the tautomeric keto form), oxo, Ci.g-alkoxy (i.e. Ci.g-alkyl-oxy), C2-s-alkenyloxy, carboxy, oxo, Ci.g- alkoxycarbonyl, Ci.g- alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)aminocarbonyl, amino-Ci.g-alkyl- aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci.g-alkylcarbonylamino, cyano, guanidino, carbamido, Ci.g-alkyl-sulphonyl-amino, aryl-sulphonyl-amino, heteroaryl- sulphonyl-amino, Ci.g-alkanoyloxy, Ci.g-alkyl-sulphonyl, Ci.g-alkyl-sulphinyl, Ci.g-alkylsulphonyloxy, nitro, Ci-s-alkylthio, halogen, where any alkyl, alkoxy, and the like representing substituents may be substituted with hydroxy, Ci.g-alkoxy, C2-s-alkenyloxy, carboxy, Ci.g-alkylcarbonylamino, halogen, Ci- s-alkylthio, Ci.g-alkyl-sulphonyl-amino, or guanidino.

[0048] In connection with the term "alkyl" the term "substituted" preferably means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from a hydroxyl group, an alkoxy group, an acyloxy group, an acylamido group, a thiol, a thioether or a phosphorus-containing functional group.

[0049] The term "contaminants", as used herein, refers to a substance, such as a chemical substance (e.g., ions, organic molecules etc.) and / or to biological material (e.g., proteins, cell debris etc.), which accompany the compound or material of interest obtained by a certain process or method.

[0050] The term "isolation", in the context of the present invention, refers to a procedure or a step of the procedure that is applied to separate the desired compound from a mixture comprising said desired compound and other compounds. In this context, the other compounds of the mixture are regarded as contaminants.

[0051] The term isolation and isolating may be used interchangeably.

[0052] The skilled person will understand that in formulas showing a specific compound, like for example formula (1), unless the chemical formula expressly describes a carbon atom having a particular stereochemical configuration, the formula is intended to cover compounds where such a stereocenter has an R or an S configuration, or wherein a double bond has a cis or a trans configuration. The skilled person would understand that when speaking of position C-l, C-2, C-3, C-4, C-5 etc., reference is herein always made to the respective carbon atoms of glycosphingolipid such as that represented by formula (1).

[0053] In the context of the present invention, the terms "about", "around", or "approximate" are applied interchangeably to a particular value (e.g. "about 70 wt.%" , "around 70 wt.%", or "approximate 70 wt.%"), to indicate a deviation from 0.1% to 10% of that particular value.

[0054] The term "glycosphingolipid", as used herein, refers to compounds that structurally consist of a glycosyl moiety and a sphingolipid moiety, or analogs thereof. The glycosyl moiety is typically linked to the sphingolipid moiety via a glycosidic bond between the anomeric carbon at the reducing end of the glycosyl moiety and the hydroxyl group at the C-l position of the sphingolipid.

[0055] The glycosyl moiety of the glycosphingolipid according to the present invention may derive from a monosaccharide or from an oligosaccharide (more than one monosaccharide units), wherein the anomeric carbon of the monosaccharide or the anomeric carbon at the reducing end of the oligosaccharide is engaged in a glycosidic bond with another chemical entity, such as a sphingolipid, and the bond, if not further specified, may be an alpha or a beta glycosidic bond. A glycosyl moiety having more than one monosaccharide unit may represent a linear or a branched structure.

[0056] The monosaccharide unit is preferably any 5-9 carbon atom sugar, comprising aldoses (e.g. D- glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g. D-fructose, D-sorbose, D-tagatose, etc.), deoxysugars (e.g. L-rhamnose, L-fucose, etc.), deoxyaminosugars (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, ketoaldonic acids (e.g. sialic acid). The monosaccharide unit can form different cyclic structures such as pyranose (six-membered) cyclic structures or furanose (five-membered) cyclic structures. In some embodiments the glycosyl moiety derives from a monosaccharide, wherein the monosaccharide is a p-galactoside. In some embodiments, the glycosyl moiety derives from an oligosaccharide, wherein the oligosaccharide carries one or more terminal p-galactopyranosyl units.

[0057] The glycosyl moieties according to the present invention may be illustrated in the following style: Gaipi-4Glcl-, wherein the dash (-) represents the point of attachment of the glycosyl moiety and wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond, preferably a beta glycosidic bond.

[0058] The sphingolipid moiety of the glycosphingolipid of the present invention preferably derives from an aliphatic amino alcohol such as a sphingoid base.

[0059] Sphingoid bases denote in the context of the present invention naturally occurring sphingoid bases, analogues thereof or derivatives thereof. Naturally occurring sphingoid bases are D-erythro-sphingosine (S), 6-Hydroxy-D-erythro-sphingosine (H), D-r / bo-phytosphingosine (P) or DL-erythro-dihydrosphingosine (DS), wherein the number of sphingoid carbons may be expressed in parenthesis following the letters S, H, P, and DS.

[0060] The letters S, H, P, and DS refer to the shorthand nomenclature developed by Motta et al. (1993) Biochim Biophys Acta. 1182:147-151 and expanded by Rabionet (2014) Biochim Biophys Acta. 1841:422-434 and by Masukawa et al., Journal of Lipid Research, 2008, 49, 1466-1476. D-erythro- dihydrosphingosine may also be represented by the letter G according to the INCI nomenclature.

[0061] Glycosphingolipid according to the present invention are typically represented by a glycosphingolipid of formula (1), or a salt thereof: wherein

[0062] W is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from a sialylated glycosyl moiety, or a neutral glycosyl moiety,

[0063] R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl,

[0064] R2is hydrogen or -OR4, wherein R4is selected from hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C2-6 acyl, preferably R4hydrogen, the bond - may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -

[0065] OR4,

[0066] R3is hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted Ci.g acyl, preferably hydrogen.

[0067] Glycosphingolipids of formula (1) lack the amide-linked fatty acyl group and may also be referred to as lysosphingolipids.

[0068] In some embodiments, for the glycosphingolipid of formula (1) R1is a saturated unsubstituted C10- C17 alkyl, R2and R3are hydrogen, and the bond - is a double bond.

[0069] In some embodiments, for the glycosphingolipid of formula (1) R1is a saturated unsubstituted C10- C17 alkyl, R2is OR4, wherein R4is hydrogen, R3is hydrogen, and the bond - is a single bond. In some embodiments, for the glycosphingolipid of formula (1) R1is a saturated unsubstituted Cio- C17 alkyl, R2and R3are hydrogen, and the bond - is a single bond.

[0070] In some embodiments, for the glycosphingolipid of formula (1) R1is a C10-C17 1-hydroxyalkyl, R2and R3are hydrogen, and the bond - is a double bond.

[0071] In some embodiments, the glycosphingolipid of formula (1) is a glycosphingolipid selected from the group consisting of glycosphingolipids of formulas (2), (3), (4), and (5), or salts thereof:

[0072] In some embodiments, W of the glycosphingolipid of formula (1), and of the glycosphingolipids of formulas (2)-(5) is a sialylated glycosyl moiety.

[0073] In some embodiments, W of the glycosphingolipid of formula (1), and of the glycosphingolipids of formulas (2)-(5) is a sialylated glycosyl moiety selected from the group consisting of the following glycosyl moieties, or salts thereof: wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond.

[0074] In some preferred embodiments, W of the glycosphingolipid of formula (1), and of the glycosphingolipids of formulas (2)-(5) is a sialylated glycosyl moiety selected from the group consisting of the following glycosyl moieties, or salts thereof: wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond. In some embodiments, the glycosphingolipid of formula (1) is selected from the group consisting of / V-lyso-GM4, / V-lyso-GM3, N-lyso-GD3, / V-lyso-GDla, and / V-lyso-GT3, or a mixture thereof.

[0075] In some preferred embodiments, the glycosphingolipid of formula (1) is / V-lyso-GM3. In some preferred embodiments, the glycosphingolipid of formula (1) is N-lyso-GD3.

[0076] In some embodiments, the glycosphingolipid of formula (1) is a mixture of / V-lyso-GM3 and N-lyso-

[0077] GD3.

[0078] In some preferred embodiments, the glycosphingolipid of formula (1) is a mixture of / V-lyso-GM3, / V-lyso-GD3, and / V-lyso-GT3.

[0079] Glycosphingolipids of formula (1), which carry a sialylated glycosyl moiety may also be referred to as sialylated glycosphingolipids. in some embodiments, W of the glycosphingolipid of formula (1), and of formulas (2)-(5) is a neutral glycosyl moiety, wherein the neutral glycosyl moiety is selected from the group consisting of the following glycosyl moieties: wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond.

[0080] In some preferred embodiments, W of the glycosphingolipid of formula (1), and of the glycosphingolipids of formulas (2)-(5) is a neutral glycosyl moiety, wherein the neutral glycosyl moiety is selected from the group consisting of the following glycosyl moieties: wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond.

