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    • 专利摘要:
    The present application describes methods for spray drying human milk oligosaccharide (HMO) or HMO precursor or a mixture of HMOs from which substantially all of the water has been removed; and from which substantially all of the residue of an organic solvent, in particular toluene or a substituted toluene and / or a lower alkanol, is thereby removed; and that the glass transition temperature of the HMOs is increased; and, furthermore, its / its stability for prolonged storage at temperatures above 25 ° C is improved; and / or its / its mammalian bioavailability is thereby further increased.
    公开号:DK201900098U1
    申请号:DK201900098U
    申请日:2019-12-02
    公开日:2020-01-16
    发明作者:Schroven Andreas；Dékany Gyula；schwarz Andrea；Erdmann Peter
    申请人:Glycom A/S；
    IPC主号:
    专利说明:

    PURIFICATION OF OLIGOSACCHARIDES
    FIELD OF THE INVENTION
    The present invention relates to a process for the purification of human milk oligosaccharides (HMOs) and their precursors and derivatives thereof, in particular as prepared by catalytic hydrogenolysis.
    BACKGROUND OF THE INVENTION
    Human milk oligosaccharides (HMOs) have attracted considerable interest in recent years as a result of their important functions in the evolution of human 10. So far, the structures of at least 115 HMOs have been established (see Urashima et al: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1), and there are probably significantly more to present in human milk. The thirteen core structures identified so far for the 115 HMOs are listed in Table 1:
    No. core Name core Structure 1 lactose (Lac) Galß1-4Glc 2 lacto-M-tetraose (LNT) Galß1-3GlcNAcß1-3Galß1-4Glc 3 lacto-M neotetraose (LNnT) Galß1-4GlcNAcß1-3Galß1-4Glc 4 lacto-M-hexaose (LNH) Galß1-3GlcNAcß1-3 (Galß1-4GlcNAcß1-6) Galß1-4Glc 5 Lacto-M-neohexaose (LNnH) Galß1-4GlcNAcß1-3 (Galß1-4GlcNAcß1-6) Galß1-4Glc 6 para-lacto-M-hexaose (paraLNH) Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glc 7 para-lacto-M-neohexaose (pa-ra-QRQ) Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glc 8 lacto-M-octaose (LNO) Galß1-3GlcNAcß1-3 (Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-6) Galß1-4Glc 9 lacto-M neooctaosis (LNnO) Galß1-4GlcNAcß1-3 (Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-6) Galß1-4Glc
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    0 iso-lacto-W-octaose (iso-LNO) Galß1-3GlcNAcß1-3 (Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-6) Galß1-4Glc 1 para-lacto-W-octaose (para-LNO) Galß1-3GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glc 2 lacto-W neodecaosis (LNnD) Galß1-3GlcNAcß1-3 [Galß1-4GlcNAcß1-3 (Galß1-4GlcNAcß1-6) Galß1-4GlcNAcß1-6] Galß1-4Glc 3 lacto-W decaose (LND) Galß1-3GlcNAcß1-3 [Galß1-3GlcNAcß1-3 (Galß1-4GlcNAcß1-6) Galß1-4GlcNAcß1-6] Galß1-4Glc Lose 1. HMO Core Structures
    Cheap methods have been sought to produce industrial quantities of as many HMOs as possible, so that their applications in nutritional and therapeutic formulations for infants, as well as possibly for children and adults, can be discovered, developed and utilized by researchers throughout world. A few HMOs have recently been chemically synthesized with high yields, for example, by hydrogenating their protected benzylated derivatives after removing other protecting groups from such protected derivatives and then crystallizing them from organic solvents. See WO 2010/115935 (2'-O-fucosyllactose or 2'-FL), WO 2011/100980 (lacto-N-neotetraose or LNnT), WO 2012/155916 (lacto-Ntetraose or LNT), WO 2011/100979 ( 6'-O-sialyllactose or 6'-SL) and WO 2012/007585 (various HMOs) and WO 2011/150939 (2'-FL polymorphs).
    However, the relatively pure crystalline HMOs synthesized according to the above methods may still be contaminated with small residues, ie. ca. 1000 to 2000 ppm, but at least 100 ppm, of one or more of the following: i) toluene from the benzyl glycoside protecting group removed from their protected precursors by hydrogenolysis; and ii) protic solvents such as (C1-C6) alcohols used. as the solvent or as co-solvent together with water in the hydrogenolysis step and / or used as the solvent or as co-solvent in
    DK 2019 00098 U1 a subsequent crystallization or recrystallization of the HMOs. In order to use the HMOs in nutritional formulations and therapeutic formulations for mammals, especially for humans, especially for infants, it has been necessary to substantially reduce such residual contaminants, e.g. down to 500 ppm or less, preferably down to 100 ppm or less. Until now, this has required additional costly treatment of the HMOs.
    Accordingly, it is an object of the present invention to provide a method for removing or substantially reducing the amount and / or concentration of organic solvent residues in and / or on HMOs, HMO precursors and mixtures, in particular mixtures of multiple HMOs. are and / or HMO precursors.
    Furthermore, crystalline HMOs have been found to be relatively unstable when stored for an extended period without cooling. They have tended to melt, thereby becoming sticky and forming agglomerates.
    Accordingly, it is also an object of the present invention to provide a method for improving the stability of HMOs, especially HMO mixtures, so that they can be stored for a longer period without cooling, for example, at temperatures up to 30 ° C or even above, preferably up to 40 ° C or even above.
