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WO2014135167A1 - Purification of oligosaccaharides by reversible derivatization - Google Patents

Purification of oligosaccaharides by reversible derivatization Download PDF

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Publication number
WO2014135167A1
WO2014135167A1 PCT/DK2014/050047 DK2014050047W WO2014135167A1 WO 2014135167 A1 WO2014135167 A1 WO 2014135167A1 DK 2014050047 W DK2014050047 W DK 2014050047W WO 2014135167 A1 WO2014135167 A1 WO 2014135167A1
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Prior art keywords
oligosaccharide
derivative
hmo
cyclic
acid
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PCT/DK2014/050047
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French (fr)
Inventor
Gyula Dekany
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Glycom A/S
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Publication of WO2014135167A1 publication Critical patent/WO2014135167A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/04—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

Definitions

  • This invention relates to a method of the purification of an hydrophilic oligosaccharide, especially an human milk oligosaccharide (HMO), produced by a fermentation or enzymatic process.
  • the invention particularly relates to forming a derivative of the oligosaccharide with different physical properties, particularly different solubility, from contaminants and impurities which are produced by the fermentation or enzymatic process.
  • HMOs human milk oligosaccharides
  • Mature human milk is the natural milk source that contains the highest concentrations of milk oligosaccharides (12-14 g/1), other milk sources are cow's milk (0.01 g/1), goat's milk and milk from other mammals.
  • HMOs Approximately 200 HMOs have been detected from human milk by means of combination of techniques including microchip liquid chromatography mass spectrometry (HPLC Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) (Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which to date at least 115 oligosaccharides have been structurally determined (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011). These human milk oligosaccharides can be grouped into 13 core units (Table 1).
  • HMOs Due to the large number of similar HMOs and their low concentrations in mammalian milk, isolation of HMOs is a difficult task even in milligram quantities. To date only analytical HPLC methodologies have been developed for the isolation of some HMOs from natural sources. It is therefore difficult to provide suitable HMO replacements in foods, particularly in infant formulae which display at least part of the entire spectrum of HMOs.
  • HMOs can be produced in high yields in aqueous media by fermentation of genetically modified bacteria, yeasts or other microorganisms. See, for example, WOOl/04341, WO2007/101862, WO2010/070104, WO2010/142305, WO2012/ 112777, Priem et al Glycobiology 12, 235 (2002), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), Han et al. Biotechnol. Adv.
  • biotechnological methods cannot provide HMOs of high purity due to the contaminants and other impurities (of bacterial or chemical origin such as enzymes, proteins, endotoxins, DNA and carbohydrate regioisomers) which can be very difficult to remove by common methods.
  • biotechnological methods involve the use of genetically modified microorganisms. As consequence of the above mentioned, the regulatory approval of HMOs produced by these methods, is likely to encounter serious obstacles in regulatory processes. There has, therefore, been a need for identifying solid, robust and cost-efficient methods for the purification of HMOs derived from a fermentation or enzymatic process. In this regard,
  • WO2012/007585 describes a method for the purification of HMOs by means of an initial anomeric Obenzylation and a subsequent reverse-phase or size exclusion chromatographic separation of the so-obtained Obenzylated HMOs, followed by their catalytic hydrogenolysis to regenerate the original HMOs.
  • alternative purification methods have been sought which would be simpler and less expensive to use on an industrial scale.
  • a method for purifying a hydrophilic oligosaccharide, advantageously a HMO, in an aqueous medium from a fermentation or enzymatic process comprising the steps of: a) optionally clarifying the aqueous medium to remove particulates and contaminants and advantageously also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b) removing substantially all proteins from the aqueous medium, advantageously after step a), to provide a protein-free oligosaccharide; c) reacting the protein-free oligosaccharide from step b) with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c); and then e) reacting the derivative of the
  • the reaction in step c) advantageously comprises one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide to form an acyl derivative of the
  • oligosaccharide iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide to form a cyclic -ketal derivative of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
  • each of the derivatizing reactions i)-iv) in step c) is advantageously followed respectively in step e) by one of the following reactions to remove the derivatizing group and regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid.
  • a method for purifying a hydrophilic oligosaccharide, advantageously an HMO, in an aqueous medium from a fermentation or enzymatic process comprising the steps of: a) reacting the oligosaccharide with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; b) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step a); and then c) reacting the derivative of the oligosaccharide to remove the derivatizing group and regenerate the oligosaccharide; wherein step a) comprises one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, particularly an acetylation, benzoylation or
  • oligosaccharide iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide to form a cyclic-ketal derivative of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
  • each of the derivatization reactions i)-iv) in step a) is advantageously followed respectively in step c) by one of the following reactions to remove the derivatizing group and regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid.
  • This invention provides a method for purifying a hydrophilic oligosaccharide, preferably an HMO, produced in an aqueous medium or broth by a fermentation or enzymatic process.
  • the method is particularly adapted to remove water soluble contaminants and impurities which are present in the aqueous medium and which are difficult to separate in a conventional manner from the oligosaccharide.
  • the method involves the chemical alteration of the oligosaccharide to produce a derivatized oligosaccharide with altered physical properties without changing significantly such physical properties of the contaminants/impurities.
  • the method involves the steps of: a) optionally clarifying the aqueous medium to remove particulates and contaminants and advantageously also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b) removing substantially all proteins from the aqueous medium, advantageously after step a), to provide a protein-free oligosaccharide; c) reacting the protein-free oligosaccharide from step b) with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c); and then e) reacting the derivative of the oligosaccharide to remove the derivatizing group.
  • the reaction in step c) preferably involves one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide; iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide.
  • a hydrophilic oligosaccharide preferably a HMO
  • a hydrophilic oligosaccharide produced in an aqueous medium or broth by a fermentation or enzymatic process
  • This invention exploits the fact that the contaminants/impurities in the aqueous medium from the fermentation or enzymatic process do not react significantly during the following chemical reactions:
  • the resulting derivatized oligosaccharide particularly a derivatized HMO
  • has altered physical properties particularly solubility in non-aqueous solvents
  • the original oligosaccharide, particularly an HMO can be recovered in pure form by removing the derivatizing group.
  • hydrophilic oligosaccharide preferably means any neutral or acidic oligosaccharide, especially an HMO, containing two to eight, preferably three to six, simple sugar or monosaccharide moieties as its components, all the hydroxyl groups of which, including its anomeric hydroxyl group, are free.
  • terms such as “derivatization”, “derivatizing” and “derivatized” preferably refer to the action, and the result, of a chemical manipulation of a hydrophilic oligosaccharide, preferably an HMO, to provide a derivative with a significantly altered chemical structure and a significantly altered physical property.
  • alkyl preferably means a linear or branched chain saturated hydrocarbon group with 1-10 carbon atoms, especially 1-6 carbon atoms, such as methyl, ethyl, n- propyl, z ' -propyl, n-butyl, z ' -butyl, s-butyl, i-butyl or n-hexyl;
  • the term "homoaryl” preferably means a homoaromatic group such as phenyl or naphthyl;
  • heteroaryl preferably means an aromatic group having one or two rings, which ring(s) contain(s) 1, 2, or 3 heteroatoms selected from the group of N, O and S, such as pyrrol, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, furan, thiophene, oxazole, isoxazole, thiazole, thiadiazole, pyr
  • the alkyl or aryl residue can either be unsubstituted or substituted with one or more groups selected from alkyl (only for aryl residues), halogen, nitro, aryl, alkoxy, amino, alkylamino, dialkylamino, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, halogenalkyl and hydroxyalkyl, giving rise to acyl groups such as chloroacetyl, trichloroacetyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4- phenylbenzoyl, 4-benzamidobenzoyl, 4-(phenylcarbamoyl)-benzoyl, glycolyl and acetoacetyl.
  • alkyl only for aryl residues
  • halogen nitro, aryl, alkoxy, amino, alkyla
  • contaminants and “impurities” preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and endotoxins which can be present in an aqueous medium from a fermentation or enzymatic process.
  • the term "clarified oligosaccharide” preferably means a hydrophilic oligosaccharide from an aqueous medium or broth from a fermentation or enzymatic process, which (medium) has been treated to remove suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the oligosaccharide, especially HMO.
  • Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a CeliteTM filter aid).
  • protein-free oligosaccharide preferably means a hydrophilic oligosaccharide from an aqueous medium or broth from a fermentation or enzymatic process, which (medium) has been treated to remove substantially all the proteins, as well as any related impurities, such as amino acids, peptides, endotoxins, glycolipids, RNA and DNA, from the process that could interfere with the eventual purification of the hydrophilic oligosaccharide, especially HMO, from the process.
  • removal of proteins, preferably substantially all proteins can be accomplished in a conventional manner by ion exchange chromatography, affinity chromatography, ultrafiltration, and size exclusion chromatography.
  • a protein-free oligosaccharide is a clarified oligosaccharide.
  • anomeric amination preferably means a selective amination of the glycosyl residue of an oligosaccharide, preferably an HMO, by replacing its anomeric OH group with an optionally substituted amine in the presence of its non-protected primary and secondary OH groups.
  • an aqueous medium which comes directly from an enzymatic or preferably a fermentation process, particularly from E. coli or yeast, and which contains a hydrophilic oligosaccharide, especially a human milk oligosaccharide (HMO), is treated according to the following purification method, which comprises the following steps: a) optional clarification of the aqueous medium to remove, from it, suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris; b) removal of proteins, as well as any amino acids, peptides, endotoxins, glycolipids, RNA and DNA from the aqueous medium, preferably from the clarified aqueous medium from step a); c) derivatization of the protein-free oligosaccharide from step b), to produce a derivative of the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the
  • the hydrophilic oligosaccharide can be clarified in a conventional manner, e.g., by centrifugation or filtration of the aqueous medium.
  • the aqueous medium is first flocculated in a conventional manner and then centrifuged or filtered to remove any remaining insoluble particulates and contaminants, as well as cells and cell components and insoluble metabolites and debris from a fermentation process.
  • the aqueous medium which contains the oligosaccharide, preferably an HMO, is separated in a conventional manner by centrifugation, filtration, or tangential flow microfiltration, preferably is firstly flocculated in a conventional manner and then centrifuged or filtered, from the insoluble particulates and contaminants, and any cells and cell components as well as any insoluble metabolites and debris
  • step b) water-soluble proteins and other related impurities, such as peptides, amino acids, RNA, DNA, endotoxins and glycolipids, can be removed in a conventional manner from the preferably clarified, hydrophilic oligosaccharide from step a).
  • Proteins and related impurities can be removed from the oligosaccharide by subjecting the aqueous medium to ultrafiltration, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration (size exclusion chromatography or "SEC”), preferably by chromatography, more preferably by ion exchange chromatography or hydrophobic interaction chromatography.
  • SEC size exclusion chromatography
  • proteins and related impurities are retained by the chromatography medium or selected membrane, while the oligosaccharide remains dissolved in the clarified aqueous medium.
  • ion exchange chromatography may not be preferred for separating proteins and related impurities, since such an oligosaccharide can be retained by the ion exchange medium.
  • use of SEC leads to retention of small molecules, such as an oligosaccharide, as well as peptides and amino acids, while larger sized molecules, such as proteins, nucleic acids and endotoxins, can be eluted first. Recovery of the small molecules carried out subsequently in a conventional manner.
  • the solvent of the protein-free aqueous medium is preferably separated from the protein-free hydrophilic oligosaccharide, preferably protein-free HMO.
  • This can be done in a conventional manner by, for example, evaporation, freeze-drying, or azeotropic distillation to provide a crude, dried oligosaccharide, which contains trace amounts of impurities and
  • step c) the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, is subjected to derivatization which leaves remaining impurities and contaminants substantially unaffected.
  • derivatization can be: i) an anomeric amination to form a glycosyl amine derivative, or ii) an acylation to form an acylated derivative or iii) a ketalation to form a ketal, preferably a cyclic ketal, derivative, or iv) an acetalation to form an acetal, preferably a cyclic acetal, derivative.
  • any hydrophilic, oligosaccharide preferably an HMO
  • step c) any hydrophilic, oligosaccharide, preferably an HMO
  • step c) can be subjected in step c) to an anomeric amination to yield an oligosaccharide derivative, preferably an HMO derivative, having its anomeric hydroxyl selectively replaced by an amino-group.
  • Such an anomeric amination can be carried out in a conventional manner by treating the oligosaccharide with an amino reagent -NHR 1 R 2 , wherein Ri and R 2 are H, alkyl, homoaryl, heteroaryl or benzyl, or Ri and R 2 together form a divalent -(CH 2 ) n - moiety wherein n is 4-8 and a methylene group can be replaced by an oxygen, a sulphur or a -N-Q moiety wherein Q is H or alkyl.
  • the amino reagent is a primary or a secondary amine.
  • Ri is H and R 2 is an optionally substituted alkyl, homoaryl, heteroaryl or benzyl, more preferably a methyl, ethyl, propyl, butyl, phenyl or benzyl.
  • Such an amination reaction can be carried out in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide in a polar solvent, such as water, a lower alkanol (e.g., methanol, ethanol, n-propanol, isopropanol, and n-butanol), nitromethane, or a mixture thereof, with the amino reagent, -NHRiR 2 .
  • a polar solvent such as water, a lower alkanol (e.g., methanol, ethanol, n-propanol, isopropanol, and n-butanol), nitromethane, or a mixture thereof.
  • a polar solvent such as water, a lower alkanol (e.g., methanol, ethanol, n-propanol, isopropanol, and n-butanol), nitromethane, or a mixture thereof.
  • the use of water as the solvent is particularly preferred, since there is no need for solvent evaporation or freeze-drying of the crude, dried hydrophilic oligosaccharide from step b), and thus, the amination can be performed directly in the aqueous medium after step b).
  • the reaction is preferably performed at 10-110 °C, preferably 23-100 °C, most preferably at room temperature or at the boiling point of the solvent.
  • the reaction can be catalysed by the addition of an acid or an acidic salt.
  • Preferred acid and acidic salt catalysts include sulfuric acid, hydrochloric acid, hydrobromic acid, acetic acid, ammonium salts, aryl ammonium salts, benzyl ammonium salts.
  • An acid or acidic salt catalyst is preferred when the amino reagent is substituted with an electron withdrawing moiety, for example a substituted aniline, preferably /?-nitroaniline.
  • their separation from the glycosyl amine oligosaccharide derivative can be readily carried out at the end of the amination reaction by cooling the reaction mixture or by prolonged stirring at ambient temperature or by adding a non-polar solvent, such as n-hexane, cyclohexane, pentane, diethyl ether, methyl ieri-butyl ether or petroleum ether, to the reaction mixture.
  • a non-polar solvent such as n-hexane, cyclohexane, pentane, diethyl ether, methyl ieri-butyl ether or petroleum ether
  • any hydrophilic oligosaccharide preferably an HMO
  • a suitable acylating agent preferably an acylating agent in the presence or absence, preferably in the presence, of a base or a basic catalyst or a Lewis acid catalyst.
  • the acylating agent is an acyl acid anhydride or an acyl halogenide, preferably acetic acid anhydride, acetic acid halogenide, levulinic acid anhydride, pivaloyl halogenide, benzoyl halogenide, or an optionally substituted benzoyl halogenide, more preferably acetic acid anhydride, benzoyl chloride, an optionally substituted benzoyl chloride or pivaloyl chloride.
  • Preferred bases and basic catalysts are pyridine,
  • the reaction can be performed in a conventional manner by initially dissolving the crude, dried, protein-free hydrophilic oligosaccharide in the base, preferably pyridine, or in a mixture of the base with a co- solvent, such as a halogenated hydrocarbon, preferably dichloromethane, at -5-25 °C, preferably 0- 2 °C, before adding the acylating agent, preferably acetic acid anhydride, to the mixture. After the addition of the acylating agent, the reaction mixture is stirred at 15-30 °C, preferably at room temperature.
  • a co- solvent such as a halogenated hydrocarbon, preferably dichloromethane
  • acylation preferably acetylation
  • reaction is catalysed by a solid such as sodium acetate or a Lewis acid
  • a suspension of the solid in the acylation agent preferably acetic acid anhydride, is recommended prior to the addition of the oligosaccharide.
