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    • 专利摘要:
    PROCESS FOR THE PURIFICATION OF A NEUTRAL HUMAN MILK OLIGOSACARIDE, NEUTRAL HUMAN MILK OLIGOSACARIDE, AND USE OF A NEUTRAL HUMAN MILK OLIGOSACARIDE. The present application discloses a process for the purification of a neutral human milk oligosaccharide (neutral HMO). The process uses simulated moving bed chromatography (SMB) which allows for the continuous purification of large quantities of high purity HMOs. Unlike neutral HMO chemical synthesis pathways, and their subsequent purification, the presented process allows the supply of HMOs free of harmful chemicals, such as, for example, trace amounts of heavy metals or organic solvents. The individual neutral HMO product can be obtained in solid form by spray drying or as a concentrated syrup. The supplied neutral HMO is well suited for use in food applications.
    公开号:BR112016007342B1
    申请号:R112016007342-8
    申请日:2014-10-02
    公开日:2020-11-17
    发明作者:Stefan Jennewein；Markus HELFRICH
    申请人:Jennewein Biotechnologie Gmbh；
    IPC主号:
    专利说明:

    [001] The present application discloses a process for the purification of a neutral human milk oligosaccharide (neutral HMO). The process uses simulated moving bed chromatography (SMB) which allows for continuous purification of large quantities of high purity HMOs. Unlike the chemical synthesis pathways of neutral HMOs, and their subsequent purification, the presented process allows the supply of HMOs free of harmful chemicals, such as, for example, trace amounts of heavy metals or organic solvents. The individual neutral HMO product can be obtained in solid form by spray drying or as a concentrated syrup. The supplied neutral HMO is well suited for use in food applications.
    [002] Human milk represents a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. Undoubtedly, the most prevalent fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides. While lactose is used as an energy source, complex oligosaccharides are not metabolized by the child. The fraction of complex oligosaccharides represents up to 1/10 of the total carbohydrate fraction and probably consists of more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and therefore cannot be found in large quantities in the milk of other mammals, such as, for example, domesticated delight-producing animals.
    [003] The existence of these complex oligosaccharides in human milk has been known for a long time, and the physiological functions of these oligosaccharides have been subjected to medical research for many decades. For some of the most abundant oligosaccharides in human milk, specific functions have already been identified.
    [004] The limited supply and difficulties in obtaining pure fractions of individual HMOs have led to the development of chemical pathways for some of these complex molecules. However, the synthesis of HMOs by chemical synthesis, enzymatic synthesis or fermentation proved to be challenging. At least large-scale quantities, as well as quantities suitable for food applications, have not been available so far. In this regard, particularly chemical synthetic pathways of specific HMOs (for example, HMO 2'-fucosyl lactose; see WO 2010/115935 Al) involve a number of harmful chemicals, which involve the risk of product contamination Final.
    [005] Due to the challenges involved in the chemical synthesis of human milk oligosaccharides, several enzymatic methods and fermentative approaches have been developed. However, these methods generate complex mixtures of oligosaccharides, i.e., the desired product is contaminated with starting material such as lactose, substrates and biosynthetic intermediates such as individual peptides and monosaccharides etc.
    [006] The processes in the prior art to purify individual oligosaccharide products from these complex mixtures are technically complex and also uneconomical for food applications. For the purification of lactose or sucrose disaccharides from complex mixtures such as whey or molasses, industrial scale processes have been developed that involve multiple crystallizations. The disadvantage of these methods is that they are designed and only lead to low yields.
    [007] For the purification of complex oligosaccharides, such as certain HMOs, gel filtration chromatography has been the method of choice until now. The disadvantage of gel filtration chromatography is that it cannot be increased effectively and is unsuitable for continuous operation. Thus, gel filtration chromatography is not economical and makes it impossible to supply certain HMOs - such as HMO 2'-fucosyl lactose - in reasonable quality and quantities and to use in human foods.
    [008] Simulated moving bed chromatography (SMB) has its origins in the mining and petrochemical industries. Currently, SMB chromatography is used by the pharmaceutical industry to separate enantiomers from racemic mixtures. SMB chromatography has already been used to separate fructose monosaccharide from fructose-glucose solutions and to separate sucrose disaccharide from sugar cane syrups or sugar beet on a large scale. However, SMB chromatography has not yet been used for the purification of HMOs, or any other complex oligosaccharide, from fermentations.
    [009] Simulated moving bed chromatography (SMB) was developed as a continuous separation process analogous to continuous chemical separation processes such as rectification. In rectification, a countercurrent is established between the liquid phase and the gas phase, which then allows the continuous application of feed and removal of product (s). In addition, countercurrent chromatographic operations, in theory, should achieve separations higher than conventional cross-current operations. However, chromatographic countercurrent operations would require the mobile and stationary phases to move in opposite directions. Thus, SMB chromatography was developed as a practical solution to the difficulties related to the concept of mobile solid chromatography material in a continuous separation chromatographic process.
    [010] The standard SMB concept involves four different zones with four externally applied currents: a feed current comprising the components to be separated, a mobile or desorbent phase current, an extract and a stream of refined material (the stream of refined material represents the least retained component (s)). These streams of liquid divide the SMB system into four different zones (each zone or section can comprise one or more columns) with the following tasks: zone I is necessary for the regeneration of the solid phase, the purpose of zone II is desorption of the less strongly desorbed material, the task of zone III is the adsorption of the strongly adsorbed material and, finally, the task of zone IV is the adsorption of the less adsorptive material. In this way, the stronger adsorption components establish a concentration wave in zone II and are transported to the extract port, while less strongly absorbed components migrate to the refined material port.
    [011] In principle, zone I and zone IV serve for the regeneration of the solid phase (regeneration zones), while zones II and III can be considered as the actual separation zones of the system (separation zones) . In addition to the four liquid current zones and the resulting zones, the system contains (for closed loop operation) a recycling pump for the mobile (desorbent) phase, which passes the mobile phase through the zones fixed in one direction. The countercurrent flow is then achieved by periodic exchange and continuous supply or withdrawal of feed, desorbent and products sequentially from one column to the next in the system.
    [012] In addition to the standard closed circuit, the 4 zone SMB system, 3 zone closed loop systems can also be used. Closed-loop 3-zone systems are economical in the event that a fresh solvent is quite inexpensive, for example, when using water or water / ethanol as the mobile phase. With the use of a 3-zone open circuit configuration, the regeneration of the liquid phase is no longer necessary, thus making zone IV obsolete.
    [013] In addition to standard SMB systems for the separation of a mixture of two components, five-zone open circuit or eight-zone closed-circuit SMB systems for the separation of more than 2 components have also been developed.
    [014] Due to the continuous mode of operation and also the possibility of using very large column sizes and mobile phase recycling, the SMB system can, in principle, be increased to production volumes of hundreds of tons.
    [015] Based on this prior art, the problem with the technique is the provision of an innovative process to provide a neutral HMO in large quantities, with high purity and free from harmful chemicals.
    [016] The problem of the technique is solved by the process according to claim 1, by the neutral HMO according to claim 14 and by the use of a neutral HMO according to claim 18. The dependent claims have advantageous modalities.
    [017] The present invention provides a process for the purification of a neutral HMO (eg 2'-fucosyl lactose) in continuous chromatography (or in a continuous manner) from a crude solution comprising the neutral HMO ( eg 2'-fucosyl lactose) and contaminants, wherein the crude solution comprising neutral HMO (eg 2'-fucosyl lactose) and contaminants comprises or consists of a solution which is selected from the group consisting of in microbial fermentation extract, biocatalysis reaction solution, chemical synthesis solution and combinations thereof, and in which the purity of the neutral HMO (for example, 2'-fucosyl lactose) in the solution is <80%. The process is distinguished by the fact that the crude solution is applied to at least one purification step using simulated moving bed chromatography. In this way, a purified solution comprising the desired neutral HMO (e.g., 2'-fucosyl lactose) with a purity of> 80% is provided.