    Furthermore, crystalline HMOs, as well as their precursors and mixtures, have been relatively difficult to dissolve in water, so that they can be readily mixed with other active ingredients and suitable adjuvants to prepare liquid and powdered nutritional formulations and therapeutic formulations. In addition, they have dissolved relatively slowly in the aqueous acid in the stomach of mammals, especially in humans, especially in infants, which has reduced their bioavailability for mammals.
    Accordingly, it is a further object of the present invention to provide a process for improving the ability of HMOs, as well as their precursors and blends, to dissolve in water and in the gastric acid of mammals.
    CN 102154163 A has recently described spray drying of an HMO, namely 3'-SL, made by fermentation.
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    SUMMARY OF THE INVENTION
    The first aspect of this invention relates to a method for improving the stability of a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture for prolonged storage at temperatures above 25 ° C, which comprises the following steps:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution, preferably about its maximum concentration, especially at a concentration of at least about 10 30-60% by weight, especially at least approx. 4050% by weight, and
    b) then spray drying the aqueous solution to remove substantially all of the water, preferably at least approx. 90%, in particular at least approx. 95%, and provide the HMO or HMO precursor or mixture with a higher glass transition temperature (Tg) of at least 40 ° C, preferably at least 50 ° C, more preferably at least 60 ° C, even more preferably at least 70 ° C. , in particular at least 80 ° C.
    An embodiment of this first aspect of the invention relates to spray drying of the aqueous solution at ca. 130-210 ° C, preferably 140-180 ° C, especially 140-160 ° C, to provide a water content of approx. 8-10% or less, preferably approx. 4-6% or less, thereby increasing its Tg to approx. 40 ° C or higher, preferably to about 40 ° C. 50 ° C or more, more preferably to about 50 ° C. 60 ° C or more, even more preferably to about 70 ° C or more, in particular to approx. 80 ° C or higher.
    The second aspect of the invention relates to a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture which is in an amorphous form and has a glass transition temperature (Tg) of at least 40 ° C, preferably at least 50 ° C, more preferably at least 60 ° C, even more preferably at least 70 ° C, especially at least 80 ° C.
    An embodiment of the second aspect relates to a human milk oligosaccharide (HMO), preferably that selected from the group consisting of LNT, LNnT, LNH, LNnH, para-LNH, para-LNnH, 2'-FL, 3-FL, DFL , LNFP I, LNFP II, LNFP III, LNFP V, F-LNnH, DF-LNH I, DF-LNH II, DF-LNH I, DF
    DK 2019 00098 U1 para-LNH, DF-para-LNnH, 3'-SL, 6'-SL, FSL, F-LST a, F-LST b, F-LST c, LST a, LST b, LST c and DS-LNT, more preferably among LNT, LNnT, 2'-FL, 3FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT, even more preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, especially among LNnT, 2'-FL and 6'-SL, which are in an amorphous form and have a glass transition temperature (Tg) of at least 40 ° C.
    Another embodiment of this second aspect relates to a human milk oligosaccharide (HMO) selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS -LNT, even more preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, especially among LNnT, 2'-FL and 6'-SL, which are in an amorphous form and has a glass transition temperature (Tg) of at least 50 ° C, preferably at least 60 ° C, more preferably at least 70 ° C, in particular at least 80 ° C.
    Yet another embodiment of the second aspect of the invention relates to a mixture comprising 2 or more, preferably 5 or more HMOs and / or HMO precursors which are in an amorphous form and have a glass transition temperature (Tg) of at least 50 ° C, preferably at least 60 ° C.
    A further embodiment of the second aspect of the invention relates to a mixture comprising 2 or more, preferably 5 or more HMOs selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL. , 6'-SL, FSL, LST a and DSLNT, preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, which are in an amorphous form and have a glass transition temperature (Tg ) of at least 60 ° C.
    The third aspect of this invention relates to a method of removing or at least substantially reducing an amount or concentration of an organic solvent residue in or on a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture, especially in or on a crystalline HMO or a crystalline HMO precursor or a crystalline HMO mixture, the process comprising the steps of:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution, preferably about its maximum concentration, especially at a concentration of at least about 10 30-60% by weight, especially at least approx. 4050% by weight, and
    DK 2019 00098 U1
    b) then spray drying the aqueous solution to remove substantially all of the water, preferably at least approx. 90%, in particular at least approx. 95% and remove substantially all residual organic solvent, preferably at least approx. 75%, in particular at least approx. 85%, from the HMO or HMO precursor or mixture.
    An embodiment of this third aspect of the invention relates to the use of the method for removing the residual organic solvent from or reducing it in an HMO or HMO precursor or HMO mixture prepared by a catalytic hydrogenolysis of a protected derivative of HMO one or the HMO precursor or mixture, which protected derivative had a protecting group which could be removed by catalytic hydrogenolysis, preferably a benzyl group. In a preferred embodiment, the remainder of the organic solvent comprises the hydrogenolysated protecting group which is toluene or a substituted toluene, preferably toluene.
    Another embodiment of the third aspect of the invention relates to the use of the process for removing the residual organic solvent from or reducing it in an HMO or HMO precursor or HMO mixture prepared by a catalytic hydrogenolysis of a protected derivative of the HMO or HMO precursor or mixture in a protic solvent, preferably a lower alkanol or an aqueous lower alkanol solvent. In a preferred embodiment, the remainder of the organic solvent comprises a residue of the protic solvent, preferably of a lower alkanol, especially methanol.
