Method for purification of lacto-n-neotetraose

Abstract

To provide a method for purification of a highly purified LNnT (lacto-N-neotetraose) from a fermentation broth.SOLUTION: The method comprises: subjecting a fermentation broth to a first membrane filtration step, thereby providing filtrate; subjecting the filtrate to a second pseudo-moving bed chromatography step to obtain a purified solution thereof; subjecting the purified solution to a third crystallization step to obtain crystals containing objective LNnT, and subjecting the crystals to a fourth and final drying step, thereby providing a highly purified LNnT powder.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for purifying LNnT (lacto-N-neotetraose, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc). More specifically, the present invention relates to the separation of by-products, impurities, and / or contaminants from LNnT obtained from a fermentation process through a purification process. [Background technology]

[0002] Human milk is considered the best diet for developing infants. It is composed of fat, protein, vitamins, minerals, trace elements, and complex oligosaccharides. Human milk, like the milks of other mammals, contains, in addition to lactose, a variety of structurally diverse oligosaccharides, also known as human milk oligosaccharides (HMOs) (Usashima T. et al. (2011) Milk 20 Oligosaccharides, Nova Biomedical Books, New York, ISBN 978-1-61122-831-1). Today, it is believed that there are over 150 structurally distinct oligosaccharides found in human milk. With very few exceptions, HMOs are characterized by a lactose disaccharide residue at their reducing end. On the other hand, many HMOs contain fucose, galactose, or N-acetylneuraminic acid residues at their nonreducing ends. Furthermore, they can be linear or branched. Generally, the monosaccharide residues of HMOs are D-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid (the latter better known as sialic acid or lactamic acid). HMOs are important in infant nutrition because their biological activities are directly linked to protecting the newborn from pathogens and supporting the development of the infant's immune system and cognitive abilities. Furthermore, HMOs serve as substrates for beneficial bacteria, such as bifidobacteria or lactobacilli.

[0003] Due to the challenges associated with chemically synthesizing human milk oligosaccharides, several enzymatic and fermentation methods have been developed. Fermentation methods, in particular, require the purification of the desired oligosaccharides from a highly complex fermentation broth containing hundreds of different individual compounds. The carbohydrate fraction of the fermentation broth alone is composed of a complex mixture of monosaccharides and oligosaccharides and their derivatives, including substrates (e.g., lactose, fructose, glucose, saccharose, and other sugars used as carbon sources), biosynthetic intermediates, individual monosaccharides (e.g., glucose, galactose, N-acetylglucosamine, fucose, and N-acetylneuraminic acid), metabolic by-products, and other oligosaccharides and polysaccharides synthesized by microorganisms. Furthermore, the structures of many of the oligosaccharides present in the fermentation broth are difficult to identify (e.g., oligosaccharides naturally produced by the synthetic host, such as cell surface glycosylation structures, or oligosaccharides produced by microorganisms as a result of stress). Therefore, purification of biotechnological products can often be much more expensive and time-consuming than their production by fermentation.

[0004] In particular, lacto-N-neotetraose fermentation is often accompanied by the over-synthesis of lacto-N-triose II (LNT II, ​​GlcNAc(β1-3)Gal(β1-4)Glc), a biosynthetic intermediate in the biosynthesis of lacto-N-neotetraose. This intermediate is often exported from the cell to the medium before further conversion to the desired LNnT. Furthermore, when LNnT is not exported but is further converted with N-acetylglucosamine (GalNAc) and glucose, para-lacto-N-neohexaose (pLNnH, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc) is often produced as a by-product, and even para-lacto-N-neooctaose (pLNnO, Gal(β1-4)GlcNAc(β1-3) Longer derivatives, such as (β1-4)Gal(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, are also detected. This latter by-product is expressed through glucosyllactose (GlcLac), galactosyllactose (GalLac), glucosylated LNnT, and galactosylated LNnT formed during fermentation. However, oligosaccharides bearing GalNAc at the non-reducing end, such as lacto-N-triose II or intermediates pLNnH and pLNnO, can be effectively removed by glycosidase treatment. Furthermore, autoclaving (heat treatment) of carbohydrates (e.g., sucrose or lactose) can result in the formation of undesired by-products, such as aldol or Maillard products. Isomerization reactions (e.g., conversion of lactose to lactulose) also generally result in even greater contamination and can generate oligosaccharide isomers. To avoid the formation of by-products from heat treatment, substrates and C sources are often sterile filtered, but this also carries the risk of adventitious growth contamination.

[0005] When producing human milk oligosaccharides by microbial fermentation, recombinant microorganisms (recombinant bacterial or yeast strains) are used. Consequently, these fermentation methods rely on genetically modified organisms (GMOs), which are considered critical in the food industry. Consequently, the desired product must be purified from residues of the GMO process, such as cells, cell fragments, endotoxins, and excess salts, to be accepted by customers and regulatory authorities. Therefore, lacto-N-neotetraose must be purified from fermentation broths containing complex mixtures of recombinant nucleic acids, such as DNA and RNA, and recombinant proteins. Furthermore, microbial fermentation, especially when Escherichia coli is used, contains significant amounts of endotoxins. However, contamination of products intended for human consumption with recombinant DNA, endotoxins, or proteins is unacceptable to both regulatory authorities and consumers. Therefore, all nucleic acids and proteins derived from the recombinant microorganisms must be removed from the desired human milk oligosaccharides.

[0006] Known methods for purifying individual oligosaccharides are technically complex and often uneconomical, especially when these raw compounds are obtained from fermentation broth as a mixture of several similarly structured compounds, and especially when the oligosaccharides are intended for food applications. For the industrial purification of food-grade disaccharides, lactose or sucrose, from complex mixtures such as whey or molasses, production-ton-scale processes involving multiple crystallization steps have been developed. However, HMOs in general, and more specifically lacto-N-neotetraose, have proven difficult to purify to date, especially when derived from fermentation broth. In the case of LNnT, synthetic production and subsequent crystallization of the resulting product have been developed to enable its use as a food ingredient. However, these methods do not contain the core product derived from fermentation, and because the method yields a nearly pure product, it is isolated as a crystalline material and further processed for economic reasons.

[0007] Early work on the isolation, characterization, and crystallization of various HMOs was carried out by Richard Kuhn and coworkers in the 1950s, when these compounds were isolated from the carbohydrate fraction of breast milk by chromatographic purification on activated charcoal / Celite columns. The first HMO crystallized by Kuhn and coworkers was lacto-N-biose I (Kuhn et al., Chem. Ber. 1954, 87(10), 1553-1560). In subsequent years, numerous other carbohydrates were isolated. Lacto-N-tetraose (Kuhn et al., Chem. Ber. 1953, 86(6), 827-830; Chem. Ber. 1954, 87(3), 289-300; Chem. Ber. 1956, 89(2), 504-511), lacto-N-fucopentaose I (Kuhn et al., Chem. Ber. 1956, 89(11), 2514- 2523), 2'-fucosyllactose (Kuhn et al., Chem. Ber. 1955, 88(8), 1135-1146; 1956, 89(11), 2513), lacto-N-triose I & II (Kuhn et al., Chem. Ber. 1956, 89(4), 1027-1033) and lacto-N-neotetraose (Kuhn et al., Chem. Ber.1962,95(11),518-522).

[0008] Chromatographic methods, particularly gel filtration chromatography, have been used to purify lacto-N-neotetraose and the closely related human milk oligosaccharide lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) (Dumon et al., 2001 Glycoconj. J. 18(6), 465-474; Priem et al., Glycobiology 2002, 12(4), 235-240; Baumgartner et al., Chem. Bio. Chem. 2014 15(13), 1896-1900, Sprenger et al., 2017, J. Biotechnol. 258, 79-91). In this regard, purification by gel filtration chromatography is not suitable for food on an industrial scale, but simulated moving bed chromatography is considered a suitable method for purifying LNnT for food use when combined with other purification steps to obtain purified LNnT as the final product.

[0009] Microbial fermentation of lacto-N-neotetraose can produce many other sugars, including not only trioses (LNT II, ​​GlcLac, GalLac) but also longer-chain oligosaccharides such as hexaose (pLNnH). Furthermore, hydrolysis products of the resulting oligosaccharides are also formed during fermentation and downstream processing. Using a purification method involving several purification steps, LNnT can be obtained in larger quantities, with higher purity and yield, making it suitable as an ingredient in food formulations or for cosmetic or medical applications.

[0010] The process of the present invention is a cost-effective alternative to prior art processes that results in a solid powder product and is particularly suitable for the purification of lacto-N-neotetraose for nutritional applications, especially infant nutrition, medical nutrition, dietary supplements or general nutrition products.