    [018] The process for purifying a neutral HMO in a continuous chromatography can be a process for purifying a neutral HMO in a continuous manner. In this regard, the neutral HMO can be 2'-fucosyl lactose or lacto-N-tetraose.
    [019] The Applicant has found that, with the developed process that involves a purification step using simulated moving bed chromatography, HMOs can be endowed with high purity, without heavy metal contaminants and in a continuous manner. In this way, large quantities of high quality HMOs can be supplied in a very convenient and economical way, for example, from a crude solution of microbial fermentation. The process of the invention has also become highly stable even without a regeneration step of the column material (e.g., cation column material) used in the simulated moving bed chromatography step. In fact, the complete process can be operated in a stable manner and continues for several months.
    [020] In a preferred embodiment, the purity of the neutral HMO in the crude solution is ^ 70%, ^ 60%, ^ 50%, ^ 40%, ^ 30%, ^ 20%, ^ 10% or ^ 5%, and / or the purified solution contains the HMO with a purity of> 80%, preferably> 90%. The term "crude solution" refers to a solution containing neutral HMO prior to the single mobile bed chromatography purification step, whereas the purified solution refers to a solution after the single mobile bed chromatography step.
    [021] At least one simulated moving bed chromatography step may have at least 4 columns, preferably at least 8 columns, more preferably at least 12 columns, at least one column comprising a weak or cation exchange resin strong, preferably, a cation exchange resin in the form of H + or in the form of Ca2 +; and / orii) four zones I, II, III and IV with different flow rates; and / oriii) an eluent comprising or consisting of water, preferably ethanol and water, more preferably, from 5 to 15% by volume of ethanol and 85 to 95% by volume of water, most preferably from 9 to 11 % by volume of ethanol and 89 to 91% by volume of water, wherein the eluent optionally comprises additional sulfuric acid, preferably, 10 mM sulfuric acid; more preferably, 5 to 5 mM sulfuric acid; and / or iv) an operating temperature of 15 to 60 ° C, preferably 20 to 55 ° C, more preferably, 25 to 50 ° C.
    [022] If the HMO to be purified is 2'-fucosyl lactose, at least one simulated moving bed chromatography step may have) four zones I, II, III and IV with different flow rates, at which flow rates they are preferably: 28 to 32 ml / min in zone I, 19 to 23 ml / min in zone II, 21 to 25 ml / min in zone III and / or 16 to 20 ml / min in zone IV; and / orii) a feed rate of 2 to 4 ml / min, preferably 3 ml / min; and / ouiii) an eluent flow rate of 10 to 13 ml / min, preferably 11.5 ml / min; and / oriv) a switching time of 16 to 20 min, preferably 17 to 19 min, more preferably, 18 min.
    [023] Preferably, at least one of the columns comprises 0.1 to 5000 kg of cation exchange resin, preferably 0.2 to 500 kg of cation exchange resin, more preferably, 0.5 to 50 kg of exchange resin cationic, with maximum preference, 1.0 to 20 kg of cation exchange resin.
    [024] Mainly, the increase in the amount of cation exchange material, the flow rate in the different zones, the feed rate, the eluent flow rate and / or the switching time is possible. The increase can be in a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all factors scaling possible among said values.
    [025] In columns, a strong cation exchange resin can be used as a stationary phase. Preferably, the cation exchange resin is a sulfonic acid resin, more preferably, a Purolite® PCR833H resin (Purolite, Ratingen, Germany), Lewatit MDS 2368 and / or Lewatit MDS 1368. If a cationic ion exchange resin is used in columns, it can be regenerated with sulfuric acid. Sulfuric acid can be used in the eluent, preferably at a concentration of sulfuric acid 10 mM or less. The cation exchange resin (strong) can be present in the form of H + or in the form of Ca2 +.
    [026] Operating temperatures above 60 ° C are not preferable during simulated cell chromatography. It was found that, especially in the presence of a strong cationic ion exchange resin (in the form of H + or in the form of Ca2 +) as a stationary phase, the applied neutral oligosaccharides were significantly destabilized, that is, depolymerized, which was detrimental to the final yield of neutral HMO.
    [027] In an advantageous embodiment of the present invention, the present invention is distinguished by the fact that the purified solution is applied to at least one additional purification step with the use of simulated moving bed chromatography, in which a purified solution comprising the neutral human milk oligosaccharide with a purity of> 90%, preferably> 92%; more preferably,> 93% is provided. In particular, the present invention yields a HMO product free of recombinant DNA, and free of host strain proteins.
    [028] Additional simulated moving bed chromatography may have at least 4 columns, preferably at least 8 columns, more preferably at least 12 columns, wherein at least one column comprises a weak or strong cation exchange resin, preferably , a cation exchange resin in the form of H + or in the form of Ca2 +; and / ouii) four zones I, II, III and IV with different flow rates, and / oriii) an eluent that comprises or consists of water, preferably ethanol and water, more preferably, from 5 to 15% by volume of ethanol and 85 to 95% by volume of water, most preferably 9 to 11% by volume of ethanol e89 to 91% by volume of water, where the eluent optionally additionally comprises sulfuric acid, preferably acid sulfuric 10 mM; more preferably, 2 to 5 mM sulfuric acid, and / or) an operating temperature of 15 to 60 ° C, preferably 20 to 55 ° C, more preferably, 25 to 50 ° C.
    [029] If the HMO to be purified is 2'-fucosyl lactose, the additional simulated moving bed chromatography step may have four) zones I, II, III and IV with different flow rates, where flow rates are preferably: 28 to 32 ml / min in zone I, 19 to 23 ml / min in zone II, 21 to 25 ml / min in zone III and / or 16 to 20 ml / min in zone IV; and / orii) a feed rate of 2 to 4 ml / min, preferably 3 ml / min; and / ouiii) an eluent flow rate of 10 to 13 ml / min, preferably 11.5 ml / min; and / ouiv) a switching time of 16 to 20 min, preferably 17 to 19 min, more preferably, 18 min.
    [030] Particularly, at least one of the columns contains 0.1 to 5000 kg of cation exchange resin, preferably 0.2 to 500 kg of cation exchange resin, more preferably, 0.5 to 50 kg of exchange resin cationic, with maximum preference, 1.0 to 20 kg of cation exchange resin.
    [031] Mainly, an increase in the amount of cation exchange material, the flow rate in the different zones, the feed rate, the eluent flow rate and / or the switching time is possible. The increase can be in a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all factors scaling possible among said values.
    [032] After a purification step using simulated moving bed chromatography, the pH of the purified solution can be adjusted to pH 7, preferably by adding a base, more preferably by adding NaOH (for example, NaOH 0.2 M).
    [033] According to the present invention, the crude solution containing the neutral HMO and contaminants comprises or consists of a solution that is selected from the group consisting of microbial fermentation, microbial fermentation extract, biocatalysis reaction solution , chemical synthesis solution and combinations thereof. Fermentation as a source of neutral HMO has the advantage that it is more cost-effective than chemical synthesis or biocatalysis, that is, enzymatic synthesis. Thus, a microbial fermentation extract is preferred.