  • the primary hydroxyl group of the oligosaccharide can be selectively acylated, particularly when a bulkier acylating agent, such as pivaloyl chloride, benzoyl chloride or optionally substituted benzoyl chloride, is used.
  • a bulkier acylating agent such as pivaloyl chloride, benzoyl chloride or optionally substituted benzoyl chloride.
  • oligosaccharide has at least 50 % (up to 100 %), preferably 60 %-95 %, even more preferably 70 %- 85 %, of its OH groups acylated.
  • an acylated oligosaccharide can be soluble in common water- immiscible organic solvents, such as a halogenated hydrocarbon (such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene or toluene, preferably dichloromethane, ethyl acetate or toluene - even when only one or a few primary hydroxyl groups on the oligosaccharide are acylated.
  • a halogenated hydrocarbon such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride
  • ethyl acetate such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride
  • ethyl acetate
  • the contaminants/impurities which are present in the crude, dried, protein-free hydrophilic oligosaccharide, remain practically non-acylated. Their physical properties, especially their solubility in polar solvents, particularly water, remain practically unchanged. As a result, their separation from the acylated oligosaccharide derivative can be readily carried out in a conventional manner with a water-immiscible organic solvent and water.
  • the acylated oligosaccharide derivative can be dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase.
  • any hydrophilic oligosaccharide preferably an HMO
  • a ketalation reaction in the presence of a ketalation agent to yield an oligosaccharide derivative, preferably an HMO derivative, which bears at least one ketal group.
  • a ketalation reaction can be performed in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, in an acid-catalysed reaction, with the ketalation agent under water exclusion conditions and in a suitable organic solvent.
  • the ketalation agent can be any suitable ketone or dialkylacetal of a ketone or any other reagent that can form at least one cyclic or acyclic ketal with the oligosaccharide.
  • the ketalation agent is selected from the group of acetone, cyclohexanone, cyclopentanone, 2,2-dimethoxypropane, 2- methyloxypropene, 1,1,2,2-tetramethoxycyclohexane, 3,3'4,4'-tetrahydro-6,6'-bis-2H-pyran and benzophenon.
  • the ketalation reaction produces a cyclic ketal such as an isopropylidene, cyclohexane-l,2-diacetal (DC A) or dispiroketal (DISPOKE) derivative.
  • the acid catalysts for the ketalation reaction can be /?-toluenesulfonic acid, (+) -camphor- 10- sulfonic acid, pyridinium p- toluenesulfonate, HC1, HBr, H 2 SO 4 , or a Lewis acid.
  • the ketalation reaction can be used where the hydrophilic oligosaccharide has at least two vicinal hydroxyl groups positioned so as to allow the formation of a ketal group (which the impurities/contaminants cannot form).
  • 1,2-cis vicinal hydroxyl groups on the oligosaccharide can form stable 1,3-dioxolane-type ketals such as isopropylidenes or cyclohexylidenes.
  • many HMOs contain at least one galactose carbohydrate unit that has free hydroxyl groups at positions 3 and 4, positioned in 1,2-cis- vicinal arrangement.
  • HMOs like the following, are particularly suitable for ketalation by means of, e.g. isopropylidenation or cyclohexylidenation, of its galactose moiety: 2'-FL, 3-FL, LDFT, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, LST b, LNH, LNnH and 6'- galactosyllactose.
  • HMOs that contain, besides a suitable galactose, a sialic acid and/or fucose unit and/or another galactose unit which is sialylated and/or fucosylated are also suitable for ketalation, particularly isopropylidenation or cyclohexylidenation, since the 3-OH and 4-OH groups of the fucose and the 8-OH and 9-OH groups of the sialic acid are positioned favourably for ketalation arrangement.
  • Such other HMOs include the following 3'-SL, FSL, LST a, F-LST a, DSLNT, FDSLNT I and FDSLNT II.
  • the one or more ketal groups on the oligosaccharide derivative alter its physical properties, especially its hydrophilic behaviour, particularly its solvent solubility, more particularly its solubility in aqueous solvents.
  • the oligosaccharide after ketalation is soluble in common water-immiscible organic solvents, such as a halogenated hydrocarbon (such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene, or toluene, preferably dichloromethane, ethyl acetate or toluene.
  • a halogenated hydrocarbon such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride
  • ethyl acetate such as dichloromethane, chloroform, trichloroethylene, 1,2-d
  • the contaminants/impurities which are present in the crude, dried, protein-free hydrophilic oligosaccharide, remain practically unreacted with the ketalation agent and therefore have unaltered physical properties, especially their solubility in polar solvents, particularly in water. Therefore, their separation from the ketalated oligosaccharide derivative can be readily carried out in a conventional manner, for example, with a water-immiscible organic solvent and water.
  • the ketalated oligosaccharide derivative can be dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase. After separation of the phases, the ketalated oligosaccharide derivative can be isolated in substantially pure form from the organic solvent phase.
  • any hydrophilic, oligosaccharide, preferably an HMO can further be subjected in step c) to an acetalation reaction, in the presence of a suitable acetalation agent to yield an oligosaccharide derivative, preferably an HMO derivative, which bears at least one acetal group.
  • a suitable acetalation agent preferably an acetalation agent to yield an oligosaccharide derivative, preferably an HMO derivative, which bears at least one acetal group.
  • Such an acetalation reaction can be carried out in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, in an acid-catalysed reaction with a suitable acetalation agent.
  • the acetalation agent can be any suitable aldehyde or dialkylacetal of an aldehyde or any other reagent that can form at least one cyclic or acyclic acetal with the oligosaccharide under water exclusion conditions and in a suitable organic solvent.
  • Preferred acetalation agents include benzaldehyde, a substituted benzaldehyde, a benzaldehyde - dialkylacetals, a substituted dialkylacetal, acrolein diacetate, acetaldehyde and, an acetaldehyde dialkylacetal, more preferably benzaldehyde, a substituted benzaldehyde, benzaldehyde
  • the resulting oligosaccharide derivative is a cyclic acetal such as a benzylidene or substituted benzylidene with 1,2-czs-oriented and 1,3-vicinal hydroxyl groups.
  • the acid catalyst for the acetalation reaction can be p- toluenesulfonic acid, (+) -camphor- 10- sulfonic acid, pyridinium /?-toluenesulfonate, HC1, HBr, H 2 SO 4 or a Lewis acid.
  • the acetalation reaction can be used where the hydrophilic oligosaccharide has at least two vicinal hydroxyl groups positioned so as to allow the formation of an acetal group (which the impurities/contaminants cannot form).
  • 1,3-vicinal hydroxyl groups on the oligosaccharide can form more stable 1,3-dioxane-type acetals, preferably benzylidene acetals, than the 1,2-dioxolane rings formed with 1,2-czs-vicinally arranged hydroxyl groups.
  • HMOs contain at least one galactose, or N-acetylglucosamine or another carbohydrate moiety, having free hydroxyl groups at positions 4 and 6 which are positioned in 1,3-vicinal arrangement and thus can form stable 1,3-dioxane-type acetals, preferably benzylidenes.
  • HMOs such as the following, which contain at least one galactose, or N-acetylglucos amine (GlcNAc) or another carbohydrate moiety - which is not bearing any other carbohydrate - are prone to be acetalated at their 4-OH and 6-OH positions: 2'-FL, 3-FL, LDFT, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, LST b, LNH and LNnH.
  • GlcNAc N-acetylglucos amine
  • HMOs that contain at least one galactose unit, sialic acid unit or sialylated galactose unit, besides a terminal unit thereof can be acetalated, since the 7-OH and 9-OH groups of the sialic acid are positioned favourably for acetalation arrangement.
  • Such HMOs which are prone to acetalation of their galactose, and/or N-acetylglucosamine, and/or sialic acid units include: 3'-SL, 6'-SL, FSL, LST a, F-LST a, DSLNT, FDSLNT I, FDSLNT II, DSLNnH and FDSLNnH.
