Process for removing residual organic solvent from crystalline oligosaccharides

Abstract

The present invention relates to a method for removing residual organic solvent from crystalline oligosaccharides by using water vapor, and pharmaceutically and nutritionally suitable crystalline oligosaccharides obtained by said method.

Description

[0001] This application is a divisional application of the invention patent application with the invention name “Method for removing residual organic solvents from crystalline oligosaccharides” and application number “201780067687.2” filed on October 31, 2017. Field of the Invention

[0002] The present invention relates to a method for removing organic solvent residues from crystalline oligosaccharides, preferably human milk oligosaccharides (HMO), and crystalline oligosaccharides, preferably crystalline HMO, having a low level of organic solvent residues obtained by the method. Background Art

[0003] In the past few years, human milk oligosaccharides (HMOs) have attracted much attention due to their beneficial effects on human postnatal development. To date, the structures of more than 140 HMOs have been confirmed (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)), and are significantly more likely to be present in human milk.

[0004] Low-cost ways to manufacture as many HMOs of industrial quality as possible have been sought so that their use in nutritional and therapeutic formulations for infants and potentially children and adults can be discovered, developed and utilized by researchers around the world. Several HMOs have recently been chemically synthesized in high quality and yield, or can be obtained biologically. The final step of these techniques is often crystallization or recrystallization of the individual HMO from an aqueous organic solvent, which is often an alcohol, typically methanol (see, e.g., WO2011 / 100980, WO2011 / 150939, WO2014 / 009921, WO2014 / 075680, WO2014 / 086373, WO2014 / 069625, WO2015 / 188834, WO2016 / 086947, WO2016 / 095924, EP-A-1405856, Kuhn et al. Chem. Ber. 95, 513 and 518 (1962); Takamura et al. Chem. Pharm. Bull. 28, 1804 (1980)).

[0005] However, high-content crystalline HMO synthesized according to the above method or similar methods may still be contaminated by residues of organic solvents used as solvents or co-solvents in the crystallization or recrystallization of the HMO, which residues cannot be removed by conventional drying even under vacuum and / or high temperature. In order to use HMO in nutritional formulations for mammals, especially humans, it is necessary to substantially reduce the level of such residual solvents.

[0006] For consumer safety, the recommended acceptable levels of residual solvents in food ingredients are much lower than in pharmaceuticals. The Commission Regulation (EU) No 231 / 2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333 / 2008 of the European Parliament and of the Council (http: / / data.europa.eu / eli / reg / 2012 / 231 / oj) sets out the purity standards for food additives with respect to solvent residues (where applicable). Although the limits for organic solvent residues may vary depending on the food additive in question, for example the limit for methanol, which is a very common crystallization / recrystallization solvent or co-solvent, is usually below 100 ppm and never exceeds 1000 ppm. For pharmaceutically active ingredients, the same limit is 3000 ppm (see Impurities: Guideline for Residual Solvents, The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), http: / / www.ich.org / products / guidelines / quality / quality-single / article / impurities-guideline-for-residual-solvents.html). Although food regulatory authorities may decide or recommend concentrations for food ingredients on a case-by-case basis (including consideration of their exposure assessment), it is likely that the limits for organic solvent residues in HMOs intended for human food will not substantially deviate from those for food additives. Thus, for example, the approved level for methanol residues in HMOs may be expected to be approximately 50-100 ppm.

[0007] WO 2013 / 185780 discloses a method for removing or at least greatly reducing the amount of organic solvent residues in HMO, which comprises the step of spray drying an aqueous solution of HMO. The method provides HMO in an amorphous form.

[0008] It is therefore 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 oligosaccharides, in particular HMOs, which provides said oligosaccharides / HMOs in crystalline form with very low levels of organic solvent residues which may be acceptable to food regulatory authorities. Summary of the invention

[0009] The present invention provides an effective method for removing the amount and / or substantially reducing the concentration of organic solvent residues in and / or on crystalline oligosaccharides. The present invention further provides crystalline oligosaccharides, in particular crystalline HMOs, obtainable by such a method, wherein the oligosaccharides / HMOs comprise an amount of organic solvent residues acceptable for food products.

[0010] Thus, a first aspect of the present invention relates to a method of removing or substantially reducing the amount or concentration of organic solvent residues in or on a crystalline oligosaccharide, the method comprising the step of exposing the crystalline oligosaccharide to water vapour.Preferably, the oligosaccharide is a crystalline hydrate.

[0011] The second aspect of the present invention relates to a crystalline oligosaccharide hydrate which is substantially free of organic solvent residues, or at least has a very low amount or concentration thereof. The low amount or concentration of organic solvent residues is preferably not more than 100 ppm, more preferably 50-100 ppm or less.

[0012] Another aspect of the invention relates to the use of water vapor to remove or substantially reduce the amount or concentration of organic solvent residues in or on oligosaccharides which are in the form of crystalline hydrates.

[0013] In one embodiment of any of the above aspects, the oligosaccharide is a HMO.

[0014] In one embodiment of the present invention, the organic solvent is a lower alcohol, ketone, ester or acid.