[0011] To overcome all these drawbacks of known methods, the present invention provides a novel, simple, cost-effective and scalable purification method for the purification of lacto-N-neotetraose obtained from fermentation processes. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Usashima T. et al. (2011), Nova Biomedical Books, New York ISBN 978-1-61122-831-1 [Non-patent document 2] Kuhn et al., Chem. Ber. 1954, 87(10), 1553-1560 [Non-patent document 3] Kuhn et al., Chem. Ber. 1953, 86(6), 827-830; Chem. Ber. 1954, 87(3), 289-300; Chem. Ber. 1956, 89(2), 504-511 [Non-patent document 4] Kuhn et al., Chem. Ber. 1956, 89(11), 2514-2523 [Non-Patent Document 5] Kuhn et al., Chem. Ber. 1955, 88(8), 1135-1146; 1956, 89(11), 2513 [Non-patent document 6] Kuhn et al., Chem. Ber. 1956, 89(4), 1027-1033 [Non-Patent Document 7] Kuhn et al., Chem. Ber. 1962, 95(11), 518-522 [Non-patent document 8] Dumon et al., 2001 Glycoconj. J. 18(6), 465-474 [Non-Patent Document 9] Priem et al., Glycobiology 2002, 12(4), 235-240 [Non-Patent Document 10] Baumgartner et al., Chem. Bio. Chem. 2014 15(13), 1896-1900 [Non-Patent Document 11] Sprenger et al., 2017, J.Biotechnol.258, 79-91 Summary of the Invention

[0013] The present invention relates to a simple and economical method for purifying lacto-N-neotetraose from microbial fermentation. The microbial fermentation of lacto-N-neotetraose can also produce several other carbohydrates, including trioses and longer-chain oligosaccharides such as hexaose. Additionally, hydrolysis products of the resulting oligosaccharides are also formed during fermentation and downstream processing.

[0014] The object of the present invention is to provide a simple, cost-effective and scalable method for obtaining purified lacto-N-neotetraose as the main product obtained from a fermentation process, while separating by-products, impurities and / or other contaminants such as lacto-N-triose II and / or para-lacto-N-neohexaose and / or para-lacto-N-neooctaose and / or glucosyllactose and / or galactosyllactose from such main product.

[0015] It is yet another object of the present invention to provide lacto-N-neotetraose having a purity of ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, and ≧95%. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows a general scheme of the steps of the purification method according to the invention. [Figure 2] FIG. 2 shows the retentate analysis of Example 1 for a retentate volume of 80 L. [Figure 3] FIG. 3 shows the retentate analysis of Example 1 for a retentate volume of 60 L. [Figure 4] FIG. 4 shows the retentate analysis of Example 1 for a retentate volume of 40 L. [Figure 5] FIG. 5 shows the permeate analysis of Example 1 for a retentate volume of 20 L of Permeate 1. [Figure 6] FIG. 6 shows the permeate analysis of Example 1 for a retentate volume of 20 L of permeate 2. [Figure 7] FIG. 7 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture used in Example 4. [Figure 8] FIG. 8 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture used in Example 4. [Figure 9] FIG. 9 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture used in Example 4. [Figure 10] FIG. 10 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometry) spectrum of the carbohydrate mixture obtained after crystallization in Example 5. [Figure 11] FIG. 11 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the carbohydrate mixture obtained after the first crystallization in Example 6. [Figure 12] FIG. 12 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the carbohydrate mixture obtained after the second crystallization in Example 6. [Figure 13] FIG. 13 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture obtained after crystallization in Example 7. [Figure 14] FIG. 14 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the seed crystals obtained by gel filtration in Example 8. [Figure 15]FIG. 15 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the LNnT starting material used in Example 9. [Figure 16] FIG. 16 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the LNnT product obtained in Example 9. [Figure 17] FIG. 17 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the LNnT product obtained in Example 10. [Figure 18] FIG. 18 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the LNnT product obtained in Example 11. [Figure 19] FIG. 19 shows the HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection) spectrum of the LNnT product obtained in Example 12. [Figure 20] FIG. 20 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture obtained after homogenization in Example 13. [Figure 21] FIG. 21 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the carbohydrate mixture obtained after homogenization in Example 14. [Figure 22] FIG. 22 shows the HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection) spectrum of the LNnT product obtained in Example 15. [Figure 23] FIG. 23 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the crystalline LNnT starting material used in Examples 16-19. [Figure 24] FIG. 24 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the homogenized LNnT product obtained in Example 17. [Figure 25] FIG. 25 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the homogenized LNnT product obtained in Example 18. [Figure 26] FIG. 26 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the homogenized LNnT product obtained in Example 19. [Figure 27] FIG. 27 shows the HILIC-CAD (hydrophilic interaction liquid chromatography coupled to a charged aerosol detector) spectrum of the homogenized LNnT product obtained in Example 20. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a simple, cost-effective and scalable method for obtaining purified lacto-N-neotetraose as a main product from a microbial fermentation process, while separating by-products, impurities and / or other contaminants such as lacto-N-triose II and / or glucosyllactose and / or galactosyllactose and / or para-lacto-N-neohexaose and / or para-lacto-N-neooctaose from the main product.

[0018] The present invention provides a batch or continuous method for purifying lacto-N-neotetraose (LNnT) from a fermentation broth obtained by a microbial fermentation process. This provides purified LNnT with a purity of greater than 80%, greater than 85%, greater than 90%, and / or greater than 95%. The fermentation broth contains neutral HMOs, biomass, medium components, contaminants, and carbohydrates other than LNnT. The purity of LNnT in the fermentation broth is ≦60%.

[0019] The inventive method uses as a starting material a fermentation broth containing LNnT and other contaminants, particularly triose, hexaose, and / or tetraose. The fermentation broth is obtained from a microbial fermentation process. The purity of LNnT in the fermentation broth is ≦60%. The fermentation broth is subjected to the following purification steps:

[0020] 1) At least one membrane filtration step of a solution, which is a fermentation broth containing a mixture of carbohydrates obtained from a microbial fermentation process, is subjected to nanofiltration after carrying out standard downstream protocols, thereby obtaining higher sugars such as pentaose and / or hexaose. The content of LNnT in the filtrate is significantly reduced to less than 10%, and the purity of LNnT in the filtrate is greater than 60%, greater than 65%, or greater than 70%; 2) at least one SMB chromatography step, with the residual hexaose content set to less than 5% to ensure proper crystallization in the next step, and the purity of LNnT in the resulting purified solution being greater than 75% or greater than 80%; 3) at least one crystallization step from water, in which the obtained crystalline mass is treated and washed with alcohol, an alcohol / water mixture, or a solvent, or a solvent / water mixture, and residual smaller saccharides are washed away by draining and setting their concentration below 3%, so that the purity of LNnT in the obtained crystals is higher than 85%, higher than 90%, or higher than 95%; 4) at least one homogenization step of the resulting drained crystalline mass, either by freeze drying, spray drying, drum / roller drying, vacuum drum / roller drying, band drying or vacuum band drying; This provides a product containing purified LNnT with a purity of ≧90%.

[0021] Membrane filtration, as the first step of the method of the present invention, is used to reduce the content of high molecular weight oligosaccharides from the target low molecular weight LNnT. There are many different types of membranes that can be used for membrane filtration, each with different carbohydrate compatibility. The most common materials used for membranes are polymeric materials and ceramic membrane modules. Polymeric membranes can be fabricated as hollow fibers or blocks due to their inherent flexibility, while ceramic membranes are limited to hollow fiber blocks. While both techniques can be combined into mixed-material membranes or processed sequentially, the industry has focused primarily on producing homogeneous materials for cost and / or efficiency reasons.

[0022] When the desired reduction of sugars having a molecular weight greater than lacto-N-neotetraose is desired, membrane filtration processes, more specifically nanofiltration processes, are used. Nanofiltration is a pressure-driven membrane process that essentially retains dissolved molecules, metal ions, and other particles above a molecular weight cut-off (MWCO) limit. Membranes used in nanofiltration have pore sizes of ≦2 nm, which distinguishes them from the coarse membranes used in other membrane filtration processes, such as ultrafiltration and microfiltration.

[0023] Compared to other membrane filtration processes, such as reverse osmosis, nanofiltration uses correspondingly coarser membranes and lower operating pressures. However, membranes used for filtration typically have limited and / or highly dependent retention properties and / or resistance to thermal shock or chemical exposure, essentially limiting the application of the method to water and aqueous mixtures. Lacto-N-neotetraose has a molecular weight of 707.6 Da, while the next largest sugars, elongated by one or two additional monosaccharides, have molecular weights of 869.3 Da (LNnT + Glc / Gal), 910.3 Da (LNnT + GlcNAc), and 1072.4 Da (LNnT + LacNAc), respectively. Therefore, nanofiltration membranes providing the smallest possible MWCO are the membranes used in the present invention. The membranes used in the present invention have a MWCO of 0.2 to 3.5 kDa, more preferably 0.2 to 2.0 kDa, and even more preferably 0.2 to 1.0 kDa.