  • the one or more acetal groups on the oligosaccharide derivative alter its physical properties, especially its hydrophilic behaviour, particularly its solvent solubility, more particularly its solubility in aqueous solvents.
  • the oligosaccharide derivative after acetalation is soluble in common water-immiscible organic solvents such as a halogenated hydrocarbon (such as
  • dichloromethane chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene or toluene, preferably dichloromethane, ethyl acetate or toluene .
  • oligosaccharide remain substantially unreacted with the acetalation agent and therefore have unaltered physical properties, especially their solubility in polar solvents, particularly in water. Therefore, their separation from the acetalated oligosaccharide derivative can be readily carried out in a conventional manner, for example, with a water- immiscible organic solvent and water.
  • the acetalated oligosaccharide derivative can be dissolved in dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase.
  • the acetalated oligosaccharide derivative can be isolated in substantially pure form from the organic solvent phase.
  • the oligosaccharide derivative preferably HMO derivative
  • the derivatizing group in order to recover the original hydrophilic oligosaccharide, preferably HMO.
  • the conditions for its removal/cleavage can vary. Acidic hydrolysis is the preferred method for the removal of the amino-group of the glycosyl amines, as well as for the cleavage of the acetal and ketal groups, while basic treatment of acylated derivatives is chosen for acyl group removal.
  • an oligosaccharide bearing an amino group or an acetal or ketal group can be treated in a conventional manner in an aqueous solvent with an acid, such as hydrochloric acid, sulfuric acid or an acidic resin to regenerate the original oligosaccharide.
  • an acid such as hydrochloric acid, sulfuric acid or an acidic resin
  • Precautions must be taken, however, during any acidic treatment, since acid- sensitive glycosidic bonds risk being cleaved.
  • Such acidic hydrolysis can be performed at -5 °C to 100 °C, preferably 20 °C to 40 °C.
  • An acylated oligosaccharide can be treated in a conventional manner with a base in a solvent to regenerate the original oligosaccharide.
  • Bases that can be used include NaOMe, K 2 CO 3 and a basic resin. Particularly when Zemplen conditions are used, anhydrous MeOH is the preferred solvent.
  • Such basic hydrolysis can be performed at -5 °C to 100 °C, preferably 20 °C to 40 °C.
  • a method for purifying a hydrophilic oligosaccharide, especially an HMO, produced by a fermentation or enzymatic process, from the contaminants and impurities which are present in the aqueous process broth and are difficult to be removed by conventional methods.
  • the method is based on the formation of an oligosaccharide derivative, such as an aminated, acylated, acetalated or ketalated oligosaccharide, preferably an aminated, acylated, acetalated or ketalated HMO, which, due to its derivatization, shows altered physical properties, particularly different solubility in organic solvents, than the contaminants and impurities which are present in the broth, thereby facilitating its purification.
  • an oligosaccharide derivative such as an aminated, acylated, acetalated or ketalated oligosaccharide, preferably an aminated, acylated, acetalated or ketalated HMO, which, due to its

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Abstract

A method of purifying a hydrophilic oligosaccharide, preferably a human milk oligosaccharide, in an aqueous medium from a fermentation or enzymatic process. The method involves: a. clarifying the aqueous medium to remove particulates and contaminants and preferably also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b. removing substantially all proteins from the clarified aqueous medium, to provide a protein-free oligosaccharide; c. reacting the protein-free oligosaccharide with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d. removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c; and then e. reacting the derivative of the oligosaccharide to remove the derivatizing group.

Description

PURIFICATION OF OLIGOSACCHARIDE DERIVATIVES
FIELD OF THE INVENTION
This invention relates to a method of the purification of an hydrophilic oligosaccharide, especially an human milk oligosaccharide (HMO), produced by a fermentation or enzymatic process. The invention particularly relates to forming a derivative of the oligosaccharide with different physical properties, particularly different solubility, from contaminants and impurities which are produced by the fermentation or enzymatic process.
BACKGROUND OF THE INVENTION
In recent years, the manufacture and commercialization of complex carbohydrates including naturally secreted oligosaccharides have increased significantly due to their roles in numerous biological processes occurring in living organisms. Secreted oligosaccharides such as human milk oligosaccharides (HMOs) are carbohydrates which have gained much interest in recent years and are becoming important commercial targets for nutrition and therapeutic industries. In particular the synthesis of these HMOs has increased significantly due to the role of HMOs in numerous biological processes occurring in humans. The great importance of HMOs is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities. Human milk oligosaccharides are found to act as prebiotics in the human intestinal system helping to develop and maintain the intestinal flora. Furthermore they have also proved to be anti-inflammatory, and therefore these compounds are attractive components in the nutritional industry for the production of, for example, infant formulas, infant cereals, clinical infant nutritional products, toddler formulas, or as dietary supplements or health functional food for children, adults, elderly or lactating women, both as synthetically composed and naturally occurring compounds and salts thereof. Likewise, the compounds are also of interest in the medicinal industry for the production of therapeutics due to their prognostic use as immunomodulators. However, the syntheses and purification of these oligosaccharides and their intermediates remained a challenging task for science.
To date, access to large volumes of human milk oligosaccharides has not been possible by using isolation, biotechnology and synthetic methodologies. The availability of naturally occurring sialylated human milk oligosaccharides is limited from natural sources. Mature human milk is the natural milk source that contains the highest concentrations of milk oligosaccharides (12-14 g/1), other milk sources are cow's milk (0.01 g/1), goat's milk and milk from other mammals. Approximately 200 HMOs have been detected from human milk by means of combination of techniques including microchip liquid chromatography mass spectrometry (HPLC Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) (Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which to date at least 115 oligosaccharides have been structurally determined (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011). These human milk oligosaccharides can be grouped into 13 core units (Table 1). Due to the large number of similar HMOs and their low concentrations in mammalian milk, isolation of HMOs is a difficult task even in milligram quantities. To date only analytical HPLC methodologies have been developed for the isolation of some HMOs from natural sources. It is therefore difficult to provide suitable HMO replacements in foods, particularly in infant formulae which display at least part of the entire spectrum of HMOs.
Figure imgf000003_0001
Table 1: 13 different core structures of human milk oligosaccharides Bio technological approaches have proved to be promising and cost-efficient for the synthesis of a variety of HMOs. Precisely, HMOs can be produced in high yields in aqueous media by fermentation of genetically modified bacteria, yeasts or other microorganisms. See, for example, WOOl/04341, WO2007/101862, WO2010/070104, WO2010/142305, WO2012/ 112777, Priem et al Glycobiology 12, 235 (2002), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), Han et al. Biotechnol. Adv. 30, 1268 (2012), Lee et al. Microb. Cell Fact. 11:48 (2012) and Baumgartner et al. Microb. Cell Fact. 12:40 (2013). However, biotechnological methods cannot provide HMOs of high purity due to the contaminants and other impurities (of bacterial or chemical origin such as enzymes, proteins, endotoxins, DNA and carbohydrate regioisomers) which can be very difficult to remove by common methods. In addition, biotechnological methods involve the use of genetically modified microorganisms. As consequence of the above mentioned, the regulatory approval of HMOs produced by these methods, is likely to encounter serious obstacles in regulatory processes. There has, therefore, been a need for identifying solid, robust and cost-efficient methods for the purification of HMOs derived from a fermentation or enzymatic process. In this regard,
WO2012/007585 describes a method for the purification of HMOs by means of an initial anomeric Obenzylation and a subsequent reverse-phase or size exclusion chromatographic separation of the so-obtained Obenzylated HMOs, followed by their catalytic hydrogenolysis to regenerate the original HMOs. However, alternative purification methods have been sought which would be simpler and less expensive to use on an industrial scale.
SUMMARY OF THE INVENTION
In accordance with this invention, a method is provided for purifying a hydrophilic oligosaccharide, advantageously a HMO, in an aqueous medium from a fermentation or enzymatic process, comprising the steps of: a) optionally clarifying the aqueous medium to remove particulates and contaminants and advantageously also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b) removing substantially all proteins from the aqueous medium, advantageously after step a), to provide a protein-free oligosaccharide; c) reacting the protein-free oligosaccharide from step b) with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c); and then e) reacting the derivative of the oligosaccharide to remove the derivatizing group and regenerate the oligosaccharide.