[0015] Another aspect of the present invention relates to a crystalline oligosaccharide hydrate obtainable by the process according to the first aspect of the present invention, comprising therein or thereon organic solvent residues in an amount of up to 100 ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below with reference to the accompanying drawings, in which:

[0017] Figure 1 Shows the powder X-ray diffraction pattern of the crystalline pLNnH hydrate prepared according to Example 6;

[0018] Figure 2 The powder X-ray diffraction pattern of the crystalline LNT hydrate prepared according to Example 7 is shown. DETAILED DESCRIPTION

[0019] The term "oligosaccharide" preferably refers to a sugar polymer with a linear or branched structure containing at least two monosaccharide units linked to each other by an interglycosidic bond. The oligosaccharide in the context of the present invention is preferably in free form, i.e., without protecting groups on any of its free anomeric, primary and secondary OH-groups (e.g. ethers, esters, acetals, etc.) and, in aminodeoxysugars, on its free NH 2 The oligosaccharide is preferably a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide or a hexasaccharide.

[0020] The term "monosaccharide" preferably refers to a sugar (carbohydrate) having 5 to 9 carbon atoms, which is an aldose (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g., D-fructose, D-sorbose, D-tagatose, etc.), a deoxysugar (e.g., L-rhamnose, L-fucose, etc.), a deoxyaminosugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), an uronic acid, an aldonic acid, a ketouronic acid (e.g., sialic acid), an aldaric acid, or a sugar alcohol.

[0021] The term "human milk oligosaccharides" or "HMOs" are trisaccharides, tetrasaccharides or higher oligosaccharides found in human breast milk (Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011; Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Preferably, the HMO is selected from a trisaccharide (e.g., 2'-FL (2'-O-fucosyllactose), 3-FL (3-O-fucosyllactose), 3'-SL (3'-O-sialyllactose), 6'-SL (6'-O-sialyllactose), β3'-GL (3'-O-β-galactolactose), β4'-GL (4'-O-β-galactolactose), β6'-GL (6'-O-β-galactolactose) or lacto-N-triose II (GlcNAc (β1-3) Gal (β1-4) Glc)), a tetrasaccharide (e.g., DFL (2',3-di-O-fucosyllactose), SFL (3-O-fucosyllactose-3'-O-sialyllactose), LNT (lactoyl-N-tetraose) or LNnT (lactoyl-N-neotetraose)), a pentasaccharide (e.g., LNFP I (lactoyl-N-fucopentaose I), LNFP II (lactoyl-N-fucopentaose II), LNFP III (lactoyl-N-fucopentaose III), LNFP V (lactoyl-N-fucopentaose V), LST a (sialyl lactoyl-N-tetraose a), LST b (sialyl lactoyl-N-tetraose b) LNH or LST c (sialyl lactoyl-N-tetraose c)), and hexasaccharides (e.g., LNH (lactoyl-N-hexaose), LNnH (lactoyl-N-p-hexaose), pLNH (p-lactoyl-N-hexaose), pLNnH (p-lactoyl-N-neohexaose), LNFDH I (lactoyl-N-difucohexaose I), LNFDH II (lactoyl-N-difucohexaose II), LNFDH III (lactoyl-N-difucohexaose III), F-LST a (fucose-sialyllactoyl-N-tetraose a), F-LST b (fucose-sialyllactoyl-N-tetraose b), F-LST c (fucose-sialyllactoyl-N-tetraose c), or DS-LNT (disialactoyl-N-tetraose)).

[0022] The term "oligosaccharide crystalline hydrate" preferably refers to an oligosaccharide with water molecules in the lattice of the crystalline phase. Thanks to its small size and the ability to participate in multi-directional hydrogen bonding, water can associate oligosaccharide molecules to form various regularly arranged 3D structures. Based on the position of water in the crystalline structure, crystalline hydrates can be divided into site hydrates, ion-associated hydrates or channel hydrates; channel hydrates can be further divided into expanded channel hydrates and planar hydrates (for example, see Datta et al. Nat. Rev. Drug. Discov. 3, 42 (2004)). The amount of crystal water can be stoichiometric or non-stoichiometric. The degree of hydration can be determined in a variety of ways, such as single crystal X-ray diffraction (SCXRD) or dynamic vapor sorption (DVS) combined with standard analytical techniques. Powder X-ray diffraction (PXRD) is suitable for characterizing the crystal form. Furthermore, in certain crystalline phases, the amount of water participating in the crystal structure can vary within a certain range without changing its 3D structure, in other words, the same crystalline oligosaccharide can be hydrated at different percentages, as can be confirmed by DVS and PXRD.

[0023] The term "lower alcohol" preferably refers to a low molecular weight alcohol, which is a mono- or dihydroxyalkane having 1 to 6 carbon atoms, such as methanol, ethanol, n-propanol, isopropanol, butanol, hexanol or ethylene glycol, more preferably a monohydroxyalkane, especially a "C 1 -C 4 alcohols", such as methanol, ethanol, isopropanol, n-propanol, n-butanol, sec-butanol and tert-butanol.

[0024] The term "crystalline oligosaccharide substantially free of organic solvent residues" preferably means that the organic solvent content in or on the crystalline oligosaccharide is below the detection level of gas chromatography, ie, below 0.001-0.0001 w / w % or 1-10 ppm.