    [034] Preferably, before applying the solution to at least one simulated moving bed chromatography step, the solution (preferably, a microbial fermentation solution) comprising the neutral HMO is filtered or centrifuged to remove the biomass and / or any insoluble material, preferably filtered with activated carbon, charcoal, celite and / or by cross-flow filtration to remove any insoluble material and organic contaminants, more preferably, filtered by cross-flow filtration, most preferably filtered by cross-flow filtration using a microfiltration membrane; and / orii) applied to at least one purification step using cation and / or anion exchange chromatography, preferably first, at least one cation exchange chromatography step and, subsequently, at least one chromatography step of anion exchange.
    [035] In an additional preferred embodiment, before applying the solution to at least one purification step using simulated moving bed chromatography or after a purification step using simulated moving bed chromatography, the solution containing the neutral HMO is subjected to electrodialysis and / or diafiltration, preferably subjected to diafiltration with a nanofiltration membrane, more preferably, subjected to diafiltration with a nanofiltration membrane that has a size exclusion limit of ^ 20 Â. Most preferably, the solution is subjected to dialysis at a conductivity of ^ 15 mS / cm, preferably, ^ 10 mS / cm, more preferably, ^ 5 mS / cm.
    [036] If the crude solution is subjected to dialysis prior to application of the solution to at least one purification step using simulated moving bed chromatography, larger contaminants depend on the origin of the neutral HMO fractions (ie, chemical synthesis , biocatalysis or fermentation). Typical contaminants are monosaccharides (for example, glucose, galactose, fucose, etc.), disaccharides (for example, lactose) and by-products (for example, lactulose). In the case where fermentation was used as a neutral HMO source, the crude solution normally comprises the carbon source employed (for example, glycerol, sucrose and / or glucose), as well as by-products of the microbes employed (for example, oligosaccharides of higher molecular weight) as contaminants. As additional contaminants, oligosaccharides that are generated due to the promiscuity of the glycosyltransferases used (for example, glycosyltransferases in the synthesis cell that convert the desired product, the substrate or an intermediate product into a contaminating oligosaccharide) may also be present. Said contaminants can be removed effectively by a purification step using simulated moving bed chromatography (SMB).
    [037] After dialysis, preferably after electrodialysis and / or diafiltration (optionally before application of the solution to the SMB chromatographic process), the solution comprising the HMO can be concentrated, in particular) to a concentration of> 50 g / 1,> 100 g / 1, preferably> 200 g / 1, more preferably> 300 g / 1; and / orii) employ a vacuum concentrator; and / or iii) by nanofiltration; and / oriv) at a temperature of 4 to 50 ° C, preferably 10 to 45 ° C, optionally 20 to 40 ° C or 30 to 35 ° C.
    [038] More preferably, the fraction comprising HMO is concentrated using nanofiltration. The nanofiltration step can additionally be used to remove contaminating salts by dialysis. In this document, the HMO fraction can first be concentrated by nanofiltration, and the resulting concentrated HMO fraction is then subsequently diluted with water, preferably bidistilled H2O (ddH2O) or deionized water, and then the fraction of Diluted HMO can again be concentrated using a nanofiltration membrane.
    [039] Nanofiltration concentration is particularly preferred due to the fact that an exposure of neutral HMOs to high temperatures can be dispensed with. Thus, the said method of concentration is less destructive to the structure of HMOs than a heat treatment, that is, it does not induce thermal damage to neutral HMOs during the concentration. An additional advantage of nanofiltration is that it can be used for both concentration and dialysis (diafiltration) of neutral HMOs. In other words, a membrane used in nanofiltration does not have to be changed if the concentration step and the dialysis step are implemented in succession in the process of the invention. In addition, the salt concentration of the solution containing neutral HMOs can be significantly reduced. This saves material and time and makes the entire process more economical. Preferably, nanofiltration is combined with electrodialysis. This combination generated excellent results in concentration and desalination.
    [040] In a preferred embodiment of the present invention, the purified solution is sterilized by filtration and / or subjected to the removal of endotoxins, preferably by filtration of the purified solution through a 3 kDa filter.
    [041] The purified solution can be spray dried, particularly spray dried at a neutral HMO concentration of 20 to 60% (w / v), preferably 30 to 50% (w / v), more preferably 35 to 45% (w / v), an inlet temperature of 110 to 150 ° C, preferably 120 to 140 ° C, more preferably, 125 to 135 ° C and / or an outlet temperature of 60 to 80 ° C, preferably , 65 to 70 ° C.
    [042] In a preferred embodiment of the process of the invention, the neutral HMO that must be purified is a neutral HMO that has more than 3 monosaccharide units, preferably a neutral human milk trisaccharide, tetrasaccharide, pentasaccharide or hexassaccharide. More preferably, the neutral HMO is selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, 2 ', 3-difucosyl lactose, lacto-N-triose II, lacto-N-tetraose, lacto-N -neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose and difucosil -lacto-N-neohexaose. Most preferably, the neutral HMO is 2'-fucosyl lactose or lacto-N-neotetraose.
    [043] Optionally, it is also possible that the neutral HMO is not 2'-fucosyl lactose.
    [044] In an advantageous embodiment of the present invention, the process of the invention is distinguished by the fact that the crude solution comprising neutral human milk contaminants and oligosaccharides comprises or consists of a microbial fermentation extract, in which the fermentation extract microbial is obtained in at least one step of microbial fermentation which is preferably followed by at least one step of a) filtration of a solution, preferably of the crude solution, to separate soluble material from insoluble material after microbial fermentation; and / orb) ion exchange chromatography, preferably, cation exchange chromatography, more preferably, cation exchange chromatography followed by anion exchange chromatography, of a solution, preferably, of a solution obtained in step a); and / orc) concentration of a solution, preferably of a solution obtained in step b), more preferably, by evaporation of water and / or by nanofiltration, optionally by concentration more than once; and / ord) dialysis of a solution, preferably of a solution obtained in step c), more preferably, by electrodialysis and / or diafiltration, with the most preference, diafiltration with a nanofiltration membrane, optionally by dialysis more than once; e / oue) chromatography of a solution using simulated moving bed chromatography, preferably of a solution obtained in step d); e / ouf) filtering a solution, preferably a solution obtained in step e), to separate the oligosaccharide from neutral human milk from colored contaminants, more preferably, by filtration through activated carbon; and / or g) spray drying a purified solution comprising the neutral human milk oligosaccharide, preferably a purified solution obtained in step f).
    [045] With the utmost preference, all steps a) to g) are implemented in succession. It has been found that the implementation of all steps a) to g) in succession is the most advantageous embodiment of the process of the invention. Said process is efficient in terms of cost and time and allows the supply of large quantities of highly pure neutral HMOs, spray-dried (ie, amorphous) from microbial fermentation (extracts). In particular, the concentration and desalination steps of the HMO solution with the use of nanofiltration represent extremely cost-effective and smooth operating steps that prevent the formation of unwanted by-products.
    [046] The present invention thus provides a neutral HMO that is feasible with the process of the invention. The neutral HMO (for example, 2'-fucosyl lactose or lacto-N-tetraose) is preferably spray dried. The purified neutral HMO has the advantage of being highly pure and free of heavy metal contaminants and / or organic solvents.
    [047] The neutral HMO according to the present invention may have a) solid granule form; and / orii) a glass transition temperature of 60 to 90 ° C, preferably 62 to 88 ° C, more preferably 64 to 86 ° C, determined by differential scanning calorimetry; and / ouiii) a particle size of 5 to 500 pm, preferably 10 to 300 pm, determined by laser diffraction; and / ouiv) an average particle size of 10 to 100 pm, preferably 20 to 90 pm, more preferably 30 to 80 pm, most preferably 40 to 70 pm, determined by laser diffraction; e / heard) an amorphous state, preferably an amorphous state without characteristic peaks of crystalline matter in powder X-ray diffraction, and / heard) a moisture content of 10%, preferably 8%, more preferably, <5% .