In this method, the reaction in step c) advantageously comprises one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide to form an acyl derivative of the
oligosaccharide; iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide to form a cyclic -ketal derivative of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
Also in this method, each of the derivatizing reactions i)-iv) in step c) is advantageously followed respectively in step e) by one of the following reactions to remove the derivatizing group and regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid.
Also in accordance with this invention, a method is provided for purifying a hydrophilic oligosaccharide, advantageously an HMO, in an aqueous medium from a fermentation or enzymatic process, comprising the steps of: a) reacting the oligosaccharide with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; b) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step a); and then c) reacting the derivative of the oligosaccharide to remove the derivatizing group and regenerate the oligosaccharide; wherein step a) comprises one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide to form an acyl derivative of the
oligosaccharide; iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide to form a cyclic-ketal derivative of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
Also in this method, each of the derivatization reactions i)-iv) in step a) is advantageously followed respectively in step c) by one of the following reactions to remove the derivatizing group and regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid. DETAILED DESCRIPTION
This invention provides a method for purifying a hydrophilic oligosaccharide, preferably an HMO, produced in an aqueous medium or broth by a fermentation or enzymatic process. The method is particularly adapted to remove water soluble contaminants and impurities which are present in the aqueous medium and which are difficult to separate in a conventional manner from the oligosaccharide. The method involves the chemical alteration of the oligosaccharide to produce a derivatized oligosaccharide with altered physical properties without changing significantly such physical properties of the contaminants/impurities. In this regard, the method involves the steps of: a) optionally clarifying the aqueous medium to remove particulates and contaminants and advantageously also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b) removing substantially all proteins from the aqueous medium, advantageously after step a), to provide a protein-free oligosaccharide; c) reacting the protein-free oligosaccharide from step b) with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c); and then e) reacting the derivative of the oligosaccharide to remove the derivatizing group.
Also in this invention, the reaction in step c) preferably involves one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide; iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide. Further in this invention, a hydrophilic oligosaccharide, preferably a HMO, produced in an aqueous medium or broth by a fermentation or enzymatic process, can be purified just by carrying out steps c) to e), using one of the aforementioned derivatization reactions i) to iv). This invention exploits the fact that the contaminants/impurities in the aqueous medium from the fermentation or enzymatic process do not react significantly during the following chemical reactions:
-forming a glycosyl amine derivative of a hydrophilic oligosaccharide, particularly an HMO, by a selective anomeric amination reaction;
- forming an acylated derivative of a hydrophilic oligosaccharide, particularly an HMO;
- forming a derivative of a hydrophilic oligosaccharide, particularly an HMO, bearing an acyclic or cyclic ketal group; or
- forming a derivative of a hydrophilic oligosaccharide, particularly an HMO, bearing an acyclic or cyclic acetal group.
Because the resulting derivatized oligosaccharide, particularly a derivatized HMO, has altered physical properties (particularly solubility in non-aqueous solvents), it can be readily separated from the contaminants and impurities which have substantially unchanged physical properties as a result of the derivatization reaction. Thereafter, the original oligosaccharide, particularly an HMO, can be recovered in pure form by removing the derivatizing group.
In this invention, the term "hydrophilic oligosaccharide" preferably means any neutral or acidic oligosaccharide, especially an HMO, containing two to eight, preferably three to six, simple sugar or monosaccharide moieties as its components, all the hydroxyl groups of which, including its anomeric hydroxyl group, are free.
Also in this invention, terms such as "derivatization", "derivatizing" and "derivatized" preferably refer to the action, and the result, of a chemical manipulation of a hydrophilic oligosaccharide, preferably an HMO, to provide a derivative with a significantly altered chemical structure and a significantly altered physical property.
Also herein, the term "alkyl" preferably means a linear or branched chain saturated hydrocarbon group with 1-10 carbon atoms, especially 1-6 carbon atoms, such as methyl, ethyl, n- propyl, z'-propyl, n-butyl, z'-butyl, s-butyl, i-butyl or n-hexyl; the term "homoaryl" preferably means a homoaromatic group such as phenyl or naphthyl; the term "heteroaryl" preferably means an aromatic group having one or two rings, which ring(s) contain(s) 1, 2, or 3 heteroatoms selected from the group of N, O and S, such as pyrrol, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, furan, thiophene, oxazole, isoxazole, thiazole, thiadiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, benzimidazole, benzoxazole, benzothiazole, indole, quinoline, isoquinoline, purine and pteridine; the term "alkyloxy" or "alkoxy" preferably means an alkyl group attached to a parent molecular moiety through an oxygen atom, such as methoxy, ethoxy or i-butoxy; "amino" preferably means a -NH2 group; "alkylamino" preferably means an alkyl group attached to a parent molecular moiety through an -NH-group, such as methylamino or ethylamino; "dialkylamino" preferably means two alkyl groups, either identical or different, attached to a parent molecular moiety through a nitrogen atom, such as dimethylamino or diethylamino; "acylamino" preferably means an acyl group attached to a parent molecular moiety through an -NH-group, such as acetylamino (acetamido) or benzoylamino (benzamido); "carboxyl" preferably means an -COOH group; "alkyloxycarbonyl" preferably means an alkyloxy group attached to a parent molecular moiety through a -C(=0)-group, such as methoxycarbonyl or i-butoxycarbonyl; "carbamoyl" preferably means an H2N-C(=0)-group; "N-alkylcarbamoyl" preferably means an alkyl group attached to a parent molecular moiety through a -HN-C(=0)-group, such as N-methylcarbamoyl; "N,N-dialkylcarbamoyl" preferably means two alkyl groups, either identical or different, attached to a parent molecular moiety through a -N-C(=0)-group, such as N,N-methylcarbamoyl; the term "optionally substituted" in connection with the alkyl, homoaryl and heteroaryl groups, preferably means that these groups can be substituted by one or more groups selected from: alkyl (only for homo- and heteroaryl residues), halogen, nitro, homoaryl, alkoxy, amino, alkylamino, dialkylamino, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, haloalkyl or hydroxyalkyl.
Also herein, the term "acyl" preferably means an R -C(=0)- group, wherein R' can be H, alkyl or aryl, such as formyl, acetyl, propionyl, butyryl or pivaloyl. The alkyl or aryl residue can either be unsubstituted or substituted with one or more groups selected from alkyl (only for aryl residues), halogen, nitro, aryl, alkoxy, amino, alkylamino, dialkylamino, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, halogenalkyl and hydroxyalkyl, giving rise to acyl groups such as chloroacetyl, trichloroacetyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4- phenylbenzoyl, 4-benzamidobenzoyl, 4-(phenylcarbamoyl)-benzoyl, glycolyl and acetoacetyl.
Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and endotoxins which can be present in an aqueous medium from a fermentation or enzymatic process.
Yet further herein, the term "clarified oligosaccharide" preferably means a hydrophilic oligosaccharide from an aqueous medium or broth from a fermentation or enzymatic process, which (medium) has been treated to remove suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the oligosaccharide, especially HMO. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a Celite™ filter aid).
Still further herein, the term "protein-free oligosaccharide" preferably means a hydrophilic oligosaccharide from an aqueous medium or broth from a fermentation or enzymatic process, which (medium) has been treated to remove substantially all the proteins, as well as any related impurities, such as amino acids, peptides, endotoxins, glycolipids, RNA and DNA, from the process that could interfere with the eventual purification of the hydrophilic oligosaccharide, especially HMO, from the process. Such removal of proteins, preferably substantially all proteins, can be accomplished in a conventional manner by ion exchange chromatography, affinity chromatography, ultrafiltration, and size exclusion chromatography. Preferably, a protein-free oligosaccharide is a clarified oligosaccharide.
Also herein, the term "anomeric amination" preferably means a selective amination of the glycosyl residue of an oligosaccharide, preferably an HMO, by replacing its anomeric OH group with an optionally substituted amine in the presence of its non-protected primary and secondary OH groups.