[0025] The above terms and definitions are general and applicable to all aspects and embodiments of the present invention. Other terms, whether general or related to specific aspects and embodiments, may be defined as follows.

[0026] It has been observed that certain crystalline oligosaccharides, particularly in the form of their crystalline hydrates, have a strong ability to adsorb organic solvents from which they are crystallized. Since these organic solvents often have relatively low boiling points, they are considered volatile and the conventional means of removing them from the crystalline material until a certain level is reached usually involves the application of vacuum and / or elevated temperatures. Nevertheless, organic solvents will often remain in the crystals, even in the range of about 5000-20000 ppm. Obviously, this will limit or even prevent the use of these crystalline oligosaccharides, particularly HMOs, in food and pharmaceutical products. The process of the present invention can overcome this problem.

[0027] It has been unexpectedly found that a modified conventional drying method including a drying step carried out in a water vapor atmosphere or a high humidity environment results in efficient removal of solvent residues and provides crystalline oligosaccharides having low levels of organic solvent residues without changing the crystalline phase.

[0028] Thus, a first aspect of the present invention relates to a method of substantially reducing the amount or concentration of organic solvent residues in or on a crystalline oligosaccharide hydrate, the method comprising the step of exposing the crystalline oligosaccharide hydrate to water vapour.

[0029] In the present context, the term "greatly reduced" means that the amount or concentration of organic solvent residues in the oligosaccharide crystals greater than 100 ppm after conventional drying is reduced to a value not greater than 100 ppm or less than 100 ppm. Preferably, the amount or concentration of organic solvent residues in the oligosaccharide crystals is reduced to a value of about 2-3 orders of magnitude, which means that the content of organic solvent residues of about 0.5 w / w% or more, for example about 5000-20000 ppm in the oligosaccharide crystals (which content cannot be reduced by conventional drying) is reduced to a value below 100 ppm, for example, below 50 ppm, 25 ppm or 10 ppm. To achieve this, according to the present invention, the crystalline oligosaccharide whose organic solvent residues are to be reduced is placed in a high humidity environment. In a preferred embodiment, the crystalline oligosaccharide hydrate is contacted with water vapor in a container, the interior space of the above container having or maintaining an elevated humidity compared to the outside of the container. The elevated humidity in the interior space of the container is advantageously a relative humidity (RH%) of about 60-100%.

[0030] It is noteworthy that, when exposed to water vapor according to the present invention, no phase change occurs in the treated crystalline oligosaccharide hydrate, in other words, the PXRD pattern of the material before and after exposure does not change. The phrase "exposed to water vapor" is interchangeable with "wet drying", "steam drying" and "steam stripping" in this article.

[0031] In one embodiment, greatly reducing the amount or concentration of organic solvent residues means removing the organic solvent residues from the oligosaccharide crystals so that the amount or concentration is reduced to below the detection level of gas chromatography (below 0.001-0.0001 w / w% or 1-10 ppm).

[0032] The container suitable for the purpose of the method can be any laboratory or industrial device for drying solid chemical substances, such as a desiccator, an oven, a drying column, a double cone dryer, a conical screw dryer, etc., or any other device suitable for drying, such as a rotary evaporator. A suitable container cannot be a closed system, but should have at least one outlet, the opening of which is used to allow water vapor to continuously escape together with trace amounts of organic solvent removed from the crystalline material. The water vapor can be generated inside or outside the container, in the latter case, the water vapor is introduced into the container via an appropriate pipe at a specified flow rate.

[0033] In one embodiment, the method is carried out at atmospheric pressure, advantageously in a container that can generate water vapor internally. In order to quickly achieve high relative humidity and / or accelerate the removal of organic solvent residues, the temperature is advantageously set at more than 40°C, such as 50-60°C, 60-70°C or 70-90°C. The method makes the crystalline oligosaccharide have a very low level of organic solvent residues in a relatively short period of time, such as in the range of a few hours to a few days. However, it should be noted that the stability of the crystalline oligosaccharide may be adversely affected by the use of high temperature for a long time. Suitable temperature ranges can be determined by those skilled in the art by trial and error, such as continuously controlling and analyzing samples during the process. If necessary, it should be set at a lower temperature, in which case the completion of the removal of solvent residues may take longer.

[0034] In a preferred embodiment, the method of the present invention is carried out under vacuum. Both internal and external water vapor generation can be applied. Under vacuum, lower temperatures are necessary to achieve the required relative humidity level, which can be advantageous in terms of product stability. In addition, vacuum generation produces a steam stream that takes trace amounts of organic solvent out. The typical temperature range for vacuum stripping is about 40-60° C., or a temperature at which the crystalline oligosaccharide remains stable.

[0035] When water vapor is generated inside a container, an open tank, such as a crystallization dish or plate, containing water and placed in the same space in the container as the crystalline oligosaccharide is typically used. By evaporating the water, its vapor saturates the interior space of the container, and the vapor contacts the crystalline oligosaccharide. The higher the internal temperature, the faster the evaporation, and the faster the removal of the organic solvent occurs. However, the temperature cannot exceed the boiling point of water at a specified pressure. It is desirable that the temperature be set to at least room temperature.