    [048] Neutral HMO can be used in medicine, preferably in the prophylaxis or therapy of gastrointestinal disorders. This can also be used in nutrition, preferably medicinal nutrition or nutrition of dairy products (for example, cereal products).
    [049] In a preferred embodiment of the present invention, the neutral human milk oligosaccharide is a neutral HMO that has more than 3 monosaccharide units, preferably a neutral human milk trisaccharide, tetrasaccharide, pentasaccharide or hexassaccharide. More preferably, the neutral HMO is selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, 2 ', 3-difucosyl lactose, lacto-N-triose II, lacto-N-tetraose, lacto-N -neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose and difucosil -lacto-N-neohexaose. Most preferably, the neutral HMO is 2'-fucosyl lactose or lacto-N-neotetraose.
    [050] Optionally, it is also possible that the neutral HMO is not 2'-fucosyl lactose.
    [051] In addition, it is proposed to use the neutral HMO according to the present invention as an additive in food, preferably as an additive in human food and / or in pet food, more preferably, as an additive in human baby food.
    [052] With reference to the following Figures and Examples, the matter according to the present invention is intended to be explained in more detail without wishing to restrict said matter to the special modalities shown in the present invention.
    [053] Figure 1 schematically illustrates a purification step using simulated moving bed chromatography. Simulated moving bed chromatography can have, for example, 12 columns in a series 1 arrangement, where the arrangement is divided into four different zones I, II, III and IV. The crude solution containing the contaminants and neutral HMO 2'-fucosyl lactose is applied between zone II and zone III at feed inlet 2. The extract is removed from outlet 4 between zone I and zone II, at the whereas the refined material is removed at the outlet 3 between zone III and zone IV. The refined material at outlet 3 contains the purified HMO 2'-fucosyl lactose, while the extract at outlet 4 contains low molecular weight contaminants (e.g., monosaccharides and disaccharides).
    [054] Figure 2 schematically illustrates two subsequent purification steps using simulated moving bed chromatography. Each simulated moving bed chromatography can have, for example, 12 columns in a 1, 1 'series arrangement, where each arrangement is divided into four different zones Ia, Ila, Ilia and IVa, or zones Ib, Ilb, Illb and IVb, respectively. The crude solution comprising the contaminants and neutral HMO 2'-fucosyl lactose is applied between zone Ila and Ilia of the first provision 1 at feed inlet 2. The extract is removed at outlet 4 between zone Ia and zone lia , while the refined material leaves the first series 1 arrangement at outlet 3 between zone Ilia, and zone IVa and is applied to the second series arrangement 1 'between zone Ilb and Illb. In the second series arrangement r, the extract is removed at outlet 6, while the refined material is removed at outlet 5 between zone Illb and zone IVb. The refined material at exit 5 contains highly purified 2'-fucosyl lactose, while the extract at exit 6 contains high molecular weight contaminants (for example, higher oligosaccharides).
    [055] Figure 3 schematically illustrates a preferred purification scheme according to the present invention. First, a solution 7 containing the contaminants and neutral HMO 2'-fucosyl lactose is applied to an electrodialysis step 8 until a conductivity of 0.5 mS / cm is obtained. Said solution is concentrated until the solution has reached a concentration of 2'-fucosyl lactose of approximately 40% (w / v). Subsequently, said solution is applied to at least one purification step using simulated moving bed chromatography 9. After simulated moving bed chromatography, a purified solution comprising high purity 2'-fucosyl lactose is obtained. Said pure solution is subjected to sterile filtration 11 (preferably, also removal of endotoxins). Prior to sterile filtration 11, an additional electrodialysis step 10 with subsequent concentration can optionally be performed. After sterile filtration 11, the purified solution comprising 2'-fucosyl lactose is subjected to spray drying 12 and 2'-fucosyl lactose dried by pure spray 13 is obtained in the form of a solid granule.
    [056] Figure 4 shows the result of a powder X-ray diffraction analysis of two samples of spray dried 2'-fucosyl lactose according to the present invention (sample n-1 and sample n-2). The two diffractograms obtained reveal that both sample n-1 and sample n2 2 are in a totally amorphous state (without characteristic peaks of crystalline matter).
    [057] Figure 5 shows the particle size distribution of spray-dried 2'-fucosyl lactose according to the present invention (sample no. 1 and sample no. 2) determined by laser diffraction. An average particle size of approximately 68 pm was determined for sample No. 1. Sample No. 2 had an average particle size of approximately 44 pm. Both values are considered to be high for a spray dried product.
    [058] Figure 6 shows the biosynthesis of the lacto-N-neotetraose 17 tetrasaccharide from human milk. Biosynthesis begins with the disaccharide lactose 14 which is converted by β-1,3-N-acetyl-glucosamintransferase 15 into the trisaccharide lacto-N-triose 16. The trisaccharide lacto-N-triose 16 is subsequently converted into the lacto-N- tetrasaccharide neotetraose 18 by the enzyme 1,4-galactosyltransferase 17. In vivo, a certain percentage of lacto-N- neotetraose 18 is additionally converted to larger oligosaccharides 19, 20, 21 by β-1,3, N-acetyl-glucosamintransferase 15 and 1,4-galactosyltransferase 17. If the purpose of the process of the invention is the purification of lacto-N-neotetraose 17, the larger oligonucleotides, as well as the smaller oligonucleotides (educts) lactose 14 and lacto-N-triosis 16 can be present as contaminants 22 in a crude solution containing lacto-N-neotetraose 17. The process of the invention allows efficient removal of said contaminants 22.
    [059] Figure 7 shows a comparison of two different nanofiltration membranes for the concentration of a 2'-Fucosyl lactose containing solution by nanofiltration (VCF: volumetric concentration factor, Flow: express the rate at which water permeates the membrane nanofiltration). The Alfa Lavai type NF99 (NF) nanofiltration membrane and the Alfa Lavai type NF99HF nanofiltration membrane were used as the nanofiltration membrane. It can be seen that in VCF 6, the nanofiltration membrane NF99HF allows a greater flow, that is, a faster concentration of the solution.
    [060] Figure 8 shows the HPLC analysis of the feed (Figure 8A) and the refined material (Figure 8B) of the SMB chromatography of Example 6 (sucrose was used as an internal standard; see Figure 8A). Like the analytical column, a ReproSil Carbohydrate column (amino-functionalized silica column; 5 -pm; 250 x 4.6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4 ml / min. As the eluent, a mixture of acetonitrile and water (68% by volume: 32% by volume) was used. The elution was isocratic. The injection volume was 20 pl. The oven temperature was 35 ° C. In SMB chromatography, a strong cation exchange resin that was present in the form of H + was used.
    [061] Figure 9 shows the HPLC analysis of the SMB chromatography extract from Example 6. As the analytical column, a ReproSil Carbohydrate column (amino functionalized silica column; 5 pm; 250 x 4.6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4 ml / min. As the eluent, a mixture of acetonitrile and water (68% by volume: 32% by volume) was used. The elution was isocratic. The injection volume was 20 pl. The oven temperature was 35 ° C. In SMB chromatography, a strong cation exchange resin that was present in the form of H + was used.
    [062] Figure 10 shows the HPLC analysis of the extract from the second SMB chromatography (= additional SMB chromatography according to the present invention) from Example 6. As the analytical column, a ReproSil Carbohydrate column (silica column functionalized with amino; 5 pm; 250 x 4.6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4 ml / min. As the eluent, a mixture of acetonitrile and water (68% by volume: 32% by volume) was used. The elution was isocratic. The injection volume was 20 pl. The oven temperature was 35 ° C. In the SMB chromatography, a strong cation exchange resin that was present in the form of H + was used.
