Method for isolating sphingoglycolipids

Abstract

The present invention describes a novel method for isolating sphingoglycolipids lacking amide-linked fatty acid acyl groups. The method comprises the steps of: preparing a solution containing the sphingoglycolipid of formula (1) or a salt thereof and one or more impurities; and diafiltration the solution using a membrane having a MWCO of 100 kDa to 300 kDa to obtain a diafiltration retaining liquid (DFR) containing the sphingoglycolipid or salt thereof, thereby isolating the sphingoglycolipid or salt thereof from one or more impurities.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating sphingoglycolipids and to a spray-dried powder containing one or more sphingoglycolipids. [Background technology]

[0002] Sphingoglycolipids (GSLs) are complex carbohydrates in which the glycan portion is linked to a 1-hydroxyl group of a ceramide or sphingoid base via glycosidic linkage.

[0003] GSLs are involved in diverse biological processes and play important structural and functional roles such as intercellular recognition, signal transduction, and intercellular adhesion. For example, sialylated sphingoglycolipids such as gangliosides are found in the brain and may play a role in neurological diseases, particularly Alzheimer's disease, Parkinson's disease, and Huntington's disease. Furthermore, certain gangliosides are found in the intestinal mucosa and may not only promote intestinal health but also act as anti-infective agents.

[0004] Therefore, sphingoglycolipids hold great potential as therapeutic agents, cosmetic products, and tools for studying important biological processes.

[0005] In order to use sphingoglycolipids as therapeutic drugs and cosmetics, it is necessary to produce such compounds in high purity.

[0006] Processes for isolating sphingoglycolipids have been reported.

[0007] The isolation of sialylated sphingoglycolipids from extracts of tissues or organs of the nervous system is described. In particular, monosialoganglioside GM1 is isolated by ultrafiltration, which is carried out in the presence of α-cyclodextrin (Patent Document 1).

[0008] Gangliosides such as GM1 are known to form micelle aggregates with molecular weights of several hundred kilodaltons in aqueous solutions (Non-Patent Literature 1), and therefore may not permeate ultrafiltration membranes. This property may prevent the direct use of ultrafiltration and / or dialysis for the isolation of sphingoglycolipids from untreated mixtures.

[0009] As described in Patent Document 1, α-cyclodextrin is used to form a complex with GM1, thereby forming a GM1-α-cyclodextrin complex that permeates an ultrafiltration membrane.

[0010] The main drawbacks associated with this method are the use of a large excess of α-cyclodextrin (4 to 6 equivalents relative to GM1) to form the ganglioside-cyclodextrin complex, and the need for multiple extractions with organic solvents to obtain free ganglioside.

[0011] A method for isolating the sodium salt of monosialoganglioside GM1 from a lipid mixture is described (Patent Document 2), comprising the step of fractionating the lipid mixture by ion exchange chromatography. The method further comprises several diafiltration steps to remove residual salts, wherein the diafiltration membrane has a molecular weight cutoff (MWCO) of 10 kDa to 100 kDa. Disadvantages associated with this method include the use of large amounts of organic solvents and the need to regenerate and equilibrate the ion exchange resin after each chromatographic separation.

[0012] Furthermore, while Patent Documents 1 and 2 describe methods for isolating gangliosides, specifically GM1, they do not describe isolation in lyso form.

[0013] Therefore, there is a need for the development of a novel methodology characterized by high technical feasibility and low cost, which enables the efficient and large-scale isolation of sphingoglycolipids. [Prior art documents]

Patent Document

[0014]

Patent Document 1

Patent Document 2

Non-Patent Document

[0015]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0016] In a first aspect, the present invention provides a compound of formula (1): TIFF2026528773000001.tif31170(wherein, W is a glycosyl moiety, preferably selected from a sialylated glycosyl moiety or a neutral glycosyl moiety, R 1 is hydrogen, aryl, or substituted or unsubstituted C 1~50 alkyl, preferably substituted or unsubstituted C 1~17 alkyl, more preferably substituted or unsubstituted C 10~17 alkyl, R 2 is hydrogen or -OR 4 where R 4 is selected from hydrogen, substituted or unsubstituted C 1~6 alkyl, or substituted or unsubstituted C 2~6 acyl, preferably, R 4 is hydrogen, bond --- is a double bond or a single bond when R 2 is hydrogen, or a single bond when R 2 is -OR 4 in which case, R 3 A method for isolating a sphingoglycolipid or salt thereof of hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl (preferably hydrogen) from a solution containing the sphingoglycolipid or salt and one or more impurities, A step of preparing a solution containing the sphingoglycolipid of formula (1) above or a salt thereof and one or more impurities, The above solution is diafiltration using a membrane having MWCO of 100kDa to 300kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the above sphingoglycolipid or a salt thereof. The present invention relates to a method for isolating the above-mentioned sphingoglycolipid or a salt thereof from one or more impurities, which includes [a specific component].

[0017] Preferably, diafiltration is performed using a membrane having an MWCO of 200 kDa to 300 kDa.

[0018] In a second embodiment, the present invention relates to one or more formulas (1): TIFF2026528773000002.tif32170 (in the formula, W is a glycosyl moiety, and the glycosyl moiety is preferably selected from a sialylated glycosyl moiety or a neutral glycosyl moiety. R 1 C is hydrogen, aryl, or substituted or unsubstituted C 1~50 Alkyl, preferably substituted or unsubstituted C 1~17 Alkyl, more preferably substituted or unsubstituted C 10~17 It is alkyl, R 2 is hydrogen or -OR 4 And R 4 C is hydrogen, substituted or unsubstituted C 1~6 Alkyl, or substituted or unsubstituted C 2~6 Selected from acyl, preferably R 4 It is hydrogen, join --- R 2In the case of hydrogen, it can be a double bond or a single bond, or R 2 ga-OR 4 In this case, it is a single bond, R 3 This relates to a spray-dried powder containing a sphingoglycolipid or salt thereof of hydrogen, a substituted or unsubstituted C1-6 alkyl group, or a substituted or unsubstituted C1-6 acyl group (preferably hydrogen).

[0019] 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 ceramides or gangliosides. [Modes for carrying out the invention]

[0020] This invention describes for the first time a method for isolating sphingoglycolipids lacking amide-linked fatty acid acyl groups. The sphingoglycolipids are preferably isolated by direct diafiltration of an untreated solution containing the sphingoglycolipid and one or more other compounds, such as proteins, sugars, and salts ("impurities"), and the diafiltration is carried out using a membrane having a molecular weight cutoff (MWCO) of 100 kDa to 300 kDa, preferably 200 kDa to 300 kDa.

[0021] Surprisingly, sphingoglycolipids lacking amide-linked fatty acid acyl groups have been found not to permeate the diafiltration membrane used in this invention. This phenomenon can be advantageously utilized for the efficient removal of organic molecules, biomaterials, and monovalent and divalent salts, thus eliminating the need for ion exchange steps. Furthermore, when using membranes with high MWCO, such as those used in this invention, a high flux can be maintained during diafiltration, thereby reducing operating time and costs.

[0022] By spray-drying a diafiltration holding solution containing sphingoglycolipids, a highly uniform and easy-to-handle spray-dried sphingoglycolipid powder can be obtained, and this spray-dried sphingoglycolipid powder can be used for the production of ceramides and gangliosides.

[0023] The method of the present invention is A step of preparing a solution containing the sphingoglycolipid of formula (1) above or a salt thereof and one or more impurities, The above solution is diafiltration using a membrane having MWCO of 100kDa to 300kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the above sphingoglycolipid or a salt thereof. This includes the ability to isolate the sphingoglycolipid or its salt from one or more impurities.

[0024] Non-limiting embodiments of various aspects of the present invention are described below and illustrated by non-limiting examples.

[0025] The terms, definitions, and embodiments described throughout this specification relate to all aspects and embodiments of the present invention.

[0026] The term "a" is grammatically singular, but it can also refer to multiple intended compounds, for example. For instance, in the expression "a glycosphingolipid," it will be understood by those skilled in the art that it can refer not only to a single sphingoglycolipid, but also to various sphingoglycolipids of the same or different types.

[0027] The term "at least" means a range of unlimited values ​​starting from the indicated value, or, in the case of a weight percentage range, a range of up to 100 weight percent starting from the indicated value.

[0028] Throughout this specification, "weight %" means the weight of the specified substance contained in 100 g of the specified composition. For example, spray-dried powder containing at least about 70 weight % of the sphingoglycolipid of formula (1) typically means that 100 g of spray-dried powder contains at least about 70 g of the sphingoglycolipid of formula (1).

[0029] As used herein, the various functional groups or substituents shown are understood to have a bond point at the atom to which the functional group or dash (-) is denoted. For example, -OR 4 In this case, the bond point is understood to be the oxygen atom. When groups without dashes are listed, their bond points are indicated by the usual meaning of that group.

[0030] As used herein, the term "alkyl" means an acyclic linear or branched hydrocarbyl group having 1 to 50 carbon atoms, which may be saturated, contain one or more double and / or triple bonds (and thus form, for example, alkenyl or alkynyl bonds), and / or be substituted or unsubstituted, as further described herein. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neopentyl, n-hexyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 3-methyl-1-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 linear saturated acyclic hydrocarbyl group having 1 to 31 carbon atoms, which may be substituted or unsubstituted.

[0031] As used herein, the term "aryl" refers to an aromatic cyclic hydrocarbyl group having 5 to 14 ring carbon atoms, which may be monocyclic or polycyclic, may contain a fused ring, preferably 1 to 3 fused or unfused rings, may contain one or more heteroatoms, and / or may be substituted or unsubstituted, as further described herein. Examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthracyl, phenanthryl, pyrrolyl, imidazolyl, thiophenyl, furanyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzofuranyl, which may each be substituted or unsubstituted. Typically, the term "aryl" refers to substituted or unsubstituted phenyl.

[0032] As used herein, the term "acyl" refers to a group derived from an oxoacid, preferably from a carboxylic acid, by removing one or more hydroxyl groups. The acyl groups according to the present invention are typically saturated or unsaturated C 2~32 It is an acyl, and may be substituted or not.

[0033] As used herein, the term “substitution” means that the group is substituted with a group that typically modifies the general chemical properties of the group. Substitutions can be used to modify the properties of a molecule, such as its stability, solubility, and crystallizing ability. Other suitable substituents with similar size and charge characteristics that can be used as substitutes in a given situation will be recognized by those skilled in the art.