In carrying out this invention, an aqueous medium, which comes directly from an enzymatic or preferably a fermentation process, particularly from E. coli or yeast, and which contains a hydrophilic oligosaccharide, especially a human milk oligosaccharide (HMO), is treated according to the following purification method, which comprises the following steps: a) optional clarification of the aqueous medium to remove, from it, suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris; b) removal of proteins, as well as any amino acids, peptides, endotoxins, glycolipids, RNA and DNA from the aqueous medium, preferably from the clarified aqueous medium from step a); c) derivatization of the protein-free oligosaccharide from step b), to produce a derivative of the oligosaccharide; d) removing water-soluble contaminants and impurities from the derivative of the
oligosaccharide; and then e) reacting the derivative of the oligosaccharide to remove the derivatizing group and
recover the oligosaccharide.
In step a), the hydrophilic oligosaccharide, can be clarified in a conventional manner, e.g., by centrifugation or filtration of the aqueous medium. Preferably the aqueous medium is first flocculated in a conventional manner and then centrifuged or filtered to remove any remaining insoluble particulates and contaminants, as well as cells and cell components and insoluble metabolites and debris from a fermentation process. To do so, the aqueous medium, which contains the oligosaccharide, preferably an HMO, is separated in a conventional manner by centrifugation, filtration, or tangential flow microfiltration, preferably is firstly flocculated in a conventional manner and then centrifuged or filtered, from the insoluble particulates and contaminants, and any cells and cell components as well as any insoluble metabolites and debris
In step b), water-soluble proteins and other related impurities, such as peptides, amino acids, RNA, DNA, endotoxins and glycolipids, can be removed in a conventional manner from the preferably clarified, hydrophilic oligosaccharide from step a). Proteins and related impurities can be removed from the oligosaccharide by subjecting the aqueous medium to ultrafiltration, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration (size exclusion chromatography or "SEC"), preferably by chromatography, more preferably by ion exchange chromatography or hydrophobic interaction chromatography. With the exception of SEC, proteins and related impurities are retained by the chromatography medium or selected membrane, while the oligosaccharide remains dissolved in the clarified aqueous medium. For an acidic oligosaccharide, ion exchange chromatography may not be preferred for separating proteins and related impurities, since such an oligosaccharide can be retained by the ion exchange medium. On the other hand, use of SEC leads to retention of small molecules, such as an oligosaccharide, as well as peptides and amino acids, while larger sized molecules, such as proteins, nucleic acids and endotoxins, can be eluted first. Recovery of the small molecules carried out subsequently in a conventional manner.
After step b), the solvent of the protein-free aqueous medium is preferably separated from the protein-free hydrophilic oligosaccharide, preferably protein-free HMO. This can be done in a conventional manner by, for example, evaporation, freeze-drying, or azeotropic distillation to provide a crude, dried oligosaccharide, which contains trace amounts of impurities and
contaminants which need to be removed.
Thus in step c), the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, is subjected to derivatization which leaves remaining impurities and contaminants substantially unaffected. Such derivatization can be: i) an anomeric amination to form a glycosyl amine derivative, or ii) an acylation to form an acylated derivative or iii) a ketalation to form a ketal, preferably a cyclic ketal, derivative, or iv) an acetalation to form an acetal, preferably a cyclic acetal, derivative.
In accordance with this invention, any hydrophilic, oligosaccharide, preferably an HMO, can be subjected in step c) to an anomeric amination to yield an oligosaccharide derivative, preferably an HMO derivative, having its anomeric hydroxyl selectively replaced by an amino-group. Such an anomeric amination can be carried out in a conventional manner by treating the oligosaccharide with an amino reagent -NHR1R2, wherein Ri and R2 are H, alkyl, homoaryl, heteroaryl or benzyl, or Ri and R2 together form a divalent -(CH2)n- moiety wherein n is 4-8 and a methylene group can be replaced by an oxygen, a sulphur or a -N-Q moiety wherein Q is H or alkyl. Preferably, the amino reagent is a primary or a secondary amine. In a primary amine, Ri is H and R2 is an optionally substituted alkyl, homoaryl, heteroaryl or benzyl, more preferably a methyl, ethyl, propyl, butyl, phenyl or benzyl. A secondary amine (wherein Ri and R2 are not H) can be a nonsymmetrical (Ri≠ R2) amine, a symmetrical (Ri = R2), a non-cyclic amine such as diethyl amine, dipropyl amine or dibenzyl amine or a symmetrical cyclic amine such as pyrrolidine, piperidine, piperazine, N-methyl piperazine, morpholine or thiomorpholine.
Such an amination reaction can be carried out in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide in a polar solvent, such as water, a lower alkanol (e.g., methanol, ethanol, n-propanol, isopropanol, and n-butanol), nitromethane, or a mixture thereof, with the amino reagent, -NHRiR2. A preferred solvent is water, a lower alkanol or a mixture thereof. The use of water as the solvent is particularly preferred, since there is no need for solvent evaporation or freeze-drying of the crude, dried hydrophilic oligosaccharide from step b), and thus, the amination can be performed directly in the aqueous medium after step b). The reaction is preferably performed at 10-110 °C, preferably 23-100 °C, most preferably at room temperature or at the boiling point of the solvent. Depending on the nucleophilicity of the selected amino reagent, the reaction can be catalysed by the addition of an acid or an acidic salt. Preferred acid and acidic salt catalysts include sulfuric acid, hydrochloric acid, hydrobromic acid, acetic acid, ammonium salts, aryl ammonium salts, benzyl ammonium salts. An acid or acidic salt catalyst is preferred when the amino reagent is substituted with an electron withdrawing moiety, for example a substituted aniline, preferably /?-nitroaniline.
The contaminants/impurities, which are present in the crude, dried, protein-free hydrophilic oligosaccharide, remain practically non-aminated and their physical properties, especially their solubility in the polar solvents remain practically unchanged. As a result, their separation from the glycosyl amine oligosaccharide derivative can be readily carried out at the end of the amination reaction by cooling the reaction mixture or by prolonged stirring at ambient temperature or by adding a non-polar solvent, such as n-hexane, cyclohexane, pentane, diethyl ether, methyl ieri-butyl ether or petroleum ether, to the reaction mixture. The non-reacted/non-aminated contaminants and impurities remain in solution in the reaction mixture, and therefore the derivatized oligosaccharide can be isolated in substantially pure form in a conventional manner, preferably by filtration.
In accordance with this invention, any hydrophilic oligosaccharide, preferably an HMO, can also be subjected in step c) to an acylation reaction in the presence of a suitable acylating agent to yield an oligosaccharide derivative, preferably a HMO derivative, having the majority of its hydroxyl groups protected by an acyl protective group. Such an acylation reaction can be carried out in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, with an acylating agent in the presence or absence, preferably in the presence, of a base or a basic catalyst or a Lewis acid catalyst. Preferably, the acylating agent is an acyl acid anhydride or an acyl halogenide, preferably acetic acid anhydride, acetic acid halogenide, levulinic acid anhydride, pivaloyl halogenide, benzoyl halogenide, or an optionally substituted benzoyl halogenide, more preferably acetic acid anhydride, benzoyl chloride, an optionally substituted benzoyl chloride or pivaloyl chloride. Preferred bases and basic catalysts are pyridine,
diisopropylethylamine, triethylamine, imidazole, lutidine, collidine or sodium acetate. The reaction can be performed in a conventional manner by initially dissolving the crude, dried, protein-free hydrophilic oligosaccharide in the base, preferably pyridine, or in a mixture of the base with a co- solvent, such as a halogenated hydrocarbon, preferably dichloromethane, at -5-25 °C, preferably 0- 2 °C, before adding the acylating agent, preferably acetic acid anhydride, to the mixture. After the addition of the acylating agent, the reaction mixture is stirred at 15-30 °C, preferably at room temperature. Furthermore, when the acylation, preferably acetylation, reaction is catalysed by a solid such as sodium acetate or a Lewis acid, a suspension of the solid in the acylation agent, preferably acetic acid anhydride, is recommended prior to the addition of the oligosaccharide.