[0024] Sugar blends are used to reduce sugars higher than LNnT, or generally higher than triose. The solution is then subjected to a filtration process by applying pressure to a membrane. Three sugars are used for the separation: LNnT, hexaose, and triose, which are also present in the fermentation broth mixture as by-products derived from the microbial fermentation process.

[0025] The purpose of this first membrane filtration step is to remove hexaose to a certain extent, resulting in a relatively low hexaose content sugar mixture, in a first approach, to make the subsequent SMB chromatography and crystallization steps as efficient as possible. Alternatively, this first membrane filtration step can be used in a second approach to relatively deplete the tetraose and hexaose content relative to the triose content, ultimately enriching the retentate with LNnT and pLNnH, so that purified LNnT can be obtained after the subsequent SMB chromatography and crystallization steps. In this method, lower saccharides are removed first, followed by higher saccharides.

[0026] An aqueous solution of at least two, preferably three or four or more, oligosaccharides containing LNnT (at least two of which have different masses, all derived from bacterial fermentation, and already purified to their sugar components using microfiltration, ultrafiltration, anion and cation exchange, activated carbon treatment and subsequent filtration, and / or diafiltration and electrodialysis (ED)) is subjected to nanofiltration by applying a pressure of 1 to 50 bar, more preferably 2 to 30 bar, more preferably 3 to 10 bar, and even more preferably 4 to 5 bar. The MWCO limit of the membrane is 0.2 to 3.5 kDa, more preferably 0.2 to 2.0 kDa, and even more preferably 0.2 to 1.0 kDa. The sugar concentration of the previously prepared solution is 0.01 to 70% dry solids content (DSC), more preferably 0.1 to 60% DSC, more preferably 1 to 50% DSC, and even more preferably 10 to 40% DSC. The dilution factor is 0.01 to 1. Therefore, the retentate must be rinsed with additional amounts of water depending on the dilution factor. The DSC of the eluate therefore varies by a factor of 0.01 to 1. Using a membrane filtration step, the hexaose content is reduced to less than 20% in the retentate, more preferably less than 15%, and even more preferably less than 10%.

[0027] The purity of LNnT in the filtrate obtained from the membrane filtration process is greater than 60%, greater than 65%, or greater than 70%. In an additional embodiment, separation of trioses from a mixture containing trioses, tetraoses, and hexaoses (these three sugars may be the major components of the sugar solution) allows for further purification through a second membrane filtration step (in this case nanofiltration) to reduce the hexaose by choosing a membrane of appropriate material and pore size, ultimately yielding a purified LNnT solution.

[0028] In an additional embodiment, separation of hexaose from a mixture containing triose, tetraose, and hexaose (these three sugars may be the major components of the sugar solution) allows for further purification through a second membrane filtration step (in this case nanofiltration) to reduce the remaining triose by choosing a membrane of appropriate material and pore size, ultimately yielding a purified LNnT solution.

[0029] The filtrate is then subjected to a second step of simulated moving bed chromatography (SMB chromatography), which is used to adjust the content of specific sugar-containing fractions, such as hexaose fractions, relative to tetraose-containing fractions using specially adapted parameters. The filtrate is the solution obtained from the membrane filtration in step 1, but its hexaose content is less than 20%, more preferably less than 15%, and even more preferably less than 10%.

[0030] The SMB chromatography step involves passing a mixture of HMOs (either a low- or high-sugar extract or raffinate containing the tetrasaccharide lacto-N-neotetraose) through successive chromatography. The second step of the present invention provides for the chromatographic (or sequential) separation of the neutral HMO-tetrasaccharide lacto-N-neotetraose from a filtrate containing the neutral HMO-tetrasaccharide lacto-N-neotetraose and other by-product contaminants, the filtrate containing a mixture of lacto-N-neotetraose and contaminants comprising or consisting of the solution obtained from membrane filtration in step 1, and the purity of the LNnT solution being less than 90%.

[0031] The filtrate is subjected to at least one purification step using simulated moving bed chromatography. This method results in two solutions, one of which contains the desired lacto-N-neotetraose. After applying SMB chromatography in this second step, LNnT can be present mainly in the extract or in the raffinate (recovery solution), by separating lower oligosaccharides such as trioses or by separating higher sugars such as hexaoses.

[0032] By using this second step of simulated moving bed chromatography, LNnT of higher purity is continuously provided. Therefore, large quantities of high-quality HMO can be provided in a very convenient and economical manner. The SMB chromatography step is very stable even without a regeneration step for the column materials (e.g., cationic column materials) used in this step. In preferred embodiments, the purity of LNnT in the filtrate is greater than 60%, greater than 65%, or greater than 70%. The term "filtrate" refers to the solution containing LNnT obtained from membrane filtration in step 1 before the single moving bed chromatography purification step, while the term "purified solution" refers to the solution after the simulated moving bed chromatography step.

[0033] At least one simulated moving bed chromatography step 2 comprises: i) at least four columns, preferably at least eight columns, more preferably at least twelve columns, at least one of which is a weak or strong cation exchange resin, preferably H + Shape, Na + shape, K + Shape or Ca 2+ and / or ii) four zones of different flow rates: I, II, III and IV; and / or iii) Water, preferably ethanol and water, more preferably 5 to 15 vol% ethanol an eluent comprising or consisting of ethanol and 85 to 95 vol% water, most preferably 9 to 11 vol% ethanol and 89 to 91 vol% water; iv) an operating temperature of 15° to 60° C., preferably 20° to 55° C., and more preferably 25° to 50° C. It has.

[0034] Since the HMO to be purified is lacto-N-neotetraose, at least one simulated moving bed chromatography step i) four zones of different flow rates, I, II, III and IV (the flow rates are preferably determined by zones I, II, III and IV) 22-32 ml / min in zone I, 17-23 ml / min in zone II, and 18-25 ml in zone III / min, and / or 14-20 ml / min in Zone IV); and / or ii) a feed rate of 0.5 to 4 ml / min, preferably 2 ml / min; and / or iii) an eluent flow rate of 6 to 12 ml / min, preferably 8 ml / min; and / or iv) A switching time of 14 to 20 minutes, preferably 16 to 18 minutes, and more preferably 17 minutes. It has.

[0035] Preferably, 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 cation exchange resin, and most preferably 1.0 to 20 kg of cation exchange resin.

[0036] Importantly, the amount of cation exchange material, the flow rates of the different zones, the feed rates, the eluent flow rates, and / or the switching times can be scaled up by 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 times or more. All possible multiples between the above values ​​are possible.

[0037] In the column, a strong cation exchange resin can be used as the stationary phase. Preferably, the cation exchange resin is a sulfonic acid resin, more preferably Purolite® PCR833H (Purolite GmbH, Ratingen, Germany), Lewatit MDS 2368 and / or Lewatit MDS 1368 resin. When a cationic ion exchange resin is used in the column, it can be regenerated with sulfuric acid. Sulfuric acid can be used in the eluent, preferably at a concentration of 10 mM sulfuric acid or less. (Strong) cation exchange resins are H + Shape or Ca 2+ It can exist in the form

[0038] During simulated moving bed chromatography, operating temperatures above 60°C are not recommended, especially for strong cation ion exchange resins (H + Shape or Ca 2+It was found that when α-LNnT was present as the stationary phase, the applied neutral oligosaccharides were significantly destabilized, i.e., depolymerized, which was detrimental to the final yield of LNnT.

[0039] In an advantageous embodiment of the present invention, the purified solution can be subjected to at least one further purification step using simulated moving bed chromatography, thereby providing a purified solution containing neutral human milk oligosaccharides with a purity of >85%, preferably >90%, and more preferably >93%. In particular, the present invention provides an HMO product that is recombinant DNA-free and host strain proteins-free.

[0040] Further simulated moving bed chromatography is i) at least four columns, preferably at least eight columns, more preferably at least twelve columns, at least one of which is a weak or strong cation exchange resin, preferably H + Shape, Na + shape, K + Shape or Ca 2+ and / or ii) four zones of different flow rates: I, II, III and IV; and / or iii) Water, preferably ethanol and water, more preferably 5 to 15 vol% ethanol an eluent comprising or consisting of ethanol and 85-95 vol% water, most preferably 9-11 vol% ethanol and 89-91 vol% water (the eluent optionally further comprising sulfuric acid, preferably ≦10 mM sulfuric acid, more preferably ≦2-5 mM sulfuric acid); and / or iv) an operating temperature of 15° to 60° C., preferably 20° to 55° C., and more preferably 25° to 50° C. It has.