[0034] In relation to the terms "alkyl," "aryl," and "acyl," the term substitution refers to the substitution of a group that is hydroxyl (which may exist in tautomeristic keto form when bonded to an unsaturated carbon atom), oxo, or C 1~6 Alkyl(i.e., C 1~6 Alkyl-oxy), C 2~6 Alkenyloxy, Carboxy, Oxo, C 1~6Alkoxycarbonyl, C 1~6 Alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono and di(C 1~6 Alkyl)amino, carbamoyl, mono and di(C) 1~6 Alkyl)aminocarbonyl, amino-C 1~6 Alkyl-aminocarbonyl, mono and di(C) 1~6 Alkyl)amino-C 1~6 Alkyl-aminocarbonyl, C 1~6 Alkylcarbonylamino, cyano, guanidino, carbamide, C 1~6 Alkyl-sulfonyl-amino, aryl-sulfonyl-amino, heteroaryl-sulfonyl-amino, C 1~6 Alkanoyloxy, C 1~6 Alkyl-sulfonyl, C 1~6 Alkyl-sulfinyl, C 1~6 Alkyl sulfonyl oxy, nitro, C 1~6 This means that the molecule is substituted once or several times, preferably one to three times, with a group selected from alkylthio and halogens (there may be more than one group), and any alkyl, alkoxy, etc. representing the substituent is hydroxy, C 1~6 Alkoxy, C 2~6 Alkenyloxy, Carboxy, C 1~6 Alkylcarbonylamino, halogen, C 1~6 Alkylthio, C 1~6 It may be substituted with alkyl-sulfonyl-amino or guanidino.

[0035] In relation to the term "alkyl," the term "substituted" preferably means that the group is substituted once or several times, preferably one to three times, with a group(s) selected from a hydroxyl group, alkoxy group, acyloxy group, acylamide group, thiol, thioether, or phosphorus-containing functional group.

[0036] When used herein, the term "impurities" refers to substances such as chemicals (e.g., ions, organic molecules, etc.) and / or biological materials (e.g., proteins, cell fragments, etc.) that are associated with the compound or material of interest obtained by a particular process or method.

[0037] In the context of this invention, the term "isolation" refers to a procedure or step in a procedure applied to separate a desired compound from a mixture containing the desired compound and other compounds. In this context, the other compounds in the mixture are considered impurities.

[0038] The terms isolation and isolating can be used interchangeably.

[0039] For example, in a formula representing a specific compound, such as formula (1), unless a carbon atom having a specific stereochemical configuration is explicitly stated in the chemical formula, it will be understood by those skilled in the art that the formula encompasses compounds in which such stereocenters have an R configuration or an S configuration, or compounds in which the double bond has a cis configuration or a trans configuration.

[0040] When referring to positions C-1, C-2, C-3, C-4, C-5, etc., it will be understood by those skilled in the art that this specification always refers to the respective carbon atoms of the sphingoglycolipid represented by formula (1), etc.

[0041] In the context of this invention, the terms “about,” “around,” or “approximate” are applied indiscriminately to indicate a deviation of 0.1% to 10% from a particular value (e.g., “about 70% by weight,” “approximately 70% by weight,” or “approximately 70% by weight”).

[0042] The term "sphingoglycolipid," as used herein, refers to a compound or analog thereof structurally composed of a glycosyl moiety and a sphingolipid moiety. The glycosyl moiety is typically linked to the sphingolipid moiety by a glycosidic bond between the anomeric carbon at the reducing end of the glycosyl moiety and the hydroxyl group at the C-1 position of the sphingolipid.

[0043] The glycosyl moiety of the sphingoglycolipid according to the present invention may be derived from a monosaccharide or oligosaccharide (two or more monosaccharide units), and the anomeric carbon of the monosaccharide or the anomeric carbon of the reducing end of the oligosaccharide is involved in glycosidic bonding with another chemical entity, such as a sphingolipid, and unless otherwise specified, the bond may be an α-glycosidic bond or a β-glycosidic bond. The glycosyl moiety having two or more monosaccharide units may represent a linear or branched structure.

[0044] The monosaccharide unit is preferably any sugar with 5 to 9 carbon atoms, including 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.), deoxy sugars (e.g., L-rhamnose, L-fucose, etc.), deoxyamino sugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acid, and ketoaldonic acid (e.g., sialic acid). The monosaccharide unit can form various cyclic structures such as a pyranose (6-membered) cyclic structure or a furanose (5-membered) cyclic structure. In some embodiments, the glycosyl portion is derived from a monosaccharide, and the monosaccharide is a β-galactoside. In some embodiments, the glycosyl portion is derived from an oligosaccharide, and the oligosaccharide has one or more terminal β-galactopyranosyl units.

[0045] The glycosyl moiety according to the present invention can be exemplified as follows: Galβ1-4Glc1-, where the dash (-) represents a bond point of the glycosyl moiety, and the glycosyl moieties may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0046] The sphingolipid portion of the sphingoglycolipid of the present invention is preferably derived from an aliphatic amino alcohol, such as a sphingoid base.

[0047] In the context of this invention, a sphingoid base refers to a naturally occurring sphingoid base, its analogue, or its derivative.

[0048] Naturally occurring sphingoid bases are D-erythro-sphingosine (S), 6-hydroxy-D-erythro-sphingosine (H), D-ribo-phytosphingosine (P), or DL-erythro-dihydrosphingosine (DS), where the number of sphingoid carbons may be indicated in parentheses after the letters S, H, P, and DS.

[0049] The letters S, H, P, and DS refer to abbreviated nomenclature developed by Motta et al. (1993) Biochim Biophys Acta. 1182:147-151 and extended by Rabionet (2014) Biochim Biophys Acta. 1841:422-434 and Masukawa et al., Journal of Lipid Research, 2008, 49, 1466-1476. D-erythrodihydrosphingosine may also be represented by the letter G according to INCI nomenclature.

[0050] The sphingoglycolipid according to the present invention is typically of formula (1): TIFF2026528773000003.tif34170 (in the formula, W is a glycosyl moiety, and the glycosyl moiety is preferably selected from a sialylated glycosyl moiety or a neutral glycosyl moiety. R 1 C is hydrogen, aryl, or substituted or unsubstituted C 1~50 Alkyl, preferably substituted or unsubstituted C 1~17 Alkyl, more preferably substituted or unsubstituted C 10~17 It is alkyl, R 2 is hydrogen or -OR 4 And R 4 C is hydrogen, substituted or unsubstituted C 1~6 Alkyl, or substituted or unsubstituted C 2~6 Selected from acyl, preferably R 4 It is hydrogen, join --- R 2 In the case of hydrogen, it can be a double bond or a single bond, or R 2 ga-OR 4 In this case, it is a single bond, R 3 It is represented by a sphingoglycolipid or salt thereof of hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl (preferably hydrogen).

[0051] The glycosphingolipid of formula (1) lacks an amide-linked fatty acid acyl group and can also be called a lysosphingolipid.

[0052] In some embodiments, with respect to the sphingoglycolipid of formula (1), R 1 is saturated unsubstituted C 10 ~C 17 It is alkyl, R 2 and R 3 It is hydrogen, and bond --- It is a double bond.

[0053] In some embodiments, with respect to the sphingoglycolipid of formula (1), R 1 is saturated unsubstituted C 10 ~C 17 It is alkyl, R 2 is OR 4 And here, R 4 is hydrogen, R 3It is hydrogen, and bond --- It is a single bond.

[0054] In some embodiments, with respect to the sphingoglycolipid of formula (1), R 1 is saturated unsubstituted C 10 ~C 17 It is alkyl, R 2 and R 3 It is hydrogen, and bond --- It is a single bond.

[0055] In some embodiments, with respect to the sphingoglycolipid of formula (1), R 1 is C 10 ~C 17 It is 1-hydroxyalkyl, R 2 and R 3 It is hydrogen, and bond --- It is a double bond.

[0056] In some embodiments, the sphingoglycolipid of formula (1) is the same as those of formulas (2), (3), (4), and (5): It is a sphingoglycolipid or a salt thereof selected from the group consisting of sphingoglycolipids of TIFF2026528773000004.tif73170.

[0057] In some embodiments, W in the sphingoglycolipid of formula (1) and W in the sphingoglycolipids of formulas (2) to (5) are sialylated glycosyl moieties.

[0058] In some embodiments, W of the sphingoglycolipid of formula (1) and W of the sphingoglycolipids of formulas (2) to (5) are as follows: A sialylated glycosyl moiety or a salt thereof selected from the group consisting of the glycosyl moiety of TIFF2026528773000005.tif144170, wherein the glycosyl moieties may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0059] In some preferred embodiments, W of the sphingoglycolipid of formula (1) and W of the sphingoglycolipids of formulas (2) to (5) are as follows: A sialylated glycosyl moiety or a salt thereof selected from the group consisting of the glycosyl moiety of TIFF2026528773000006.tif37170, wherein the glycosyl moieties may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0060] In some embodiments, the sphingoglycolipid of formula (1) is selected from the group consisting of N-lyso-GM4, N-lyso-GM3, N-lyso-GD3, N-lyso-GD1a, and N-lyso-GT3, or is a mixture thereof.

[0061] In some preferred embodiments, the sphingoglycolipid of formula (1) is N-lyso-GM3.

[0062] In some preferred embodiments, the sphingoglycolipid of formula (1) is N-lyso-GD3.

[0063] In some embodiments, the sphingoglycolipid of formula (1) is a mixture of N-lyso-GM3 and N-lyso-GD3.

[0064] In some preferred embodiments, the sphingoglycolipid of formula (1) is a mixture of N-lyso-GM3, N-lyso-GD3, and N-lyso-GT3.

[0065] Sphingoglycolipids of formula (1) having a sialylated glycosyl moiety can also be called sialylated sphingoglycolipids.

[0066] In some embodiments, W in the sphingoglycolipids of formulas (1) and (2) to (5) is a neutral glycosyl moiety, where the neutral glycosyl moiety is defined as follows: Selected from the group consisting of glycosyl moieties of TIFF2026528773000007.tif94170, where the glycosyl moieties may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0067] In some preferred embodiments, W in the sphingoglycolipid of formula (1) and W in the sphingoglycolipids of formulas (2) to (5) are neutral glycosyl moieties, where the neutral glycosyl moiety is defined as follows: Selected from the group consisting of glycosyl portions of TIFF2026528773000008.tif21170, where the glycosyl portions may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0068] In some embodiments, the sphingoglycolipid of formula (1) is selected from the group consisting of lactosyl D-erythro-sphingosine, lactosyl D-erythro-dihydrosphingosine, lactosyl D-ribo-phytosphingosine, galactosyl D-erythro-sphingosine, galactosyl D-erythro-dihydrosphingosine, galactosyl D-ribo-phytosphingosine, glucosyl D-erythro-sphingosine, glucosyl D-erythro-dihydrosphingosine, and glucosyl D-ribo-phytosphingosine, or is a mixture thereof.