    Yet another embodiment of the third aspect of the invention relates to the use of the method of removing the residues of two or more organic solvents from or reducing them in an HMO or HMO precursor or HMO mixture prepared by a catalytic hydrogenolysis in a protic solvent, preferably a lower alkanol or aqueous lower alkanol solvent, of a protected derivative of the HMO or HMO precursor or mixture, said protected derivative having a protecting group removable by catalytic hydrogenolysis, preferably a benzyl group. In a preferred embodiment, a residue of one comprises organic
    The hydrogenolysated protecting group which is toluene or a substituted toluene, preferably toluene, and a residue of another organic solvent comprises a residue of the protic solvent, preferably of a lower alkanol, especially methanol.
    A further embodiment of the third aspect of the invention relates to the use of the method of removing the residue (s) of one or more organic solvents from or reducing it (s) in a mixture of 2 or more, preferably 5 or more HMOs and / or HMO precursors which are crystallized separately from one or more organic solvents.
    A still further embodiment of the third aspect of the invention relates to the use of the method for removing a residue of an organic solvent from an HMO or an HMO precursor or derivative or an HMO mixture crystallized from an organic solvent. In one embodiment of this aspect, the remainder of the organic solvent comprises a lower alkanol, preferably methanol.
    The fourth aspect of this invention relates to a method of increasing the bioavailability of mammals, especially humans, especially infants, of an HMO or HMO precursor or HMO mixture, the method comprising the steps of:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution, preferably about its maximum concentration, especially at a concentration of at least about 10 30-60% by weight, especially at least approx. 4050% by weight, and
    b) then spray drying the aqueous solution to remove substantially all of the water, preferably at least approx. 90%, in particular at least approx. 95%, and provide the HMO or HMO precursor or derivative or mixture with an amorphous physical state.
    DETAILED DESCRIPTION OF THE INVENTION
    According to this invention, an aqueous solution of a human milk oligosaccharide (HMO) or its precursor or a mixture of HMOs and / or HMO precursors, preferably in a single spray drying step, is spray dried to:
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    removing substantially all of the water, preferably at least approx. 90%, in particular at least approx. 95%, from it; and thereby
    removing substantially all the residue of an organic solvent, preferably at least approx. 75%, especially at least approx. 85%, preferably residue of a lower alkanol, more preferably methanol, from it; and besides
    - improve its stability for longer periods of storage at temperatures above 25 ° C; and / or additionally
    - increase its / its bioavailability for mammals, especially humans, especially infants.
    In addition, in this invention, the term "protecting group removable by catalytic hydrogenolysis" refers to a group whose C-O bond is cleaved by the addition of hydrogen in the presence of a hydrogenolysis catalyst. The hydrogen oil catalyst is used in the presence of pressurized hydrogen gas. The hydrogenolysis catalyst may be, for example, palladium, Raney nickel, coal palladium or palladium black or another suitable metal catalyst known for use in hydrogenolysis. The hydrogenolysis results in restoration of the OH group which was protected. The protecting groups of this type are known (see, e.g., P.G.M. Wuts and T.W. Greene: Protective Groups in Organic Synthesis, John Wiley & Sons (2007)). Suitable protecting groups include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl or triphenylmethyl (trityl) groups, each of which may be optionally substituted by one or more groups selected from: alkyl, alkoxy, phenyl, amino , acylamino, alkylamino, dialkylamino, nitro, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl, N, N-dialkylcarbamoyl, azido, haloalkyl or halogen. Such a substitution, if present, is preferably present on the aromatic ring (s). A particularly preferred protecting group is benzyl which is optionally substituted with one or more groups selected from alkyl or halogen. More preferably, the protecting group is selected from unsubstituted benzyl, 4-chlorobenzyl and 4-methylbenzyl. These particularly preferred and more preferred protecting groups have the advantage that the by-products of the hydrogenolysis (such as the hydrogenolysed protecting groups) are exclusively toluene or subDK 2019 00098 U1 substituted toluene. Such by-products, even in the order of several tonnes, can be easily removed from water-soluble oligosaccharide products via evaporation and / or extraction processes.
    In addition, in this invention, the term "lower alkanol" preferably denotes a hydroxy or dihydroxyalkanol having 1 to 6 carbon atoms such as methanol, ethanol, n-propanol, i-propanol, ethylene glycol, diethylene glycol, 1,2-propanediol, 1,3-propanediol, butanol and an i-hexanol, especially a “(C1-C4) alcohol” such as methanol, ethanol, isopropanol, 1,2-propanediol and t-butanol, especially methanol.
    In addition, in this invention, the term "HMO" refers to tri-, tetra- and oligosaccharides found in breast milk (see Urashima et al: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978- 1-61122-831-1). An HMO is preferably selected from the group consisting of LNT, LNnT, LNH, LNnH, para-LNH, para-LNnH, 2'-FL, 3-FL, DFL, LNFP I, LNFP II, LNFP III, LNFP V, F- LNnH, DF-LNH I, DF-LNH II, DF-LNH I, DFpara-LNH, DF-para-LNnH, 3'-SL, 6'-SL, FSL, F-LST a, F-LST b, F -LST c, LST a, LST b, LST c and DS-LNT, more preferably among LNT, LNnT, 2'-FL, 3FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT, even more preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, particularly among LNnT, 2'-FL and 6'-SL.