[0036] In the case of external steam generation, water vapor is typically introduced into the container via a tube to contact the crystalline oligosaccharide. In one embodiment, water vapor is introduced by means of a carrier gas stream. The carrier gas should be inert, that is, the carrier gas cannot react with water vapor or crystalline oligosaccharides. Some non-limiting examples of carrier gases include air, nitrogen or argon. Typically, an inert gas is bubbled through water and a humidified gas stream is introduced into the container by applying positive or negative pressure.

[0037] In order to speed up the process of the present invention and / or make it suitable for larger quantities, in some embodiments, the process of the present invention can be carried out by the following steps. After crystallization from a solvent system including an aqueous organic solvent, the oligosaccharide crystals collected by filtration ("wet cake") often contain a large amount of organic solvent (30-50%) and water. The amount of organic solvent can be reduced to about 0.5-2% by implementing a pre-drying step, which is typically a conventional drying method in an appropriate container (e.g., using vacuum and / or high temperature). Conventional drying in this context refers to a mass transfer process consisting of removing organic solvent and water from the crystallized oligosaccharide by evaporating them, for example, by heating the container wall and / or applying a vacuum, in the following manner: both water (moisture) and organic solvent diffuse / move along the direction of their decreasing concentration (i.e., from the crystalline solid to the internal space of the container and then leaving the container). In, for example, an oven, pre-dried wet cakes often result in the formation of a lumpy crystalline solid, which needs to be ground and / or sieved to increase the surface of the particles for more effective contact with steam. Particles having a diameter of about 1-2 mm (or less) are generally suitable for the method of the present invention.

[0038] Therefore, some embodiments of the method include the following steps:

[0039] a) crystallizing an oligosaccharide hydrate from a mixture of water and a suitable organic solvent,

[0040] b) subjecting the crystals obtained in step a) to conventional pre-drying to remove most of the organic solvent, and

[0041] c) exposing the crystals obtained in step b) to remove the remaining organic solvent until the concentration thereof decreases to 100 ppm or less.

[0042] Moreover, in some cases, the process can provide crystalline materials with very low levels of organic solvent residues but supersaturated with water vapor. In order to meet the water content requirements of specific oligosaccharides, the process of the present invention can be followed by an additional drying step, wherein excess or unnecessary water is removed by conventional drying (vacuum and / or high temperature) in the absence of water vapor and in the presence of conventional desiccants.

[0043] The organic solvent whose residual amount is to be removed by the method of the present invention is one of the antisolvents which are usually used in oligosaccharide crystallization and are completely or partially miscible with water. Frequently suitable solvents include lower alcohols, ketones (such as acetone and methyl ethyl ketone), acetic acid, pyridine, dimethyl sulfoxide, DMF, ethyl acetate or ethers (such as diethyl ether, THF, dioxane and ethylene glycol mono- or diethyl ether). Preferably, the organic solvent to be removed by the method of the present invention is C 1 -C 4 Alkanol which is methanol, ethanol, isopropanol, n-propanol, n-butanol, sec-butanol or tert-butanol, in particular methanol.

[0044] In order to achieve a relatively short stripping time, the crystalline oligosaccharide should be a hydrate. In this case, the kinetics of removing the residual organic solvent is exponential and, depending on the arrangement of the apparatus used in the process and / or the bulkiness of the oligosaccharide crystals, oligosaccharide crystals substantially free of organic solvent or with a very low amount thereof can be obtained in a period of several hours to 1-2 days. However, crystalline oligosaccharide anhydrates do not appear to be suitable for the purpose of organic solvent stripping according to the process of the invention, since the kinetics of removing the residual organic solvent from these crystalline materials is linear with a low gradient, and therefore the extrapolated time to achieve complete or at least substantial removal of the organic solvent residues would be unrealistically long.

[0045] In one embodiment, the method of the present invention is carried out in a drying oven at ambient pressure or vacuum. The steam source is a large crystallization dish or dish containing water placed at the bottom of the oven. As the treatment time proceeds, water slowly evaporates from the dish. This embodiment is suitable for processing oligosaccharide crystals in the gram to kilogram range.

[0046] In another embodiment, water vapor is passed through a fluidized bed of crystallized oligosaccharides, for example using a drying column. This arrangement ensures that mass transfer of water vapor to the crystals is enhanced, which increases stripping efficiency and reduces the time to complete the process (typically taking less than 24 hours).

[0047] In another embodiment, the method of the present invention is carried out in a conical screw dryer (suitable for industrial applications). Water vapor is introduced into the conical dryer at the bottom via a sleeve and an injection valve. The conical dryer is equipped with a rotating mixing screw that continuously mixes the crystallized mass in an intensive, but gentle manner, ensuring good mass transfer of the steam. The mechanical forces of the mixer destroy the bulk material lumps that occasionally form in the previous crystallization and / or pre-drying steps, so that a separate grinding / sieving step before carrying out the method of the present invention can be advantageously avoided. Both ambient and vacuum stripping can be carried out in a conical screw dryer.