[0041] If the HMO to be purified is lacto-N-neotetraose, a further simulated moving bed chromatography step is i) four zones of different flow rates, I, II, III and IV (the flow rates are preferably determined by zones I, II, III and IV) 22-32 ml / min in zone I, 18-23 ml / min in zone II, and 19-25 ml in zone III / min, and / or 15-20 ml / min in Zone IV); and / or ii) a feed rate of 1 to 4 ml / min, preferably 2 ml / min; and / or iii) an eluent flow rate of 6 to 12 ml / min, preferably 9 ml / min; and / or iv) A switching time of 16 to 22 minutes, preferably 18 to 20 minutes, and more preferably 19 minutes. It has.

[0042] In particular, 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 cation exchange resin, and most preferably 1.0 to 20 kg of cation exchange resin.

[0043] Important factors include the amount of cation exchange material, the flow rates of the different zones, the feed rate, the eluent flow rate, and The expansion of the switching time and / or the switching time can be performed by 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 times, or any other possible factor between the above values. After the purification step using simulated moving bed chromatography, the pH of the purified solution can be adjusted to pH 7, preferably by the addition of a base, more preferably by the addition of NaOH (e.g., 0.2 M NaOH).

[0044] The purity of LNnT in the purified solution obtained from the simulated moving bed chromatography step is greater than 75% or greater than 80%. This purified solution is then subjected to the third step of crystallization, which is used to obtain highly pure LNnT. The purified solution obtained from SMB chromatography in step 2 has a hexaose content of less than 20%, more preferably less than 15%, and even more preferably less than 10%.

[0045] The crystallization process is a) obtaining a mixture of at least two, more preferably at least three or more oligosaccharides, one of which is a trisaccharide or a sugar lower than a random tetraose, one of which is lacto-N-neotetraose and one of which is para-lacto-N-neohexaose or a sugar higher than a random tetraose of the mentioned oligosaccharides (after steps 1 and 2, a purified solution of oligosaccharides, one of which is lacto-N-neotetraose, is obtained, preferably at a concentration of at least about 10% DSC, in particular at least about 30% DSC, in particular at least about 50% DSC, and with an LNnT purity of this mixture of at least 15%, preferably at least 25%, in particular at least about 40%, in particular at least about 60%), b) crystallizing the mixture of oligosaccharides from a starting temperature of at least 20-30°C, preferably at least about 30-40°C, preferably at least about 40-50°C, more preferably at least about 50-60°C, and crystallizing the saturated mass by cooling to at least 40-50°C, preferably at least about 30-40°C, preferably at least about 20-30°C, preferably at least about 10-20°C, more preferably at least about 0-10°C, in order to obtain a homogeneous crystalline mass; c) treating the obtained crystalline mass thoroughly with a mixture of water and an alcohol solvent such as methanol, ethanol, isopropanol, glycol, glycerol, or any other water-miscible alcohol or solvent (the alcohol or solvent content of the solution is 10-90 vol%, preferably 30-80 vol%, more preferably 50-70 vol%) to produce a mixture of alcohol or solvent-containing solution and crystalline mass; d) filtering off the alcohol or solvent-containing, now sugar-containing, fraction of the crystal mass in order to reduce the content of low-grade sugars from the crystal mass (preferably, the washing step can be repeated at least twice in order to reduce the content of low-grade sugars below a certain limit, i.e., below 10%, preferably below 5%, more preferably below 3%, before subjecting the crystal mass to a fourth homogenization step). This includes:

[0046] The amount of alcohol or solvent used to wash the crystalline mass is not critical, but at least about 40-100 L, preferably about 30-70 L, of alcohol or solvent is used per 10 kilograms of crystalline mass. Preferably, the alcohol or solvent solution is then filtered to remove lower oligosaccharide by-products. The filtered alcohol or solvent solution, now containing only a small amount of lacto-N-neotetraose, can be used in another crystallization cycle, if desired, to provide more lacto-N-neotetraose. However, not only lacto-N-neotetraose can be purified in this manner, but also trisaccharides and higher sugars such as pentaose, hexaose, and octaose derived from the fermentation broth or from chemical synthesis or biocatalysis can be purified from the crystallization mass by washing the lower oligosaccharides with an aqueous mixture containing alcohol or solvent.

[0047] The purity of LNnT in the crystals obtained from the crystallization process is greater than 85%, greater than 90%, and greater than 95%. The crystals are then subjected to a fourth step, homogenization, which is used to obtain a highly homogenous and purified LNnT product with a residual moisture content of 20% or less.

[0048] Crystal homogenization was used to obtain dried lacto-N-neotetraose from raw crystalline mass. Drying is generally used to effect a mass transfer process that involves the removal of water or another solvent from a solid, semi-solid, or liquid by evaporation. This process is often used as a final manufacturing step. A heat source and chemicals are often required to remove the vapors produced by the process. In bioproducts such as foods, grains, and pharmaceuticals such as vaccines, the solvent being removed is almost exclusively water. Desiccation can be considered synonymous with or an extreme form of drying.

[0049] In the most common example, a gas stream, e.g., air, adds heat by convection and removes the vapor as moisture. Another possibility is vacuum drying, where heat is supplied by conduction or radiation (or microwaves), but the vapors thus produced are removed by a vacuum system. Another indirect technique is drum drying, where a heated surface is used to provide the energy and an aspirator draws the vapor out of the chamber.

[0050] In the case of the crystallized lacto-N-neotetraose of the present invention, several methods can be used to provide a homogenized dry material of suitable product specificity. One of the most common methods used is oven drying or vacuum oven drying. However, in the case of raw crystalline mass, In this case, oven drying or vacuum oven drying can only provide a homogeneous material by combining drying and mixing. Therefore, the drained crystal mass obtained from the crystallization purification process must be evenly distributed on the surface to obtain the maximum surface area (from which the solvent can evaporate during the process). When applying the oven drying process, the temperature is set to 20-200°C, preferably 25-100°C, and even more preferably 30-50°C to evaporate the solvent. The solvent is removed by airflow. When applying vacuum oven drying, the temperature is set to 20-200°C, preferably 25-100°C, and even more preferably 30-50°C to evaporate the solvent. The solvent is removed by a pump set at a vacuum in the range of 0.01-1000 mbar, preferably 0.1-100 mbar, and even more preferably 1-10 mbar. In both cases, drying is carried out for 1 hour to 7 days. The materials are mixed before or after drying.

[0051] In the second embodiment, final homogenization is not required because the drying of the material is performed by applying oven drying or vacuum oven drying after the second crystallization (which is performed after the first crystallization and subsequent drying steps). With the second and third crystallizations as here, no washing steps are required and no heterogeneous material is formed. Generally, in the second and third crystallizations, the crystals grow better due to the higher purity obtained, so the crystals are easier to dry after the additional crystallization steps.

[0052] In a third embodiment, the homogenization method is freeze-drying. In this method, the crystals obtained after draining the crystallization mass are dissolved in water and then subjected to a freeze-drying process. Here, LNnT crystals are dissolved in water at a concentration ranging from 0.1 to 70% DSC, preferably 1 to 50% DSC, and more preferably 5 to 25% DSC, then frozen and subjected to a freeze-drying apparatus. In this apparatus, the water is removed, resulting in a foam-to-gel type dry homogenous sugar product.

[0053] Ideally, the application of lyophilization, in contrast to most other drying methods, results in a completely anhydrous or substantially anhydrous product. This allows indirect determination of the water content of the sample, especially for carbohydrates such as HMOs (of which the LNnT used was one), which can lead to the determination of residual water. This is difficult because, firstly, they are poorly soluble in anhydrous solvents, and secondly, the alcohol functional group (OH group) has spectroscopic properties similar to or identical to those of water.

[0054] In a fourth embodiment, the homogenization method is spray drying. A spray dryer takes in a liquid stream and separates the solution or suspension into solids and the solvent into vapor. The solids are typically collected in a drum or cyclone. The liquid input stream is atomized through a nozzle into a hot vapor stream that is vaporized. Nozzles are typically used to minimize droplet size, maximizing heat transfer and water evaporation rate. Spray dryers can dry products very quickly compared to other drying methods. Spray dryers also convert solutions or slurries into dry, homogenous powders in a single step.

[0055] Spray drying is preferably applied by setting the sugar solution concentration to 1-70% DSC, preferably 10-60% DSC, preferably 20-50% DSC, and more preferably 30-40% DSC. The sugar-containing solution is then passed under pressure through a spray dryer nozzle with an inlet temperature setting of 100-160°C, preferably 110-150°C, and more preferably 120-140°C. The flow is adjusted to maintain an outlet temperature of 50-80°C, preferably 60-70°C, and more preferably 66-67°C. Using these settings, a homogeneous spray-dried powder is obtained.