    In this invention, however, an HMO precursor preferably denotes mono-, di- and trisaccharides which are the moieties of HMOs which are glucose, galactose, nacetyl-glucosamine, fucose, sialic acid, lactose, lacto-N-biose (Galp13GlcNAc ), N-acetyl-lactosamine (Galp1-4GlcNAc) and lacto-N-triose (GlcNAcp1-3Galp1-4Glc). Preferred HMO precursors are selected from the group consisting of fucose, sialic acid, lacto-N-biose, N-acetyl-lactosamine and lactoN-triose.
    In addition, in this invention, the term "blend" or "HMO blend" refers to a blend of two or more, preferably 5 or more HMOs and / or HMO precursors. The mixture preferably consists of two or more, preferably 5 or more HMOs selected from LNT, LNnT, LNH, LNnH, para-LNH, para-LNnH, 2'-FL, 3-FL, DFL, LNFP I, LNFP II, LNFP III, LNFP V, FDK 2019 00098 U1
    LNnH, DF-LNH I, DF-LNH II, DF-LNH I, DF-para-LNH, DF-para-LNnH, 3'-SL, 6'-SL, FSL, F-LST a, F-LST b , F-LST c, LST a, LST b, LST c and DS-LNT, more preferably among LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST α and DS-LNT, even more preferably among LNT, LNnT, 2′-FL, 3-FL, 3′SL and 6′-SL.
    In carrying out this invention, a protected derivative of an HMO or an HMO precursor or an HMO mixture, said protected derivative having a protecting group removable by catalytic hydrogenolysis, preferably a benzyl group, can be catalytically hydrogenated in a protic solvent in conventional manner. , preferably a lower alkanol or aqueous lower alkanol solvent, or in an aqueous solution with a protic solvent, to obtain a solution of the HMO or HMO precursor or mixture. See WO 2012/007585, which is incorporated herein by reference. The resulting solution - containing residual organic solvent, which is often approx. 1000 to 2000 ppm of toluene or a substituted toluene prepared during the hydrogenolysis, and often approx. Preferably, 1000 to 2000 ppm of the protic solvent used in the hydrogenolysis is heated to partially evaporate the protic solvent, to concentrate the solution, and also to partially evaporate the toluene or substituted toluene and the protic solvent. The resulting aqueous solution is then preferably diluted one or more times with water and concentrated again to substantially reduce the content of each such organic solvent residue. Consequently, the resulting aqueous solution preferably has a protic solvent content of not more than about. 500 ppm, especially not more than approx. 100 ppm, in particular not more than approx. 50 ppm and a content of toluene or a substituted toluene not exceeding approx. 200 ppm, preferably not more than approx. 50 ppm, in particular not more than approx. 20 ppm. In addition, the resulting aqueous solution has a concentration of an HMO or an HMO precursor or an HMO mixture of approx. 10-50% by weight, preferably approx. 2040% by weight, in particular approx. 25-35% by weight.
    In carrying out this invention, a dry HMO or dry HMO precursor or dry HMO mixture, preferably in crystalline form, may alternatively
    DK 2019 00098 U1 is dissolved in water in a conventional manner. The water is preferably heated slightly, for example up to approx. 40-50 ° C, to promote the dissolution of the HMO or HMO precursor or mixture in the resulting aqueous solution, preferably up to its maximum concentration (e.g., about 10-50% by weight), especially to a concentration of approx. 20-40% by weight, in particular approx. 25-35% by weight.
    The resulting aqueous solution can then be spray dried in conventional manner with hot air or hot inert gas, preferably hot air, at approx. 130-210 ° C, preferably 140-180 ° C, especially 140-160 ° C, to produce a substantially dry, amorphous, free-flowing powder of the HMO or HMO precursor or mixture. In this regard, any conventional spray dryer can be used, such as a co-stream or multi-stage spray dryer, preferably a two-stage spray dryer (with fluid bed connection). Also, the choice of nebulizer or spray nozzle in the spray dryer is not considered critical and any conventional rotary disk or high pressure vortex chamber nozzle can be suitably used.
    Preferably, a spray-dried powder of the HMO or HMO precursor or mixture is obtained, wherein substantially all of the water content, preferably at least approx. 90%, in particular at least approx. 95%, is removed and substantially all residual organic solvent, especially at least approx. 75%, in particular at least approx. 85%, especially residues of toluene or substituted toluene, preferably toluene, and / or of a lower alkanol, preferably methanol, which was present in the feed solution prior to spray drying, are removed. The glass transition temperature (Tg) of the spray-dried powder is a function of its water content, but a water content of approx. 8-10% or less, preferably approx. 4-6% or less, can provide a Tg of at least 40 ° C, preferably at least 50 ° C, more preferably at least 60 ° C, even more preferably at least 70 ° C, in particular at least 80 ° C. Such Tg can be readily obtained by spray drying the HMO or HMO precursor or mixture at ca. 130-210 ° C, preferably 140-180 ° C, especially 140-160 ° C. A Tg can advantageously be increased to approx. 80 ° C or higher when an HMO selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT, preferably from LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, more preferably among LNnT, 2'-FL and 6'-SL, are spray dried. Also advantageously, a Tg can, in case
    DK 2019 00098 U1 of spray drying an HMO mixture comprising 2 or more HMOs and / or HMO precursors, preferably 5 or more HMOs selected from the group consisting of LNT, LNnT, 2'-FL, 3- FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT, even more preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6 ' SL, increases to approx. 60 ° C or above. The resulting amorphous powder of an HMO or an HMO precursor or an HMO mixture, when Tg is at least 60 ° C, can be stored at not more than about 100 ° C. 50 ° C, and when Tg is at least 80 ° C, it can be stored at no more than approx. 60 ° C, and such a powder can be stored for a long time without significantly altering its composition and without significant decomposition while retaining its amorphous state.