[0048] Crystalline oligosaccharide hydrates, including crystalline HMO hydrates, can be prepared according to crystallization methods disclosed in the prior art. For example, 2'-FL (2'-O-fucosyllactose, Fucpα1-2Galpβ1-4Glc) 3 / 2 hydrate can be crystallized from aqueous methanol, ethanol, propanol or acetone (WO 2014 / 009921), LNnT (lactoyl-N-neotetraose, Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc) polymorph II can be crystallized from aqueous acetone (EP-A-1405856), LNnT polymorph III can be crystallized from aqueous methanol (WO 2011 / 100980), and DFL (difucosyllactose, Fucpα1-2Galpβ1-4[Fucpα1-3]Glc) can be crystallized from aqueous ethanol (WO 2016 / 086947). Other crystalline HMO hydrates are described herein as follows.

[0049] A second aspect of the present invention relates to a crystalline oligosaccharide hydrate wherein or thereon comprises an organic solvent residue having a content of not more than 100 ppm. The crystalline oligosaccharide of the second aspect can advantageously be obtained by applying the method according to the first aspect of the present invention. The amount or concentration of the organic solvent residue in or on the crystalline oligosaccharide hydrate can be determined by standard methods, for example, by gas chromatography. An embodiment of the second aspect of the present invention includes a crystalline oligosaccharide wherein or thereon wherein the organic solvent residue is lower than a level of 50, 25 or 10 ppm.

[0050] In certain embodiments, the crystalline oligosaccharide hydrate comprises about 50-100 ppm, 25-75 ppm, 25-50 ppm, 10-50 ppm, or 10-25 ppm of organic solvent residues.

[0051] Another embodiment of the second aspect of the invention relates to crystalline oligosaccharide hydrates having substantially no organic solvent residues therein or thereon, ie, the amount or concentration of organic solvent is virtually undetectable (less than about 1-10 ppm) when determined by standard gas chromatography methods.

[0052] Exemplary embodiments of the crystalline oligosaccharide hydrate are trehalose dihydrate, raffinose pentahydrate and HMO hydrate. A preferred embodiment is HMO hydrate.

[0053] More preferably, the crystalline HMO hydrate is selected from 2'-FL polymorph A (3 / 2 hydrate) characterized in WO 2014 / 009921, LNnT polymorph II characterized in EP-A-1405856, LNnT polymorph III characterized in WO 2011 / 100980, DFL hydrate characterized in WO 2016 / 086947, LNT (lactoyl-N-tetraose, Galpβ1-3GlcNAcpβ1-3Galpβ1-4Glc) hydrate and pLNnH (p-lactoyl-N-neohexose, Galpβ1-4GlcNAcpβ1-3Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc) hydrate, and even more preferably LNnT polymorph III, DFL hydrate, LNT hydrate and pLNnH hydrate. Preferred embodiments are: LNnT polymorph III comprising ethanol or isopropanol as an organic solvent residue in an amount not greater than 100 ppm; LNnT polymorph III comprising methanol as an organic solvent residue in an amount not greater than 25, preferably 15 ppm; crystalline difucosyllactose hydrate comprising methanol or ethanol as an organic solvent residue in an amount not greater than 100, preferably 50, more preferably 25 ppm; crystalline LNT hydrate comprising methanol or ethanol as an organic solvent residue in an amount not greater than 100, preferably 50, more preferably 25 ppm; crystalline pLNnH hydrate comprising methanol or ethanol as an organic solvent residue in an amount not greater than 100, preferably 50, more preferably 25 ppm.

[0054] Crystalline oligosaccharide hydrates in which or on which organic solvent residues are substantially absent or in which the level is very low (less than 100 ppm) represent a new quality product for crystalline oligosaccharides, which are not available by conventional isolation and / or separation methods. It is worth noting that, although crystalline oligosaccharide crystals are easily obtained by crystallization thereof from aqueous organic solvents, the residual amount of organic solvent after drying the crystals under conventional conditions, e.g. at elevated temperature and / or under vacuum, has been shown to be quite high. The consequence of this disadvantage is that their commercial use as active substances or ingredients in pharmaceutical and nutritional compositions is greatly limited or even actually hindered.

[0055] The crystalline oligosaccharide hydrate according to the second aspect of the invention, wherein or thereon therein substantially no organic solvent residue or its level is very low (less than 100 ppm), advantageously obtained by the method according to the first aspect of the invention, can be beneficially used in the manufacture of pharmaceutical preparations, nutritional preparations (such as foods, beverages or feeds), food supplements, digestive health functional foods or other consumer products, which are intended for animals, such as pets such as dogs and cats, or humans, such as infants, children, adults or the elderly.

[0056] Thus, a third aspect of the present invention relates to a nutritional formulation comprising a crystalline oligosaccharide hydrate according to the second aspect of the present invention wherein or thereon the level of organic solvent residues is less than 100 ppm.

[0057] In a preferred embodiment, the crystalline oligosaccharide hydrate is a crystalline HMO hydrate in a nutritional formulation.

[0058] In one embodiment, the nutritional formulation comprising a crystalline oligosaccharide hydrate wherein or on which the organic solvent residue level is less than 100 ppm can be a food supplement. Such a food supplement can preferably contain ingredients defined for the above-mentioned nutritional food, for example, vitamins, minerals, trace elements and other micronutrients, etc. The food supplement can be in the form of, for example, tablets, capsules, lozenges or liquids. The supplement can contain conventional additives selected from, but not limited to, adhesives, coatings, emulsifiers, solubilizers, encapsulating agents, film formers, adsorbents, carriers, fillers, dispersants, wetting agents, gelling agents, gelling agents, etc.