[0081] In some embodiments, the glycosphingolipid of formula (1) is selected from the group consisting of lactosyl D-erytbro-sphingosine, lactosyl D-erytbro-dihydrosphingosine, lactosyl D-ribo- phytosphingosine, galactosyl D-erytbro-sphingosine, galactosyl D-erytbro-dihydrosphingosine, galactosyl D-r / bo-phytosphingosine, glucosyl D-erytbro-sphingosine, glucosyl D-erythro- dihydrosphingosine, and glucosyl D-r / bo-phytosphingosine, or a mixture thereof.

[0082] In some preferred embodiments, the glycosphingolipid of formula (1) is selected from the group consisting of lactosyl D-erytbro-sphingosine, lactosyl D-erytbro-dihydrosphingosine, lactosyl D-ribo- phytosphingosine.

[0083] In some embodiments, the glycosphingolipid of formula (1) is selected from the group consisting of galactosyl D-erytbro-sphingosine, galactosyl D-erytbro-dihydrosphingosine, galactosyl D-ribo- phytosphingosine.

[0084] In some embodiments, the glycosphingolipid of formula (1) is selected from the group consisting of glucosyl D-erytbro-sphingosine, glucosyl D-erytbro-dihydrosphingosine, and glucosyl D-ribo- phytosphingosine.

[0085] In some preferred embodiments, the glycosphingolipid of formula (1) is a mixture of one or / V-lyso- GM3, lactosyl D-erytbro-sphingosine, and glucosyl D-erytbro-sphingosine.

[0086] The present invention describes a method for the isolation of a glycosphingolipid of formula (1), which is preferably produced via enzymatic or biotechnological process (as described in further embodiments below).

[0087] Typically, the glycosphingolipid according to the present invention is isolated from a solution comprising one or more contaminants, wherein said solution is preferably an aqueous solution, and wherein said contaminants are typically, but not limited to, ions, small organic molecules (e.g., sugars, nucleotides etc.), and proteins.

[0088] The solution, comprising the glycosphingolipid and the one or more contaminants, is subjected to a diafiltration (DF) step wherein water is preferably utilized as the diafiltration medium.

[0089] The solution is typically subjected to DF using from about 2 volumes to about 10 volumes of the DF medium. In some embodiments, the solution is subjected to DF using about 2, 3, 4, 5, 6, 7 , 8, 9 or 10 volumes of the DF medium.

[0090] The DF step, according to the present invention, is conducted at a constant temperature, preferably between about 10 and 80° C, preferably between about 15-45 °C, more preferably between about 20-25 °C.

[0091] A preferred applied pressure in the nanofiltration separation is about 2-50 bars, such as 8-20 bars, or such as 8-10 bars.

[0092] The DF step is conducted using membranes such as hollow-fiber membranes, spiral-wound membranes, or ceramic membranes with suitable molecular weight cut-off (MWCO).

[0093] Preferably, the membrane having a MWCO between about 100 to 300 kDa.

[0094] In some preferred embodiments, the diafiltration membranes having a MWCO of about 200-250 kDa, or about 250-300 kDa e.g., about 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, or 300 kDa.

[0095] During the DF step according to the present invention, the glycosphingolipid is retained in the diafiltration retentate (DFR), whereas any permeable contaminant will pass through the membrane and accumulate in the diafiltration permeate (DFP).

[0096] Permeable contaminants, in the context of the present invention, are molecules having a molecular weight up to about 280 KDa, such as e.g., carbohydrates, salts, nucleotides, proteins etc.

[0097] It should be mentioned that the diafiltration membranes used in the present invention have a MWCO well above the molecular weight of the glycosphingolipid.

[0098] It has been described that ganglioside GM1, can form micellar aggregates in aqueous solutions having molecular weights between about 250 kDa and 450 kDa, wherein the size of the micellar aggregate will depend on the length of the fatty acid chain in the constituent molecule (D.B. Gammak, Biochem J 1963, 88, 373). This property may render gangliosides, such as GM1 not permeable through ultrafiltration membranes having a MWCO higher than that of GM1.

[0099] However, surprisingly, the present inventors have found that also / V-lyso forms of gangliosides, as well as glycosylated sphingoid bases, which lack a fatty acid chain are not permeable through diafiltration membranes having a MWCO higher than that of the lysosphingolipid. Accordingly, without being bound by theory, micellar aggregation may surprisingly occur independently from the presence of a fatty acid chain in the molecule.

[0100] An advantage connected to the use of membranes with a high MWCO (i.e., at least 100 kDa) is that a high flux can be maintained during the diafiltration even when applying a low transmembrane pressure, which reduces the operation time and costs. Particularly, when using membranes having a MWCO between about 200 and 300 kDa a high flux of about 15.5-18.1 (l / m2h) could be maintained at a transmembrane pressure of about 8-10 bar. On the other end, when using membranes having MWCO between about 300-500 Da a flux of about 9.6 (l / m2h) could be reached only when applying a transmembrane pressure of about 30 bar.

[0101] Another advantage connected to the use of membranes with a high MWCO (i.e., at least 100 kDa) is the efficient removal of organic molecules, biological material, and monovalent as well as divalent salts. Particularly, when using membranes having a MWCO between about 200 and 300 kDa a reduction of about 90-99 % in the content of small organic molecules (e.g. sugars such as monosaccharides, disaccharides, trisaccharides etc.), as well as salts is achieved.

[0102] In some embodiments, the method further comprising a step of concentrating the DFR.

[0103] Accordingly, in some embodiments, the present invention describing a method for the isolation of a glycosphingolipid of formula (1), the method comprising the steps of:

[0104] - providing solution comprising said glycosphingolipid of formula (1) and one or more contaminants,

[0105] - diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa,

[0106] - concentration of the DFR, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

[0107] The concentration of the DFR is typically performed using the same membrane used during the diafiltration step, and for a period of time required to reduce the volume of the DFR to the desired final volume.

[0108] The DFR comprising the glycosphingolipid of formula (1), is spray dried or spray granulated.

[0109] In some preferred embodiments, the DFR comprising the glycosphingolipid of formula (1), is spray dried.

[0110] In some embodiments, the present invention describing a method for the isolation of a glycosphingolipid of formula (1), the method comprising the steps of:

[0111] - providing solution comprising said glycosphingolipid of formula (1) and one or more contaminants, - diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa,

[0112] - spray drying the DFR comprising the glycosphingolipid, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

[0113] In some embodiments, the present invention describing a method for the isolation of a glycosphingolipid of formula (1), the method comprising the steps of:

[0114] - providing solution comprising said glycosphingolipid of formula (1) and one or more contaminants,

[0115] - diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa,

[0116] - concentration of the DFR,

[0117] - spray drying the DFR comprising the glycosphingolipid, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

[0118] The spray drying step, according to the present invention, is typically conducted with a fast-rotating disk or a nozzle which generates small particles. The particles can then fall, under gravity, towards the bottom of a spray drying tower. Here, a fluid bed may be provided, which can use hot air to effect drying (suitably at around 80° C to around 95° C). Here, agglomeration can take place, and the particles can stick together. Following this, the agglomerated (granular) particles are subjected to drying, for example on a belt drying bed or on a sub-fluidized bed.

[0119] Another technique is to use a fluidized bed agglomeration. Here, powder can be fluidized in a gas flow. In the particle bed a fluid is sprayed with water that wets the powder and enhances the agglomeration. This combination of spray-drying in combination with a fluid bed after dryer is suited for the agglomeration of many different types of solutions.

[0120] Drying can occur under air or under an inert gas, such as nitrogen. With fluidized and sub-fluidized bed drying, the temperature in the bed can be adjusted to pre-set values. These values can range widely, for example, from 35° to 120°C, such as 50 to 90°C, e. g. from 60 to 80°C.

[0121] The spray-dried powder, obtained following the method of the present invention, will typically have a water content from about 0.5 wt.% to about 6 wt.%, preferably from about 2 wt.% to around 3 wt.%. The spray-dried powder, obtained following the method of the present invention, will typically have a median particles diameter between about 15 pm and about 30 pm, e.g., preferably between about 15 and 20 pm e.g., about 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, or 20 pm.

[0122] The Span of the particles will typically be less than about 3, preferably less than about 2. In some preferred embodiments, the Span of the particles is between about 1 and 2 e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

[0123] The Span of the particle is a dimensionless parameter indicative of the uniformity of the particle size distribution, and it is defined as: [D(0.9) - D(0.1)] / D(0.5), wherein D(0.9), D(0.1), and D(0.5) represent the cutoff size below which 10%, 50%, and 90% (by volume) of particles are distributed, respectively.

[0124] Generally, a low Span (i.e., less than 3) is characteristic of a narrow particle size distribution, resulting in improved flow characteristics of the spray-dried powder.

[0125] The spray-dried powder, obtained following the method of the present invention, will typically have a specific volume of less than about 4 mL / g, preferably less than about 3 ml / g.

[0126] In some embodiments, the spray-dried powder has a specific volume between about 2.0 ml / g and about 3.0 ml / g. Accordingly in some embodiments, the spray-dried powder has a specific volume of about 2.0 ml / g, 2.1 ml / g, 2.2 ml / g, 2.3 ml / g, 2.4 ml / g, 2.5 ml / g, 2.6 ml / g, 2.7 ml / g, 2.8 ml / g, 2.9 ml / g, or 3.0 ml / g.

[0127] Spray-dried powders with such low specific volumes (i.e., less than 4 ml / g) are generally preferred as they have good flow characteristics.