    Also preferably, a spray-dried powder of the HMO or HMO precursor or mixture obtained by the process of this invention contains only small amounts or low concentration of organic solvent residues. Some HMOs, HMO mixtures or precursors may have high affinity to adsorb organic solvents which can only be partially removed by conventional drying methods. Crystallized fucosylated HMOs, especially 2'-FL, bind strongly to lower alcohols, especially methanol, the amount of which cannot be reduced below a certain size. This may prevent the use of such HMOs for food and pharmaceutical applications. This problem can be overcome by spray drying, rather than crystallizing, the HMO or HMO precursor or mixture, especially 2'-FL, which has a high concentration of residual organic solvent after its chemical or chemo-enzymatic synthesis.
    The amorphous powder of an HMO or HMO precursor or HMO mixture obtained by the process of the invention can be advantageously used in the preparation of nutritional formulations (such as foods, beverages or feed), supplements, functional foods to promote digestive health or other products. for ingestion intended for use in infants, children, adults or the elderly.
    Accordingly, a further aspect of the invention relates to a pharmaceutical composition for the treatment of infants, children, adults and / or the elderly, and in particular persons with special needs (eg having metabolic diseases),
    The composition comprises the amorphous powder of an HMO or an HMO precursor or an HMO mixture according to the second aspect. The amorphous powder of an HMO or HMO precursor or HMO mixture of the second aspect may be added to pharmaceutically acceptable carriers such as conventional additives, adjuvants, excipients and diluents (water, gelatin, talc, sugar, starch, gum arabic, vegetable gums, vegetable oils, polyalkylene glycols, flavorings, preservatives, stabilizers, emulsifiers, lubricants, colorants, fillers, wetting agents, etc.). Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art. When the amorphous powder of an HMO or HMO precursor or HMO mixture of the second aspect is added to the pharmaceutically acceptable carriers, a dosage may be prepared in the form of, but not limited to, for example, tablets, powders, granules, suspensions, emulsions, infusions, capsules, injections, liquids, elixirs, extracts and tincture. Further, to the above formulations, probiotics, e.g. lactic acid bacteria, Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soybean, whey or skimmed milk, carbohydrates such as lactose, sucrose, maltodextrin, starch or mixtures thereof, lipids (e.g. vitamins and minerals that are essential in a daily diet.
    Pharmaceutical compositions comprising the amorphous powder of an HMO or an HMO precursor or an HMO mixture according to the second aspect can be prepared by any conventional method known in the art, e.g. as described in the latest issue of Remington's Pharmaceutical Sciences, a standard reference text in the field.
    A still further aspect of the invention is a nutritional formulation such as a food, beverage or feed containing the amorphous powder of an HMO or an HMO precursor or HMO mixture of the second aspect and conventional edible micronutrients, vitamins and minerals. . The amounts of such ingredients may vary, depending on whether the product for ingestion is intended for use in infants, children, adults,
    DK 2019 00098 U1 elderly or persons with special needs (who have metabolic diseases, for example). Micronutrients include, for example, edible oils, fats or fatty acids (such as coconut oil, soybean oil, monoglycerides, diglycerides, palm oil, sunflower oil, fish oil, linoleic acid, linolenic acid, etc.), carbohydrates (such as glucose, fructose, sucrose, corn starch, starch, starch, maltodextrin, starch, and proteins from casein, soybean, whey or skimmed milk, or hydrolysates of these proteins, but protein from another source (either intact or hydrolyzed) may also be used. Vitamins such as A, B1, B2, B5, B6, B12, C, D, E, H, Vitamin K, folic acid, inositol and nicotinic acid may be selected. The nutritional formulation may contain the following minerals and trace elements: Ca, P, K, Na, Cl, Mg, Mn, Fe, Cu, Zn, Se, Cr or I. Further probiotics may be added, e.g. lactic acid bacteria, Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soybean, whey or skimmed milk, carbohydrates such as lactose, sucrose, maltodextrin, starch or mixtures thereof, lipids (e.g. minerals that are essential in a daily diet.
    In a preferred embodiment, the nutritional formulation which comprises the amorphous powder of an HMO or an HMO precursor or HMO mixture according to the second aspect may be a dietary supplement. Such a dietary supplement preferably contains ingredients as defined for foods above, e.g. vitamins, minerals, trace elements and other micronutrients, etc. For example, the dietary supplement may be in the form of tablets, capsules, lozenges or a liquid. The supplement may contain conventional additives selected from, but not limited to, binders, coatings, emulsifiers, solubilizers, encapsulants, film forming agents, adsorbents, carriers, fillers, dispersants, wetting agents, gelling agents, gelling agents, etc.