[0059] In another embodiment, a nutritional formulation comprising a crystalline oligosaccharide hydrate having an organic solvent residue level of less than 100 ppm therein or thereon can be a digestive health functional food, since administration of dietary oligosaccharides, particularly HMOs, can provide a beneficial effect on digestive health. The digestive health functional food is preferably a processed food intended for improving and maintaining digestive health by utilizing a mixture of oligosaccharides according to the present invention (in the form of tablets, capsules, powders, etc.) as a physiologically functional ingredient or component. In addition, different terms such as dietary supplements, nutraceuticals, designer foods, and health products can also be used to refer to digestive health functional foods.

[0060] In another embodiment, the nutritional formulation including crystalline oligosaccharide hydrate according to the second aspect of the present invention can be food, beverage or feed also containing conventional edible micronutrients, vitamins and minerals.The amount of these compositions can vary depending on the target user classification, that is, the consumer product is intended for animals such as pets such as dogs or cats, or people such as infants, children, adults, the elderly, or the subject with special needs (such as suffering from metabolic diseases).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, maltodextrin, starch, hydrolyzed corn starch, etc.) and from casein, soybean, whey or skim milk protein or the hydrolyzate of these proteins, but protein from other sources (complete or hydrolyzed) can also be used. Vitamins can be selected, such as vitamin A, B1, B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid. The nutritional preparation may contain the following minerals and trace elements: Ca, P, K, Na, Cl, Mg, Mn, Fe, Cu, Zn, Se, Cr or I. Furthermore, additional probiotics may be added, such as lactic acid bacteria, Bifidobacterium species, and prebiotics such as oligofructose and oligogalactose, proteins from casein, soy, whey or skim milk, carbohydrates such as lactose, sucrose, maltodextrin, starch or mixtures thereof, lipids (e.g., palm oil, sunflower oil, safflower oil) and vitamins and minerals necessary for the daily diet.

[0061] The above-mentioned nutritional formulation can be prepared by any conventional means. For example, it can be prepared by mixing the micronutrient components in appropriate proportions. Then, vitamins and minerals are added, but, in order to avoid thermal decomposition or degradation, heat-sensitive vitamins can be added after homogenization. Lipophilic vitamins can be dissolved in a fat source before mixing. Water with a temperature of preferably about 50-80° C. is used to form a liquid mixture to facilitate the dissolution or dispersion of the ingredients. Crystallized oligosaccharide crystals according to the second aspect can be added at this stage. The resulting mixture is then homogenized by rapid heating to about 80-150° C. by means of steam injection, heat exchanger or autoclave. This heat treatment also significantly reduces the bacterial load. The hot mixture is then rapidly cooled to about 60-80° C. If necessary, further homogenization can be performed at this temperature under a high pressure of about 2-30 MPa. After cooling, heat-sensitive ingredients can be added at this stage, and pH and solid content can be conveniently adjusted. The resulting mixture is then dried by conventional methods such as spray drying or freeze drying into a powder. Probiotics can be added by dry mixing at this time.

[0062] A fourth aspect of the present invention relates to a pharmaceutical formulation comprising a crystalline oligosaccharide hydrate according to the second aspect of the present invention wherein or thereon the level of organic solvent residues is less than 100 ppm.

[0063] In a preferred embodiment, the crystalline oligosaccharide hydrate in the pharmaceutical composition is a crystalline HMO hydrate.

[0064] In one embodiment, the pharmaceutical composition is intended for treating people, such as infants, children, adults and / or the elderly, and specific subjects with special needs (e.g., subjects suffering from metabolic or mental illness, infection, cardiovascular disease, etc.). Crystalline oligosaccharide crystals according to the second aspect can be added to a pharmaceutically acceptable carrier, such as conventional additives, adjuvants, excipients and diluents (water, gelatin, talc, sugar, starch, gum arabic, vegetable gum, vegetable oil, polyalkyl alcohol, flavoring, preservative, stabilizer, emulsifier, lubricant, colorant, filler, wetting agent, etc.), to manufacture a pharmaceutical composition. Suitable carriers are described in the latest version of the standard reference textbook Remington's Pharmaceutical Sciences in this area. When the crystalline oligosaccharide hydrate according to the second aspect is added to a pharmaceutically acceptable carrier, the following dosage forms can be prepared: for example, but not limited to, tablets, powders, granules, suspensions, emulsions, infusions, capsules, injections, liquids, elixirs, extracts and tinctures, or prepared in other forms, for example, in a specific formulation suitable for a specific application method, patient category or disease. If necessary, probiotics, such as lactic acid bacteria, bifidobacterium species, prebiotics, such as oligofructose and oligogalactose, proteins from casein, soy, whey or skim milk, carbohydrates, such as lactose, sucrose, maltodextrin, starch or mixtures thereof, lipids (e.g., palm oil, sunflower oil, safflower oil) and / or vitamins and minerals necessary for daily diet can also be added to the above preparations.

[0065] The pharmaceutical compositions of the fourth aspect of the invention may be manufactured by any conventional means known in the art, for example as described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference textbook in the field.