[0128] The spray-dried powder, obtained following the method of the present invention, will typically have a glycosphingolipid content of at least about 65 wt.%, usually of at least about 70 wt.%, preferably of at least about 75 wt.%, more preferably of at least about 85 wt.%.

[0129] In some embodiments, the present invention describing a spray dried powder comprising one or more glycosphingolipids of formula (1).

[0130] In some embodiments, the spray-dried powder comprising one glycosphingolipid of formula (1).

[0131] In some embodiments, the spray-dried powder comprising more than one glycosphingolipid of formula (1).

[0132] In some embodiments, the spray-dried powder comprising a glycosphingolipid selected from the group consisting of / V-lyso-GM4, / V-lyso-GM3, N-lyso-GD3, / V-lyso-GDla, and N-lyso-GT3, or a mixture thereof.

[0133] In some embodiments, the spray-dried powder comprising a glycosphingolipid selected from the group consisting of lactosyl d-erythro-sphingosine, lactosyl d-erythro-dihydrosphingosine, lactosyl d-r / bo-phytosphingosine, galactosyl d-erythro-sphingosine, galactosyl d-erythro- dihydrosphingosine, galactosyl d-r / bo-phytosphingosine, glucosyl d-erythro-sphingosine, glucosyl d- erythro-dihydrosphingosine, and glucosyl d-r / bo-phytosphingosine, or a mixture thereof.

[0134] In some embodiments, the spray-dried powder comprising at least about 70 wt.% of / V-lyso-GM3, or at least about 75 wt.% of / V-lyso-GM3, or at least about 80 wt.%> of / V-lyso-GM3.

[0135] In some embodiments, the spray-dried powder comprising about 75-80 wt.% of / V-lyso-GM3, and wherein the spray dried-powder further comprising about 7-9 wt.% of lactosyl D-erythro- sphingosine, and about 0.1-1.0 wt.% of glucosyl D-erythro-sphingosine.

[0136] In some embodiments, the spray-dried powder comprising at least about 50 wt.% of a mixture of N- lyso-GD3 and / V-lyso-GM3, or at least about 60 wt.% of a mixture of / V-lyso-GD3 and / V-lyso-GM3, or at least about 70 wt.% of a mixture of / V-lyso-GD3 and / V-lyso-GM3, or at least about 80 wt.% of a mixture of / V-lyso-GD3 and / V-lyso-GM3, and wherein the weight ratio between / V-lyso-GD3 and N- lyso-GM3 in said mixture is from about 1:10 to about 10:1.

[0137] In some embodiments, the weight ratio between / V-lyso-GD3 and / V-lyso-GM3 in said mixture is about 1:10.

[0138] In some embodiments, the weight ratio between / V-lyso-GD3 and / V-lyso-GM3 in said mixture is about 1:3.

[0139] In some embodiments, the weight ratio between / V-lyso-GD3 and / V-lyso-GM3 in said mixture is about 1:1.

[0140] In some embodiments, the ratio weight between / V-lyso-GD3 and / V-lyso-GM3 in said mixture is about 4:1.

[0141] In some embodiments, the spray-dried powder comprising about 40-55 wt.% of / V-lyso-GD3 and about 10-15 wt.% of / V-lyso-GM3, and wherein the spray-dried powder further comprising about 3- 6 wt.% of / V-lyso-GT3, about 4-6 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D-erythro-sphingosine.

[0142] In some embodiments, the spray-dried powder comprising about 15-20 wt.% of / V-lyso-GD3 and about 50-60 wt.% of / V-lyso-GM3, and wherein the spray-dried powder further comprising about 0.1-0.5 wt.% of / V-lyso-GT3, about 4-7 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D-erythro-sphingosine.

[0143] In some embodiments, the spray-dried powder comprising about 35-40 wt.% of / V-lyso-GD3 and about 25-40 wt.% of / V-lyso-GM3, and wherein the spray-dried powder further comprising about 5- 6 wt.% of lactosyl D-erythro-sphingosine, and about 0.5-1.0 wt.% of glucosyl D-erythro-sphingosine. In some embodiments, the spray dried powder is obtainable by the method of the present invention.

[0144] In some embodiments, the spray-dried powder according to the present invention, is utilized for the production of gangliosides or ceramide according to methods such that described in WO2023099478 Al.

[0145] In some embodiments, the spray-dried powder according to the present invention, is utilized for the production of a ganglioside or a ceramide according to a method comprising: reacting a spray-dried powder comprising one or more glycosphingolipids of formula (1) with an ester of formula (6):

[0146] (6), wherein

[0147] R5is selected from a substituted or unsubstituted C1-31 alkyl which may be saturated or unsaturated, preferably a substituted or unsubstituted C9-31 alkyl which may be saturated or unsaturated;

[0148] R6is a C1-C4 alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl, or ethyl, in the presence of a base.

[0149] Typically, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of a base such as an alkoxide, an amine, a carbonate, or a bicarbonate.

[0150] In some embodiments, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of an amine, wherein the amine is preferably selected from triethylamine, / V, / V-diisopropylethylamine, and pyridine.

[0151] In some embodiments, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of a carbonate, wherein the carbonate is preferably selected from Na2CO3, K2CO3, CaCO3, Li2CO3, (NH4)2CO3.

[0152] In some embodiments, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of a bicarbonate, wherein the bicarbonate is preferably selected from NaHCO3, KHCO3, Ca(HCO3)2, LiHCO3, NH4HCO3. In some preferred embodiments, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of an alkoxide, and wherein the alkoxide is an alkoxide of formula (12):

[0153] R7— O'Q

[0154] (12), wherein

[0155] R7is a C1-C4 alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl, or ethyl;

[0156] Q+is a cation selected from Na+, K+, Li+or NH4+, preferably Na+.

[0157] In some more preferred embodiments, the spray-dried glycosphingolipid of formula (1) and the ester of formula (6) are reacted in the presence of sodium methoxide.

[0158] The base may be used in catalytic amounts, equimolar amounts or in excess.

[0159] In some embodiments, the spray-dried glycosphingolipid of formula (1) is in the free-base form and the base is used in a catalytic amount from about 0.1 to about 0.5 molar equivalents based on the amount of the spray-dried glycosphingolipid.

[0160] In some embodiments, the spray-dried glycosphingolipid of formula (1) is in a salt form and the base is used in an amount from about 1.0 to about 1.7 molar equivalents based on the amount of the spray-dried glycosphingolipid.

[0161] In some preferred embodiments, the spray-dried glycosphingolipid of formula (1) is in a salt form and the base is used in an amount from about 1.2 to 1.3 molar equivalents based on the amount of the spray-dried glycosphingolipid.

[0162] In some embodiments, the spray-dried glycosphingolipid of formula (1), the ester of formula (6), and the base are reacted in a polar solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol.

[0163] In some preferred embodiments, the reaction is performed in methanol.

[0164] In some embodiments the reaction is performed in a mixture of one or more polar solvents, such as a mixture of methanol and ethanol, methanol and propanol, methanol and isopropanol, methanol and butanol, methanol and isobutanol, or the mixture of water and an aliphatic alcohol. In some embodiments, the reaction is performed in acetonitrile. In some embodiments, the spray-dried glycosphingolipid of formula (1), the ester of formula (6), and the base are reacted in C5-C10 hydrocarbon solvent, preferably wherein the hydrocarbon solvent is heptane.

[0165] In some embodiments, the reaction is performed solvent-free.

[0166] The spray-dried glycosphingolipid of formula (1), the ester of formula (6), and the base are typically reacted at a temperature from about 50° C to about 125° C. Preferably at a temperature from about 60° C to about 65° C. Accordingly, in some preferred embodiments, the reaction is performed at a temperature of about 60° C, 61° C, 62° C, 63° C, 64° C, or 65° C.

[0167] The components of the reaction may be combined in any order, and it will be appreciated that the order of combining the reactants may be adjusted as needed.

[0168] The spray-dried glycosphingolipid of formula (1), the ester of formula (6), and the base, as well as any other reagent used during the reaction may be added to the reaction either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the reaction.

[0169] Glycosphingolipids according to the present invention are typically produced via enzymatic or biotechnological methods.

[0170] A method for the production of glycosphingolipids carrying a neutral glycosyl moiety, such as lactosyl d-erythro-sphingosine is described in WO2023118378 Al, wherein said glycosphingolipids are obtained via the coupling of a glycosyl fluoride with a sphingoid base in the presence of an endoglycoceramidase glycosynthase.

[0171] A biotechnological method for the synthesis of complex glycosphingolipids has been described in WO2021170624 A2, wherein a glycosylated sphingoid bases, such as lactosyl D-erythro-sphingosine, are internalized by a cell and further glycosylated.

[0172] Glycosphingolipids carrying a sialylated glycosyl moiety, such as / V-lyso-GM3 or / V-lyso-GD3, may be produced via the enzymatic processes described in the embodiments below.