[0056] In a fifth embodiment, the homogenization method for obtaining lacto-N-neotetraose is roller or drum drying or vacuum roller drying. Roller drying or drum drying is a method used to drain liquid from heterogeneous raw materials. In the drying process, the raw ingredients are dried at relatively low temperatures on rotating, high-volume rolls, producing sheets of dry, homogeneous product. Drum drying techniques result in dried materials that are readily reconstituted and retain much of their original flavor, color, and nutritional value. Some advantages of roller drying include its ability to dry viscous, heterogeneous solutions that cannot be easily dried by other methods, and the fact that drum dryers are easy to clean, operate, and maintain.

[0057] If roller drying is applied, the temperature of the rolls is set at 20 to 200°C, preferably 50 to 150°C, more preferably 75 to 125°C to evaporate the solvent. The solvent is removed by airflow. If vacuum roller drying is applied, the temperature is set at 20 to 200°C, more specifically 50 to 150°C, even more specifically 75 to 125°C to evaporate the solvent. The solvent is removed by a pump set at a vacuum in the range of 0.1 to 1000 mbar, preferably 1 to 500 mbar, more preferably 10 to 100 mbar. In both cases, the rolls rotate at a speed of 0.1 to 100 rev / min, preferably 1 to 10 rev / min.

[0058] In a sixth embodiment, the homogenization method for obtaining lacto-N-neotetraose is band or vacuum band drying. Band or vacuum band drying is a method used to drain liquid from raw materials. In the drying process, raw materials are dried at relatively low temperatures on a moving, large-volume band to produce a dry, homogenous product.

[0059] The final powder product has a purity of LNnT in the purified preparation of ≧80%, preferably ≧85%, more preferably ≧90% based on the dry matter of the purified preparation. The present invention will be described with respect to particular embodiments and with reference to the drawings but the present invention is not limited thereto but only by the claims. Moreover, terms such as "first," "second," etc. in the description and claims are used to distinguish between similar elements and do not necessarily describe an order in time, space, ranking, or any other manner. It should be understood that terms so used are interchangeable under appropriate circumstances, and that the aspects of the invention described herein are capable of operating in other orders than those described or illustrated herein.

[0060] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. Thus, it should be interpreted as embodying the presence of a specified referenced feature, integer, step or element, but without excluding the presence or addition of one or more other features, integers, steps or elements, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting of only elements A and B. It means that, in the context of the present invention, only the relevant elements of the device are A and B.

[0061] Throughout this specification, references to "one embodiment" or "embodiment" do not necessarily imply that the particular feature, structure, or characteristic described in connection with that embodiment is at least part of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may be. Furthermore, particular features, structures, or characteristics may be used in more than one embodiment, as would be apparent to one skilled in the art from this disclosure. Or multiple aspects can be combined in any suitable manner.

[0062] Similarly, in describing exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, drawing, or description thereof to streamline the disclosure and to facilitate understanding of one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected in the following claims, inventive aspects lie in fewer than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the invention.

[0063] Furthermore, although some embodiments described herein include some features (but not other features) included in other embodiments, combinations of features from different embodiments are meant to be within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0064] Furthermore, some of the embodiments are described herein as methods or combinations of elements of methods that can be implemented by a processor of a computer system or other means for implementing that function. Thus, a processor with the necessary instructions for implementing such a method or element of a method forms a means for implementing the method or element of a method. Furthermore, elements described herein in apparatus aspects are examples of means for performing the functions performed by the element to implement the invention.

[0065] Numerous specific details are set forth in the descriptions and drawings provided herein. However, it should be understood that aspects of the present invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0066] The present invention will now be described by describing in detail certain aspects of the present invention. Other aspects of the present invention can be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and it is apparent that the present invention is limited only by the terms of the appended claims. [Example]

[0067] Example 1: An 80 L volume of LNnT-containing carbohydrate mixture containing two major by-products was separated by nanofiltration, resulting in the removal of higher sugars such as pLNnH and the accumulation of lower sugars such as triose and LNnT.

[0068] 80 L of the aqueous sugar mixture was subjected to 200 Da nanofiltration to remove pLNnH. The dry solids content (DSC) of this sugar solution was 5.51%, and the ratios of the three sugars considered were expressed as triose: 4.0%, LNnT: 62.0%, and pLNnH: 20.8%. This corresponds to a total sugar content of 4.41 kg and an LNnT content of 2.73 kg. The sugar mixture was fed to the circulation of the filtration plant at a pressure of approximately 4 bar using a feed pump. A circulation pump was used to pump the sugar solution in a circular motion around the 200 Da ceramic membrane to be filtered at a pressure of 5 bar. On the permeate side, the filtered sugar solution exited the membrane at a pressure of 1 bar. After successive filtrations of 20 liters each, the retentate and permeate were analyzed for sugar composition and sugar concentration.

[0069] After 20 liters, the retentate changes as follows: The 60 L retentate is now characterized by a DSC of 6.85% and a composition of 3.9% triose, 61.7% LNnT, and 22.3% pLNnH. This corresponds to 4.11 kg total sugars and 2.54 kg LNnT. The permeate has the following composition: 4.5% triose, 67.2% LNnT, and 11.7% pLNnH at 1.88% DSC. This corresponds to 380 g total sugars and 253 g LNnT.

[0070] After another 20 liters, the retentate changes as follows: The remaining 40 L of retentate is now characterized by a dry weight of 9.95% DSC with a composition of 3.9% triose, 62.5% LNnT, and 24.6% pLNnH. This corresponds to 3.98 kg total sugars and 2.49 kg LNnT. The 20 L of permeate has the following composition: 2.44% DSC with a composition of 4.6% triose, 67.5% LNnT, and 12.1% pLNnH. This corresponds to 490 g total sugars and 329 g LNnT.

[0071] These results allow the calculation of the permeability coefficient for each sugar component: triose permeates with a coefficient of approximately 1.15, LNnT with a coefficient of 1.09, and pLNnH with a coefficient of 0.55.

[0072] The results are shown in Tables 1, 2 and 3.

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3]

[0076] Example 2: A 35 L volume of LNnT-containing carbohydrate mixture containing two major by-products was separated by nanofiltration, and an additional 10 L of water was added to the feed solution, achieving the removal of higher sugars such as pLNnH and the accumulation of lower sugars such as triose and LNnT.

[0077] A 35-liter aqueous solution of the sugar mixture was subjected to 200 Da nanofiltration to remove pLNnH. The sugar solution had a dry solids content (DSC) of 7.88%, and the ratios of the three sugars considered were expressed as triose: 2.8%, LNnT: 58.0%, and pLNnH: 26.1%. This corresponds to a total sugar content of 2.76 kg and LNnT: 1.60 kg. The sugar mixture was fed to the circulation of the filtration plant at a pressure of approximately 4 bar using a feed pump. A circulation pump was used to pump the sugar solution in a circular motion around the 200 Da ceramic membrane to be filtered at a pressure of 5 bar. On the permeate side, the filtered sugar solution exited the membrane at a pressure of 1 bar. The sugar solution was pumped through the membrane in 5-liter increments, and the remaining retentate and the resulting permeate were analyzed for sugar composition and sugar concentration. After the first 10 L, 5 L of fresh water was added twice to the feed solution.

[0078] After the first 5 liters, the retentate changes as follows: 30 L of retentate is now characterized by a DSC of 8.34% and a composition of 2.2% triose, 57.5% LNnT, and 26.1% pLNnH. This corresponds to 2.50 kg total sugars and 1.44 kg LNnT. The permeate has the following composition: 2.37% DSC and a composition of 3.5% triose, 59.6% LNnT, and 16.5% pLNnH. This corresponds to 120 g total sugars and 71 g LNnT.

[0079] After another 5 L, the retentate changes as follows: The remaining 25 L of retentate is now characterized by a dry weight DSC of 9.07% with a composition of 2.3% triose, 57.1% LNnT, and 26.9% pLNnH. This corresponds to 2.27 kg total sugars and 1.30 kg LNnT in the retentate. The combined 10 L of permeate has the following composition: 2.21% DSC, 3.5% triose, 61.1% LNnT, and 26.9% pLNnH. The composition is H: 16.3%, which corresponds to a total sugar content of 220 g and an LNnT content of 135 g.

[0080] At this point, 5 L of water is added to the feed before further filtration. After the additional 5 L, the retentate changes as follows: The remaining 25 L of retentate is now characterized by a dry weight DSC of 8.95% and a composition of 2.2% triose, 56.1% LNnT, and 26.3% pLNnH. This corresponds to 2.24 kg of total sugars and 1.26 kg of LNnT in the retentate. The combined 15 L of permeate has the following composition: 2.24% DSC and a composition of 3.6% triose, 60.4% LNnT, and 16.6% pLNnH. This corresponds to 340 g of total sugars and 203 g of LNnT.