Under certain conditions, e.g. high dilution and low temperature (below 0 °C), the primary hydroxyl group of the oligosaccharide can be selectively acylated, particularly when a bulkier acylating agent, such as pivaloyl chloride, benzoyl chloride or optionally substituted benzoyl chloride, is used. With a larger oligosaccharide, such as a higher and more complex HMO, complete acylation of all its hydroxyl groups can often not be possible; as a result, a mixture of partially acylated oligosaccharides is likely to be obtained. In accordance with this invention, an acylated
oligosaccharide has at least 50 % (up to 100 %), preferably 60 %-95 %, even more preferably 70 %- 85 %, of its OH groups acylated. The acyl groups on the derivatized oligosaccharide, preferably derivatized HMO, alter the physical properties and especially the solvent solubility, particularly the solubility in aqueous solvents, of the oligosaccharide, as compared to the non-acylated
oligosaccharide. In this regard, an acylated oligosaccharide can be soluble in common water- immiscible organic solvents, such as a halogenated hydrocarbon (such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene or toluene, preferably dichloromethane, ethyl acetate or toluene - even when only one or a few primary hydroxyl groups on the oligosaccharide are acylated.
The contaminants/impurities, which are present in the crude, dried, protein-free hydrophilic oligosaccharide, remain practically non-acylated. Their physical properties, especially their solubility in polar solvents, particularly water, remain practically unchanged. As a result, their separation from the acylated oligosaccharide derivative can be readily carried out in a conventional manner with a water-immiscible organic solvent and water. Thus, the acylated oligosaccharide derivative can be dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase. After separation of the phases, the acylated oligosaccharide derivative can be isolated in substantially pure form from the organic solvent phase. In accordance with this invention, any hydrophilic oligosaccharide, preferably an HMO, can also be subjected in step c) to a ketalation reaction, in the presence of a ketalation agent to yield an oligosaccharide derivative, preferably an HMO derivative, which bears at least one ketal group. Such a ketalation reaction can be performed in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, in an acid-catalysed reaction, with the ketalation agent under water exclusion conditions and in a suitable organic solvent. The ketalation agent can be any suitable ketone or dialkylacetal of a ketone or any other reagent that can form at least one cyclic or acyclic ketal with the oligosaccharide. Preferably, the ketalation agent is selected from the group of acetone, cyclohexanone, cyclopentanone, 2,2-dimethoxypropane, 2- methyloxypropene, 1,1,2,2-tetramethoxycyclohexane, 3,3'4,4'-tetrahydro-6,6'-bis-2H-pyran and benzophenon. The ketalation reaction produces a cyclic ketal such as an isopropylidene, cyclohexane-l,2-diacetal (DC A) or dispiroketal (DISPOKE) derivative. The acid catalysts for the ketalation reaction can be /?-toluenesulfonic acid, (+) -camphor- 10- sulfonic acid, pyridinium p- toluenesulfonate, HC1, HBr, H2SO4, or a Lewis acid. The ketalation reaction can be used where the hydrophilic oligosaccharide has at least two vicinal hydroxyl groups positioned so as to allow the formation of a ketal group (which the impurities/contaminants cannot form). In this regard, 1,2-cis vicinal hydroxyl groups on the oligosaccharide can form stable 1,3-dioxolane-type ketals such as isopropylidenes or cyclohexylidenes. In particular, many HMOs contain at least one galactose carbohydrate unit that has free hydroxyl groups at positions 3 and 4, positioned in 1,2-cis- vicinal arrangement. Hence, HMOs, like the following, are particularly suitable for ketalation by means of, e.g. isopropylidenation or cyclohexylidenation, of its galactose moiety: 2'-FL, 3-FL, LDFT, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, LST b, LNH, LNnH and 6'- galactosyllactose. Moreover, other HMOs that contain, besides a suitable galactose, a sialic acid and/or fucose unit and/or another galactose unit which is sialylated and/or fucosylated are also suitable for ketalation, particularly isopropylidenation or cyclohexylidenation, since the 3-OH and 4-OH groups of the fucose and the 8-OH and 9-OH groups of the sialic acid are positioned favourably for ketalation arrangement. Such other HMOs include the following 3'-SL, FSL, LST a, F-LST a, DSLNT, FDSLNT I and FDSLNT II.
The one or more ketal groups on the oligosaccharide derivative alter its physical properties, especially its hydrophilic behaviour, particularly its solvent solubility, more particularly its solubility in aqueous solvents. Hence, the oligosaccharide after ketalation is soluble in common water-immiscible organic solvents, such as a halogenated hydrocarbon (such as dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene, or toluene, preferably dichloromethane, ethyl acetate or toluene. The contaminants/impurities, which are present in the crude, dried, protein-free hydrophilic oligosaccharide, remain practically unreacted with the ketalation agent and therefore have unaltered physical properties, especially their solubility in polar solvents, particularly in water. Therefore, their separation from the ketalated oligosaccharide derivative can be readily carried out in a conventional manner, for example, with a water-immiscible organic solvent and water. Thus, the ketalated oligosaccharide derivative can be dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase. After separation of the phases, the ketalated oligosaccharide derivative can be isolated in substantially pure form from the organic solvent phase.
In accordance with this invention, any hydrophilic, oligosaccharide, preferably an HMO, can further be subjected in step c) to an acetalation reaction, in the presence of a suitable acetalation agent to yield an oligosaccharide derivative, preferably an HMO derivative, which bears at least one acetal group. Such an acetalation reaction can be carried out in a conventional manner by treating the crude, dried, protein-free hydrophilic oligosaccharide, preferably HMO, in an acid-catalysed reaction with a suitable acetalation agent. The acetalation agent can be any suitable aldehyde or dialkylacetal of an aldehyde or any other reagent that can form at least one cyclic or acyclic acetal with the oligosaccharide under water exclusion conditions and in a suitable organic solvent.
Preferred acetalation agents include benzaldehyde, a substituted benzaldehyde, a benzaldehyde - dialkylacetals, a substituted dialkylacetal, acrolein diacetate, acetaldehyde and, an acetaldehyde dialkylacetal, more preferably benzaldehyde, a substituted benzaldehyde, benzaldehyde
dimethylacetal and a substituted benzaldehyde-dimethylacetal. The resulting oligosaccharide derivative is a cyclic acetal such as a benzylidene or substituted benzylidene with 1,2-czs-oriented and 1,3-vicinal hydroxyl groups. The acid catalyst for the acetalation reaction can be p- toluenesulfonic acid, (+) -camphor- 10- sulfonic acid, pyridinium /?-toluenesulfonate, HC1, HBr, H2SO4 or a Lewis acid. The acetalation reaction can be used where the hydrophilic oligosaccharide has at least two vicinal hydroxyl groups positioned so as to allow the formation of an acetal group (which the impurities/contaminants cannot form). In this regard, 1,3-vicinal hydroxyl groups on the oligosaccharide can form more stable 1,3-dioxane-type acetals, preferably benzylidene acetals, than the 1,2-dioxolane rings formed with 1,2-czs-vicinally arranged hydroxyl groups. In particular, many HMOs contain at least one galactose, or N-acetylglucosamine or another carbohydrate moiety, having free hydroxyl groups at positions 4 and 6 which are positioned in 1,3-vicinal arrangement and thus can form stable 1,3-dioxane-type acetals, preferably benzylidenes. In other words, HMOs, such as the following, which contain at least one galactose, or N-acetylglucos amine (GlcNAc) or another carbohydrate moiety - which is not bearing any other carbohydrate - are prone to be acetalated at their 4-OH and 6-OH positions: 2'-FL, 3-FL, LDFT, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, LST b, LNH and LNnH. Moreover, other HMOs that contain at least one galactose unit, sialic acid unit or sialylated galactose unit, besides a terminal unit thereof can be acetalated, since the 7-OH and 9-OH groups of the sialic acid are positioned favourably for acetalation arrangement. Such HMOs which are prone to acetalation of their galactose, and/or N-acetylglucosamine, and/or sialic acid units include: 3'-SL, 6'-SL, FSL, LST a, F-LST a, DSLNT, FDSLNT I, FDSLNT II, DSLNnH and FDSLNnH.