[0069] In some preferred embodiments, the sphingoglycolipid of formula (1) is selected from the group consisting of lactosyl D-erythro-sphingosine, lactosyl D-erythro-dihydrosphingosine, and lactosyl D-ribo-phytosphingosine.

[0070] In some preferred embodiments, the sphingoglycolipid of formula (1) is selected from the group consisting of galactosyl D-erythro-sphingosine, galactosyl D-erythro-dihydrosphingosine, and galactosyl D-ribo-phytosphingosine.

[0071] In some embodiments, the sphingoglycolipid of formula (1) is selected from the group consisting of glucosyl D-erythro-sphingosine, glucosyl D-erythro-dihydrosphingosine, and glucosyl D-ribo-phytosphingosine.

[0072] In some preferred embodiments, the sphingoglycolipid of formula (1) is one or a mixture of N-lyso-GM3, lactosyl D-erythro-sphingosine, and glucosyl D-erythro-sphingosine.

[0073] The present invention describes a method for isolating a sphingoglycolipid of formula (1), which is preferably produced by an enzymatic process or a biotechnological process (as described in further embodiments below).

[0074] Typically, the sphingoglycolipid according to the present invention is isolated from a solution containing one or more impurities, where the solution is preferably an aqueous solution, and the impurities are typically, but not limited to, ions, small organic molecules (e.g., sugars, nucleotides, etc.), and proteins.

[0075] A solution containing sphingoglycolipids and one or more impurities is subjected to a diafiltration (DF) step, where water is preferably used as the diafiltration medium.

[0076] The solution is typically supplied to the DF using approximately 2 to 10 volumes of DF medium.

[0077] In some embodiments, the solution is supplied to the DF using about 2, 3, 4, 5, 6, 7, 8, 9, or 10 volumes of DF medium.

[0078] The DF process according to the present invention is carried out at a constant temperature, preferably about 10°C to 80°C, preferably about 15°C to 45°C, and more preferably about 20°C to 25°C.

[0079] The preferred applied pressure in nanofiltration separation is approximately 2 to 50 bar, for example, 8 to 20 bar, or for example, 8 to 10 bar.

[0080] The DF process is carried out using a membrane such as a hollow fiber membrane, spiral membrane, or ceramic membrane having an appropriate molecular weight cutoff (MWCO).

[0081] A membrane having an MWCO of approximately 100 kDa to 300 kDa is preferred.

[0082] In some preferred embodiments, the diafiltration membrane has an MWCO of about 200 kDa to 250 kDa, or about 250 kDa to 300 kDa, for example, about 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, or 300 kDa.

[0083] During the DF process according to the present invention, sphingoglycolipids are retained in the diafiltration retaining solution (DFR), but all permeable contaminants pass through the membrane and accumulate in the diafiltration permeate (DFP).

[0084] In the context of the present invention, the permeable impurities are molecules having a molecular weight of up to approximately 280 kDa, such as carbohydrates, salts, nucleotides, and proteins.

[0085] It should be noted that the diafiltration membrane used in the present invention has a MWCO (Motor Waste Coefficient) that is well greater than the molecular weight of the sphingoglycolipid.

[0086] It has been reported that ganglioside GM1 can form micelle aggregates with a molecular weight of approximately 250 kDa to 450 kDa in aqueous solution, and the size of the micelle aggregates depends on the length of the fatty acid chains of the constituent molecules (Non-Patent Literature 1). Due to this property, gangliosides such as GM1 may not permeate ultrafiltration membranes that have a larger MWCO than GM1.

[0087] However, surprisingly, the inventors have found that gangliosides in the N-lysyl form lacking fatty acid chains and glycosylated sphingoid bases do not permeate through a diafiltration membrane having a MWCO larger than lysosphingolipids. Thus, without being bound by theory, micelle aggregation can surprisingly occur regardless of the presence of fatty acid chains within the molecule.

[0088] An advantage associated with using a membrane having a high MWCO (i.e., at least 100 kDa) is that a high flux can be maintained during diafiltration even when the transmembrane differential pressure applied is low, thereby reducing the operating time and cost. In particular, when using a membrane having a MWCO of about 200 kDa to 300 kDa, a high flux of about 15.5 to 18.1 (l / m 2 ·h) can be maintained at a transmembrane differential pressure of about 8 bar to 10 bar. On the other hand, when using a membrane having a MWCO of about 300 Da to 500 Da, a flux of about 9.6 (l / m 2 ·h) can be achieved only when a transmembrane differential pressure of about 30 bar is applied.

[0089] Another advantage associated with using a membrane having a high MWCO (i.e., at least 100 kDa) is that organic molecules, biomaterials, and monovalent and divalent salts can be efficiently removed. In particular, when using a membrane having a MWCO of about 200 kDa to 300 kDa, a reduction of about 90% to 99% in the content of organic small molecules (e.g., sugars such as monosaccharides, disaccharides, trisaccharides, etc.) and salts is achieved.

[0090] In some embodiments, the method further includes a step of concentrating the DFR.

[0091] Thus, in some embodiments, the present invention is a method for isolating a sphingoglycolipid of formula (1), a step of preparing a solution containing the sphingoglycolipid of formula (1) and one or more contaminants, The process involves diafiltration of the above solution using a membrane having MWCO of 100kDa to 300kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the above sphingoglycolipid or a salt thereof, The process of concentrating DFR, The present invention describes a method for isolating the above-mentioned sphingoglycolipid or a salt thereof from one or more impurities, which includes the above-mentioned sphingoglycolipid.

[0092] DFR enrichment is typically carried out using the same membrane used during the diafiltration process, for the period necessary to reduce the volume of the DFR to the desired final volume.

[0093] DFR containing the sphingoglycolipid of formula (1) is spray-dried or spray-granulated.

[0094] In some preferred embodiments, the DFR containing the sphingoglycolipid of formula (1) is spray-dried.

[0095] In some embodiments, the present invention relates to a method for isolating a sphingoglycolipid of formula (1), The process involves preparing a solution containing the sphingoglycolipid of formula (1) above and one or more impurities, The process involves diafiltration of the above solution using a membrane having MWCO of 100kDa to 300kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the above sphingoglycolipid or a salt thereof, A step of spray-drying a DFR containing sphingoglycolipids, The present invention describes a method for isolating the above-mentioned sphingoglycolipid or a salt thereof from one or more impurities, which includes the above-mentioned sphingoglycolipid.

[0096] In some embodiments, the present invention relates to a method for isolating a sphingoglycolipid of formula (1), The process involves preparing a solution containing the sphingoglycolipid of formula (1) above and one or more impurities, The process involves diafiltration of the above solution using a membrane having MWCO of 100kDa to 300kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the above sphingoglycolipid or a salt thereof, The process of concentrating DFR, A step of spray-drying a DFR containing sphingoglycolipids, The present invention describes a method for isolating the above-mentioned sphingoglycolipid or a salt thereof from one or more impurities, which includes the above-mentioned sphingoglycolipid.

[0097] The spray drying process according to the present invention is typically carried out using a high-speed rotating disk or nozzle, thereby generating small particles. The particles can then descend towards the bottom of a spray drying tower under gravity. A fluidized bed may be provided here, where drying can be brought about using hot air (appropriately at approximately 80°C to approximately 95°C). Here, agglomeration can occur, causing the particles to adhere to each other. The agglomerated (granular) particles are then subjected to drying, for example, on a belt drying bed or a subfluidized bed.

[0098] Another technique involves using fluidized bed agglomeration. Here, the powder can be fluidized in a gas stream. In the particle bed, water is sprayed into the fluid to moisten the powder and promote agglomeration. This combination of spray drying and a fluidized bed post-dryer is suitable for agglomeration of many different types of solutions.

[0099] Drying can be carried out under air or under an inert gas such as nitrogen. In fluidized bed drying and subfluidized bed drying, the bed temperature can be adjusted to a preset value. These values ​​can range over a wide range, for example, from 35°C to 120°C, from 50°C to 90°C, or from 60°C to 80°C.

[0100] The water content of the spray-dried powder obtained according to the method of the present invention is typically about 0.5% by weight to about 6% by weight, preferably about 2% by weight to about 3% by weight.

[0101] The median particle size of the spray-dried powder obtained according to the method of the present invention is typically about 15 μm to about 30 μm, for example, preferably about 15 to 20 μm, for example, about 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm.

[0102] The particle span is typically less than about 3, preferably less than about 2. In some preferred embodiments, the particle span is about 1 to 2, for example, about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

[0103] The particle span is a dimensionless parameter that indicates the uniformity of the particle size distribution, and is defined as [D(0.9)-D(0.1)] / D(0.5), where D(0.9), D(0.1), and D(0.5) represent the cutoff sizes of less than 10%, less than 50%, and less than 90% (by volume) of the particles, respectively.

[0104] Generally, a low span (i.e., less than 3) is characteristic of a narrow particle size distribution, which leads to improved flow characteristics of spray-dried powders.

[0105] The spray-dried powder obtained according to the method of the present invention typically has a specific volume of less than about 4 mL / g, preferably less than about 3 mL / g.

[0106] In some embodiments, the spray-dried powder has a specific volume of about 2.0 ml / g to about 3.0 ml / g. Therefore, 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.

[0107] Spray-dried powders with such low specific volume (i.e., less than 4 ml / g) are generally preferred because they have good flow characteristics.

[0108] The spray-dried powder obtained according to the method of the present invention typically has a sphingoglycolipid content of at least about 65% by weight, usually at least about 70% by weight, preferably at least about 75% by weight, and more preferably at least about 85% by weight.

[0109] In some embodiments, the present invention describes a spray-dried powder containing one or more sphingoglycolipids of formula (1).

[0110] In some embodiments, the spray-dried powder contains a sphingoglycolipid of formula (1).

[0111] In some embodiments, the spray-dried powder contains two or more sphingoglycolipids of formula (1).

[0112] In some embodiments, the spray-dried powder contains sphingoglycolipids selected from the group consisting of N-lyso-GM4, N-lyso-GM3, N-lyso-GD3, N-lyso-GD1a, and N-lyso-GT3, or mixtures thereof.

[0113] In some embodiments, the spray-dried powder contains a sphingoglycolipid selected from the group consisting of lactosyl d-erythro-sphingosine, lactosyl d-erythro-dihydrosphingosine, lactosyl d-ribo-phytosphingosine, galactosyl d-erythro-sphingosine, galactosyl d-erythro-dihydrosphingosine, galactosyl d-ribo-phytosphingosine, glucosyl d-erythro-sphingosine, glucosyl d-erythro-dihydrosphingosine, and glucosyl d-ribo-phytosphingosine, or a mixture thereof.

[0114] In some embodiments, the spray-dried powder comprises at least about 70% by weight of N-lyso-GM3, or at least about 75% by weight of N-lyso-GM3, or at least about 80% by weight of N-lyso-GM3.