    In another preferred embodiment, the nutritional formulation comprising the amorphous powder of an HMO or HMO precursor or HMO mixture according to the second aspect may be a functional food for promoting digestive health since administration of an HMO or HMO
    DK 2019 00098 U1 precursor or an HMO mixture provides a beneficial effect on digestive health. Preferably, a functional digestive health food is a processed food which is used to improve and maintain digestive health using the mixture of oligosaccharides of the present invention as physiologically functional ingredients or ingredients in the form of a tablet, a capsule, a powder. etc. Various terms, such as supplements, nutraceuticals, designed foods or health products, may also be used to refer to a functional food to promote digestive health. The nutritional formulation comprising the amorphous powder of an HMO or an HMO precursor or an HMO mixture according to the second aspect can be prepared in any conventional manner. For example, it can be prepared by mixing micronutrient constituents in appropriate proportions. Then, the vitamins and minerals are added, but to avoid thermal degradation or decomposition, heat-sensitive vitamins can be added after homogenization. Lipophilic vitamins can be dissolved in the fat source before mixing. A liquid mixture is formed using water, the temperature of which is preferably approx. is 50-80 ° C, to promote dissolution or dispersion of the ingredients. The amorphous powder of an HMO or an HMO precursor or an HMO mixture according to the second aspect may be added at this stage. The resulting mixture is then homogenized by flash heating to ca. 80150 ° C by steam injection, heat exchanger or autoclave. This heat treatment also significantly reduces the amount of bacteria. The hot mixture is then quickly cooled to ca. 60-80 ° C. If necessary, further homogenization can be carried out at this temperature under high pressure of approx. 2-30 MPa. After cooling, heat sensitive constituents can be added at this stage and the pH and solids content adjusted accordingly. The resulting mixture is then dried by a conventional method such as spray drying or freeze drying to powder. Probiotics can be added at this time by dry mixing.
    EXAMPLES
    The calorimetric experiments to determine the glass transition DK 2019 00098 U1 temperature of the amorphous samples were performed using the Perkin Elmer DSC-7 at a heating rate of 10 ° C / min; the samples (5-7 mg) were placed in open aluminum crucibles.
    Example 1 - Lacto-N-neotetraose (LNnT) g (14.1 mmol) of the benzyl glycoside of LNnT (synthesized according to WO 2011/100980) was dissolved in 40 ml of water, 0.3 g of Pd-C and 80 µl of acetic acid were added. and the mixture was stirred at room temperature under H 2 atmosphere (about 40 bar) for 2 days. The reaction mixture was diluted with deionized water (10.0 ml) and the catalyst was filtered off. The mixture was concentrated to 20 ml and then diluted with deionized water (30.0 ml) and concentrated to 20 ml (22 g; 41% LNnT).
    The concentrated solution was used for spray drying on a Büchi Mini Spray Dryer B290 under the following conditions:
    Inlet temperature: 160 ° C
    Outlet temperature: 75 ° C
    Aspirator: 38 m 3 / h
    Compressed air: 600 l / h
    Pump: 8 ml / min% of the methanol in the feed solution was removed and the spray-dried LNnT became an amorphous, non-sticky material which exhibited excellent shelf life at a storage temperature below 40 ° C. Tg: 81.6 ° C. This indicates that there is a limit of about 60 ° C for the storage temperature of this LNnT material. Stability tests at two months (40 ° C, relative humidity of 75%) showed that there were no changes in the analysis data and the X-ray powder diagram of the material.
    Comparison of the above obtained amorphous spray dried LNnT sample with the crystalline LNnT sample prepared according to WO 2011/100980:
    MeOH (at GC) water amorphous spray-dried LNnT 14 ppm 3.2% crystalline LNnT 34 ppm 8.5%
    DK 2019 00098 U1
    The data shows that substantially lower amounts of organic solvent and water residues can be found in the spray-dried amorphous material.
    Example 2 - 2'-O-fucosyllactose (2'-FL) (2-O-benzyl-αL-fucopyranosyl) - (1'2) -β-D-galactopyranosyl- (1-4) D-glucose (synthesized WO 2010/115935) (10.0 g) was suspended in methanol (60 ml) and Pd / C (10%, 500 mg) suspended in methanol (5.0 ml) was added. The reaction mixture was then stirred under H 2 pressure (3 bar) for 40 hours. The reaction mixture was diluted with deionized water (35 ml) and the catalyst was filtered off. The colorless solution was concentrated to 30 ml and then diluted with deionized water (35 ml) and concentrated to 20 ml (22 g; 38% 2'-FL).
    The concentrated solution was used for spray drying on a Büchi Mini Spray Dryer B290 under the following conditions:
    Inlet temperature: 140 ° C Outlet temperature: 67 ° C Aspirator: 38 m 3 / h
    Compressed air: 600 l / h
    Pump: 8 ml / min% of the methanol in the feed solution was removed. The spray-dried 2'-FL became an amorphous, non-sticky material containing 2.8% water and exhibited excellent durability at a storage temperature below 40 ° C. Tg: 83.9 ° C. This suggests that there is a limit of about 60 ° C for the storage temperature of this 2'-FL material. Stability tests at two months (40 ° C, relative humidity of 75%) showed that there were no changes in the analysis data and the X-ray powder diagram of the material.
    Comparison of the above obtained amorphous spray-dried 2'-FL sample with the crystalline 2'-FL sample prepared according to WO 2010/115935:
    MeOH (at GC) amorphous spray dried 2'-FL 9 ppm
    DK 2019 00098 U1
    crystalline 2'-FL 190 ppm
    The data shows that substantially lower amount of organic solvent residue can be found in the spray-dried amorphous material.
    Example 3 - 6'-O-sialyllactose (6'-SL)
    The sodium salt of the benzyl glycoside of 6'-SL (synthesized according to WO 2011/100979, 10.0 g) was solubilized in deionized water (15.0 ml) and 1.5 M aqueous HCl (2.20 m) was added followed by Pd / C (10%, 580 mg). The mixture was stirred under H 2 pressure (5 bar) for 20 hours at 40 ° C. The catalyst was filtered off and the filtrate was concentrated to 10 ml. Deionized water (15.0 ml) was added and the mixture was concentrated to 15 ml (17 g; 52% 6'-SL). The solution was used for spray drying on a Büchi Mini Spray Dryer B290 under the following conditions:
    Inlet temperature: 140 ° C Outlet temperature: 57 ° C Aspirator: 38 m 3 / h
    Compressed air: 600 l / h
    Pump: 8 ml / min% of the methanol in the feed solution was removed. The spray-dried 6'-SL became an amorphous, non-sticky material containing 6.9% water and exhibited excellent durability at a storage temperature below 40 ° C. Tg: 84.0 ° C. This suggests that there is a limit of about 60 ° C for the storage temperature of this 6'-SL material.