    [063] Figure 11 shows the HPLC analysis of the feed (Figure 11A) and the refined material (Figure 11B) of the SMB chromatography of Example 7. As the analytical column, a ReproSil Carbohydrate column (amino-functionalized silica column; 5 pm; 250 x 4, 6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4 ml / min. As the eluent, a mixture of acetonitrile and water (68% by volume: 32% by volume) was used. The elution was isocratic. The injection volume was 20 pl. The oven temperature was 35 ° C. In this SMB chromatography, a strong cation exchange resin that was present in the form of Ca was used.
    [064] Figure 12 shows the HPLC analysis of the SMB chromatography extract of Example 7. As the analytical column, a ReproSil Carbohydrate column (amino functionalized silica column; 5 pm; 250 x 4.6 mm; Dr. Maisch GmbH; Ammerbuch) was employed with a flow rate of 1.4 ml / min. As the eluent, a mixture of acetonitrile and water (68% by volume: 32% by volume) was used. The elution was isocratic. The injection volume was 20 pl. The oven temperature was 35 ° C. In this SMB chromatography, a strong cation exchange resin that was present in the form of Ca2 + was used. EXAMPLE 1: PURIFICATION OF 2'-FUCOSILLACTOSECOMING USED SIMPLIFIEDELECTED MOBILE (SMB CHROMATOGRAPHY)
    [065] A clear particle-free solution containing 2'-fucosyl lactose in a concentration of 250 g / 1 was subjected to electrodialysis at 0.5 mS / cm using a PC-Cell BED 1-3 electrodialysis device ( PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack. Said pile comprised the following membranes: CEM exchange membrane CEM: PC SK and the AEM exchange membrane AEM: PcAcid60 which has a size exclusion limit of 60 Da.
    [066] For the purification of SMB, the 2'-fucosyl lactose solution was concentrated to 300 g / 1 using a vacuum concentrator at 40 ° C. For the SMB chromatography, a closed circuit SMB system equipped with 12 columns (Prosep® columns with dimensions: 40 mm x 740 mm (Latek, Eppelheim, Germany)) arranged in 4 zones. Each column comprised 760 g of Purolite® PCR833H + strong cation-exchange resin (Purolite, Ratingen, Germany).
    [067] The system was operated at a temperature of 25 ° C with the following configuration of flow parameters: the flow rate of zone I was 30.00 ml / min, the flow rate of zone II was set to 21 .00 ml / min, zone III flow rate was 21.48 ml / min, zone IV flow rate was set at 18.44 ml / min, feed was set at 3.00 ml / min , the eluent flow was set at 11.56 ml / min, and the switching time was set at 17.92 min. As the eluent, water with 10% (v / v) ethanol for food products was used.
    [068] Larger contaminants such as lactose, monosaccharides such as fucose, glucose, galactose and glycerol, were fractionated in the extract. 2'-fucosyl lactose and larger oligosaccharide contaminants (eg, difucosyl lactose) were fractionated in the refined material.
    [069] 2'-fucosyl lactose was slightly diluted through the SMB purification step - the concentration of 2'-fucosyl lactose in the refined material was determined at 200 g / 1. The pH of the refined material was adjusted to pH 7 using 0.2 N NaOH. In the configurations described, SMB systems can be continuously operated for at least 3 months.
    [070] Then, the obtained solution was again subjected to electrodialysis until a conductivity of less than 0.5 mS / cm was obtained and concentrated to obtain a 40% (w / v) solution of 2'-fucosyl lactose.
    [071] The solution was then subjected to sterile filtration and endotoxin removal by passing the solution through a 3 kDa filter (Pall Microza SEP-2013 hollow fiber ultrafiltration module, Pali Corporation, Dreieich).
    [072] Using this protocol, 2'-fucosyl lactose with a purity of 90.4% can be obtained. Major contaminants were 3'-fucosyl lactose (2.6%), difucosyl lactose (1.5%) and lactose (1.4%). The purification yield was approximately 80%. EXAMPLE 2: PURIFICATION OF 2 "-FUCOSIL LACTOSE WITH THE USE OF MULTIPLE COMPONENT SMB CHROMATOGRAPHIC SEPARATION
    [073] A clear particle-free solution comprising 2'-fucosyl lactose in a concentration of 250 g / 1 was subjected to electrodialysis at 0.5 mS / cm using a PC-Cell BED electrodialysis device 1-3 (PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack. Said pile comprised the following membranes: CEM exchange membrane CEM: Pc SK and anion exchange membrane AEM: Pc Acid 60 with a size exclusion limit of 60 Da.
    [074] For the purification of SMB, the 2'-fucosyl lactose solution was concentrated to 300 g / 1 using a vacuum concentrator at 40 ° C. For SMB chromatography, a closed circuit multi-component SMB system equipped with 24 columns (Prosep® columns with dimensions: 40 mm x 740 mm (Latek, Eppelheim, Germany)) arranged in 2 x 4 zones. Each column contained 760 g of Purolite® PCR833H + strong cation-exchange resin (Purolite, Ratingen, Germany).
    [075] The system was operated at 25 ° C with the following configuration of flow parameters: the flow rate of zone Ia was 30.00 ml / min, the flow rate of zone lia was set to 21.00 ml / min, the flow rate of the Ilia zone was set to 21.48 ml / min, the flow rate of the zone IVa was set to 18.44 ml / min, the feed was set to 3.00 ml / min, the eluent flow was set at 11.56 ml / min, and the switching time was set at 17.92 min.
    [076] The refined material from the first separation was passed at a flow rate of 3.04 ml / min to a second separation step. The flow rate of zone Ib was maintained at 30 ml / min, the flow rate of zone Ilb was set to 19.61 ml / min, the flow rate of zone Illb was set to 21.63 ml / min, at flow rate of zone IVb was set at 18.46 ml / min, eluent flow was set up similarly to 11.56 ml / min, and the switching time of zones Ib to IVb was 10.46 min.
    [077] As the eluent, water with 10% (v / v) deethanol for food products was used.
    [078] In the separation of multiple components, contaminants such as lactose, monosaccharides such as fucose, glucose, galactose and glycerol were found in the extract from the first separation step and larger oligosaccharide contaminants (for example, difucosyl lactose) were fractionated in the refined material the second separation step.
    [079] 2'-fucosyl lactose was fractionated in the refined material of the first separation step and in the extract of the second separation step and was thus free of low and high molecular weight contaminants. 2'-fucosyl lactose was only slightly diluted through the SMB purification step - the concentration of 2'-fucosyl lactose in the extract from the second purification step was determined at 200 g / 1.
    [080] The pH of the refined material after the first separation step was adjusted to pH 7 using 0.2 N NaOH.
    [081] Using this protocol, 2'-fucosyl lactose with a purity of 93.0% can be obtained. Major contaminants were 3'-fucosyl lactose (1.1%), difucosyl lactose (0.9%) and lactose (1.0%). EXAMPLE 3: OBTAINING 2'-FUCOSIL LACTOSE IN SOLID FORM THROUGH ASPERSION DRYING
    [082] The 2'-fucosyl lactose fractions obtained by SMB chromatography were again subjected to electrodialysis treatment until a conductivity of less than 0.5 mS / cm was obtained. The fractions were then concentrated in vacuo to obtain fractions of 2'-fucosyl lactose which contain 40% (w / v) of 2'-fucosyl lactose. The solutions were subsequently subjected to sterile filtration and endotoxin removal by passing the solution through a 3 kDa filter (hollow fiber module of Pali Microza SEP-2013 ultrafiltration, Pali Corporation, Dreieich, Germany).