[0173] In one embodiment, the sphingolipid of formula (1) is a sialylated glycosphingolipid, wherein the sialylated glycosphingolipid is produced via a method comprising steps of:

[0174] - providing: a glycosphingolipid acceptor of formula (7): (7) wherein X is Gaipi-, or a glycosyl moiety carrying one or more terminal p-galactopyranosyl units, R1, R2, R3and the bond - are as defined as for the glycosphingolipid of formula (1), a sialic acid donor, an enzyme having a trans-sialidase activity, and an enzyme having p-galactosidase activity,

[0175] - mixing said glycosphingolipid acceptor with said sialic acid donor in the presence of said enzyme having trans-sialidase activity, thereby producing said sialylated glycosphingolipid, and sequentially

[0176] - adding the enzyme having a p-galactosidase activity,

[0177] - nanofiltration of the reaction mixture, and wherein the sialic acid donor is 3'-sialyllactose.

[0178] In some embodiments, X of the glycosphingolipid acceptor of formula (6) is selected from the group consisting of Gall-, Glcl-, Gaipi-4Glcl-, wherein the glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond.

[0179] In some embodiments, the glycosphingolipid acceptor of formula (6) is selected from the group consisting of lactosyl D-erytbro-sphingosine, lactosyl D-erytbro-dihydrosphingosine, lactosyl D-ribo- phytosphingosine, galactosyl D-erytbro-sphingosine, galactosyl D-erytbro-dihydrosphingosine, galactosyl D-r / bo-phytosphingosine, glucosyl D-erytbro-sphingosine, glucosyl D-erythro- dihydrosphingosine, and glucosyl D-r / bo-phytosphingosine, or a mixture thereof.

[0180] In some preferred embodiments, the glycosphingolipid acceptor of formula (6) is lactosyl D-erythro- sphingosine.

[0181] In some embodiments, X of the glycosphingolipid acceptor of formula (6) is Gaipi-3GalNAcpi- 4(Neu5Aca2-3)Gaipi-4Glcl-, wherein the glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond.

[0182] In some embodiments, the glycosphingolipid acceptor of formula (6) is / V-lyso-GMla.

[0183] The term "sialic acid donor", as used herein, refers to a compound carrying a sialic acid unit that can be transferred to a suitable acceptor, such as a glycosphingolipid. Sialic acid donors, suitable for use in the context of the present invention, are typically a-sialylated compounds which can derive from natural sources or can be chemically synthesized. a-Sialylated compounds deriving from natural sources are for example 3'-sialyllactose, sialic acid rich protein, and colominic acid. Chemically synthesized a-sialylated compounds include but are not limited to p-nitrophenyl / V- acetylneuraminic acid (Neu5AcapNP), methylumbelliferyl / V-acetylneuraminic acid (Neu5AcaMU) and derivatives thereof.

[0184] In a preferred embodiment, the sialic acid donor is 3'-sialyllactose.

[0185] The term "an enzyme having a trans-sialidase activity" may be interchangeably used with the term "trans-sialidase" and denotes, in the context of the present invention, an enzyme belonging to the glycoside hydrolase family 33 (GH33) which typically catalyses the reversible transfer of a glycosidically linked sialic acid from sialic acid donors such as for example oligosaccharides, glycoproteins, glycolipids, and colominic acid to acceptor molecules containing a terminal - galactopyranosyl unit. In the absence of a suitable acceptor molecule, these enzymes may act as sialidases and transfer the glycosidically linked sialic acid to a water molecule. However, their hydrolytic activity is typically low.

[0186] The trans-sialidase in its wild-type form, may originate from parasitic euglenoids, such as Trypanosoma cruzi, Trypanosoma congolense, or Trypanosome brucei.

[0187] In some embodiments, the enzyme having trans-sialidase activity is a wild-type trans-sialidase originating from Trypanosoma cruzi. The amino acid sequence of the wild-type trans-sialidase originating from Trypanosoma cruzi correspond to the amino acid sequence having accession No: Q26966 (https: / / www.uniprot.org / ).

[0188] The trans-sialidase originating from Trypanosoma cruzi may also be referred to as TcTS.

[0189] In some embodiments, the enzyme having trans-sialidase activity is a mutant of the wild-type trans-sialidase originating from Trypanosoma cruzi (Q26966).

[0190] In some embodiments, the amino acids sequence of the mutant trans-sialidase comprises the following mutations / modifications compared to the wild-type amino acid sequence Q26966: Ser263Thr, Arg477His, Val485Leu, Glu559Val, Ser496Lys, / V-terminal His-tag, deletion of 7 amino acids A636-642 (the numbering corresponding to alignment of the mutant amino acid sequence with the amino acid sequence of Q26966).

[0191] The mutant trans-sialidase, according to the present invention, may be produced by methods known to the skilled person. A method for the expression and purification of a mutant trans- sialidase is for example described in Paris et al., Glycobiology 2001, 11, 305-311, or in Buschiazzo et al., Molecular Cell 2002, 10, 757-768.

[0192] The term "an enzyme having a p-galactosidase activity" may be interchangeably used with the term "P-galactosidase" and denotes, in the context of the present invention, an enzyme belonging to the glycoside hydrolase family 35 (GH35) which typically catalyses the hydrolysis of terminal nonreducing p-D-galactose residues in p-D-galactosides.

[0193] In the context of the present invention a p-galactosidase may also be referred to as lactase.

[0194] In some embodiments, the enzyme having p-galactosidase activity is a wild-type p-galactosidase originating from Aspergillus orizyae, or a functional analogue thereof. The amino acid sequence of the wild-type p-galactosidase originating from Aspergillus orizyae correspond to the amino acid sequence having accession No: Q2UCU3 (https: / / www.uniprot.org / , accession).

[0195] In some preferred embodiments, the enzyme having p-galactosidase activity is a truncated variant of the wild-type p-galactosidase originating from Aspergillus orizyae (Q2UCU3).

[0196] The truncated variant of the p-galactosidase, according to the present invention, can be purchased from established manufacturers, e.g. Calza Clemente, or produced by methods known to the skilled person such as that described in M.M. Maksimainen et al., International Journal of Biological Macromolecules 2013, 60, 109-115.

[0197] The step of adding an enzyme having a p-galactosidase activity may advantageously be used to hydrolyze the lactose formed during the sialyltransferase catalyzed sialylation of the glycosphingolipid acceptor of formula (6).

[0198] The step of adding the enzyme having a p-galactosidase activity is typically performed after a certain conversion of 3'-sialyllactose is reached. Preferably, the step of adding the enzyme having a P-galactosidase activity is performed when a conversion of at least about 50% of 3'-sialyllactose is reached, preferably when a conversion of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of 3'-sialyllactose is reached. The conversion of 3'-sialyllactose can be determined by standard techniques known to the skilled person. Typically, the conversion of 3'-sialyllactose is determined by HPLC and may be given in mol.% or wt.%.

[0199] The nanofiltration (NF) step may be used to concentrate the mixture containing the sialylated glycosphingolipid, to remove ions, mainly monovalent ions, and / or to remove organic materials having a molecular weight lower than that of the sialylated glycosphingolipid, such as monosaccharides. In a preferred embodiment, the nanofiltration step is used to remove galactose and glucose from the mixture containing the sialylated glycosphingolipid.

[0200] Typically, the nanofiltration membrane has a molecular weight cut-off (MWCO) that ensures the retention of the sialylated glycosphingolipid of interest. As an example, a nanofiltration membrane having a MWCO of about 200-500 Da, is suitable for retaining the sialylated glycosphingolipid. In this regard the sialylated glycosphingolipid is accumulated in the NF retentate (NFR). Nanofiltration can be combined with diafiltration (DF) with water in order to remove permeable molecules more effectively, e.g. until the conductivity of the permeator shows no or very low presence of salts.

[0201] The NF step according to the present invention is conducted, with or without the optional DF step, at a constant temperature, preferably between about 15-45 °C, more preferably between about 20- 35 °C. The NF step, with or without diafiltration, is continued until reaching the desired concentration of the sialylated glycosphingolipid in the NFR. Other technical parameters like setting in the flux and pressure is a matter of routine skills.

[0202] The sialylation method described above comprises a step of mixing a glycosphingolipid acceptor with a sialic acid donor in the presence of an enzyme having trans-sialidase activity, followed by a step of adding a p-galactosidase to the reaction mixture.

[0203] The enzyme(s) and substrates may be added in any order, and it is appreciated that the order of combining the reactants may be adjusted as needed.

[0204] For example, the sialic acid donor may be added to a solution of the glycosphingolipid, followed by the addition of the trans-sialidase.

[0205] The sialic acid donor, the glycosphingolipid, the trans-sialidase, and the p-galactosidase, as well as any other component used during the sialylation reaction may be added to the reaction mixture either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the process.

[0206] The temperature at which the above process is carried out can range from just above freezing to the temperature at which the most sensitive enzyme denatures. That temperature range is preferably from about 0 °C to about 45 °C, and more preferably from about 20 °C to 37 °C.

[0207] The glycosphingolipid acceptor and the sialic acid donor are for a period of time sufficient to obtain the desired high yield of the desired sialylated glycosphingolipid.

[0208] Typically, the reaction is allowed to proceed for between about 1 to about 24 hours, preferably between about 5 to about 10 hours. In some embodiments, reaction is allowed to proceed for about 5, 6, 7, 8, 9, or 10 hours.