    Example 4 - Sialic Acid (Neu5Ac)
    Anhydrous Neu5Ac (5.00 g) was solubilized in deionized water (25.0 ml) by gentle heating. The solution was filtered hot, 2000 ppm methanol was added and the solution was spray dried immediately on a Büchi Mini Spray Dryer B290 under the following conditions: Inlet temperature: 150 ° C
    DK 2019 00098 U1
    Outlet temperature: 64 ° C
    Aspirator: 38m
    Compressed air: 600l / h
    Pump: 8 ml / min% of the methanol in the feed solution was removed under the conditions indicated. The spray-dried Neu5Ac became an amorphous, non-sticky material which exhibited excellent durability at a storage temperature below 40 ° C.
    Example 5 - HMO mixture
    The following HMOs were dissolved by gentle heating in deionized water (9.50 ml):
    2'-FL 3.38 g
    3-FL 0.87 g
    LNT 1.20 g
    LNnT 0.35 g
    3'-SL 0.27 g
    6'-SL 0.72 g
    2000 ppm methanol was added to the aqueous solution of HMOs and the solution was spray dried immediately on a Büchi Mini Spray Dryer B290 under the following conditions:
    Inlet temperature: 150 ° C Outlet temperature: 63 ° C
    Aspirator: 38 m 3 / h
    Compressed air: 600 l / h
    Pump: 8 ml / min% of the methanol in the solution was removed. The spray-dried mixture of HMOs became an amorphous, non-sticky material which exhibited excellent durability at a storage temperature below 40 ° C. Tg: 63.6 ° C. This indicates that there is a limit of about 50 ° C for the storage 2019 00098 U1 annealing temperature of this blend material.
    Further Embodiments:
    A method for improving the stability of a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture for prolonged storage at temperatures above 25 ° C, the method comprising the following steps:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution; and
    b) then spray drying the aqueous solution to remove at least approx. 90% of the water and provide the HMO or HMO precursor or mixture with a higher glass transition temperature (Tg) of at least 40 ° C.
    The process of Embodiment 1 wherein the aqueous solution is spray dried at ca. 130-210 ° C to provide a water content of approx. 8-10% or less, thereby increasing its Tg to approx. 40 ° C or higher.
    The method of any one of embodiments 1 and 2, wherein the HMO is selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL , LST a and DS-LNT, and Tg is increased to approx. 80 ° C or higher.
    The method of embodiment 3 wherein the HMO is selected from LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, preferably from LNnT, 2'-FL and 6'-SL .
    The method of any one of embodiments 1 and 2, wherein the HMO mixture comprises 2 or more HMOs and / or HMO precursors, and Tg is increased to ca. 60 ° C or above.
    The method of embodiment 5, wherein the HMO mixture comprises 2 or more HMOs, preferably 5 or more HMOs, from the group consisting of LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'- SL, 6'-SL, FSL, LST a and DS-LNT.
    7. A human milk oligosaccharide (HMO) or an HMO precursor or an HMO mixture which is in an amorphous form and has a glass surface.
    DK 2019 00098 U1 operating temperature (Tg) of at least 40 ° C.
    An HMO of Embodiment 7 selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT and has a Tg of at least 80 ° C.
    An HMO according to embodiment 8 selected from LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, FSL, LST a and DS-LNT, preferably from LNnT, 2 ' -FL and 6'-SL.
    An HMO mixture according to Embodiment 7 comprising 2 or more HMOs and / or HMO precursors and having a Tg of at least 60 ° C.
    An HMO mixture according to Embodiment 10, comprising 2 or more HMOs, preferably 5 or more HMOs, from the group consisting of LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3 '-SL, 6'-SL, FSL, LST a and DS-LNT.
    A method of removing or at least substantially reducing an amount or concentration of an organic solvent residue in or on a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture, the method comprising the steps of:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution; and
    b) then spray drying the aqueous solution to remove at least approx. 90% of the water and remove at least approx. 75% of the remainder of the organic solvent from the HMO or HMO precursor or mixture.
    The process of embodiment 12, wherein the HMO or HMO precursor or mixture is prepared by a catalytic hydrogenolysis of a protected derivative of the HMO or HMO precursor or mixture, which protected derivative had a removable protecting group. by catalytic hydrogenolysis and wherein the removed organic solvent is the hydrogenolysed protecting group.
    The process of embodiment 12, wherein the HMO or HMO precursor or mixture is prepared by a catalytic hydrogenolysis of a protected derivative of the HMO or HMO precursor or mixture in a protic solvent and wherein the organic solvent removed is the protic solvent.
    DK 2019 00098 U1
    The process of embodiment 14, wherein the organic solvent comprises a lower alkanol, especially methanol.
    The process of embodiment 12, wherein the remainder of an organic solvent is removed from a mixture of 2 or more, preferably 5 or more HMOs and / or HMO precursors which are crystallized separately from one or more organic solvents.
    The process of embodiment 12 wherein the HMO is crystallized from an organic solvent.