[0081] At this point, another 5 L of water is added to the feed before further filtration. After the additional 5 L, the retentate changes as follows: The remaining 25 L of retentate is now characterized by a dry weight DSC of 7.86% and a composition of 2.2% triose, 56.3% LNnT, and 26.9% pLNnH. This corresponds to 1.97 kg total sugars and 1.11 kg LNnT in the retentate. The combined 20 L of permeate has the following composition: 1.94% DSC and a composition of 3.4% triose, 61.3% LNnT, and 16.5% pLNnH. This corresponds to 390 g total sugars and 238 g LNnT.

[0082] After an additional 5 L, the retentate changes as follows: The remaining 20 L of retentate is now characterized by a dry weight DSC of 8.15% and a composition of 2.2% triose, 56.4% LNnT, and 27.4% pLNnH. This corresponds to 1.63 kg of total sugars and 919 g of LNnT in the retentate. The combined 25 L of permeate has the following composition: 1.94% DSC and a composition of 3.4% triose, 61.9% LNnT, and 17.5% pLNnH. This corresponds to 490 g of total sugars and 300 g of LNnT.

[0083] After the final 5 L, the combined 30 L of permeate had the following composition: 2.51% DSC, triose: 3.5%, LNnT: 59.7%, and pLNnH: 16.5%, corresponding to 750 g total sugars and 450 g LNnT.

[0084] From these results, the permeability coefficients for each sugar component can be calculated: triose permeates with a coefficient of approximately 1.55, LNnT with a coefficient of 1.05, and pLNnH with a coefficient of 0.62. The results are shown in Tables 4, 5 and 6.

[0085] [Table 4]

[0086] [Table 5]

[0087] [Table 6]

[0088] Example 3: By separating an LNnT-containing carbohydrate mixture containing two major by-products by simulated moving bed chromatography, higher sugars such as pLNnH were reduced to less than 3% and lower sugars such as triose and LNnT were accumulated.

[0089] For SMB purification, the clear, particle-free solution containing the three oligosaccharides was concentrated to approximately 300 g / L using a vacuum concentrator at 45 °C. For SMB chromatography, a closed-loop multicomponent SMB system was used, equipped with 12 columns (Prosep® columns, dimensions: 40 mm × 740 mm (Lartek, Eppelheim, Germany)) arranged in a 2 × 4 zone configuration. Each column contained 760 g of Purolite® PCR833H+ (Purolite, Ratingen, Germany) strong cation exchange resin.

[0090] The system was operated at 25°C with the following flow parameters set: Zone I flow rate was 30.00 ml / min, Zone II flow rate was set at 21.00 ml / min, Zone III flow rate was set at 21.48 ml / min, and Zone IV flow rate was set at 18.44 ml / min. The feed rate was set at 3.00 ml / min, the eluent flow rate at 11.56 ml / min, and the switching time at 17.92 min. Water containing 10% (v / v) food-grade ethanol was used as the eluent. Sugars smaller than pentaose, e.g., trioses and LNnT, smaller hydrolysis products, lactose, lacto-N-biose, glucose, galactose, and N-acetylgalactosamine, were primarily fractionated in the extract. Higher oligosaccharides, more precisely, sugars larger than tetraose, e.g., pentaose, hexaoses such as pLNnH, and larger oligosaccharides, e.g., heptaose or octaose, as well as residual salt contaminants from the eluent or resin loading, were fractionated in the raffinate. As described, With the selected settings, the SMB system was able to operate continuously for at least three months.

[0091] Using this protocol, the purity of LNnT could be significantly increased. In this first example, the following LNnT specifications were used: 12.7% DSC, composition: triose: 3.5%, LNnT: 59.7%, and pLNnH: 16.5%. After SMB chromatography, the extract had the following composition: triose: 4.9%, LNnT: 70.4%, and pLNnH: 2.2%. The raffinate had the following composition: triose: 0.7%, LNnT: 1.1%, and pLNnH: 59.5%. The purification yield was approximately 80%.

[0092] The results are shown in Table 7.

[0093] [Table 7]

[0094] Example 4: By separating an LNnT-containing carbohydrate mixture containing two major by-products by simulated moving bed chromatography, higher sugars such as pLNnH were reduced to less than 3% and lower sugars such as triose and LNnT were accumulated.

[0095] For SMB purification, the clear, particle-free solution containing the three oligosaccharides was concentrated to approximately 300 g / L using a vacuum concentrator at 45 °C. For SMB chromatography, a closed-loop multicomponent SMB system was used, equipped with 12 columns (Prosep® columns, dimensions: 40 mm × 740 mm (Lartek, Eppelheim, Germany)) arranged in a 2 × 4 zone configuration. Each column contained 760 g of Purolite® PCR833H+ (Purolite, Ratingen, Germany) strong cation exchange resin.

[0096] The system was operated at 25°C with the following flow parameters set: Zone I flow rate was 30.00 ml / min, Zone II flow rate was set at 21.00 ml / min, Zone III flow rate was set at 21.48 ml / min, and Zone IV flow rate was set at 18.44 ml / min. The feed rate was set at 3.00 ml / min, the eluent flow rate at 11.56 ml / min, and the switching time at 17.92 min. Water containing 10% (v / v) food-grade ethanol was used as the eluent. Sugars smaller than pentaose, e.g., trioses and LNnT, as well as smaller hydrolysis products such as lactose, lacto-N-biose, glucose, galactose, and N-acetylgalactosamine, were primarily fractionated in the extract. Higher oligosaccharides, more precisely, sugars larger than tetraose, e.g., pentaose and hexaoses such as pLNnH, and larger oligosaccharides, e.g., heptaose and octaose, as well as residual salt contaminants from the eluate or resin loading, were fractionated in the raffinate. With the described settings, the SMB system could be operated continuously for at least three months.

[0097] Using this protocol, the purity of LNnT could be significantly increased. In this second example, the following LNnT specifications were used: triose: 3.7%, LNnT: 67.6%, and pLNnH: 15.4%. After SMB chromatography, the extract had the following composition: triose: 4.3%, LNnT: 80.3%, and pLNnH: 0.6%. The raffinate had the following composition: triose: 5.3%, LNnT: 2.1%, and pLNnH: 47.9%. The purification yield was approximately 80%.

[0098] The results are shown in Table 8.

[0099] [Table 8]

[0100] Example 5: By separating an LNnT-containing carbohydrate mixture containing one major by-product by crystallization, a reduction of lower sugars such as trioses to less than 3% was achieved. A 385.3 g sugar mixture with the following composition was subjected to a crystallization process: triose: 4.9%, LNnT: 70.4%, pLNnH: 2.2%. The material was dissolved in approximately 0.4 L of deionized water and its DSC was set to 70% using a rotary evaporator under reduced pressure. The sugar mixture was removed from the rotary evaporator and stored in an environment that ensured a slow cooling rate of approximately 10° / h. This resulted in a solid layer forming on the surface of the mixture, and after 3 days, a completely crystalline mass was finally obtained. After complete crystallization, the crystalline mass was thoroughly mixed with two parts of a 70 vol% ethanol solution (70 vol% EtOH / 30 vol% HO). Finally, the resulting washing solution was removed under reduced pressure using a funnel frit filter. The washing process was repeated twice to obtain a purified LNnT crystalline mass that was drained and dried in a vacuum oven at 3 mbar and 35°C. The yield of the crystallization was determined to be 228.0 g (59.2%). The composition of the compound was determined to be triose content: 0.5%, LNnT content: 94.4%, and pLNnH content: 1.2%. However, considering the purity of the LNnT starting material and that of the crystalline product, the yield increased to 79.3% (94.4%). * 228g) / (70.4% * 385.3g).

[0101] The results are shown in Table 9.

[0102] [Table 9]

[0103] Example 6: By separating an LNnT-containing carbohydrate mixture containing one major by-product by crystallization, a reduction of lower sugars such as trioses to less than 3% was achieved. A 462.3 g sugar mixture with the following composition was sent to the crystallization process: triose: 4.3%, LNnT: 80.3%, pLNnH: 0.6%. The LNnT content was determined to be 371.1 g. The material was dissolved in approximately 0.5 L of deionized water and its DSC was adjusted to 69% by using a rotary evaporator under reduced pressure. The sugar mixture was removed from the rotary evaporator and stored in an environment that ensured a slow cooling rate of approximately 10° / h. This allowed a solid layer to begin growing on the surface of the mixture, and after two days, a completely crystalline mass was finally obtained. After complete crystallization, the crystalline mass was thoroughly mixed with two parts of a 70 vol% ethanol solution (70 vol% EtOH / 30 vol% HO), and finally, the resulting washing solution was removed under reduced pressure using a funnel frit filter. The washing process was repeated to obtain a purified LNnT crystalline mass that was drained and dried in a vacuum oven at 3 mbar and 35°C. The yield of the crystallization was determined to be 244.0 g (52.8%). The composition of the compound was determined to be triose content: 6.6%, LNnT content: 89.7%, and pLNnH content: <0.5%. Purification by crystallization was unsuccessful. However, the product was used in another crystallization.