[0066] A fifth aspect of the present invention relates to the use of water vapor in greatly reducing the amount or concentration of organic solvent residues in or on crystalline oligosaccharide hydrates, preferably in or on crystalline HMO hydrates. When water vapor is contacted with crystalline oligosaccharide hydrates containing organic solvent residues, the use of water vapor unexpectedly reduces the amount or concentration of organic solvent residues remaining in the oligosaccharide hydrate crystal mass that cannot be eliminated by conventional drying methods. Advantageously, the contact is carried out in a container, and the concentration of water vapor in the above container is higher than the concentration outside the container. This specific use of water vapor provides crystalline oligosaccharide hydrates of improved quality, which means that their amount or concentration of organic solvent residues is very low, less than 100 ppm, for example, less than 50 ppm, 25 ppm or 10 ppm, and is therefore suitable for use in the manufacture of pharmaceutical or nutritional compositions intended for human consumption.

[0067] A sixth aspect of the present invention relates to a crystalline oligosaccharide hydrate comprising 10-100 ppm or less of organic solvent residues therein or thereon, which can be obtained by the method according to the first aspect of the present invention. The amount or concentration of organic solvent residues in or on the crystalline oligosaccharide hydrate can be determined by standard methods, for example, by gas chromatography. Embodiments of the crystalline oligosaccharide hydrate obtainable by the method according to the first aspect of the present invention include crystalline oligosaccharide hydrates wherein or thereon the level of organic solvent residues is less than 50, 25 or 10 ppm, for example, about 50-100 ppm, 25-75 ppm, 25-50 ppm, 10-50 ppm or 10-25 ppm.

[0068] A preferred embodiment of the sixth aspect of the present invention is HMO hydrate, more preferably, the crystalline HMO hydrate is selected from 2'-FL polymorph A (3 / 2 hydrate), LNnT polymorph II, LNnT polymorph III, DFL hydrate, LNT hydrate and pLNnH hydrate, and even more preferably LNnT polymorph III, DFL hydrate, LNT hydrate and pLNnH hydrate. Preferred embodiments are: LNnT polymorph III comprising no more than 100 ppm of organic solvent residual ethanol or isopropanol; LNnT polymorph III comprising no more than 25, preferably 15 ppm of organic solvent residual methanol; crystalline difucosyllactose hydrate comprising no more than 100, preferably 50, more preferably 25 ppm of organic solvent residual methanol or ethanol; crystalline LNT hydrate comprising no more than 100, preferably 50, more preferably 25 ppm of organic solvent residual methanol or ethanol; crystalline pLNnH hydrate comprising no more than 100, preferably 50, more preferably 25 ppm of organic solvent residual methanol or ethanol.

[0069] Example

[0070] Example 1

[0071] LNnT polymorph III crystallized from aqueous methanol (see WO 2011 / 100980) may contain 2.0-9.3 w / w % water with the same powder X-ray diffraction pattern.

[0072] The wet cake of crystalline LNnT was pre-dried in a rotary evaporator at 60°C and 30 mbar for 2.5 hours. GC analysis of the sample showed a methanol content of 18240 ppm. The pre-dried material was then placed in a drying oven with a vessel containing water, set at 90°C. After drying for 24 and 60 hours, the residual methanol was stripped to 15 and 5 ppm, respectively.

[0073] Example 2

[0074] According to WO 2011 / 100980, LNnT polymorph III is crystallized from aqueous isopropanol. The wet crystals are dried in a vacuum oven (60°C, 50mbar) for 24 hours, and the residual isopropanol content is 2.19%. The crystals are then ground and further dried for 20 hours in an oven at 90°C, ambient pressure conditions, in the presence of a vessel containing water. The temperature is then reduced to 60°C, the vessel containing water is taken out, vacuum (50mbar) is applied, and a constant humid internal atmosphere is ensured by an air inlet bubbled in water at room temperature. After 17 hours, the isopropanol content is reduced to 94ppm.

[0075] Example 3

[0076] According to WO 2011 / 100980, LNnT polymorph III is crystallized from aqueous methanol. The wet crystals are dried in a vacuum oven (60°C, 50mbar) with a residual methanol content of 15804ppm. After grinding and sieving (2mm), the crystals are placed in a drying column, and the bottom of the drying column is connected to a tank filled with water via a plastic tube. The top of the column is connected to a vacuum pump. The water tank and the drying column containing the crystallized LNnT polymorph III are maintained at 25°C. The water vapor generated by the vacuum (17-20mbar) passes through the LNnT crystal bed. After the specified time, a sample is taken from the fluidized bed and its methanol content is determined by GC:

[0077] time 0 1h 15m 2h 45m 4h 5m MeOH (ppm) 15804 737 19 16

[0078] Example 4

[0079] In the same experimental design as in Example 3, the temperature of the drying column was maintained at 50° C. and the water tank was heated to 40-45° C. The vacuum was first set at 100 mbar and then at 60 mbar.

[0080] The following values ​​were measured from a sample of LNnT polymorph III.