    The process of embodiment 17, wherein the HMO is 2'FL and the organic solvent is a lower alkanol, especially methanol.
    A method for increasing mammalian bioavailability of a human milk oligosaccharide (HMO) or an HMO precursor or HMO mixture, the method comprising the steps of:
    a) providing the HMO or HMO precursor or mixture in an aqueous solution; and
    b) then spray drying the aqueous solution to remove at least approx. 90% of the water and provide the HMO or HMO precursor or mixture with an amorphous physical state.
    权利要求:
    Claims (13)
    [1]
    1. Amorphous, spray-dried, human Oligosaccharide (HMO) powder consisting of one or more HMOs selected from LNT, LNnT, LNH, LNnH, para-LNH, para-LNnH, 2'-FL, 3-FL, DFL, LNFP I, LNFP II, LNFP III, LNFP V, F-LNnH, DF-LNH I, DF-LNH II, DF-LNH I, DF-para-LNH, DF-para-LNnH, 3'-SL, 6'-SL, FSL, F-LST a, F-LST b, F-LST c, LST a, LST b, LST c and DS-LNT, characterized in that the HMO powder has a water content of approx. 8-10% or less, preferably approx. 4-6% or less, and a glass transition temperature (Tg) of at least 40 ° C.
    [2]
    The HMO powder of claim 1, comprising one or more HMOs selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3'-SL, 6'-SL, FSL, LST a and DS-LNT, even more preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, especially among LNT, LNnT, 2'-FL and 6'-SL.
    [3]
    HMO powder according to any one of claims 1 or 2, further comprising one or more organic impurities selected from (C1-C3) carboxylic acids such as formic acid, acetic acid and propionic acid, (C1-C4) alcohols such as methanol, ethanol, 2-propanol, 1,2-propanediol and t-butanol, and (C1-C3) alkylbenzenes such as toluene, 4-chlorotoluene and 4-methyltoluene.
    [4]
    HMO powder according to any one of the preceding claims having a glass transition temperature (Tg) of at least 50 ° C, preferably at least 60 ° C, even more preferably at least 70 ° C, in particular at least 80 ° C.
    [5]
    HMO powder according to any one of the preceding claims, consisting of 2 or more, preferably 5 or more HMOs selected from LNT, LNnT, 2'-FL, 3-FL, DFL, LNFP I, 3 '-SL, 6'-SL, FSL, LST a and DS-LNT, preferably among LNT, LNnT, 2'-FL, 3-FL, 3'-SL and 6'-SL, which HMOs have a glass transition temperature (Tg) of at least 60 ° C.
    [6]
    HMO powder according to any one of the preceding claims, obtained by spray drying a solution of the HMO or HMOs at a temperature of from 130 ° C to 210 ° C.
    [7]
    HMO powder according to any one of the preceding claims, which HMO powder can be obtained by a process comprising the following:
    DK 2019 00098 U1 the steps:
    a. providing the HMO or HMOs in an aqueous solution containing one or more organic impurities selected from (C1-C3) carboxylic acids such as formic acid, acetic acid and propionic acid, (C1-C4) alcohols such as methanol , ethanol, 2-propanol, 1,2-propanediol and t-butanol, and (C1-C3) alkylbenzenes such as toluene, 4-chlorotoluene and 4-methyltoluene, followed by
    b. subjecting the aqueous solution to additional concentration steps to obtain an aqueous HMO concentrate containing not more than 50 ppm of the organic impurities specified in (a), and a concentration of the HMO or HMOs of approx. . 10-50% by weight, followed by
    c. spray drying the aqueous HMO concentrate to remove at least approx. 90%, in particular at least approx. 95%, of the water in which the HMO powder contains not more than 15 ppm, more preferably not more than 10 ppm, in particular not more than 5 ppm, of the organic impurity or organic impurities listed in step a).
    [8]
    HMO powder according to any one of the preceding claims, containing one or more organic impurities selected from (C1-C3) carboxylic acids, such as formic acid, acetic acid and propionic acid, at a level not exceeding 10 ppm, in particular not exceeding 5 ppm. .
    [9]
    The HMO powder according to any one of the preceding claims, which contains one or more organic impurities selected from (C1-C3) carboxylic acids, such as formic acid, acetic acid and propionic acid, at a level of 15 ppm.
    [10]
    HMO powder according to any one of the preceding claims for use as a nutritional supplement or as a component of a nutritional composition.
    [11]
    HMO powder according to any one of claims 1-10 for use as a functional food for the promotion of digestive health.
    [12]
    The HMO powder according to any one of claims 1-10 for use as a drug, preferably in the prevention or treatment of a gastrointestinal disease.
    DK 2019 00098 U1
    [13]
    13. Pharmaceutical composition or nutritional composition for the treatment of infants, children, adults and / or the elderly, and in particular persons with special needs, such as e.g. persons having metabolic disorders, the composition comprising the HMO powder according to any one of claims 1 to 10.
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    同族专利:
    公开号 | 公开日
    CN104428307A|2015-03-18|
    EP2861609A1|2015-04-22|
    US9896470B2|2018-02-20|
    US20150183814A1|2015-07-02|
    EP2861609A4|2015-11-04|
    DE202013012827U1|2020-02-13|
    DE202013012829U1|2020-03-04|
    WO2013185780A1|2013-12-19|
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    法律状态:
    2020-01-16| UAT| Utility model published|Effective date: 20191202 |
    优先权:
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    DKPA201270329|2012-06-14|
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