[0104] 244.0 g of the sugar mixture was sent to the crystallization process. The amount of LNnT was determined to be 218.9 g. The material was dissolved in approximately 0.25 L of deionized water and then set to 70% DSC using a rotary evaporator under reduced pressure. The sugar mixture was removed from the rotary evaporator and stored in an environment that ensured a slow cooling rate of approximately 10° / h. This allowed a solid layer to begin growing on the surface of the mixture, and after 5 days, a completely crystalline mass was finally obtained. After complete crystallization, the crystalline mass was thoroughly mixed with two parts of 70 vol% ethanol solution, and finally, the resulting washing solution was removed under reduced pressure using a funnel frit filter. The washing process was repeated twice to obtain a drained and purified LNnT crystalline mass, which was then dried in a vacuum oven at 3 mbar and 35°C. The crystallization yield was determined to be 204.6 g (83.9%). The composition of the compound was determined to be triose content: 0.7%, LNnT content: 97.4%, and pLNnH content: 0.2%. Considering the purity of the LNnT starting material and that of the crystalline product, the yield increased to 91.1% (97.4%). * 204.6g) / (89.7% * 244.0g).

[0105] The results are shown in Table 10.

[0106] [Table 10]

[0107] Example 7: By separating a pLNnH-containing carbohydrate mixture containing one major by-product by crystallization, a reduction of lower sugars such as LNnT to less than 4% was achieved. Approximately 250 g of a sugar mixture with the following composition was subjected to a crystallization process: triose: <0.5%, LNnT: 2.1%, pLNnH: 47.9%. The remaining solids were essentially composed of residual salts. The material was dissolved in approximately 0.5 L of deionized water and its DSC was set to 55% using a rotary evaporator under reduced pressure. The sugar mixture was removed from the rotary evaporator and stored in an environment that ensured a slow cooling rate of approximately 10° / h. This resulted in the formation of a solid layer on the surface of the mixture, and after 5 days, a completely crystalline mass was finally obtained. After complete crystallization, 50 g of the crude crystalline mass was thoroughly mixed with two parts of a 70 vol% ethanol solution (70 vol% EtOH / 30 vol% HO). Finally, the resulting washing solution was removed under reduced pressure using a funnel frit filter. The washing process was repeated twice, and purified pLNnH crystalline mass was obtained after drainage. This was dried in a vacuum oven at 3 mbar and 35°C. The yield of the crystallization was determined to be 13.0 g (26.0%). The composition of the compound was determined to be triose content: 0%, LNnT content: 3.75%, pLNnH content: 80.6%. However, taking into account the pLNnH purity of the starting material and that of the crystalline product, and considering the humidity of the drained crystal mass at 25%, the yield increases to 58.3% (80.6%). * 13.0g) / (0.75 * 47.9% * 50g).

[0108] Example 8: Preparation of LNnT seed crystals from deionized water The 56% LNnT mixture obtained from the fermentation process was purified by repeated gel filtration steps using purified water as the solvent (Biorad Bio-Gel® P-2, fine granules). After volume reduction using a rotary evaporator, LNnT seed crystals were finally obtained. 10.0 g of the spray-dried starting material was diluted with purified water to 50% DSC and filtered three times. After each filtration step, the fractions containing primarily LNnT were combined, reconcentrated under vacuum, and diluted again to 50% DSC for the next gel filtration. After concentration under reduced pressure, 2.8 g of a gel-like material was obtained, which crystallized upon standing. The product was analyzed by HPAEC PAD chromatography, revealing that the purity of the unwashed crystals was 83.1% LNnT, with 6.3% unseparated triose as the main by-product.

[0109] Example 9: Crystallization of 200 g of LNnT by adding NaCl To prevent gel formation during LNnT crystallization, add 1 mass percent of a food-grade salt, such as NaCl, to the solution. Dissolve 200 g of LNnT (triose: 15.0%, LNnT: 65.9%, pLNnH: 4.4%; this is equivalent to 131.8 g of pure LNnT) and 2.0 g of NaCl in 200 mL of water. The solution is then set to 70% DSC. Add some seed crystals to the high-viscosity slurry, and allow the LNnT to crystallize at room temperature for 1 day. After complete crystallization, mix the crystalline mass with two parts of an 80 vol% ethanol solution (80 vol% EtOH / 20 vol% H2O) and centrifuge at 6000 rpm for 15 minutes. The precipitated carbohydrate mixture is then separated. The resulting precipitate was suspended in 1 part of an 80 vol% ethanol solution and centrifuged again. This process was repeated. After three centrifugations, the precipitate was freeze-dried. After the first crystallization, 163.6 g of LNnT was obtained. The purity of LNnT was increased by approximately 11% from 65.9% to 76.5% (triose: 6.5%, pLNnH: 5.2%), and 125.1 g of pure LNnT was obtained (yield: 94.9%).

[0110] Example 10: Second Crystallization of 200 g of LNnT 158.6 g of LNnT obtained from Example 9 is crystallized. Therefore, the starting material is dissolved in 159 mL of deionized water and concentrated under reduced pressure to 70% DSC. Seed crystals are added to the thick slurry, and the LNnT is crystallized at room temperature for 1 day. After complete crystallization, the crystalline mass is mixed with two parts of an 80 vol% ethanol solution (80 vol% EtOH / 20 vol% H2O) and centrifuged at 6000 rpm for 15 minutes. The precipitated carbohydrate mixture is suspended in another part of an 80 vol% ethanol solution and centrifuged again. This process is repeated. After three centrifugations, the precipitate is freeze-dried. After the second crystallization, 115.2 g of LNnT is obtained. The purity of LNnT was increased by approximately 6%, from 76.5% to 82.4% (triose: 2.4%, pLNnH: 5.7%), and 94.9 g of pure LNnT was obtained (yield: 78.2%).

[0111] Example 11: Crystallization of 100 g of LNnT by adding NaCl 100 g of LNnT (triose: 15.0%, LNnT: 65.9%, pLNnH: 4.4%; equivalent to 131.8 g of pure LNnT) and 1.0 g of NaCl were dissolved in 100 ml of water and then concentrated to 70% DSC under reduced pressure. Seed crystals were added to the high-viscosity slurry, and the LNnT was allowed to crystallize at room temperature for 1 day. After complete crystallization, the crystalline mass was mixed with two portions of an 80 vol% ethanol solution (80 vol% EtOH / 20 vol% H2O) and centrifuged at 6000 rpm for 15 minutes. The precipitated product was suspended in another volume of 80% EtOH and centrifuged again. This process was repeated. The precipitate was then freeze-dried. After the first crystallization, 67.0 g of LNnT was obtained. The purity of the material was increased by approximately 15% from 65.9% to 81.7% (triose: 6.2%, hexose: 6.0%), and 54.7 g of pure LNnT was obtained (yield 83.1%).

[0112] Example 12: Second crystallization of 100 g of LNnT 50.0 g of LNnT obtained from Example 11 was crystallized. The starting material was dissolved in 100 ml of water and concentrated to 70% DSC at 60°C. Seed crystals were added to the thick slurry, and the LNnT was allowed to crystallize at room temperature for 1 day. After complete crystallization, the crystalline mass was mixed with two volumes of an 80% ethanol solution (80% EtOH / 20% H2O) and centrifuged at 6000 rpm for 15 minutes. The precipitated carbohydrate mixture was suspended twice with another volume of 80% EtOH and centrifuged again. The precipitate was then freeze-dried. After the second crystallization, 36.0 g of LNnT was obtained. The purity of the material was increased by approximately 3% from 81.7% to 84.3% (triose: 2.9%, hexose: 6.4%), yielding 30.3 g of pure LNnT (74.3% yield).

[0113] Example 13: Homogenization of LNnT by using spray drying 228.0 g of LNnT having the following composition was homogenized by spray-drying the product in a Buchi B-290 apparatus (triose: 0.5%, LNnT: 94.4%, pLNnH: 1.2%). The material was then dissolved in 1.5 L of water (15.2% DSC), sterile filtered (45 mm pore size), and spray-dried at an inlet temperature of 130°C and an outlet temperature of 66°C. 178.0 g (78.1%) of a white, homogenous powder was obtained, and the residual water content was determined to be 8.6% by Karl Fischer titration. The purity of the material was determined to be 0.4% triose, 94.4% LNnT, and 1.1% pLNnH, yielding 168.0 g of pure, homogenous spray-dried LNnT.