[0081] time 0 1h 10m 2h 30m MeOH (ppm) 15804 602 13 Vacuum(mbar) - 100 60

[0082] Example 5

[0083] DFL was crystallized from aqueous ethanol according to WO 2016 / 086947. The wet crystallized material was dried in a vacuum oven (8 mbar) at 60 ° C for 11 hours and at 40 ° C for 13 hours. Its residual ethanol content was 1.03%. Drying was continued in the same vacuum oven (30 mbar, 50 ° C), but in the presence of a water container. Samples were taken after the specified time and their ethanol content was determined by GC:

[0084] time 0 15h 23h 38h EtOH (ppm) 10284 117 49 17

[0085] Example 6

[0086] A clear solution of pLNnH in water (BRIX 14.2) was concentrated to a suspension at 70°C in vacuum (60 mbar) by evaporating 5 / 6 of the water. The suspension was allowed to cool to room temperature while stirring overnight. The solid was filtered, washed with methanol / water (1:1), methanol / water (2:1) and methanol, and then dried in a vacuum oven for 27 hours (60°C, 5 mbar). Sample analysis showed that the solid obtained was crystalline (see Figure 1 ), containing 7.9% water (determined by Karl-Fisher titration) and 870 ppm methanol (determined by GC). The crystalline solid was further dried in a vacuum oven for 3 days (50° C., 30 mbar) in the presence of a water container. The methanol content of the obtained crystals was reduced to below the detection level of GC (less than 1 ppm).

[0087] Example 7

[0088] A) LNT (9.7 g) dissolved in water (33 ml) was heated to 60°C. Methanol (66 ml) was slowly added over 35 minutes. The solution was stirred at 60°C and spontaneous nucleation occurred after 15 minutes. The suspension was stirred at 60°C for 2 hours and then slowly cooled to 40°C over 1.5 hours. The suspension was further stirred at 40°C for 14 hours. The suspension was then cooled to 25°C and stirred at this temperature for a further 2 hours. The crystals were filtered off and washed with 10 ml of methanol. The wet crystals were dried at 50°C at 50 mbar for 21 hours to give a crystalline material (see PXRD pattern Figure 2 ), its water content was 9.4% (by Karl-Fisher titration).

[0089] B) The aqueous solution containing LNT (34 g) was concentrated to brix 24°. At 35°C, a first portion of MeOH (150 ml) was added. The solution was seeded. Further MeOH (150 ml) was added in 25 ml portions over a period of 1 hour. The suspension was stirred at 35°C for 3 hours, then heated to 55°C, stirred at 55°C for 6 hours, then cooled to 35°C and stirred at 35°C for 4 hours. The solid was filtered off, washed with 60 ml MeOH and dried in a vacuum oven at 60°C at 50 mbar for 5 hours to give crystalline material (29 g) which was identical to the sample obtained above according to PXRD, with a water content of 9.1% (by Karl-Fisher titration) and a methanol content of 5234 ppm (by GC). The solid was further dried in an oven adjacent to a water vessel at 70°C and ambient pressure for 65 hours to give 29 g of crystals whose methanol content had dropped below 10 ppm (by GC).

Claims

1. A method for reducing the amount or concentration of organic solvent residues in or on a crystalline oligosaccharide hydrate, comprising the step of exposing the crystalline oligosaccharide hydrate to water vapor.

2. The method of claim 1, wherein the step of exposing the oligosaccharides to water vapor is performed in a container, the interior space of the container having or maintained at an elevated humidity compared to the exterior of the container.

3. The method of claim 2, wherein the elevated humidity in the interior space of the container is or is maintained in the range of 60-100 RH%.

4. A process according to any one of the preceding claims, wherein the amount or concentration of organic solvent residues in or on the crystalline oligosaccharide hydrate is reduced to 100 ppm or less.

5. The method according to any one of the preceding claims, wherein the method is carried out under vacuum.

6. The method according to any one of the preceding claims, wherein the oligosaccharide is a human milk oligosaccharide.

7. The method of claim 6, wherein the human milk oligosaccharide is selected from LNnT polymorph III, DFL hydrate, LNT hydrate and pLNnH hydrate.

8. The process according to any one of the preceding claims, wherein the organic solvent is a lower alcohol, a ketone, acetic acid, pyridine, dimethyl sulfoxide, DMF, ethyl acetate or ether.

9. The process according to claim 8, wherein the lower alcohol is a C1-C4 alkanol, in particular methanol.

10. A crystalline oligosaccharide hydrate, wherein or thereon, comprises an organic solvent residue in an amount of not more than 100 ppm. The crystalline oligosaccharide hydrate according to claim 10 , which is a crystalline human milk oligosaccharide hydrate.

12. The crystalline human milk oligosaccharide hydrate according to claim 11, which is selected from LNnT polymorph III, DFL hydrate, LNT hydrate and pLNnH hydrate.

13. A nutritional formulation comprising the crystalline oligosaccharide hydrate according to any one of claims 10 to 12.

14. A pharmaceutical preparation comprising the crystalline oligosaccharide hydrate according to any one of claims 10 to 12.

15. Use of water vapour for reducing the amount or concentration of organic solvent residues contained in or on a crystalline oligosaccharide hydrate, preferably a human milk oligosaccharide hydrate.

16. A crystalline oligosaccharide hydrate comprising therein or thereon an organic solvent residue in an amount of up to 100 ppm, which is obtained by the process according to claim 1. The crystalline oligosaccharide hydrate according to claim 16 , which is a crystalline human milk oligosaccharide hydrate.

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