    [083] The sterile 2'-fucosyl lactose solutions obtained in this way were then spray dried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany). For spray drying of 2'-fucosyl lactose, the 40% (w / v) solution was passed under pressure through the spray drying nozzles with an inlet temperature set at 130 ° C. The flow was adjusted to maintain an outlet temperature between 66 ° C and 67 ° C.
    [084] Using these settings, a spray-dried powder with less than 5% moisture can be obtained. The moisture content was determined by Karl-Fischer titration. EXAMPLE 4: CHARACTERIZATION OF DRY 2'-FUCOSIL LACTOSE BY ASPERSION DIFFERENTIAL SCAN CALORIMETRY (DSC)
    [085] A Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine the thermal events of two samples (sample # 1 and sample # 2) of spray dried 2'-fucosyl lactose.
    [086] Approximately 25 mg of a spray-dried sample was analyzed in Al corrugated crucibles (Mettler Toledo, Giessen, Germany). The samples were cooled to 0 ° C to -263.15 ° C / min (10 K / min) and reheated to 100 ° C at a scan rate of -263.15 ° C / min (10 K / min). After cooling the samples to 0 ° C in a second heating cycle, the samples were heated to 150 ° C. The midpoint of the baseline endothermic change during the heating scan was taken as Tg. Exothermic and endothermic peaks are reported using the peak temperature and normalized energy of the event. The results of the DSC analysis of the samples are summarized in na: not detected Table 1.

    [087] DSC analysis of sample # 1 revealed a major glass transition (Tg) at 67.4 ° C in the 2nd heating scan. A second small Tg was also detected at 122.6 ° C in the 2nd heating scan. The main glass transition was detected in the first heating scan followed by an exothermic and endothermic event at temperatures above Tg. These events are attributed to relaxation effects in the sample.
    [088] DSC analysis of sample # 2 showed a substantially higher glass transition (Tg) at 84.6 ° C in the 2nd heating scan that was also detected in the 1st heating scan. This may point to a lower residual water content of sample n-2 compared to sample n2 1. Since the glass transition was detected close to the final temperature of the 1st heating scan, potential relaxation phenomena may not be detected. In this sample, a second glass transition may not be detected, although a small exothermic peak at 125.9 ° C was visible on the 2nd heating scan. X-RAY DIFFERENCE
    [089] Wide angle powder X-ray diffraction (XRD) was used to study the morphology of samples n- 1 and n- 2. The Empyrean X-ray diffractometer (Panalytical, Almelo, Netherlands) equipped with an anode of copper (45 kV, 40 mA, Kαi emission at a wavelength of 0.154 nm) and a PIXcel3D detector was used. Approximately 100 mg of the spray dried samples were analyzed in reflection mode in the angular range of 5 to 45 ° 2θ, with a step size of 0.04 ° 20 and a counting time of 100 seconds per step.
    [090] XRD analysis of samples # 1 and # 2 revealed the completely amorphous state of both samples and showed no characteristic peaks in crystalline matter (see Figure 4 for an overlap of both diffractograms). LASER DIFFERENCE
    [091] Laser diffraction measurements were performed using a Partica LA-950 Laser Diffraction Particle Size Distribution Analyzer (Horiba, Kyoto, Japan) equipped with a 605 nm laser diode to detect particles > 500 nm and 405 nm blue light-emitting diode (LED) to detect particles <500 nm.Isoctane was used as a dispersion medium (refractive index 1.391). Since the refractive index of the samples was unknown, the refractive index of sugar particles (disaccharide) was used (1.530). The samples were dispersed in isoctane by ultrasonication for up to 5 minutes. Before the measurement, the system was suppressed with isoctane. The dispersion of each sample was measured 3 times and the mean values and standard deviation are reported.
    [092] The average particle size (weighted average of particle sizes by volume) and the mode particle size (peak distribution) were recorded. In addition to the particle distribution by volume (q%), the results were recorded as: d (v, 10): particle diameter corresponding to 10% of the cumulative pass-through distribution in volumed (v, 50): particle diameter corresponding to 50% of the cumulative distribution of the passer in volume (v, 90): particle diameter corresponding to 90% of the cumulative distribution of the passer in volume
    [093] The particle size distribution for sample No. 1 and No. 2 is shown in Figure 5. The mode size, which represents the particle size of the highest intensity, is comparable for both samples. In general, the average particle size of 67.85 pm (sample no. 1) and 43.65 pm (sample no. 2), respectively, is considered to be unusually high for spray-dried particles. The fraction detected at larger particle diameters is probably caused by agglomerated dust particles.
    [094] Table 2 summarizes the particle size characteristics of sample # 1 and # 2.
    TABLE 2 EXAMPLE 5: VOLUME REDUCTION AND DESALINATION OF 2'-FUCOSIL LACTOSE THAT UNDERSTANDS SUPERB WITH THE USE OF NANOFILTRATION
    [095] For the concentration and desalination of 2'-fucosyl lactose containing culture supernatant, an Alfa Laval M-20 membrane filtration module equipped with an NF99 (Alfa Laval NF99) or NF99HF (Alfa Laval NF99HF) nanofiltration membrane was employed. The used 2'-fucosyl lactose containing culture supernatant (which contains 25 g / 1 of 2'-fucosyl lactose) was separated from the microbial fermentation biomass using cross-flow filtration. The molecular cross-section of the cross-flow filtration membrane was 150 kDa (StrassBurger Filter Micro Cross Module® FS10LFC-FUS1582).
    [096] The cell-free filtered material was then treated with a cation ion exchanger (Lewatit S2568 in proton form (Lanxess)) and anion ion exchanger (Lewatit S6368 in carbonate form (Lanxess)) before being subjected to nanofiltration. The solution was neutralized after each step of the ion exchanger using sodium hydroxide solution or hydrochloric acid, respectively. The inlet pressure of the membrane module was 4.2 MPa (42 bar), and the outlet pressure was 4.0 MPa (40 bar), the flow rate of the feed in the membrane module was 8 liters / min. The permeate material was removed from the process, while the trapped material was fed back into the reservoir and membrane module. The volume of the reservoir connected to the membrane stack was 6 liters. During the concentration of the solution comprising 2'-fucosyl lactose, the reservoir was continuously filled with 2'-fucosyl lactose culture supernatant solution until an 8-fold concentrated solution was obtained.
    [097] Figure 7 shows the flow obtained (L / m2 / h) plotted against the volume concentration factor (VCF) of the two membranes employed. Having concentrated the concentration of 2'-fucosyl lactose 8 times to approximately 200 g / 1 of 2'-fucosyl lactose, the concentrated solution was subjected to diafiltration for desalination by adding deionized water at the same rate as the permeate material was obtained of the membrane module. With the use of the diafiltration step, the conductivity of the concentrated material of 2'-fucosyl lactose can decrease from 25 mS to less than 7 mS with the use of the HF99HF membrane.
    [098] Using this nanofiltration approach, the 2'-fucosyl lactose solution can be concentrated 8 times to a concentration of 2'-fucosyl lactose of ^ 200 g / 1 2'-fucosyl lactose under mild conditions (avoiding thermal exposure), with the diafiltration step, a large fraction of the salt content can be removed. A concentrated material of 2'-fucosyl lactose was submitted to electrodialysis to further reduce the salt content. EXAMPLE 6; PURIFICATION OF LACTO-N-TETRAOSE WITH THE USE OF TWO STEPS OF SIMULATED MOBILE BED CHROMATOGRAPHY EUMA H + IONIC EXCHANGE RESIN
    [099] A clear particulate lacto-N-tetraose solution (30 g / 1) obtained from bacterial fermentation was subjected to electrodialysis at a conductivity of 0.5 mS / cm using a PC-Cell electrodialysis device ( see above). For SMB chromatography, the lacto-N-tetraose solution was concentrated at 50 g / 1 under vacuum at 40 ° C. Alternatively, the solution containing lacto-N-tetraose can be desalted and concentrated using a nanofiltration membrane (for example, HF99HF nanofiltration membrane available from Alfa Lavai).