[0209] The glycosphingolipid, the trans-sialidase and the p-galactosidase, may be combined by admixture in an aqueous reaction medium. The medium generally has a pH value of about 5 to about 7.5. The selection of the medium is based on the ability of the medium to maintain the pH value at the desired level. Accordingly, in some embodiments the medium is buffered to a pH value of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In some preferred embodiments, the medium is buffered to a pH value of about 5.5 to 6.5. Accordingly, in some preferred embodiments, the medium is buffered to a pH value of about

[0210] 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

[0211] Suitable buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, MOPS, HEPES, PBS, sodium acetate buffer, sodium citrate buffer. Preferably, sodium acetate buffer. If a buffer is not used, the pH of the medium should be maintained at about 5 to about 7.5 by the use of a base or an acid. A suitable base is NaOH, a suitable acid is HCI.

[0212] In another embodiment, the sphingolipid of formula (1) is a sialylated glycosphingolipid, wherein the sialylated glycosphingolipid is produced via a method comprising mixing a glycosphingolipid acceptor of formula (8): wherein Y is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from the group consisting of Gall-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal / V-acetyl-galactosamine units and / or one or more terminal sialic acid units, R1, R2and R3and the bond - are as defined as for the glycosphingolipid of formula (1), with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extract(s) of a microorganism, said microorganism comprising one or more endogenous polypeptide(s) having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein the said one or more cell-free extract(s) comprise:

[0213] - at least one polypeptide having cytidine monophosphate kinase activity,

[0214] - at least one polypeptide having / V-acylneuraminate citydyltransferase activity, and

[0215] - at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside acceptor.

[0216] In some preferred embodiments, Y of the glycosphingolipid acceptor of formula (7) is a glycosyl moiety selected from the group consisting of the following glycosyl moieties Neu5Aca2-3Gaipi-4Glc- , Gaipi-3GalNAcpi-4(Neu5Aca2-3)Gaipi-4Glcl-, or salts thereof, and wherein the glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably via a beta glycosidic bond. In some preferred embodiments, the glycosphingolipid acceptor of formula (7) is selected form the group consisting of / V-lyso-GM3, and / V-Lyso-GMla.

[0217] In some embodiments, Y of the glycosphingolipid acceptor of formula (7) is selected from the group consisting of Gall-, and Gaipi-4Glcl-. Accordingly, in some preferred embodiments, the glycosphingolipid acceptor is galactosyl D-erythro-sphingosine, or lactosyl D-erythro-sphingosine.

[0218] The sialylation of the glycoside acceptor of formula (7) is typically carried out as part of a sialyltransferase cycle, which comprises a CMP-sialic acid recycling system, wherein CMP-sialic acid is generated / regenerated from sialic acid and CMP.

[0219] CMP-Sialic acid is a relatively expensive sugar nucleotide. Therefore, the in-situ generation and regeneration of the sialic acid donor is of economic advantage and enables the scale up of the process.

[0220] The sialyltransferase cycle described in the present invention, typically comprises sialic acid, cytidine monophosphate (CMP), a nucleoside triphosphate, and the use of one or more cell-free extract(s) of a microorganism, wherein the one or more cell-free extracts comprising the enzymatic activities needed for the sialyltransferase cycle. The enzymatic activities needed for the sialyltransferase cycle comprise:

[0221] - at least one phosphotransferase enzymatic activity

[0222] - at least one inorganic diphosphatase enzymatic activity,

[0223] - at least one cytidine monophosphate kinase enzymatic activity,

[0224] - at least one / V-acylneuraminate cytidylyltransferase enzymatic activity, and

[0225] - at least one sialyl transferase enzymatic activity.

[0226] Nucleoside triphosphates suitable for use in the context of the present invention are adenosine-5'- triphosphate (ATP), uridine-5'-triphosphate (UTP), guanosine-5'-triphosphate (GTP), inosine triphosphate (ITP) and thymidine-5'-triphosphate (TTP).

[0227] In some preferred embodiments, the nucleoside triphosphate is adenosine-5'-triphosphate (ATP).

[0228] Accordingly in some preferred embodiments the sialyltransferase cycle comprises / V-acetyl- neuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), and one or more cell-free extract of a microorganism, wherein the one or more cell-free extract comprising one polypeptide having cytidine monophosphate kinase activity (CMK) (for the phosphorylation of CMP), one polypeptide having phosphotransferase activity (for the phosphorylation of CDP), one polypeptide having / V-acylneuraminate cytidyltransferase activity (CSS) (for the transfer of CMP from CTP to Neu5Ac), one polypeptide having sialyltransferase activity (for the transfer of Neu5Ac from CMP-Neu5Ac to the acceptor substrate), and one polypeptide having inorganic diphosphatase activity (PPase) (to degrade the inorganic pyrophosphate (PPi) formed during the cycle), and wherein, the polypeptide having phosphotransferase activity, and the polypeptide having inorganic diphosphatase activity (PPase) are endogenously expressed by the microorganism.

[0229] In some preferred embodiments, the one or more cell-free extract(s) are of a microorganism, wherein said microorganism comprising at least one endogenous polypeptide having phosphotransferase enzymatic activity, and at least one endogenous polypeptide having diphosphatase enzymatic activity, and wherein the said microorganism is genetically engineered for the expression of one or more polypeptides selected from the group consisting of:

[0230] -at least one heterologous polypeptide having cytidine monophosphate kinase activity, -at least one heterologous polypeptide having N-acylneuraminate citydyltransferase activity, - at least one heterologous polypeptide having sialyl-transferase activity.

[0231] The microorganism according to method described in present invention preferably comprises reduced or no p-galactosidase activity. The reduction or knock-out of the p-galactosidase activity may be achieved, e.g., by genetic manipulation of a gene encoding a polypeptide with p- galactosidase activity, e.g., by introduction of a mutation leading to expression of inactive enzyme, to a gene knock out or by other means.

[0232] The microorganism according to the method described in the present invention may be a yest or a bacterium, preferably a bacterium.

[0233] In some embodiments, the microorganism is Escherichia coli (E. coli).

[0234] In some embodiments, the microorganism is an E. coli BL21 (DE3) strain.

[0235] The person skilled in the art will understand that in the context of the present invention, the term "a microorganism" is meant to encompass a living cell, such as a bacterial or yeast cell, and may comprise one or more variations of said living cell also called herein "strain(s)" of said microorganism. The term "strains" also includes variants of a microorganism artificially (recombinantly) created by genetic engineering of the said microorganism.

[0236] In some embodiments, the one or more strain(s) of the microorganism are created by genetic engineering of the E. coli BL21 (DE3) strain.

[0237] E. coli BL21 (DE3) cells can be obtain from established manufacturer such as ThermoFischer Scientific. Typically, the one or more strain(s) of the microorganism are utilized for the production of the one or more cell-free extract(s) via methods known to the skilled person (e.g. Cole et al. Synthetic and Systems Biotechnology 2020, 5, 252-267).

[0238] In some embodiments, the method comprises the use of three cell-free extracts of a microorganism, wherein the first cell-free extract comprising a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract comprising a polypeptide having N- acylneuraminate citydyltransferase activity, and the third cell-free extract comprising a polypetide having sialyltransferase activity, and wherein the three cell-free extracts are of a microorganism which endogenously express a polypeptide having inorganic diphosphatase activity, and at least a polypeptide having phosphotransferase activity.

[0239] The term "a polypeptide having a sialyltransferase activity" may be interchangeably used with the term "sialyltransferase" and denotes, in the context of the present invention, an enzyme belonging to the EC class 2.4.99.-.

[0240] Suitable sialyltransferases for use in the context of the present invention are sialyltransferases capable of catalyzing the transfer of a sialic acid residue to the 0-3 of a p-linked galactose residue of a glycoside acceptor and / or to the 0-8 of an a-2-3-linked sialic acid residue of a glycoside acceptor.

[0241] In some embodiments, the polypeptide having sialyltransferase activity is the wild-type a-2,3 / a-2,8- sialyltransferase originating from Campylobacter jejuni, strain OX=197. The amino acid sequence of the wild-type a-2,3 / a-2,8-sialyltransferase originating from Campylobacter jejuni, strain OX=197 corresponds to the amino acid sequence having accession NO: Q9LAK3 (https: / / www.ncbi.nlm.nih.gov / protein /

[0242] In some embodiments, the polypeptide having sialyltransferase activity is a mutant of the wild-type a-2,3 / a-2,8-sialyltransferase Q9LAK3, wherein the mutant comprising the following mutations / modifications compared to the wild-type: He53Ser, deletion of 32 amino acids at the C- terminus (the numbering corresponding to alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3).

[0243] In some embodiments, the a-2,3 / a-2,8-sialyltransferase is a mutant of the wild-type a-2,3 / a-2,8- sialyltransferase Q9LAK3, wherein the mutant comprising the following mutations / modifications compared to the wild-type: He53Ser, / V-terminal-histidine tag, deletion of 32 amino acids at the C- terminus (the numbering corresponding to alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3). In some embodiments, the a-2,3 / a-2,8-sialyltransferase a mutant of the wild-type a-2,3 / a-2,8- sialyltransferase Q9LAK3, wherein the mutant comprising the following mutations / modifications compared to the wild-type: He53Gly, / V-terminal-histidine tag, deletion of 32 amino acids at the C- terminus (the numbering corresponding to alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3).