[0114] Example 14: Homogenization of LNnT by using spray drying 204.0 g of LNnT having the following composition was homogenized by spray-drying the product in a Buchi B-290 apparatus (triose: 0.7%, LNnT: 97.4%, pLNnH: 0.2%). The material was then dissolved in 1.2 L of water (14.6% DSC), sterile filtered (45 mm pore size), and spray-dried at an inlet temperature of 130°C and an outlet temperature of 66°C. 129.0 g (63.2%) of a white, homogenous powder was obtained, and the residual water content was determined to be 7.1% by Karl Fischer titration. The purity of the material was determined to be triose: 0.3%, LNnT: 96.3%, and pLNnH: <0.25%, yielding 124.2 g of pure, homogenous spray-dried LNnT.

[0115] Example 15: Homogenization of LNnT by using spray drying 110.7 g of LNnT having the following composition was homogenized by spray drying the product in a Buchi apparatus B-290 (triose: 2.4%, LNnT: 82.4%, pLNnH: 5.7%). The material was then dissolved in 300 ml of water (27% DSC), then sterile filtered (pore size 45 mm) and spray dried at an inlet temperature of 130°C and an outlet temperature of 66°C. 71.9 g (65.0%) of a white homogeneous powder was obtained, which showed nearly the same spectrum as the starting material of Example 10, demonstrating the suitability of the resulting material for spray drying.

[0116] Example 16: Drying of LNnT by using oven drying 9.629 g of crystalline LNnT was oven-dried. After 7 days at 35°C, its weight decreased to 9.612 g, which corresponds to 99.8% purity. The LNnT purity was determined to be 88.7% before drying and 88.2% after drying, demonstrating the suitability of oven-drying at 35°C.

[0117] Example 17: Drying of LNnT by using vacuum oven drying 10.307 g of crystalline LNnT was oven-dried. After 7 days at 35°C, its weight decreased to 10.219 g, which corresponds to 99.8% purity. The LNnT purity was determined to be 88.7% before drying and 90.3% after drying, demonstrating the suitability of oven-drying at 35°C.

[0118] Example 18: Homogenization of LNnT by use of freeze-drying 10.265 g of crystalline LNnT was freeze-dried in a Christ BETA 2-8 LD plus apparatus. The HMO was then dissolved in approximately 90 mL of deionized water, frozen at -80°C, and then lyophilized to 2.0 mL. * 10 -3 The homogenized LNnT was subjected to a freeze-drying process at a pressure of 1000 mbar and cooled to -85°C. After 7 days, the weight was reduced to 9.668 g, which corresponds to 94.2% purity. The LNnT purity was determined to be 88.7% before drying and 90.2% after drying, demonstrating that freeze-drying is a suitable homogenization method.

[0119] Example 19: Homogenization of LNnT by use of freeze-drying 15.404 g of crystalline LNnT was freeze-dried in a Christ BETA 2-8 LD plus apparatus. The HMO was then dissolved in approximately 60 mL of deionized water, frozen at -80°C, and then lyophilized to 2.0 mL. * 10 -3 The homogenized LNnT was subjected to a freeze-drying process at a pressure of 1000 mbar and cooled to -85°C. After 7 days, the weight was reduced to 14.566 g, which corresponds to 94.6% purity. The LNnT purity was determined to be 88.7% before drying and 89.5% after drying, demonstrating that freeze-drying is a suitable homogenization method.

Claims

1. 1. A method for purifying LNnT (lacto-N-neotetraose) from a fermentation broth, the method comprising: - providing a fermentation broth containing LNnT, biomass, medium components, contaminants and carbohydrates other than LNnT; - subjecting the fermentation broth to at least one membrane filtration step using a nanofiltration membrane to provide a filtrate containing LNnT; - subjecting said filtrate to at least one simulated moving bed chromatography step to provide a purified solution containing LNnT; - subjecting said purified solution to at least one crystallization step to provide a crystalline mass; and - subjecting said crystalline mass to at least one homogenization step to provide homogenized, dried and purified LNnT. wherein the purified preparation contains LNnT having a purity of ≧90%.

2. 2. The method of claim 1, wherein the membrane filtration step is nanofiltration using a membrane with a molecular weight cutoff of 0.2 to 3.5 kDa.

3. 10. The method of any preceding claim, wherein the membrane has a molecular weight cut-off of 0.2 to 2.0 kDa.

4. 10. The method of any preceding claim, wherein the membrane has a molecular weight cut-off of 0.2 to 1.0 kDa.

5. 10. The method of any of the preceding claims, wherein the filtrate obtained from the membrane filtration step contains LNnT having a purity of greater than 60%, greater than 65%, or greater than 70%.

6. The simulated moving bed chromatography process comprises: i) at least four columns, preferably at least eight columns, more preferably at least twelve columns, at least one of which is a weak or strong cation exchange resin, preferably H ＋ Shape, Na ＋ shape, K ＋ Shape or Ca ２＋ and / or cation exchange resins of the form ii) four zones of different flow rates, I, II, III and IV; and / or iii) Water, preferably ethanol and water, more preferably 5 to 15 vol % ethanol an eluent comprising or consisting of ethanol and 85-95 vol% water, most preferably 9-11 vol% ethanol and 89-91 vol% water; iv) an operating temperature of 15 to 60°C, preferably 20 to 55°C, more preferably 25 to 50°C The method of claim 1 , comprising:

7. The simulated moving bed chromatography process comprises: i) four zones, I, II, III and IV, with different flow rates (the flow rates are preferably 22-32 ml / min in Zone I, 17-23 ml / min in Zone II, and 18-25 ml / min in Zone III / min, and / or 14-20 ml / min in Zone IV); and / or ii) a feed rate of 0.5 to 4 ml / min, preferably 2 ml / min; and / or iii) an eluent flow rate of 6 to 12 ml / min, preferably 8 ml / min; and / or iv) A switching time of 14 to 20 minutes, preferably 16 to 18 minutes, and more preferably 17 minutes. Preferably, 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 cation exchange resin. kg of cation exchange resin, most preferably 1.0 to 20 kg of cation exchange resin.

8. 10. The method of any of the preceding claims, wherein the purified solution obtained from the simulated moving bed chromatography contains LNnT having a purity of more than 75% or more than 80%.

9. The method according to claims 1, 6 to 8, wherein the purified solution obtained from at least one simulated moving bed chromatography step can be subjected to at least one other further purification step using simulated moving bed chromatography, providing a purified solution comprising LNnT having a purity of >85%, preferably >90%; more preferably >93%.

10. The crystallization step comprises: obtaining a mixture of at least two oligosaccharides containing at least LNnT at a concentration of at least about 1% DSC, preferably at least 10% DSC, preferably at least 30% DSC, more preferably at least about 50% DSC, and wherein the purity of said LNnT in said mixture is at least about 15%, preferably at least about 25%, preferably at least about 40%, more preferably at least about 60%; - crystallizing the purified solution obtained from the simulated moving bed chromatography step from an initial temperature of at least about 20-30°C, preferably at least about 30-40°C, preferably at least about 40-50°C, more preferably at least about 50-60°C; - crystallizing the saturated mass by cooling to at least 40-50°C, preferably at least about 30-40°C, preferably at least about 20-30°C, preferably at least about 10-20°C, more preferably at least about 0-10°C to obtain a homogeneous crystalline mass; - treating the crystalline mass with an alcohol, a solvent, or a mixture of a water-miscible alcohol or solvent with water (the alcohol or solvent content of the solution is 10-90 vol%, preferably 30-80 vol%, more preferably 50-70 vol%) to obtain a mixture of an alcohol- or solvent-containing solution and the crystalline mass; and - washing the alcohol or solvent-containing fraction or the sugar-containing fraction of the crystalline mass at least once to reduce the content of lower sugars from the crystalline mass; The method of claim 1 , comprising:

11. 11. The method of claims 1 and 10, wherein washing of the alcohol or solvent-containing fraction or the sugar-containing fraction of the crystals can be repeated at least twice to reduce the content of lower sugars.

12. 12. The method according to claims 1, 10 and 11, wherein the content of low chain sugars is less than 10%, preferably less than 5%, more preferably less than 3%.

13. 10. The method of any of the preceding claims, wherein the crystals obtained from the crystallization step contain LNnT with a purity of greater than 85%, greater than 90%, and greater than 95%.

14. 2. The method of claim 1, wherein the homogenization step can be selected from freeze drying, spray drying, roller or drum drying, and / or band or vacuum band drying.

15. 10. The method according to any one of the preceding claims, wherein the purity of LNnT in the purified preparation is ≧80%, preferably ≧85%, more preferably ≧90%, based on the dry matter of the purified preparation.

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