[0115] In some embodiments, the spray-dried powder comprises about 75% to 80% by weight of N-lyso-GM3, where the spray-dried powder further comprises about 7% to 9% by weight of lactosyl D-erythro-sphingosine and about 0.1% to 1.0% by weight of glucosyl D-erythro-sphingosine.

[0116] In some embodiments, the spray-dried powder comprises at least about 50% by weight of a mixture of N-lyso-GD3 and N-lyso-GM3, or at least about 60% by weight of a mixture of N-lyso-GD3 and N-lyso-GM3, or at least about 70% by weight of a mixture of N-lyso-GD3 and N-lyso-GM3, or at least about 80% by weight of a mixture of N-lyso-GD3 and N-lyso-GM3, wherein the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the mixture is about 1:10 to about 10:1.

[0117] In some embodiments, the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the above mixture is approximately 1:10.

[0118] In some embodiments, the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the above mixture is approximately 1:3.

[0119] In some embodiments, the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the above mixture is approximately 1:1.

[0120] In some embodiments, the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the above mixture is approximately 4:1.

[0121] In some embodiments, the spray-dried powder comprises about 40% to 55% by weight of N-lyso-GD3 and about 10% to 15% by weight of N-lyso-GM3, and further comprises about 3% to 6% by weight of N-lyso-GT3, about 4% to 6% by weight of lactosyl D-erythro-sphingosine, and about 0.1% to 1.0% by weight of glucosyl D-erythro-sphingosine.

[0122] In some embodiments, the spray-dried powder comprises about 15% to 20% by weight of N-lyso-GD3 and about 50% to 60% by weight of N-lyso-GM3, and further comprises about 0.1% to 0.5% by weight of N-lyso-GT3, about 4% to 7% by weight of lactosyl D-erythro-sphingosine, and about 0.1% to 1.0% by weight of glucosyl D-erythro-sphingosine.

[0123] In some embodiments, the spray-dried powder comprises about 35% to 40% by weight of N-lyso-GD3 and about 25% to 40% by weight of N-lyso-GM3, further comprising about 5% to 6% by weight of lactosyl D-erythro-sphingosine and about 0.5% to 1.0% by weight of glucosyl D-erythro-sphingosine.

[0124] In some embodiments, spray-dried powder can be obtained by the method of the present invention.

[0125] In some embodiments, the spray-dried powder according to the present invention is used to produce gangliosides or ceramides in accordance with a method such as that described in International Publication No. 2023099478.

[0126] In some embodiments, A spray-dried powder containing one or more sphingoglycolipids of formula (1) is prepared using formula (6): TIFF2026528773000009.tif27170 (in the formula, R 5 This can be saturated or unsaturated substituted or unsubstituted C 1~31 Alkyl, preferably saturated but may be substituted or unsubstituted C 9~31 Selected from alkyl groups, R 6 The spray-dried powder according to the present invention is used for the production of gangliosides or ceramides by a method comprising reacting the ester of a C1-C4 alkyl group (preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl or ethyl) in the presence of a base.

[0127] Typically, the spray-dried sphingoglycolipid 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.

[0128] In some embodiments, the spray-dried sphingoglycolipid of formula (1) and the ester of formula (6) are reacted in the presence of an amine, where the amine is preferably selected from triethylamine, N,N-diisopropylethylamine, and pyridine.

[0129] In some embodiments, the spray-dried sphingoglycolipid of formula (1) and the ester of formula (6) are reacted in the presence of a carbonate, where the carbonate is preferably selected from Na2CO3, K2CO3, CaCO3, Li2CO3, (NH4)2CO3.

[0130] In some embodiments, the spray-dried sphingoglycolipid of formula (1) and the ester of formula (6) are reacted in the presence of a bicarbonate, where the bicarbonate is preferably selected from NaHCO3, KHCO3, Ca(HCO3)2, LiHCO3, NH4HCO3.

[0131] In some preferred embodiments, the spray-dried sphingoglycolipid of formula (1) and the ester of formula (6) are reacted in the presence of an alkoxide, where the alkoxide has the formula (12): R 7 -O - Q + (12) (where R 7 is C1-C4 alkyl, preferably selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, more preferably selected from methyl or ethyl, Q + is a cation selected from Na + 、K + 、Li + or NH 4+ and preferably Na +It is an alkoxide of (which is).

[0132] In some more preferred embodiments, the spray-dried sphingoglycolipid of formula (1) and the ester of formula (6) are reacted in the presence of sodium methoxide.

[0133] The base can be used in catalytic, equimolar, or excess amounts.

[0134] In some embodiments, the spray-dried sphingoglycolipid of formula (1) is in free base form, and the base is used in a catalytic amount of about 0.1 to 0.5 molar equivalents based on the amount of spray-dried sphingoglycolipid.

[0135] In some embodiments, the spray-dried sphingoglycolipid of formula (1) is in salt form, and the base is used in an amount of about 1.0 molar equivalent to about 1.7 molar equivalents based on the amount of spray-dried sphingoglycolipid.

[0136] In some preferred embodiments, the spray-dried sphingoglycolipid of formula (1) is in salt form, and the base is used in an amount of about 1.2 to 1.3 molar equivalents based on the amount of spray-dried sphingoglycolipid.

[0137] In some embodiments, a spray-dried sphingoglycolipid of formula (1), an ester of formula (6), and a base are reacted in a polar solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol.

[0138] In some preferred embodiments, the reaction is carried out in methanol.

[0139] In some embodiments, the reaction is carried out in a mixture of one or more polar solvents, such as a mixture of methanol and ethanol, a mixture of methanol and propanol, a mixture of methanol and isopropanol, a mixture of methanol and butanol, a mixture of methanol and isobutanol, or a mixture of water and an aliphatic alcohol. In some embodiments, the reaction is carried out in acetonitrile.

[0140] In some embodiments, a spray-dried sphingoglycolipid of formula (1), an ester of formula (6), and a base are used in a C5-C 10 The reaction is carried out in a hydrocarbon solvent, where heptane is preferred.

[0141] In some preferred embodiments, the reaction is carried out without a solvent.

[0142] The spray-dried sphingoglycolipid of formula (1), the ester of formula (6), and the base are typically reacted at a temperature of about 50°C to about 125°C, preferably about 60°C to about 65°C. Therefore, in some preferred embodiments, the reaction is carried out at a temperature of about 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C.

[0143] It will be understood that the components of a reaction can be combined in any order, and the order in which the reactants are combined can be adjusted as needed.

[0144] The spray-dried sphingoglycolipid of formula (1), the ester of formula (6), and the base, as well as any other reagents used in the reaction, may be added to the reactants either as solids or dissolved in a solvent, and in any quantity and form effective for the intended outcome of the reaction.

[0145] The sphingoglycolipids according to the present invention are typically produced by enzymatic or biotechnological methods.

[0146] A method for producing sphingoglycolipids having a neutral glycosyl moiety, such as lactosyl d-erythro-sphingosine, is described in International Publication No. 2023118378, in which the sphingoglycolipid is obtained by coupling a fluorinated glycosyl with a sphingoid base in the presence of endoglycoceramidase glycosynthase.

[0147] A biotechnological method for synthesizing complex sphingoglycolipids is described in International Publication No. 2021170624, in which a glycosylated sphingoid base, such as lactosyl D-erythro-sphingosine, is translocated into the cell and further glycosylated.

[0148] Sphingoglycolipids having a sialylated glycosyl moiety, such as N-lyso-GM3 or N-lyso-GD3, can be produced by the enzymatic process described in the embodiments below.

[0149] In one embodiment, the sphingolipid of formula (1) is a sialylated sphingoglycolipid, where the sialylated sphingoglycolipid is Formula (7): TIFF2026528773000010.tif20170(wherein X is a Galβ1- or glycosyl moiety having one or more terminal β-galactopyranosyl units, R 1 , R 2 , R 3 and combination --- The sphingoglycolipid receptor is defined as follows (with respect to the sphingoglycolipid in formula (1)): Sialic acid donor, Enzymes having trans-sialidase activity, and Enzymes possessing β-galactosidase activity, The process of preparing, The above sphingoglycolipid receptor is mixed with the above sialic acid donor and the above enzyme having trans-sialidase activity, thereby generating the above sialylated sphingoglycolipid, and the process is carried out sequentially, A step of adding an enzyme having β-galactosidase activity, The process involves nanofiltration of the reaction solution, It is produced by a method that includes and the sialic acid donor is 3'-sialyl lactose.

[0150] In some embodiments, X of the sphingoglycolipid receptor of formula (6) is selected from the group consisting of Gal1-, Glc1-, and Galβ1-4Glc1-, where the glycosyl moiety may be linked via an α-glycosidic bond or a β-glycosidic bond, preferably a β-glycosidic bond.

[0151] In some embodiments, the sphingoglycolipid receptor of formula (6) is selected from or a mixture thereof from lactosyl D-erythro-sphingosine, lactosyl D-erythro-dihydrosphingosine, lactosyl D-ribo-phytosphingosine, galactosyl D-erythro-sphingosine, galactosyl D-erythro-dihydrosphingosine, galactosyl D-ribo-phytosphingosine, glucosyl D-erythro-sphingosine, glucosyl D-erythro-dihydrosphingosine, and glucosyl D-ribo-phytosphingosine.

[0152] In some preferred embodiments, the sphingoglycolipid receptor of formula (6) is lactosyl D-erythro-sphingosine.

[0153] In some embodiments, X of the sphingoglycolipid receptor of formula (6) is Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, where the glycosyl moiety may be linked via an α-glycosidic bond or a β-glycosidic bond, preferably a β-glycosidic bond.

[0154] In some embodiments, the sphingoglycolipid receptor of formula (6) is N-lyso-GM1a.

[0155] The term "sialic acid donor," as used herein, refers to a compound having a sialic acid unit that can be transferred to a suitable receptor, such as a sphingoglycolipid. Suitable sialic acid donors for use in the context of the present invention are typically α-sialylated compounds, which may be derived from natural sources or chemically synthesized. Examples of α-sialylated compounds derived from natural sources include 3'-sialyl lactose, sialic acid-rich proteins, and colomic acid. Examples of chemically synthesized α-sialylated compounds include, but are not limited to, p-nitrophenyl N-acetylneuraminic acid (Neu5AcαpNP), methylumbelliferyl N-acetylneuraminic acid (Neu5AcαMU), and their derivatives.

[0156] In a preferred embodiment, the sialic acid donor is 3'-sialyl lactose.