The one or more acetal groups on the oligosaccharide derivative alter its physical properties, especially its hydrophilic behaviour, particularly its solvent solubility, more particularly its solubility in aqueous solvents. Hence, the oligosaccharide derivative after acetalation is soluble in common water-immiscible organic solvents such as a halogenated hydrocarbon (such as
dichloromethane, chloroform, trichloroethylene, 1,2-dichloroethane or carbon tetrachloride), ethyl acetate, benzene or toluene, preferably dichloromethane, ethyl acetate or toluene . The
contaminants/impurities, which are present in the crude, dried, protein-free hydrophilic
oligosaccharide, remain substantially unreacted with the acetalation agent and therefore have unaltered physical properties, especially their solubility in polar solvents, particularly in water. Therefore, their separation from the acetalated oligosaccharide derivative can be readily carried out in a conventional manner, for example, with a water- immiscible organic solvent and water. Thus, the acetalated oligosaccharide derivative can be dissolved in dissolved in the organic solvent phase, such as dichloromethane or ethyl acetate, while the contaminants/impurities can be dissolved in the water phase. After separation of the phases, the acetalated oligosaccharide derivative can be isolated in substantially pure form from the organic solvent phase.
In step d), the oligosaccharide derivative, preferably HMO derivative, is subjected to cleavage of the derivatizing group in order to recover the original hydrophilic oligosaccharide, preferably HMO. Depending on the derivatizing group type, the conditions for its removal/cleavage can vary. Acidic hydrolysis is the preferred method for the removal of the amino-group of the glycosyl amines, as well as for the cleavage of the acetal and ketal groups, while basic treatment of acylated derivatives is chosen for acyl group removal. In particular, an oligosaccharide bearing an amino group or an acetal or ketal group can be treated in a conventional manner in an aqueous solvent with an acid, such as hydrochloric acid, sulfuric acid or an acidic resin to regenerate the original oligosaccharide. Precautions must be taken, however, during any acidic treatment, since acid- sensitive glycosidic bonds risk being cleaved. Such acidic hydrolysis can be performed at -5 °C to 100 °C, preferably 20 °C to 40 °C.
An acylated oligosaccharide, can be treated in a conventional manner with a base in a solvent to regenerate the original oligosaccharide. Bases that can be used include NaOMe, K2CO3 and a basic resin. Particularly when Zemplen conditions are used, anhydrous MeOH is the preferred solvent. Such basic hydrolysis can be performed at -5 °C to 100 °C, preferably 20 °C to 40 °C.
In accordance with this invention, a method is provided for purifying a hydrophilic oligosaccharide, especially an HMO, produced by a fermentation or enzymatic process, from the contaminants and impurities which are present in the aqueous process broth and are difficult to be removed by conventional methods. The method is based on the formation of an oligosaccharide derivative, such as an aminated, acylated, acetalated or ketalated oligosaccharide, preferably an aminated, acylated, acetalated or ketalated HMO, which, due to its derivatization, shows altered physical properties, particularly different solubility in organic solvents, than the contaminants and impurities which are present in the broth, thereby facilitating its purification.

Claims

1. A method of purifying a hydrophilic oligosaccharide, preferably an HMO, in an aqueous medium from a fermentation or enzymatic process, comprising the steps of: a. optionally clarifying the aqueous medium to remove particulates and contaminants and preferably also cell components and any insoluble metabolites and debris from the fermentation or enzymatic process and thereby provide a clarified oligosaccharide; b. removing substantially all proteins from the aqueous medium, preferably after step a), to provide a protein-free oligosaccharide; c. reacting the protein-free oligosaccharide from step b) with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; d. removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step c); and then e. reacting the derivative of the oligosaccharide to remove the derivatizing group and regenerate the oligosaccharide.
2. The method of claim 1, wherein the derivatizing reaction in step c) comprises one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, preferably an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide to form an acyl derivative of the
oligosaccharide; iii) a cyclic-ketalation, preferably an isopropylidenation, of one or more 1,2-cis-vicinal hydroxyl groups of the oligosaccharide to form a cyclic -ketal derivative of the
oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
3. The method of claim 2, wherein in reaction ii) 50- 100 %, preferably 60-95 %, more preferably 70-85 %, of the hydroxyl groups of the oligosaccharide, especially the HMO, are acylated.
4. The method of claim 2, wherein in reaction iii) at least a galactose of the oligosaccharide, preferably the HMO, is ketalated.
5. The method of claim 4, wherein the ketalated galactose of the oligosaccharide, preferably the HMO, does not bear any other carbohydrate moiety.
6. The method of claim 4 or 5, wherein the oligosaccharide, preferably the HMO, bears a sialic acid and/or fucose moiety which is optionally ketalated, preferably isopropylidenated.
7. The method of claim 6, wherein any galactose of the oligosaccharide, preferably the HMO, bears a sialic acid and/or fucose moiety which is optionally ketalated, preferably isopropylidenated.
8. The method of claim 2, wherein in reaction iv), at least a galactose or a N-acetylglucos amine of the oligosaccharide, preferably the HMO, is acetalated, preferably benzylidenated.
9. The method of claim 8, wherein the acetalated galactose or N-acetylglucos amine of the oligosaccharide, preferably the HMO, does not bear any other carbohydrate moiety.
10. The method of claim 8 or 9, wherein the oligosaccharide, preferably the HMO, bears a sialic acid which is optionally acetalated, preferably benzylidenated.
11. The method of claim 10, wherein any galactose of the oligosaccharide, preferably the HMO, bears a sialic acid moiety which is optionally acetalated, preferably benzylidenated.
12. The method of any one of claims 1 to 11, wherein the derivatization reaction in step c) provides the derivative of the oligosaccharide, preferably the HMO, with significantly different physical properties, particularly significantly different solubility, quite particularly significantly different solubility in the protein-free aqueous medium, from contaminants and impurities which are produced by the fermentation or enzymatic process.
13. The method of claim 12, wherein the derivative of the oligosaccharide, preferably the HMO, precipitates from the reaction mixture of step c) i) or iv).
14. The method of claim 13, wherein in step d), the derivative of the oligosaccharide, preferably the HMO, can be dissolved in an organic solvent while contaminants and impurities which are produced by the fermentation or enzymatic process remain dissolved in the protein-free aqueous medium.
15. The method of any one of claims 1 to 14, wherein each of the derivatization reactions i)-iv) in step c) are advantageously followed respectively in step e) by one of the following reactions to remove the derivatizing group and regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid.
16. A method for purifying a hydrophilic oligosaccharide, preferably an HMO, in an aqueous medium from a fermentation or enzymatic process, comprising the steps of: a) reacting the oligosaccharide with a derivatizing group to form a derivative of the oligosaccharide having different physical properties from the oligosaccharide; b) removing water-soluble contaminants and impurities from the derivative of the oligosaccharide from step a); and then c) reacting the derivative of the oligosaccharide to remove the derivatizing group; wherein step a) is one of the following derivatization reactions: i) an anomeric amination of the oligosaccharide to form a glycosyl amine derivative of the oligosaccharide; ii) an acylation, particularly an acetylation, benzoylation or pivaloylation, of at least a primary hydroxyl group of the oligosaccharide to form an acyl derivative of the
oligosaccharide; iii) a cyclic-ketalation, advantageously an isopropylidenation, of one or more 1,2-cis- vicinal hydroxyl groups of the oligosaccharide to form a cyclic-ketal derivative of the oligosaccharide; or iv) a cyclic-acetalation, preferably a benzylidenation, of one or more 1,3-vicinal hydroxyl groups of the oligosaccharide to form a cyclic-acetal derivative of the oligosaccharide.
17. The method of claim 16, wherein each of the derivatization reactions i)-iv) in step a) is advantageously followed respectively in step c) by one of the following reactions to regenerate the oligosaccharide: i) the glycosyl amine derivative of the oligosaccharide is treated in an aqueous solvent with an acid; ii) the acyl derivative of the oligosaccharide is treated with a base in a solvent; iii) the cyclic-ketal derivative of the oligosaccharide is treated in an aqueous solvent with an acid; or iv) the cyclic-acetal derivative of the oligosaccharide is treated in an aqueous solvent with an acid.
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