[0244] The a-2,3 / a-2,8-sialyltransferase originating from Campylobacter jejuni, or the functional analogues thereof may also be referred to as CST-IL

[0245] In some embodiments, the polypeptide having sialyltransferase activity is the wild-type a-2,3 sialyltransferase originating from Bibersteinia trehalosi, strain DSM 23101. The amino acid sequence of the wild-type Bibersteinia trehalosi a-2,3 sialyltransferase corresponds to the amino acid sequence having accession NO: WP_025267256 (https: / / www.ncbi.nlm.nih.gov / protein).

[0246] In some embodiments, the a-2,3 sialyltransferase is a recombinant a-2,3 sialyltransferase derived from the wild-type a-2,3 sialyltransferase WP_025267256, wherein the recombinant sialyltransferase comprising the following mutations / modifications compared to the wild-type: N- terminal histidine tag MGHHHHHH.

[0247] The a-2,3-sialyltransferase originating from Bibersteinia trehalose, or the functional analogues thereof may also be referred to as BtSiaT.

[0248] The term "a polypeptide having cytidine monophosphate kinase activity" may be interchangeably used with the term "CMP kinase" or "CMK" and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.25., which typically catalyses the phosphorylation of CMP (or dCMP), using ATP as the preferred phosphoryl donor.

[0249] In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a CMP kinase originating from Mycobacterium tuberculosis, or a functional analogue thereof.

[0250] In some embodiments, the polypeptide having CMK kinase activity is the wild-type CMK kinase originating form Mycobacterium tuberculosis. The amino acid sequence of the wild-type CMP kinase originating from Mycobacterium tuberculosis corresponds to the amino acid sequence having accession NO: WP_129368399 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0251] In some embodiments, the CMP kinase is a recombinant CMP kinase deriving from the wild-type CMP kinase WP_129368399, wherein the recombinant CMP kinase comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH.

[0252] The CMP kinase originating from Mycobacterium tuberculosis, or its functional analogues thereof may also be referred to as MtCMK. The term "a polypeptide having / V-acylneuraminate cytidyltransferase activity" may be interchangeably used with the term " / V-acylneuraminate cytidylyltransferase" or "CSS" and denotes, in the context of the present invention, an enzyme of the EC class 2.7.7.43, which catalyses the transfer of CMP from CTP to / V-acetyl-neuraminic acid (Neu5Ac).

[0253] In some embodiments, the polypeptide having / V-acylneuraminate cytidyltransferase activity is the wild-type CSS originating from Neisseria meningitidis. The amino acid sequence of the wild-type CSS originating from Neisseria meningitidis corresponds to the amino acid sequence having accession NO: WP_061726245 (https: / / www.ncbi.nlm.nih.gov / genbank / )

[0254] In some embodiments, the CSS is a recombinant CSS derived from the wild-type CSS WP_061726245 comprising the following mutations / modifications compared to the wild-type: N- terminal histidine tag MGHHHHHH.

[0255] The / V-acylneuraminate cytidyltransferase originating from Neisseria meningitidis, or its functional analogues thereof may also be referred to as NmCSS.

[0256] The term a "polypeptide having inorganic diphosphatase activity" may be interchangeably used with the term "inorganic diphosphatase" or "PPase" and denotes, in the context of the present invention, an enzyme of the EC class 3.6.1.1., which catalyses the hydrolysis of pyrophosphate (PPi).

[0257] In some preferred embodiments, the polypeptide having inorganic diphosphatase activity is the wild-type PPase originating from Escherichia coli. The amino acid sequence of the wild-type inorganic diphosphatase originating from Escherichia coli corresponds to the amino acid sequence having accession No: WP_073849715 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0258] The inorganic diphosphatase originating from Escherichia coli may also be referred to as EcPPase.

[0259] In some embodiments, the polypeptide having phosphotransferase activity" is a polypeptide having nucleoside diphosphate kinase activity.

[0260] The term "a polypeptide having nucleoside diphosphate kinase activity" may be interchangeably used with the term "nucleoside-diphosphate kinase" or "NDK" and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.6., which catalyses the phosphorylation of a nucleoside diphosphate.

[0261] In some preferred embodiments, the polypeptide nucleoside diphosphate kinase activity is the wild-type NDK originating from Escherichia coli, strain BL21(DE3). The amino acid sequence of the wild-type NDK originating from Escherichia coli, strain BL21(DE3), corresponds to the amino acid sequence having accession NO: ACT44230 (https: / / www.ncbi.nlm.nih.gov / genbank / ). The nucleoside diphosphatase originating from Escherichia coli, strain BL21(DE3) may also be referred to as EcNDK.

[0262] In some embodiments, the polypeptide having phosphotransferase activity is a polypeptide having myokinase activity.

[0263] The term "a polypeptide having myokinase activity" may be interchangeably used with the term "myokinase", "adenylate kinase", or "ADK" and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.3., which catalyses the interconversion of the various adenosine phosphates (e.g. ATP, ADP, and AMP).

[0264] In some preferred embodiments, the polypeptide having myokinase activity is the wild-type myokinase originating from Escherichia coli, strain BL21(DE3). The amino acid sequence of the wildtype ADK originating from Escherichia coli, strain BL21(DE3), corresponds to the amino acid sequence having accession NO: ACT42324 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0265] The myokinase originating from Escherichia coli, strain BL21(DE3) may also be referred to as EcADK.

[0266] In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK (GenBank Accession NO: WP_129368399), the second cell-free extract comprising the recombinant NmCSS (wild-type: WP_061726245, modification: / V-terminal histidine tag MGHHHHHH ), and the third cell- free extract comprising a mutant CSTII (wild-type Q9LAK3, mutation / modifications: He53Ser or He53Ser, / V-terminal-histidine tag, deletion of 32 amino acids at the C-terminus ), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715), EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324).

[0267] In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK (GenBank Accession NO: WP_129368399), the second cell-free extract comprising the recombinant NmCSS (wild-type: WP_061726245, modification: N-terminal histidine tag MGHHHHHH ), and the third cell- free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715), EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324).

[0268] In some embodiments, the method comprises the use of four cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK ( GenBank Accession NO: WP_129368399), the second cell-free extract comprising the recombinant NmCSS (wild-type: WP_061726245, modification: N-terminal histidine tag MGHHHHHH ), the third cell-free extract comprising a mutant CSTII (wild-type Q9LAK3, mutation / modifications: He53Ser or He53Ser, / V-terminal-histidine tag, deletion of 32 amino acids at the C-terminus ), and the fourth cell-free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715) , EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324).

[0269] For the sialyltransferase cycles, the concentrations or amounts of the various reactants used in the processes depend upon numerous factors including reaction conditions such as temperature and pH value, and the choice and amount of acceptor glycoside to be sialylated. Because the sialylation process permits regeneration of activating nucleotides, activated donor sugars, and scavenging of produced PPi in the presence of catalytic amounts of the enzymes, the process is limited by the concentrations or amounts of the stoichiometric substances. The upper limit for the concentrations of reactants that can be used in accordance with the method of the present invention is determined by the solubility of such reactants. Preferably, the concentrations of activating nucleotides, phosphate donor, the donor sugar and enzymes are selected such that glycosylation proceeds until the acceptor is consumed.

[0270] The sialyl transferase cycle according to the method of the present invention, can also include other ingredients that facilitate the sialyltransferase activity. These ingredients can include a divalent cation (e.g., Mg+2or Mn+2), materials necessary for ATP regeneration, phosphate ions etc. The reaction medium may also comprise solubilizing detergents (e.g., Triton or SDS) and organic solvents such as methanol or ethanol, or a cyclodextrin.

[0271] In a preferred embodiment the reaction medium comprises a cyclodextrin.

[0272] In some embodiments, the cyclodextrin is selected from the group consisting of p-cyclodextrin, hydroxypropyl-p-cyclodextrin, randomly methylated p-cyclodextrin, or sulfobutylether-p- cyclodextrin. In some preferred embodiments, the cyclodextrin is p-cyclodextrin.

[0273] The cyclodextrin is typically used in an amount between about 0.1 equivalents to about 1 equivalent based on the amount of the glycosphingolipid acceptor. In some preferred embodiments the cyclodextrin is used in an amount between about 0.1 equivalents to about 0.5 equivalents based on the amount of the glycosphingolipid acceptor. Accordingly, in some preferred embodiments, the cyclodextrin is used in an amount of about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the amount of the glycosphingolipid acceptor.

[0274] The use of a cyclodextrin provides several advantages such as high yields, and eliminates the need for the use of a detergent or organic solvent to increase accessibility to the glycosyl moiety of the glycosphingolipid acceptor. However, detergents or organic solvents can also be used in the method of the invention.

[0275] In optimized reactions, the above ingredients can be combined by admixture in an aqueous reaction medium (solution) which has a pH value of about 6 to about 8.5. The medium is devoid of chelators that bind enzyme cofactors such as Mg+2or Mn+2. The selection of a medium is based on the ability of the medium to maintain pH value at the desired level. Thus, in some embodiments, the medium is buffered to a pH value at about 6.5 to about 8.5. If a buffer is not used, the pH of the medium should be maintained at about 6.5 to 8.0, preferably about 7.3 to 8.0, by the addition of base. A suitable base is NaOH. Accordingly, in some preferred embodiments the pH is buffered or kept at a value of about 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0

[0276] The temperature at which the above process is carried out can range from just above freezing to the temperature at which the most sensitive enzyme denature. That temperature range is preferably at about 0 °C to about 45 °C, and more preferably at about 20 °C to 37 °C.