[0157] The term "enzyme having trans-sialidase activity" can be used interchangeably with the term "trans-sialidase" and, in the context of this invention, refers to enzymes belonging to the glycoside hydrolase family 33 (GH33), which typically catalyze the reversible transfer of glycosidicated sialic acid from sialic acid donors, such as oligosaccharides, glycoproteins, glycolipids, and colomic acid, to receptor molecules containing a terminal β-galactopyranosyl unit. In the absence of a suitable receptor molecule, these enzymes can act as sialidases to transfer glycosidicated sialic acid to water molecules. However, the hydrolytic activity of these enzymes is typically low.

[0158] The wild-type transsialidase may originate from parasitic Euglena, such as Trypanosoma cruzi, Trypanosoma congolense, or Trypanosome brucei.

[0159] In some embodiments, the enzyme possessing trans-sialidase activity is wild-type trans-sialidase originating from Trypanosoma cruzi. The amino acid sequence of wild-type trans-sialidase originating from Trypanosoma cruzi corresponds to the amino acid sequence with accession number Q26966 (https: / / www.uniprot.org / ).

[0160] Trans-sialidases originating from Trypanosoma cruzi can also be referred to as TcTS.

[0161] In some embodiments, the enzyme possessing trans-sialidase activity is a variant of wild-type trans-sialidase (Q26966) originating from Trypanosoma cruzi.

[0162] In some embodiments, the amino acid sequence of the mutant trans-sialidase includes the following mutations / modifications compared to the wild-type amino acid sequence Q26966: Ser263Thr, Arg477His, Val485Leu, Glu559Val, Ser496Lys, an N-terminal His tag, and seven amino acid deletions Δ636-642 (numbering corresponding to the alignment between the mutant amino acid sequence and the amino acid sequence of Q26966).

[0163] The mutant trans-sialidase according to the present invention can be produced by methods known to those skilled in the art. Methods for expressing and purifying the mutant trans-sialidase are described, for example, in Paris et al., Glycobiology 2001, 11, 305-311, or in Buschiazzo et al., Molecular Cell 2002, 10, 757-768.

[0164] The term "enzyme having β-galactosidase activity" can be used interchangeably with the term "β-galactosidase" and, in the context of the present invention, refers to an enzyme belonging to the glycoside hydrolase family 35 (GH35), which typically catalyzes the hydrolysis of the terminal unreduced β-D-galactose residue of β-D-galactosides.

[0165] In the context of the present invention, β-galactosidase may also be referred to as lactase.

[0166] In some embodiments, the enzyme possessing β-galactosidase activity is wild-type β-galactosidase originating from Aspergillus oryzae or a functional analog thereof. The amino acid sequence of wild-type β-galactosidase originating from Aspergillus oryzae corresponds to the amino acid sequence with deposit number: Q2UCU3 (https: / / www.uniprot.org / , deposited).

[0167] In some preferred embodiments, the enzyme having β-galactosidase activity is a shortened variant of wild-type β-galactosidase (Q2UCU3) originating from Aspergillus oryzae.

[0168] The shortened variant of β-galactosidase according to the present invention may be purchased from an established manufacturer, such as Calza Clemente, or may be prepared by methods known to those skilled in the art, such as those described in MM Maksimainen et al., International Journal of Biological Macromolecules 2013, 60, 109-115.

[0169] By advantageously using the step of adding an enzyme having β-galactosidase activity, the lactose formed during sialylation catalyzed by the sialyltransferase of the sphingoglycolipid receptor of formula (6) can be hydrolyzed.

[0170] The step of adding an enzyme having β-galactosidase activity is typically performed after a certain conversion rate of 3'-sialyl lactose has been achieved. The step of adding the enzyme having β-galactosidase activity is preferably performed when a conversion rate of at least about 50% of 3'-sialyl lactose has been achieved, preferably when a conversion rate of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of 3'-sialyl lactose has been achieved. The conversion rate of 3'-sialyl lactose can be determined by standard techniques known to those skilled in the art. Typically, the conversion rate of 3'-sialyl lactose is determined by HPLC and can be expressed in units of mol% or weight%.

[0171] Nanofiltration (NF) steps can be used to concentrate mixtures containing sialylated sphingoglycolipids in order to remove ions, mainly monovalent ions, and / or organic materials having a molecular weight smaller than that of the sialylated sphingoglycolipids, such as monosaccharides. In a preferred embodiment, nanofiltration steps are used to remove galactose and glucose from a mixture containing sialylated sphingoglycolipids.

[0172] Typically, nanofiltration membranes have a molecular weight cutoff (MWCO) that ensures the retention of the desired sialylated sphingoglycolipid. For example, nanofiltration membranes with an MWCO of approximately 200 Da to 500 Da are suitable for retaining sialylated sphingoglycolipids. In this regard, sialylated sphingoglycolipids accumulate in the NF retention solution (NFR). To more effectively remove permeable molecules, for example, until the conductivity of the permeate indicates the absence or very low presence of the salt, nanofiltration can be combined with water-based diafiltration (DF).

[0173] The NF process according to the present invention is carried out at a constant temperature, preferably about 15°C to 45°C, more preferably about 20°C to 35°C, with or without an optional DF process. The NF process, with or without diafiltration, continues until the sialylated sphingoglycolipid in the NFR reaches the desired concentration. Other technical parameters, such as the setting of permeation flux and pressure, are standard technical matters.

[0174] The above sialylation method includes the steps of mixing a sphingoglycolipid receptor with a sialic acid donor in the presence of an enzyme having trans-sialidase activity, and the subsequent step of adding β-galactosidase to the reaction mixture.

[0175] It is understood that enzymes (or multiple enzymes) and substrates can be added in any order, and the order in which the reactants are combined can be adjusted as needed.

[0176] For example, a sialic acid donor may be added to a solution of sphingoglycolipids, and then trans-sialidase may be added.

[0177] Sialic acid donors, sphingoglycolipids, trans-sialidases, and β-galactosidases, as well as any other components used in the sialylation reaction, can be added to the reaction mixture either as a solid or dissolved in a solvent, and in any quantity and form effective for the intended outcome of the process.

[0178] The temperature at which the above process is carried out can range from slightly above the freezing point to the temperature at which the most sensitive enzyme denatures. The temperature range is preferably about 0°C to about 45°C, more preferably about 20°C to 37°C.

[0179] The sphingoglycolipid receptor and sialic acid donor are reacted for a period of time sufficient to obtain the desired sialylated sphingoglycolipid in the desired high yield.

[0180] Typically, the reaction is allowed to proceed for about 1 to 24 hours, preferably about 5 to 10 hours. In some embodiments, the reaction is allowed to proceed for about 5, 6, 7, 8, 9, or 10 hours.

[0181] Sphingoglycolipids, trans-sialidase, and β-galactosidase can be combined by mixing them in an aqueous reaction medium. The pH of the medium is generally about 5 to about 7.5. The choice of medium is based on the medium's ability to maintain the pH at a desired level. Therefore, in some embodiments, the pH of the medium is buffered to 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, and 7.5. In some preferred embodiments, the pH of the medium is buffered to about 5.5 to 6.5. Therefore, in some preferred embodiments, the pH value of the medium is buffered to approximately 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5.

[0182] Suitable buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, MOPS, HEPES, PBS, sodium acetate buffer, and sodium citrate buffer. Sodium acetate buffer is preferred. If no buffer is used, the pH of the medium should be maintained at approximately 5 to 7.5 by using a base or acid. A suitable base is NaOH, and a suitable acid is HCl.

[0183] In another embodiment, the sphingolipid of formula (1) is a sialylated sphingoglycolipid, where the sialylated sphingoglycolipid is of formula (8): TIFF2026528773000011.tif31170 (in the formula, Y is a glycosyl moiety, and the glycosyl moiety is preferably selected from the group consisting of Gal1-, or a glycosyl moiety having one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units. R 1 , R 2 and R 3 and combination --- The sphingoglycolipid receptor of formula (1) is defined as follows: The method comprises sialylation of the glycoside receptor by mixing it with sialic acid, cytidine monophosphate, nucleoside triphosphate, and one or more cell-free extracts of microorganisms, wherein the microorganisms include one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity. One or more of the above cell-free extracts At least one polypeptide having cytidine monophosphate kinase activity, At least one polypeptide having N-acyl ilaminate cytidyltransferase activity, At least one polypeptide having sialyltransferase activity, It is generated by a method that includes [this method].

[0184] In some preferred embodiments, Y of the sphingoglycolipid receptor of formula (7) is a glycosyl moiety selected from the group consisting of the following glycosyl moieties: Neu5Acα2-3Galβ1-4Glc-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, or salts thereof, where the glycosyl moieties may be linked via α-glycosidic bonds or β-glycosidic bonds, preferably β-glycosidic bonds.

[0185] In some preferred embodiments, the sphingoglycolipid receptor of formula (7) is selected from the group consisting of N-lyso-GM3 and N-lyso-GM1a.

[0186] In some embodiments, Y of the sphingoglycolipid receptor of formula (7) is selected from the group consisting of Gal1- and Galβ1-4Glc1-. Therefore, in some preferred embodiments, the sphingoglycolipid receptor is galactosyl D-erythro-sphingosine or lactosyl D-erythro-sphingosine.

[0187] The sialylation of the glycoside receptor of formula (7) typically occurs as part of a sialyltransferase cycle, which involves a CMP-sialic acid recycling system in which CMP-sialic acid is generated / regenerated from sialic acid and CMP.

[0188] CMP-sialic acid is a relatively expensive sugar nucleotide; therefore, in-situ generation and regeneration of sialic acid donors offer economic advantages and enable process scale-up.

[0189] The sialyltransferase cycle described in the present invention typically comprises the use of sialic acid, cytidine monophosphate (CMP), nucleoside triphosphate, and one or more cell-free extracts of microorganisms, wherein the one or more cell-free extracts contain the enzyme activity required for the sialyltransferase cycle. The enzyme activity required for the sialyltransferase cycle is, At least one phosphotransferase enzyme activity, Activity of at least one inorganic diphosphatase enzyme, At least one cytidine monophosphate kinase enzyme activity, At least one N-acyl ilaminate cytidylyl transferase enzyme activity, and At least one sialyltransferase enzyme activity, Includes.

[0190] 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).

[0191] In some preferred embodiments, the nucleoside triphosphate is adenosine-5'-triphosphate (ATP).

[0192] Therefore, in some preferred embodiments, the sialyltransferase cycle comprises N-acetylneuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), and one or more cell-free extracts of microorganisms, wherein the one or more cell-free extracts include one polypeptide having cytidine monophosphate kinase activity (CMK) (for phosphorylation of CMP), one polypeptide having phosphotransferase activity (for phosphorylation of CDP), and N-acyl neuramine cytidyltransferase activity ( The polypeptide comprises one polypeptide having 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 receptor substrate), and one polypeptide having inorganic diphosphatase activity (PPase) (for the degradation of inorganic pyrophosphate (PPi) formed during the cycle). The polypeptide having phosphotransferase activity and the polypeptide having inorganic diphosphatase activity (PPase) are endogenously expressed by microorganisms.