    [0100] For SMB chromatography, a closed circuit SMB system equipped with 12 columns (Prosep® glass columns with dimensions: 40 mm x 740 mm (Lartek, Eppelheim, Germany) arranged in 4 zones was used. contained 7 60 g of strong ion exchange resin Purolite® PCR833H + The strong cation exchange resin was present in the form of H +.
    [0101] The system was operated at 25 ° C with the following configuration parameters: the zone I flow rate was set at 30.00 ml / min, the zone II flow rate was set at 19.07 ml / min, at zone IV flow rate was set at 18.44 ml / min. The feed was maintained at 1 ml / min and the eluent flow was set at 11.56 ml / min with a switching time of 17.92 min. As the eluent, water with 10% (v / v) ethanol for food products was used.
    [0102] Under these parameters, lacto-N-tetraose and larger neutral oligosaccharides were fractionated in the refined material. The purity of lacto-N-tetraose was 86.3% instead of 33.4% in the SMB feed (see Figure 7A for the HPLC analysis of the SMB feed and Figure 7B for the HPLC analysis of the refined material SMB). Contaminants such as lactose, monosaccharides and glycerol were found in the SMB extract fraction (see Figure 8 for the HPLC analysis of the SMB extract fraction). The refined material containing lacto-N-tetraose was adjusted to neutral pH using a 0.2 N NaOH solution.
    [0103] The pH of the extract containing the obtained lacto-N-tetraose was adjusted to pH 7 using 0.2 N NaOH. Then, the obtained solution was subjected to electrodialysis until a conductivity of less than 0, 5 mS was obtained again. The solution was then concentrated in vacuo to approximately 50 g / 1 lacto-N-tetraose and then sterilized by filtration by passing through a 3 kDa cross-flow filter (hollow fiber module of Ultra Micro filtration Pall Microza SEP- 2013, Pali Corporation, Dreieich).
    [0104] In order to separate the larger oligosaccharide contaminants from lacto-N-tetraose, a second separation by SMB chromatography was performed. Using the same SMB system, the parameters were changed as follows: the flow rate of zone I was 30 ml / min, the flow rate of zone II was 19.27 ml / min, the rate of zone IV flow was 17.30 ml / min. The feed was set at 2.08 ml / min, and the eluent flow was 12.70 ml / min. The refined material flow was 4.04 ml / min, and the extract flow was 10.73 ml / min. The switching time of the SMB separation was set at 10.4 6 min. As the eluent, again a 9: 1 (v / v) water / ethanol mixture was employed. HPLC analysis of the extract from the second lacto-N-tetraose separation by SMB chromatography is shown in Figure 10.
    [0105] Under these conditions, lacto-N-tetraose was fractionated in the extract, and 5 to 10% of the total amount of major neutral oligosaccharides was found in the refined material. With the use of this protocol (silica column functionalized with amino after the second stage of SMB), lacto-N-tetraose with a purity of 93.1% can be obtained. EXAMPLE 7; PURIFICATION OF LACTO-N-TETRAOSE WITH THE USE OF MOBILE BED CHROMATOGRAPHY SIMULATED WITH A CA2 + ION EXCHANGE RESIN
    [0106] A clear particle-free lacto-N-tetraose solution (30 g / 1) obtained from bacterial fermentation was subjected to electrodialysis at a conductivity of 0.5 mS / cm using a PC electrodialysis device - Cell (see above).
    [0107] For the chromatography of SMB with calcium as the counterion, the lacto-N-tetraose solution was concentrated at 50 g / 1 under vacuum at 40 ° C. For SMB chromatography, a closed circuit SMB system equipped with 12 columns (Prosep® glass columns with dimensions: 40 mm x 740 mm (Lartek, Eppelheim, Germany)) arranged in 4 zones.
    [0108] Each column contained 760 g of Purolite® PCR833H + strong cation exchange resin. The cation ion exchange resin was washed with 50 liters of 200 mM CaCl2 to exchange H ions for Ca ions. Thus, the strong cation exchange resin was present in the form of Ca2 +.
    [0109] The system was operated at 25 ° C with the following configuration parameters: the zone I flow rate was set to 30.00 ml / min, the zone II flow rate was set to 20.07 ml / min min, the zone IV flow rate was set at 17.04 ml / min. The feed was maintained at 2.5 ml / min, and the eluent flow was set to 11.56 ml / min with a switching time of 17.92 min. As the eluent, water with 10% (v / v) ethanol for food products was used. EXAMPLE 8: OBTAINING LACTO-N-TETRAOSE IN SOLID FORM THROUGH ASPERSION DRYING.
    [0110] Fractions containing lacto-N-tetraose were concentrated in vacuo to obtain a lacto-N-tetraose concentration of 25% (w / v). The solutions were then for sterilization by filtration and removal of endotoxins, passed through a 3 kDa filter (hollow fiber ultrafiltration module Pall Microza SEP-1013, Pali Corporation, Dreieich, Germany).
    [0111] The sterile lacto-N-tetraose solution obtained in this way was then spray-dried using a B-290 Mini Spray Dryer (Büchi Labortechnik GmbH, Essen, Germany). For spray drying the lacto-N-tetraose, the solution was passed under pressure through the spray dryer nozzles with an outlet temperature between 120 ° C and 130 ° C, and the flow was adjusted to maintain an escape temperature between 66 ° C and 67 ° C. Using these settings, a spray-dried powder with 7 to 9% moisture and a spray-dried yield of 72% lacto-N-tetraose can be obtained. The moisture content was determined by Karl-Fischer titration.
    权利要求:
    Claims (13)
    [0001]
    1. PROCESS FOR THE PURIFICATION OF A NEUTRAL HUMAN MILK OLIGOSACARIDE FROM A CRUDE SOLUTION, comprising: i. obtain a crude solution from microbial fermentation, chemical synthesis, enzymatic biocatalysis and / or combinations thereof, in which the crude solution comprises contaminants and neutral human milk oligosaccharides, in which the contaminants in the crude solution comprise oligosaccharides generated by a glycosyltransferase used in the synthesis of the neutral human milk oligosaccharide, and in which the purity of the neutral human milk oligosaccharide in the solution is <80%, characterized by still comprising. application of a crude solution to simulated moving bed chromatography, in which each column in the simulated moving bed chromatography comprises a cation exchange resin, eiii. obtaining a purified solution comprising the neutral human milk oligosaccharide with a purity of> 80% in a raffinate flow.
    [0002]
    2. PROCESS, according to claim 1, characterized in that the purity of the neutral human milk oligosaccharide in the crude solution is ^ 70%, ^ 60%, ^ 50%, ^ 40%, <30%, <20%, < 10% or <5%, and / or the purified solution contains the neutral human milk oligosaccharide with a purity of> 85%, preferably> 90%.
    [0003]
    PROCESS, according to either of claims 1 or 2, characterized in that the simulated mobile chromatography (teri) four zones I, II, III, IV with different flow rates; ii) an eluent comprising or consisting of water, preferably, ethanol and water, more preferably, from 5 to 15% by volume of ethanol and 85 to 95% by volume of water, with the most preference, from 9 to 11% by volume of ethanol and 89 to 91% by volume of ethanol water, in which the eluent optionally additionally contains sulfuric acid, preferably, 10 mM sulfuric acid or less; and / oriii) an operating temperature of 15 to 60 ° C, preferably 20 to 55 ° C, more preferably, 25 to 50 ° C.