[0277] The reaction mixture so formed is maintained for a period of time sufficient for the sialyltransferase to sialylate a high percentage of the acceptors. Typically, the reaction will often be allowed to proceed for about 8 to about 240 hours, preferably between about 24 and 48 hours.

[0278] The / V-acetyl-neuraminic acid (Neu5Ac), the cytidine monophosphate (CMP), the adenosine 5'- triphosphate (ATP), polyphosphate, the cell-free extract(s), as well as any other component used during the cycle may be added to the reaction mixture either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the process.

[0279] In the context of the present invention, cell-free extract enzymatic activities are expressed in activity Units, which is a measure of the initial rate of catalysis. One activity Unit catalyses the formation of 1 pmol of product per minute at a given pH and temperature. The cell-free extract(s) enzymatic activities can be measured according to procedures known to the skilled person.

[0280] The glycosphingolipids according to the present invention may be produced or utilized in the form of salts, preferably in the form of pharmaceutical acceptable salts.

[0281] In some embodiments, the salts may be formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphoric acid, acetic acid, camphor sulfonic acid, p-toluene sulfonic acid, methane sulfonic acid, trifluoromethanesulfonic acid, perchloric acid.

[0282] Examples

[0283] The working examples below describe non-limiting embodiments of the invention and are given only to illustrate the invention.

[0284] General methods and materials The glycosphingolipid content of the spray-dried powders was determined by HPLC via peak area analysis using external standards. HPLC analyses were performed on a Dionex Ultimate 3000 HPLC system coupled with a Corona Veo Charged Aerosol Detector using an Accucore aQ. (150 mm x 4.6 mm, 2.6 pm) column. Methods are described in Example 14.

[0285] Lactosyl D-erythro-sphingosine was synthesized as described in WO2023118378 Al, or by

[0286] Vaughan et al., J. Am. Chem. Soc. 2006, 128, 6300-6301.

[0287] Solution comprising N-lyso-GM3 or mixtures of N-lyso-GM3 and N-lyso-GD3 were prepared according to the procedures described in Examples 11 and 12, respectively.

[0288] Mutant TcTS (wild-type Q26966, mutations / modifications: Ser263Thr, Arg477His, Val485Leu, Glu559Val, Ser496Lys, / V-terminal His-tag, deletion of 7 amino acids A636-642 ) was expressed from Escherichia coli (E. coli) strains following methods described in Paris et al., Glycobiology 2001, 11, 305-311, or in Buschiazzo et al., Molecular Cell 2002, 10, 757-768.

[0289] The following cell-free extracts were produced from E. coli expression strains: (i) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for the expression of MtCMK, accession: WP_129368399;

[0290] (ii) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for the expression of recombinant NmCSS (wild-type: WP_061726245, modification: / V-terminal histidine tag MGHHHHHH);

[0291] (iii) Cell-free extract from E. coli BL21 (DE3) dLacZ genetically engineered for expression a mutant CSTII (wild-type Q9LAK3, mutation / modifications: He53Ser or He53Gly, / V-terminal-histidine tag, deletion of 32 amino acids at the C-terminus).

[0292] Cell-free extracts were prepared as described in Example 13.

[0293] Example 1. General Diafiltration Procedure

[0294] A feed solution comprising a glycosphingolipid was subjected to diafiltration (DF). The DF was performed by applying 250 kDa spiral-wound membranes having a membrane area of about 0.668 m2, a flow rate of about 10 l / h, a transmembrane pressure of about 8-10 bar, a temperature between about 20-25 °C, and around 2-10 DF volumes relative to the volume of the feed solution. During diafiltration, a high flux of about 15.3-18.1 l / m2h was maintained.

[0295] The DF retentate (DFR), comprising the glycosphingolipid, is preferably concentrated, and then spray dried as described in Example 2.

[0296] Example 2. Spray drying of the DFR

[0297] The diafiltration retentate obtained in Example 1 was spray dried on a Mobile Minor ® (GEA) spray drier under the following conditions:

[0298] Inlet flow rate: 30-50 g / min Atomizer speed: 20,000 rpm

[0299] Inlet temperature: 150-160 °C

[0300] Outlet temperature: 85-92 °C

[0301] Following this procedure, a spry-dried powder comprising about 65-90 wt.% of one or more glycosphingolipid was obtained.

[0302] Example 3. Particle size analysis of the spray-dried powder

[0303] The mean particle diameter, as well as the D(0.1), D(0.5), and D(0.9) values were measured by laser diffraction particle size analysis using a Malvern Mastersizer 3000 (Malvern Instruments). The spray- dried powder obtained in Example 2 was dispersed in cyclohexane including 0.1 % soy lecithin. The samples were sonicated before size measurement to disperse the aggregated particles.

[0304] Example 4. Water content of the spray-dried powder

[0305] The water content of the spray-dried powder obtained in Example 2 was determined by thermogravimetry (TG) and differential scanning calorimetry (DSC), or via Karl Fisher titration. TG and DSC measurements were performed on a Setaram LabsysEvo (Setaram). The spray-dried powder typically contains between about 2-3 wt.% of water.

[0306] Example 5. Specific volume of the spray-dried powder

[0307] A sample of the spray-dried powder obtained in Example 2 was poured into a graduated cylinder, the tare weight of which had been measured, and the volume and weight of the sample recorded.

[0308] Example 6. Spray-dried powder comprising / V-lyso-GM3

[0309] A DFR comprising / V-lyso-GM3, obtained following the procedure of Example 1, was spray-dried under the condition of Example 2 to afford a spray-dried powder having the following characteristics: Example 7. Spray-dried powders comprising a 4:1 mixture of / V-lyso-GD3 and / V-lyso-GM3

[0310] A DFR comprising a 4:1 mixture / V-lyso-GD3 and / V-lyso-GM3, obtained following the procedure of Example

[0311] 1, was spray-dried under the condition of Example 2 to afford a spray-dried powder having the following characteristics:

[0312] Example 8. Spray-dried powders comprising a 1:3 mixture of / V-lyso-GD3 and / V-lyso-GM3

[0313] A DFR comprising a 1:3 mixture / V-lyso-GD3 and / V-lyso-GM3, obtained following the procedure of Example

[0314] 1, was spray-dried under the condition of Example 2 to afford a spray-dried powder having the following characteristics:

[0315] Example 9. Spray-dried powders comprising a 1:1 mixture of / V-lyso-GD3 and / V-lyso-GM3

[0316] A DFR comprising a 1:1 mixture / V-lyso-GD3 and / V-lyso-GM3, obtained following the procedure of

[0317] Example 1, was spray-dried under the condition of Example 2 to afford a spray-dried powder having the following characteristics:

[0318] Example 10. Spray-dried powders comprising lactosyl D-erythro-sphingosine

[0319] A DFR comprising lactosyl D-erythro-sphingosine, obtained following the procedure of Example 1, was spray-dried under the condition of Example 2 to afford a spray-dried powder having the following characteristics:

[0320] Example 11. Production of / V-lyso-GM3

[0321] The aqueous solution comprising / V-lyso-GM3 was obtained via the TcTS catalyzed sialylation of lactosyl D-erythro-sphingosine. A typical reaction mixture contained the lactosyl D-erythro-sphingosine (1 eq.), 3'-sialyllactose (3.5 eq.), and 2,3-transialidase (TcTS, 0.4 g / L). The reaction mixture was stirred for about 3-6 hours at a temperature of about 37 °C, then the p-galactosidase was added (0.13 g / L) and the reaction mixture was nanofiltered. The nanofiltration of the reaction mixture was performed applying 300-500 Da membranes, a pressure of 15-20 bar and a temperature of about 30-40 °C for about 6-8 hours. The NF retentate (NFR) was heated at a temperature between about 60-95 °C for about 10-60 minutes, and then diafiltered as described in Example 1.

[0322] Example 12. Production of mixtures of / V-lyso-GD3 and / V-lyso-GM3

[0323] The aqueous solution comprising / V-lyso-GD3 was obtained via the sialyltransferase cycle from / V-lyso- GM3. The sialyltransferase cycle was performed in an aqueous solution at a pH between about 7.0 to about 7.5, the temperature ranged between about 25 °C to about 37 °C. A typical reaction mixture contained the glycoside acceptor (1 eq.), / V-acetylneuraminic acid (Neu5Ac, 1.2-2.5 eq.), p-cyclodextrin (0.5 eq.), ATP (2.0-3.5 eq.), CMP (0.1-0.3 eq.), MgCL (0.5 M), and the following three cell-free extracts: cell-free extract (i) (5-12 g / L), cell-free extract (ii) (1-2 g / L), cell-free extract (iii) (2.5-5 g / L). The sialylation cycle was monitored by LCMS.