[0193] In some preferred embodiments, one or more cell-free extracts are cell-free extracts of microorganisms, the microorganisms comprising at least one endogenous polypeptide having phosphotransferase enzyme activity and at least one endogenous polypeptide having diphosphatase enzyme activity, the microorganisms At least one heterologous polypeptide having cytidine monophosphate kinase activity, At least one heterologous polypeptide having N-acyl ilaminate cytidyltransferase activity, At least one heterologous polypeptide having sialyl-transferase activity, The genes are genetically engineered to express one or more polypeptides selected from the group consisting of [specific groups].

[0194] The microorganisms produced by the methods described in the present invention preferably contain reduced β-galactosidase activity or do not contain β-galactosidase activity. The reduction or knockout of β-galactosidase activity can be achieved, for example, by genetic manipulation of the gene encoding a polypeptide having β-galactosidase activity, for example by introducing a mutation that results in the expression of an inactive enzyme and knocking out the gene, or by other means.

[0195] The microorganisms produced by the method described in the present invention may be yeast or bacteria, preferably bacteria.

[0196] In some embodiments, the microorganism is Escherichia coli (E. coli).

[0197] In some embodiments, the microorganism is Escherichia coli BL21(DE3) strain.

[0198] In the context of the present invention, the term "microorganism" includes living cells such as bacterial cells or yeast cells, and may also include one or more variations (hereinafter also referred to as "strains") of the above-mentioned living cells of the above-mentioned microorganism. The term "strain" also includes variants of microorganisms that are artificially created (by recombination) by genetically modifying the above-mentioned microorganisms.

[0199] In some embodiments, one or more strains of microorganisms are created by genetically modifying the E. coli BL21(DE3) strain.

[0200] E. coli BL21(DE3) cells can be obtained from established manufacturers such as ThermoFischer Scientific.

[0201] Typically, one or more strains of microorganisms are used to produce one or more cell-free extracts by methods known to those skilled in the art (e.g., Cole et al. Synthetic and Systems Biotechnology 2020, 5, 252-267).

[0202] In some embodiments, the method comprises the use of three cell-free extracts of a microorganism, wherein the first cell-free extract comprises a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract comprises a polypeptide having N-acyl furanate cytidyltransferase activity, and the third cell-free extract comprises a polypeptide having sialyltransferase activity. These three cell-free extracts are cell-free extracts of a microorganism that intrinsically expresses a polypeptide having inorganic diphosphatase activity and at least one polypeptide having phosphotransferase activity.

[0203] The term "polypeptide having sialyltransferase activity" can be used interchangeably with the term "sialyltransferase" and, in the context of the present invention, refers to an enzyme belonging to EC class 2.4.99.

[0204] A sialyltransferase suitable for use in the context of the present invention is a sialyltransferase capable of catalyzing the transfer of a sialic acid residue from the O-3 of the β-linked galactose residue of a glycoside receptor and / or from the O-8 of the α-2,3-linked sialic acid residue of a glycoside receptor.

[0205] In some embodiments, the polypeptide possessing sialyltransferase activity is wild-type α-2,3 / α-2,8-sialyltransferase originating from Campylobacter jejuni OX=197 strain. The amino acid sequence of wild-type α-2,3 / α-2,8-sialyltransferase originating from Campylobacter jejuni OX=197 strain corresponds to the amino acid sequence with accession number: Q9LAK3 (https: / / www.ncbi.nlm.nih.gov / protein).

[0206] In some embodiments, the polypeptide having sialyltransferase activity is a mutant of the wild-type α-2,3 / α-2,8-sialyltransferase Q9LAK3, the mutant containing the following mutations / modifications compared to the wild-type: Ile53Ser, deletion of 32 amino acids at the C-terminus (numbering corresponding to the alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3).

[0207] In some embodiments, the α-2,3 / α-2,8-sialyltransferase is a variant of the wild-type α-2,3 / α-2,8-sialyltransferase Q9LAK3, which includes the following mutations / modifications compared to the wild-type: Ile53Ser, an N-terminal histidine tag, and a deletion of 32 amino acids at the C-terminus (numbered to correspond to the alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3).

[0208] In some embodiments, α-2,3 / α-2,8-sialyltransferase is a variant of wild-type α-2,3 / α-2,8-sialyltransferase Q9LAK3, the variant containing the following mutations / modifications compared to the wild type: Ile53Gly, an N-terminal histidine tag, and a deletion of 32 amino acids at the C-terminus (numbered to correspond to the alignment of the mutant amino acid sequence with the amino acid sequence of Q9LAK3).

[0209] α-2,3 / α-2,8-sialyltransferase or its functional analogue, originating from Campylobacter jejuni, may also be referred to as CST-II.

[0210] In some embodiments, the polypeptide possessing sialyltransferase activity is wild-type α-2,3-sialyltransferase originating from Bibersteinia trehalosi strain DSM23101. The amino acid sequence of wild-type Bibersteinia trehalosi α-2,3-sialyltransferase corresponds to the amino acid sequence with accession number: WP_025267256 (https: / / www.ncbi.nlm.nih.gov / protein).

[0211] In some embodiments, the α-2,3 sialyltransferase is a recombinant α-2,3 sialyltransferase derived from the wild-type α-2,3 sialyltransferase WP_025267256, wherein the recombinant sialyltransferase includes the following mutation / modification compared to the wild-type: N-terminal histidine tag MGHHHHHH.

[0212] α-2,3-sialyltransferase or its functional analogue, originating from Bibersteinia trehalose, may also be referred to as BtSiaT.

[0213] The term "cytidine monophosphate kinase activity polypeptide" can be used interchangeably with the terms "CMP kinase" or "CMK" and, in the context of the present invention, refers to an EC class 2.7.4.25 enzyme that catalyzes the phosphorylation of CMP (or dCMP), for which ATP is typically used as the preferred phosphoryl donor.

[0214] In some embodiments, the polypeptide having cytidine monophosphate kinase activity is CMP kinase or a functional analog thereof derived from Mycobacterium tuberculosis.

[0215] In some embodiments, the polypeptide possessing CMK kinase activity is a wild-type CMK kinase originating from Mycobacterium tuberculosis. The amino acid sequence of the wild-type CMK kinase originating from Mycobacterium tuberculosis corresponds to the amino acid sequence with accession number: WP_129368399 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0216] In some embodiments, the CMP kinase is a recombinant CMP kinase derived from the wild-type CMP kinase WP_129368399, and the recombinant CMP kinase includes the following mutation / modification compared to the wild-type: N-terminal histidine tag MGHHHHHH.

[0217] CMP kinases or their functional analogues originating from Mycobacterium tuberculosis may also be referred to as MtCMK.

[0218] The term "polypeptide having N-acyl neuramine cytidyltransferase activity" can be used interchangeably with the terms "N-acyl neuramine cytidylyltransferase" or "CSS," and in the context of the present invention, refers to an EC class 2.7.7.43 enzyme that catalyzes the transfer of CMP from CTP to N-acetylneuraminic acid (Neu5Ac).

[0219] In some embodiments, the polypeptide having N-acyl-noiraminate cytidyltransferase activity is wild-type CSS originating from Neisseria meningitidis. The amino acid sequence of wild-type CSS originating from Neisseria meningitidis corresponds to the amino acid sequence with accession number: WP_061726245 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0220] In some embodiments, the CSS is a recombinant CSS derived from the wild-type CSS WP_061726245, and the recombinant CSS includes the following mutation / modification compared to the wild-type: N-terminal histidine tag MGHHHHHH.

[0221] Neisseria meningitidis-derived N-acylneuraminic acid cytidyltransferase or a functional analog thereof may also be referred to as NmCSS.

[0222] The term "polypeptide having inorganic diphosphatase activity" can be used interchangeably with the term "inorganic diphosphatase" or "PPase", and in the context of the present invention, refers to an enzyme of EC class 3.6.1.1 that catalyzes the hydrolysis of pyrophosphate (PPi).

[0223] In some preferred embodiments, the polypeptide having inorganic diphosphatase activity is wild-type PPase derived from Escherichia coli. The amino acid sequence of wild-type inorganic diphosphatase derived from Escherichia coli corresponds to the amino acid sequence having accession number: WP_073849715 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0224] Inorganic diphosphatase derived from Escherichia coli may also be referred to as EcPPase.

[0225] In some embodiments, the "polypeptide having phosphotransferase activity" is a polypeptide having nucleoside diphosphate kinase activity.

[0226] The term "polypeptide having nucleoside diphosphate kinase activity" can be used interchangeably with the term "nucleoside diphosphate kinase" or "NDK", and in the context of the present invention, refers to an enzyme of EC class 2.7.4.6 that catalyzes the phosphorylation of nucleoside diphosphate.

[0227] In several preferred embodiments, the polypeptide nucleoside diphosphate kinase activity is that of a wild-type NDK originating from the E. coli BL21(DE3) strain. The amino acid sequence of the wild-type NDK originating from the E. coli BL21(DE3) strain corresponds to the amino acid sequence with accession number ACT44230 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0228] Nucleoside diphosphatases originating from the E. coli BL21(DE3) strain can also be referred to as EcNDK.

[0229] In some embodiments, the polypeptide having phosphotransferase activity is a polypeptide having myokinase activity.

[0230] The term "polypeptide having myokinase activity" can be used interchangeably with the terms "myokinase," "adenylate kinase," or "ADK," and in the context of the present invention, refers to an EC class 2.7.4.3 enzyme that catalyzes the interconversion of various adenosine phosphates (e.g., ATP, ADP, and AMP).

[0231] In some preferred embodiments, the polypeptide having myokinase activity is a wild-type myokinase originating from the E. coli BL21(DE3) strain. The amino acid sequence of wild-type ADK originating from the E. coli BL21(DE3) strain corresponds to the amino acid sequence with accession number ACT42324 (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0232] Myokinases originating from the E. coli BL21(DE3) strain can also be referred to as EcADK.

[0233] In some embodiments, the method involves the use of three cell-free extracts of the E. coli BL21(DE3) strain, where the first cell-free extract contains wild-type MtCMK (GenBank accession number: WP_129368399), the second cell-free extract contains recombinant NmCSS (wild-type: WP_061726245, modified: N-terminal histidine tag MGHHHHHH), and the third cell-free extract contains mutant CSTII (wild-type The E. coli BL21(DE3) strain contains Q9LAK3 (mutation / modification: Ile53Ser or Ile53Ser, N-terminal histidine tag, deletion of 32 amino acids at the C-terminus) and endogenously expresses the following enzymes: EcPPase (GenBank accession number: WP_073849715), EcNDK (GenBank accession number: ACT44230), and EcADK (GenBank accession number: ACT42324).