    [0004]
    PROCESS according to any one of claims 1 to 3, characterized in that the purified solution is applied to at least one additional purification step using simulated moving bed chromatography, wherein a purified solution comprising the milk oligosaccharide neutral human at> 92% purity is provided.
    [0005]
    5. PROCESS according to claim 4, characterized by the additional simulated moving bed chromatography teri) four zones I, II, III and IV with different flow rates; ii) an eluent comprising or consisting of water, preferably ethanol and water, more preferably, from 5 to 15% in volume of ethanol and 85 to 95% in volume of water, with the most preference, from 9 to 11% in volume of ethanol and 89 to 91% in volume of water, in whereas the eluent optionally additionally contains sulfuric acid, preferably, 10 mM sulfuric acid or less; and / oriii) an operating temperature of 15 to 60 ° C, preferably 20 to 55 ° C, more preferably, 25 to 50 ° C.
    [0006]
    A PROCESS according to any one of claims 1 to 5, characterized in that the crude solution comprises or consists of a microbial fermentation extract, wherein the microbial fermentation extract is obtained in at least one microbial fermentation step which is preferably followed by at least one step of a) filtering a solution, preferably from the crude solution, to separate soluble material from insoluble material after microbial fermentation; b) ion exchange chromatography, preferably, cation exchange chromatography, more preferably, chromatography cation exchange followed by anion exchange chromatography, of a solution, preferably of a solution obtained in step a); c) concentration of a solution, preferably of a solution obtained in step b), more preferably, by evaporation of water and / or by nanofiltration, optionally by concentration more than once; d) dialysis of a solution, preferably of a solution obtained in step c), more preferably, by electrodialysis and / or diafiltration, with the most preference, diafiltration with a nanofiltration membrane, optionally by dialysis more than once; e) chromatography of a solution using bed chromatography simulated mobile, preferably, of a solution obtained in step d); f) filtration of a solution, preferably a solution obtained in step e), to separate the oligosaccharide from neutral human milk from colored contaminants, more preferably, by filtration through carbon activated; and g) spray drying a purified solution comprising the neutral human milk oligosaccharide, preferably a purified solution obtained in step f), in which, most preferably, all steps a) to g) are implemented in succession.
    [0007]
    PROCESS according to any one of claims 1 to 6, characterized in that before applying the solution to the simulated moving bed chromatography, the solution comprising the neutral human milk oligosaccharide is filtered to remove any insoluble material, preferably filtered with activated carbon, charcoal and / or celite to remove any insoluble material and organic contaminants, more preferably, filtered by cross-flow filtration, with the most preference, filtered by cross-flow filtration using a microfiltration membrane; and / orii) applied to at least one purification step using cation and / or anion exchange chromatography, preferably first, at least one cation exchange chromatography step and, subsequently, at least one exchange chromatography step anionic.
    [0008]
    PROCESS according to any one of claims 1 to 7, characterized in that before applying the solution to simulated moving bed chromatography or after simulated moving bed chromatography, the solution comprising the neutral human milk oligosaccharide is subjected to electrodialysis and / or subjected to diafiltration, preferably subjected to diafiltration with a nanofiltration membrane, more preferably, subjected to diafiltration with a nanofiltration membrane that has a size exclusion limit of ^ 2 0 Â, where, with the utmost preference , the solution is subjected to dialysis at a conductivity of ^ 15 mS / cm, preferably, ^ 10 mS / cm, more preferably, ^ 5 mS / cm.
    [0009]
    9. PROCESS according to claim 8, characterized in that after dialysis, the solution comprising the neutral human milk oligosaccharide is concentrated to a concentration of> 100 g / 1, preferably> 200 g / 1, more preferably ,> 300 g / 1; and / orii) employ a vacuum concentrator; and / or iii) by nanofiltration; and / oriv) at a temperature of 4 to 50 ° C, preferably 10 to 45 ° C, optionally 20 to 40 ° C or 30 to 35 ° C.
    [0010]
    PROCESS according to any one of claims 1 to 9, characterized in that the purified solution is sterilized by filtration and / or subjected to the removal of endotoxins, preferably by filtration of the purified solution through a 3 kDa filter.
    [0011]
    PROCESS according to any one of claims 1 to 10, characterized in that the purified solution is spray dried, particularly spray dried at the concentration of neutral human milk oligosaccharides of 20 to 60% (w / v), preferably 30 to 50% (w / v), more preferably 35 to 45% (w / v), a nozzle temperature of 110 to 150 ° C, preferably 120 to 140 ° C, more preferably, 125 to 135 ° C and / or an outlet temperature of 60 to 80 ° C, preferably 65 to 70 ° C.
    [0012]
    12. PROCESS according to any one of claims 1 to 11, characterized in that the neutral human milk oligosaccharide is a neutral human milk oligosaccharide that has more than 3 monosaccharide units, preferably a trisaccharide, tetrasaccharide, pentasaccharide or hexassaccharide of neutral human milk, more preferably, the neutral HMO is selected from the group consisting of 2'-fucosyl lactose, 3-fucosyl lactose, 2 ', 3-difucosyl lactose, lacto-N-triose II, lacto-N-tetraose , lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose HI, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto -N-difucohexaose I, lacto-N- difucohexaose II, 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N -neohexaose and difucosyl-lacto-N- neohexaose, with the most preference, the neutral HMO is 2'- fucosyl lactose or lacto-N-tetraose.
    [0013]
    13. PROCESS according to any one of claims 1 to 12, characterized in that the process for the purification of a neutral human milk oligosaccharide is a process for the purification of a neutral human milk oligosaccharide in a continuous manner, wherein the oligosaccharide of neutral human milk is preferably 2'-fucosyl lactose or lacto-N-tetraose.
    类似技术:
    公开号 | 公开日 | 专利标题
    BR112016007342B1|2020-11-17|process for the purification of a neutral human milk oligosaccharide from a crude solution
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    同族专利:
    公开号 | 公开日
    KR102298281B1|2021-09-07|
    RU2016109548A3|2018-06-25|
    PH12016500594A1|2016-06-13|
    CN105814070A|2016-07-27|
    US20160237104A1|2016-08-18|
    RU2016109548A|2017-11-09|
    DK3063159T3|2021-05-10|
    RU2685537C2|2019-04-22|
    EP2857410A1|2015-04-08|
    ES2868249T3|2021-10-21|
    WO2015049331A1|2015-04-09|
    CN109705175A|2019-05-03|
    MX2016004288A|2017-01-18|
    JP2016535724A|2016-11-17|
    US10435427B2|2019-10-08|
    KR20160090791A|2016-08-01|
    EP3063159A1|2016-09-07|
    US20190292211A1|2019-09-26|
    BR112016007342A2|2017-08-01|
    EP3063159B1|2021-04-07|
    JP6666243B2|2020-03-13|
    PL3063159T3|2021-07-19|
    US11168105B2|2021-11-09|
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    法律状态:
    2017-12-12| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07H 3/06 (2006.01), A61P 1/14 (2006.01) |
    2018-05-02| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|
    2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-04-28| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: C07H 3/06 , A61P 1/14 Ipc: A23L 33/15 (2016.01), A23L 33/16 (2016.01), A61K 3 |
    2020-07-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/10/2014, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP13187369.7|2013-10-04|
    EP13187369.7A|EP2857410A1|2013-10-04|2013-10-04|Process for purification of 2´-fucosyllactose using simulated moving bed chromatography|
    PCT/EP2014/071145|WO2015049331A1|2013-10-04|2014-10-02|Process for purification of a neutral human milk oligosaccharide using simulated moving bed chromatography|
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