[0324] Example 13. Preparation of the cell-free extract

[0325] Genes encoding the enzymes are usually ordered as codon-optimized synthetic genes for optimal expression in the E. coli host strain. The synthetic constructs contain overhangs with Bsal restriction sites for golden gate cloning into a pET28a-based expression vector (carrying introduced Bsal restriction sites and a fluorescent drop-out cassette). The resulting plasmids were used for transformation of E. coli BL21 (DE3) dLacZ.

[0326] A preculture of the expression strain was prepared in lOmL LB medium supplemented with the respective antibiotic and incubated at 37°C shaking overnight. The culture of the expression strain was started by a l:100-fold dilution of the preculture into TB medium supplemented with the respective antibiotic. The culture was incubated at 37°C until an ODgoo of 0.7-1.0 was reached. The culture was cooled to the desired expression temperature, induced with 0.5 mM IPTG, and incubated for the desired expression time.

[0327] Cells were harvested by centrifugation and resuspended in water. Cell lysis was achieved by sonication. The resulting lysed cell suspension was centrifuged to separate the cell-free extract, comprising soluble enzymes, from the debris. The supernatant, containing the cell-free extract, was freeze-dried to dryness.

[0328] Example 14. HPLC Analysis

[0329] The glycosphingolipid content of spray-dried powders comprising / V-lyso-GM3 and lactosyl d-erythro- sphingosine was determined under the following conditions:

[0330] HPLC eluent profile: solvent A: 1 L water + 0.5 mL formic acid + 4 mmol ammonium formate, and solvent B: 1 L MeOH + 1 L acetonitrile + 4 mL formic acid + 4 mmol ammonium formate.

[0331] A gradient of 50-100% B in A was applied over 13 min, followed by an isocratic of 100% B for 18 min, followed by an isocratic of 50% B in A for 40 min. The glycosphingolipid content of the powder was quantified via peak area analysis using external standards.

[0332] The glycosphingolipid content of the spray-dried powders comprising mixtures of N-lyso-GD3 and N- lyso-GM3 was determined under the following conditions:

[0333] HPLC eluent profile: solvent A: 1 L water + 2.0 mL formic acid + 2 mmol ammonium formate, and solvent B: 1.5 L MeOH + 0.5 L acetonitrile + 4 mL formic acid + 4 mmol ammonium formate.

[0334] A gradient of 70-100% B in A was applied over 8 min., followed by an isocratic of 100% B for 11 min., followed by an isocratic of 70% B in A for 25 min. The glycosphingolipid content of the powders was quantified via peak area analysis using external standards. The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0335] The above described embodiments are combinable.

[0336] The following claims further set out particular embodiments of the disclosure.

Claims

CLAIMS1. Method for the isolation a glycosphingolipid of formula (1):whereinW is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from a sialylated glycosyl moiety, or a neutral glycosyl moiety,R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl,R2is hydrogen or -OR4, wherein R4is selected from hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C2-6 acyl, preferably R4hydrogen, the bond - may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR4,R3is hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted Ci.g acyl, preferably hydrogen, or a salt thereof, from a solution comprising said glycosphingolipid or said salt, and one or more contaminants, the method comprising the steps of:- providing a solution comprising said glycosphingolipid of formula (1) or a salt thereof and one or more contaminants,- diafiltration of said solution, thereby obtaining a diafiltration retentate (DFR) comprising said glycosphingolipid, or salt thereof, by using a membrane having a MWCO of 100-300 kDa, thereby isolating said glycosphingolipid or salt thereof from the one or more contaminants.

2. The method according to claim 1, wherein the diafiltration is performed by using a membrane having a MWCO of 200-300 kDa.

3. The method according to claims 1 or 2, wherein W of the one or more glycosphingolipid of formula (1) is a sialylated glycosyl moiety, and wherein the sialylated glycosyl moiety is preferably selected from the group consisting of Neu5Aca2-3Gall-, Neu5Aca2-3Gaipi-4Glc-,Neu5Aca2-8Neu5Aca2-3Gaipi-4Glcl-, Neu5Aca2-3Gaipi-3GalNAcpi-4(Neu5Aca2-3)Gaipi- 4Glcl-, and Neu5Aca2-8Neu5Aca2-8 Neu5Aca2-3Gaipi-4Glcl-.

4. The method according to claim 3, wherein the glycosphingolipid of formula (1) is a sialylated glycosphingolipid selected from the group consisting of / V-lyso-GM4, / V-lyso-GM3, N-lyso-GD3, / V-lyso-GDla, and / V-lyso-GT3, or a mixture thereof.

5. The method according to claims 1 or 1, wherein W of the glycosphingolipid of formula (1) is a neutral glycosyl moiety, and wherein the neutral glycosyl moiety is preferably selected from the group consisting of Gall-, Glcl-, and Gaipi-4Glcl-.

6. The method according to claim 5, wherein the glycosphingolipid of formula (1) is selected from the group consisting of lactosyl D-erytbro-sphingosine, lactosyl D-erytbro-dihydrosphingosine, lactosyl D-r / bo-phytosphingosine, galactosyl D-erytbro-sphingosine, galactosyl D-erythro- dihydrosphingosine, galactosyl D-r / bo-phytosphingosine, glucosyl D-erytbro-sphingosine, glucosyl D-erytbro-dihydrosphingosine, and glucosyl D-r / bo-phytosphingosine, or a mixture thereof.

7. The method according to any of claims 1 to 6, wherein the method further comprising a step of spray drying or spray granulating the DFR comprising the glycosphingolipid of formula(l), or a salt thereof.

8. The method according to any one of claims 1 to 7, wherein the method further comprising a step of spray drying the DFR comprising the glycosphingolipid of formula (1), or a salt thereof.

9. A spray-dried powder comprising one or more glycosphingolipids of formula (1), or salts thereof:whereinW is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from a sialylated glycosyl moiety, or a neutral glycosyl moiety,R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl,R2is hydrogen or -OR4, wherein R4is selected from hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C2-6 acyl, preferably R4is hydrogen, the bond - may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR4,R3is hydrogen, a substituted or unsubstituted Ci.g alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen.

10. The spray-dried powder according to claim 9, wherein W of the one or more glycosphingolipids of formula (1), or salts thereof, is a sialylated glycosyl moiety, and wherein the sialylated glycosyl moiety is preferably selected from the group consisting of Neu5Aca2-3Gaipi-, Neu5Aca2- 3Gaipi-4Glc-, Neu5Aca2-8Neu5Aca2-3Gaipi-4Glcl-, Neu5Aca2-3Gaipi-3GalNAcpi- 4(Neu5Aca2-3)Gaipi-4Glcl-, and Neu5Aca2-8Neu5Aca2-8 Neu5Aca2-3Gaipi-4Glcl-.

11. The spray-dried powder according to claim 9, wherein W of the one or more glycosphingolipids of formula (1), or salts thereof, is a neutral glycosyl moiety, and wherein the neutral glycosyl moiety is preferably selected from the group consisting of Gall-, Glucl-, and Gaipi-4Glcl-.

12. The spray-dried powder according to claims 9 or 10, wherein the spray-dried powder comprising at least about 70 wt.% of / V-lyso-GM3.

13. The spray-dried powder according to claim 12, wherein the spray-dried powder comprising about 75-80 wt.% of / V-lyso-GM3, and wherein the spray dried-powder further comprising about 7-9 wt.%> of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.%> of glucosyl D-erythro- sphingosine.

14. The spray-dried powder according to claims 9 or 10, wherein the spray-dried powder comprising at least about 60 wt.%> of a mixture of / V-lyso-GD3 and / V-lyso-GM3, and wherein the weight ratio between / V-lyso-GD3 and / V-lyso-GM3 in said mixture is from about 1:10 to about 10:1.

15. The spray-dried powder according to claim 14, wherein the spray-dried powder comprising about 40-55 wt.%> of / V-lyso-GD3 and about 10-15 wt.%> of / V-lyso-GM3, and wherein the spray- dried powder further comprising about 3-6 wt.%> of / V-lyso-GT3, about 4-6 wt.%> of lactosyl d- erythro-sphingosine, and about 0.1-1.0 wt.%> of glucosyl D-erythro-sphingosine.

16. The spray-dried powder according to claim 14, wherein the spray-dried powder comprising about 15-20 wt.%> of / V-lyso-GD3 and about 50-60 wt.%> of / V-lyso-GM3, and wherein the spray- dried powder further comprising about 0.1-0.5 wt.%> of / V-lyso-GT3, about 4-7 wt.%> of lactosyl d- erythro-sphingosine, and about 0.1-1.0 wt.%> of glucosyl D-erythro-sphingosine.

17. The spray-dried powder according to claim 14, wherein the spray-dried powder comprising about 35-40 wt.% of / V-lyso-GD3 and about 25-40 wt.% of / V-lyso-GM3, and wherein the spray- dried powder further comprising about 5-6 wt.% of lactosyl d-erythro-sphingosine, and about 0.5-1.0 wt.%> of glucosyl D-erythro-sphingosine.

18. The spray-dried powder according to claim 11, wherein the spray-dried powder comprising 80-90 wt.%> lactosyl d-erythro-sphingosine.

19. Use of the spray-dried powder according to any one of claims 9 to 18, for the production of a ceramide or a ganglioside.

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