[0234] In some embodiments, the method involves the use of three cell-free extracts of the E. coli BL21(DE3) strain, where the first cell-free extract contains wild-type MtCMK (GenBank accession number: WP_129368399), the second cell-free extract contains recombinant NmCSS (wild-type: WP_061726245, modified: N-terminal histidine tag MGHHHHHH), and the third cell-free extract contains wild-type BtSiaT (GenBank accession number: WP_025267256), and the E. coli BL21(DE3) strain endogenously expresses the following enzymes: EcPPase (GenBank accession number: WP_073849715), EcNDK (GenBank accession number: ACT44230), and EcADK (GenBank accession number: ACT42324).

[0235] In some embodiments, the method involves the use of four cell-free extracts of the Escherichia coli BL21(DE3) strain, where the first cell-free extract contains wild-type MtCMK (GenBank accession number: WP_129368399), the second cell-free extract contains recombinant NmCSS (wild-type: WP_061726245, modified: N-terminal histidine tag MGHHHHHH), and the third cell-free extract contains mutant CSTII (wild-type Q9LAK3, mutant / modified: Ile53Ser or Il The fourth cell-free extract contains wild-type BtSiaT (GenBank accession number: WP_025267256), and the E. coli BL21(DE3) strain endogenously expresses the following enzymes: EcPPase (GenBank accession number: WP_073849715), EcNDK (GenBank accession number: ACT44230), and EcADK (GenBank accession number: ACT42324).

[0236] Regarding the sialyltransferase cycle, the concentrations or amounts of the various reactants used in the process depend on numerous factors, including reaction conditions such as temperature and pH, as well as the selection and amount of glycosidic receptors to be sialized. In the sialylation process, in the presence of a catalytic amount of enzyme, the regeneration of activated nucleotides and activated donor sugars, and the removal of the generated PPi are possible, and the process is limited by the concentration or amount of stoichiometric substances. The upper limit of the concentrations of reactants that can be used according to the method of the present invention is determined by the solubility of such reactants. The concentrations of activated nucleotides, phosphate donors, donor sugars, and enzymes are preferably selected so that glycosylation proceeds until the receptor is consumed.

[0237] The sialyltransferase cycle according to the present invention may also include other components that promote sialyltransferase activity. These components may include divalent cations (e.g., Mg +2 or Mn +2Examples of materials necessary for ATP regeneration include phosphate ions, etc. The reaction medium may also include a solubilizing surfactant (e.g., Triton or SDS) and an organic solvent such as methanol or ethanol, or cyclodextrin.

[0238] In a preferred embodiment, the reaction medium includes cyclodextrin.

[0239] In some embodiments, the cyclodextrin is selected from the group consisting of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin. In some preferred embodiments, the cyclodextrin is β-cyclodextrin.

[0240] Cyclodextrin is typically used in amounts of about 0.1 to 1 equivalent based on the amount of sphingoglycolipid receptors. In some preferred embodiments, cyclodextrin is used in amounts of about 0.1 to 0.5 equivalents based on the amount of sphingoglycolipid receptors. Therefore, in some preferred embodiments, cyclodextrin is used in amounts of about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the amount of sphingoglycolipid receptors.

[0241] The use of cyclodextrin offers several advantages, including high yield, and eliminates the need to use surfactants or organic solvents to enhance access to the glycosyl moiety of the sphingoglycolipid receptor. However, surfactants or organic solvents can also be used in the method of the present invention.

[0242] In the optimized reaction, the above components can be combined by mixing them in an aqueous reaction medium (solution) having a pH value of approximately 6 to 8.5. The medium is Mg +2 or Mn +2It lacks chelating agents that bind enzyme cofactors such as etc. The selection of the medium is based on the ability of the medium to maintain the pH value at a desired level. Thus, in some embodiments, the medium is buffered to a pH value of about 6.5 to about 8.5. When not using a buffer solution, the pH of the medium should be maintained at about 6.5 to 8.0, preferably about 7.3 to 8.0, by adding a base. A suitable base is NaOH. Thus, in some preferred embodiments, the pH is buffered or maintained at values of about 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0243] The temperature at which the above process is carried out can range from a temperature slightly above the freezing point to the temperature at which the most sensitive enzyme denatures. That temperature range is preferably about 0°C to about 45°C, more preferably about 20°C to 37°C.

[0244] The reaction mixture thus formed is maintained for a period sufficient for a high percentage of receptors to be sialylated by sialyltransferase. Usually, in many cases, the reaction is allowed to proceed for about 8 hours to about 240 hours, preferably about 24 hours to 48 hours.

[0245] N-acetyl-neuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphoric acid, cell-free extract (in some cases), and any other components used during the cycle can be added to the reaction mixture either as a solid or dissolved in a solvent, and in any quantity and manner effective for the intended result of the process.

[0246] In the context of the present invention, cell-free extract enzyme activity is expressed in activity units, which is a measure of the initial rate of catalysis. 1 activity unit catalyzes the formation of 1 μmol of product per minute at a given pH and temperature. Cell-free extract (in some cases) enzyme activity can be measured according to procedures known to those skilled in the art.

[0247] The sphingoglycolipids according to the present invention can be produced or utilized in the form of a salt, preferably in the form of a pharmaceutically acceptable salt.

[0248] In some embodiments, the salt can be formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphate, acetic acid, camphor sulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, and perchloric acid. [Examples]

[0249] The following embodiments describe non-limiting embodiments of the present invention and are provided merely to illustrate the present invention.

[0250] General methods and materials The sphingoglycolipid content of the spray-dried powder was determined by HPLC using peak area analysis with an external standard. HPLC analysis was performed using a Dionex Ultimate 3000 HPLC system combined with a Corona Veo Charged Aerosol Detector and an Accucore aQ (150 mm × 4.6 mm, 2.6 μm) column. The method is described in Example 14.

[0251] Lactosyl D-erythro-sphingosine was synthesized as described in International Publication No. 2023118378 or Vaughan et al., J. Am. Chem. Soc. 2006, 128, 6300-6301.

[0252] Solutions containing N-lyso-GM3 or a mixture of N-lyso-GM3 and N-lyso-GD3 were prepared according to the procedures described in Examples 11 and 12, respectively.

[0253] Mutant TcTS (wild-type Q26966, mutation / modification: Ser263Thr, Arg477His, Val485Leu, Glu559Val, Ser496Lys, N-terminal His tag, 7 amino acid deletions Δ636~642) was expressed in Escherichia coli strains according to the method described in Paris et al., Glycobiology 2001, 11, 305-311 or Buschiazzo et al., Molecular Cell 2002, 10, 757-768.

[0254] The following cell-free extracts were produced from E. coli expression strains: (i) Cell-free extract from E. coli BL21(DE3)dLacZ genetically engineered to express MtCMK (accession number: WP_129368399); (ii) Cell-free extract from E. coli BL21(DE3)dLacZ genetically engineered to express recombinant NmCSS (wild type: WP_061726245, modification: N-terminal histidine tag MGHHHHHH); (iii) Cell-free extract from E. coli BL21(DE3)dLacZ genetically engineered to express mutant CSTII (wild type Q9LAK3, mutation / modification: Ile53Ser or Ile53Gly, N-terminal histidine tag, deletion of 32 amino acids at the C-terminus).

[0255] Cell-free extracts were prepared as described in Example 13.

[0256] Example 1. General Diafiltration Procedure The supply solution containing sphingoglycolipids was subjected to diafiltration (DF). The DF was approximately 0.668 m 2 The procedure was carried out using a 250 kDa spiral membrane with a membrane surface area, a flow rate of approximately 10 l / hour, a membrane pressure differential of approximately 8 to 10 bar, a temperature of approximately 20°C to 25°C, and applying a DF volume of approximately 2 to 10 relative to the volume of the supply solution. During diafiltration, approximately 15.3 l / m³ 2 ·Time~18.1l / m 2 • Maintained high permeation flux over time.

[0257] The DF-retaining solution (DFR) containing sphingoglycolipids is preferably concentrated and then spray-dried as described in Example 2.

[0258] Example 2. Spray drying of DFR The diafiltration retaining liquid obtained in Example 1 was spray-dried using a Mobile Minor® (GEA) spray dryer under the following conditions: Inlet flow rate: 30g / min~50g / min Sprayer speed: 20,000 rpm Inlet temperature: 150℃~160℃ Outlet temperature: 85℃~92℃

[0259] Following this procedure, a spray-dried powder containing approximately 65% ​​to 90% by weight of one or more sphingoglycolipids was obtained.

[0260] Example 3. Particle size analysis of spray-dried powder The average particle size, 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 containing 0.1% soy lecithin. The sample was sonicated before size measurement to disperse any aggregated particles.

[0261] Example 4. Water content of spray-dried powder The water content of the spray-dried powder obtained in Example 2 was determined by thermogravimetric analysis (TG) and differential scanning calorimetry (DSC), or by Karl Fischer titration. TG and DSC measurements were performed using Setaram LabsysEvo (Setaram). Spray-dried powder typically contains about 2% to 3% by weight of water.

[0262] Example 5. Specific volume of spray-dried powder The spray-dried powder sample obtained in Example 2 was poured into a graduated cylinder whose tare weight had been measured, and the volume and weight of the sample were recorded.

[0263] Example 6. Spray-dried powder containing N-lyso-GM3 The DFR containing N-lyso-GM3 obtained according to the procedure of Example 1 was spray-dried under the conditions of Example 2 to obtain a spray-dried powder having the following characteristics:

[0264] TIFF2026528773000012.tif45170

[0265] Example 7. Spray-dried powder containing a 4:1 mixture of N-lyso-GD3 and N-lyso-GM3. A DFR containing a 4:1 mixture of N-lyso-GD3 and N-lyso-GM3 obtained according to the procedure of Example 1 was spray-dried under the conditions of Example 2 to obtain a spray-dried powder having the following characteristics:

[0266] TIFF2026528773000013.tif55170

[0267] Example 8. Spray-dried powder containing a 1:3 mixture of N-lyso-GD3 and N-lyso-GM3. A DFR containing a 1:3 mixture of N-lyso-GD3 and N-lyso-GM3 obtained according to the procedure of Example 1 was spray-dried under the conditions of Example 2 to obtain a spray-dried powder having the following characteristics:

[0268] TIFF2026528773000014.tif55170

[0269] Example 9. Spray-dried powder containing a 1:1 mixture of N-lyso-GD3 and N-lyso-GM3. A DFR containing a 1:1 mixture of N-lyso-GD3 and N-lyso-GM3 obtained according to the procedure of Example 1 was spray-dried under the conditions of Example 2 to obtain a spray-dried powder having the following characteristics:

[0270] TIFF2026528773000015.tif45170

[0271] Example 10. Spray-dried powder containing lactosyl D-erythro-sphingosine The DFR containing lactosyl D-erythro-sphingosine obtained according to the procedure of Example 1 was spray-dried under the conditions of Example 2 to obtain a spray-dried powder having the following characteristics:

[0272] TIFF2026528773000016.tif27170

[0273] Example 11. Production of N-lyso-GM3 An aqueous solution containing N-lyso-GM3 was obtained by sialylation of lactosyl D-erythro-sphingosine catalyzed by TcTS. A typical reaction mixture contained lactosyl D-erythro-sphingosine (1 equivalent), 3'-sialyl lactose (3.5 equivalents), and 2,3-trans-sialidase (TcTS, 0.4 g / L). The reaction mixture was stirred at a temperature of approximately 37°C for approximately 3 to 6 hours, then β-galactosidase (0.13 g / L) was added, and the reaction mixture was nanofiltered. Nanofiltration of the reaction mixture was performed by applying a 300 Da to 500 Da membrane, a pressure of 15 to 20 bar, and a temperature of approximately 30°C to 40°C for approximately 6 to 8 hours. The NF holding solution (NFR) was heated at a temperature of approximately 60°C to 95°C for approximately 10 to 60 minutes, and then diafiltration was performed as described in Example 1.

[0274] Example 12. Production of a mixture of N-lyso-GD3 and N-lyso-GM3 An aqueous solution containing N-lyso-GD3 was obtained by a sialyltransferase cycle from N-lyso-GM3. The sialyltransferase cycle was carried out in an aqueous solution with a pH of approximately 7.0 to 7.5 and a temperature range of approximately 25°C to 37°C. A typical reaction mixture contained glycoside acceptor (1 equivalent), N-acetylneuraminic acid (Neu5Ac, 1.2 to 2.5 equivalents), β-cyclodextrin (0.5 equivalents), ATP (2.0 to 3.5 equivalents), CMP (0.1 to 0.3 equivalents), MgCl2 (0.5M), and the following three cell-free extracts: cell-free extract (i) (5 g / L to 12 g / L), cell-free extract (ii) (1 g / L to 2 g / L), and cell-free extract (iii) (2.5 g / L to 5 g / L). The sialylation cycle was monitored by LC-MS.

[0275] Example 13. Preparation of cell-free extracts The enzyme-encoding gene is typically purchased as a codon-optimized synthetic gene for optimal expression in an E. coli host strain. The synthetic construct contains an overhang with a BsaI restriction site for golden gate cloning into a pET28a-based expression vector (which has an introduced BsaI restriction site and a fluorescent drop-out cassette). The resulting plasmid was used to transform E. coli BL21(DE3)dLacZ.

[0276] Pre-cultures of the expression strains were prepared in 10 mL of LB medium supplemented with each antibiotic and incubated overnight at 37°C with shaking. Culturing of the expression strains was started using 1:100 dilutions of the pre-cultures in TB medium supplemented with each antibiotic. The cultures were incubated at 37°C. 600 The culture was incubated until it reached 0.7–1.0. The culture was cooled to the desired expression temperature, induced with 0.5 mM IPTG, and incubated for the desired expression time.

[0277] Cells were recovered 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 containing soluble enzymes from the debris. The supernatant containing the cell-free extract was freeze-dried to dryness.

[0278] Example 14. HPLC analysis The sphingoglycolipid content of spray-dried powders containing N-lyso-GM3 and lactosyl d-erythro-sphingosine was determined under the following conditions: HPLC eluent profiles: 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.

[0279] A gradient of B from 50% to 100% in A was applied for 13 minutes, followed by 18 minutes with a fixed composition of 100% B, and then 40 minutes with a fixed composition of 50% B in A. The sphingoglycolipid content of the powder was quantified by peak area analysis using an external standard.

[0280] The sphingoglycolipid content of a spray-dried powder containing a mixture of N-lyso-GD3 and N-lyso-GM3 was determined under the following conditions: 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.

[0281] A gradient from 70% A to 100% B was applied over 8 minutes, followed by 11 minutes with the standard 100% B composition, and then 25 minutes with the standard 70% B composition in A. The sphingoglycolipid content of the powder was quantified by peak area analysis using an external standard substance.

[0282] This disclosure should not be considered in any way limited to the embodiments described herein, and those skilled in the art will foresee many of the possible modifications.

[0283] The embodiments described above can be combined with each other.

[0284] Specific embodiments of the present disclosure are further defined in the attached claims.

Claims

1. Formula (1): (In the formula, W is a glycosyl moiety, and the glycosyl moiety is preferably selected from a sialylated glycosyl moiety or a neutral glycosyl moiety. R １ C is hydrogen, aryl, or substituted or unsubstituted C １～５０ Alkyl, preferably substituted or unsubstituted C １～１７ Alkyl, more preferably substituted or unsubstituted C １０～１７ It is alkyl, R ２ is hydrogen or -OR ４ where R ４ is hydrogen, substituted or unsubstituted C １～６ alkyl, or substituted or unsubstituted C ２～６ acyl, and preferably R ４ is hydrogen The bond is R ２ In the case of hydrogen, it can be a double bond or a single bond, or R ２ ga- OR ４ In this case, it is a single bond, R ３ C is hydrogen, substituted or unsubstituted C １ ~ ６ Alkyl, or substituted or unsubstituted C １ ~ ６ A method for isolating a sphingoglycolipid (preferably hydrogen) or a salt thereof from a solution containing the sphingoglycolipid or salt and one or more impurities, A step of preparing a solution containing the sphingoglycolipid of formula (1) or a salt thereof and one or more impurities, The process involves diafiltration of the aforementioned solution using a membrane having MWCO of 100 kDa to 300 kDa, thereby obtaining a diafiltration holding liquid (DFR) containing the sphingoglycolipid or a salt thereof. A method comprising, wherein the sphingoglycolipid or a salt thereof is isolated from one or more impurities.

2. The method according to claim 1, wherein the diafiltration is carried out using a membrane having an MWCO of 200 kDa to 300 kDa.

3. The method according to claim 1 or 2, wherein W of one or more sphingoglycolipids of formula (1) is a sialylated glycosyl moiety, and it is preferable that the sialylated glycosyl moiety is selected from the group consisting of Neu5Acα2-3Gal1-, Neu5Acα2-3Galβ1-4Glc-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3GalNAacβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, and Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-.

4. The method according to claim 3, wherein the sphingoglycolipid of formula (1) is a sialylated sphingoglycolipid selected from the group consisting of N-lyso-GM4, N-lyso-GM3, N-lyso-GD3, N-lyso-GD1a, and N-lyso-GT3, or a mixture thereof.

5. The method according to claim 1 or 2, wherein W of the sphingoglycolipid in formula (1) is a neutral glycosyl moiety, and it is preferable that the neutral glycosyl moiety is selected from the group consisting of Gal1-, Glc1-, and Galβ1-4Glc1-.

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

7. The method according to any one of claims 1 to 6, further comprising the step of spray-drying or spray-granulating a DFR containing a sphingoglycolipid of formula (1) or a salt thereof.

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

9. One or more formulas (1): (In the formula, W is a glycosyl moiety, and the glycosyl moiety is preferably selected from a sialylated glycosyl moiety or a neutral glycosyl moiety. R １ C is hydrogen, aryl, or substituted or unsubstituted C １～５０ Alkyl, preferably substituted or unsubstituted C １～１７ Alkyl, more preferably substituted or unsubstituted C １０～１７ It is alkyl, R ２ is hydrogen or -OR ４ And R ４ C is hydrogen, substituted or unsubstituted C １～６ Alkyl, or substituted or unsubstituted C ２～６ Selected from acyl, preferably R ４ It is hydrogen, The bond is R ２ In the case of hydrogen, it can be a double bond or a single bond, or R ２ ga- OR ４ In this case, it is a single bond, R ３ C is hydrogen, substituted or unsubstituted C １ ~ ６ Alkyl, or substituted or unsubstituted C １ ~ ６ A spray-dried powder containing a sphingoglycolipid (preferably hydrogen) or a salt thereof.

10. The spray-dried powder according to claim 9, wherein W of one or more sphingoglycolipids represented by formula (1) or a salt thereof is a sialylated glycosyl moiety, and it is preferable that the sialylated glycosyl moiety is selected from the group consisting of Neu5Acα2-3Galβ1-, Neu5Acα2-3Galβ1-4Glc-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-3Galβ1-3GalNAacβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, and Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-.

11. The spray-dried powder according to claim 9, wherein W of one or more sphingoglycolipids of formula (1) or a salt thereof is a neutral glycosyl moiety, and it is preferable that the neutral glycosyl moiety is selected from the group consisting of Gal1-, Gluc1-, and Galβ1-4Glc1-.

12. The spray-dried powder according to claim 9 or 10, comprising at least about 70% by weight of N-lyso-GM3.

13. The spray-dried powder according to claim 12, comprising approximately 75% to 80% by weight of N-lyso-GM3, and further comprising approximately 7% to 9% by weight of lactosyl D-erythrose-sphingosine, and approximately 0.1% to 1.0% by weight of glucosyl D-erythrose-sphingosine.

14. The spray-dried powder according to claim 9 or 10, comprising at least about 60% by weight of a mixture of N-lyso-GD3 and N-lyso-GM3, wherein the weight ratio of N-lyso-GD3 to N-lyso-GM3 in the mixture is about 1:10 to about 10:

1.

15. The spray-dried powder according to claim 14, comprising approximately 40% to 55% by weight of N-lyso-GD3 and approximately 10% to 15% by weight of N-lyso-GM3, further comprising approximately 3% to 6% by weight of N-lyso-GT3, approximately 4% to 6% by weight of lactosyl d-erythrose-sphingosine, and approximately 0.1% to 1.0% by weight of glucosyl D-erythrose-sphingosine.

16. The spray-dried powder according to claim 14, comprising approximately 15% to 20% by weight of N-lyso-GD3 and approximately 50% to 60% by weight of N-lyso-GM3, further comprising approximately 0.1% to 0.5% by weight of N-lyso-GT3, approximately 4% to 7% by weight of lactosyl d-erythrose-sphingosine, and approximately 0.1% to 1.0% by weight of glucosyl D-erythrose-sphingosine.

17. The spray-dried powder according to claim 14, comprising approximately 35% to 40% by weight of N-lyso-GD3 and approximately 25% to 40% by weight of N-lyso-GM3, further comprising approximately 5% to 6% by weight of lactosyl d-erythrose-sphingosine and approximately 0.5% to 1.0% by weight of glucosyl D-erythrose-sphingosine.

18. The spray-dried powder according to claim 11, comprising 80% to 90% by weight of lactosyl d-erythrose-sphingosine.

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

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