A method for purifying sialylated oligosaccharides

Abstract

The present application relates to the technical field of substance purification, in particular to a sialylated oligosaccharide purification method. The sialylated oligosaccharide purification method provided by the present application is simple and easy to operate, reduces the dissociation and browning of sialylated oligosaccharide in the purification process, effectively reduces the loss of sialylated oligosaccharide, and improves the yield of sialylated oligosaccharide. The sialylated oligosaccharide prepared by the purification method can achieve high purity and yield, and has good application prospect in industrial production.

Description

Technical Field

[0001] This invention relates to the field of substance purification technology, and in particular to a method for purifying sialylated oligosaccharides. Background Technology

[0002] Sialylated human milk oligosaccharides (SHMOs) are important oligosaccharides in breast milk. Their molecules contain sialic acid (SA), usually in the form of N-acetylneuraminic acid (Neu5Ac), which plays a crucial role in infant brain development, neurotransmission, and synapse formation, and also has immunomodulatory functions. Sialyllactose (SL) is a sialylated human milk oligosaccharide with important physiological functions. Among them, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are two particularly important sialylated human milk oligosaccharides in breast milk.

[0003] Currently, the main methods for synthesizing sialylated human milk oligosaccharides include chemical synthesis and biosynthesis. Chemical synthesis typically involves enzymatic reactions to synthesize sialylated human milk oligosaccharides, while biosynthesis utilizes microorganisms such as *E. coli*. In both methods, purifying the target product from the fermentation broth or enzymatic reaction solution is a crucial step in the preparation of sialylated human milk oligosaccharides. The currently used purification process mainly involves: hydrolyzing lactose to galactose using glycosidases, removing excess salt compounds using anion and cation exchange columns, and then removing galactose and other impurities through two nanofiltration or chromatographic separations. For example, the method disclosed in Chinese patent CN111447845B first hydrolyzes lactose to form galactose using glycosidases, and then uses 200L of H₂O₂ to purify the galactose. +A strong cation exchanger is used to remove cationic contaminants; then, NaOH is used to adjust the pH of the resulting solution to 7.0; next, a strong anion exchanger in chloride form is used to remove anions and colorant impurities from the solution. After nanofiltration and decolorization, other contaminating sugars are removed by chromatographic separation. The purpose of purification with the strong anion exchanger in chloride form is to remove excess impurity anions. This method requires the simultaneous addition of substances that inhibit the adsorption of sialylated oligosaccharides by the anion exchanger (e.g., co-anions). The removal of impurity sugars is not addressed in the ion exchange purification step; these are primarily removed in the chromatographic step. Similarly, the purification methods disclosed in CN112203519B and CN116964066A both employ this process route, except that they use membrane filtration for impurity sugar removal. Although the method disclosed in patent application WO2017152918A1 does not use glycosidase for lactose hydrolysis, based on its description of "allowing negatively charged materials to be absorbed onto anion exchange resins, including sialylated oligosaccharides / sialylated lactose. The liquid obtained after contacting the anion exchange resin as the eluent mainly contains water, cations, and neutral carbohydrates," it can be inferred that this process does not separate lactose and sialylated lactose in the anion exchange resin purification step, and it does not have special requirements for the anion exchange resin. Furthermore, based on the alternative process route it provides (purification using only a cation exchange column), it can be seen that the purpose of the ion exchange purification step in this process is mainly to prepare the product into a salt.

[0004] In summary, the current purification process for sialylated human milk oligosaccharides is still quite complex, requiring chromatographic separation to remove impurities, which increases the complexity of the process and the purification cost. Summary of the Invention

[0005] This invention provides a method for purifying sialylated oligosaccharides.

[0006] To simplify the purification process of sialylated oligosaccharides and improve purification efficiency, this invention attempts to remove excess acid, lactose, and small molecule salts using a simplified ion exchange process. During this process, the inventors unexpectedly discovered that while removing excess acid, lactose, and small molecule salts using ion exchange technology, approximately 10-15% of sialyl lactose is lost. The main reason for this is that sialyl lactose undergoes a prolonged elution time in the anion exchange resin, leading to its dissociation and browning. This results in a lower sialyl lactose content in the purified product or necessitates a further decolorization step to address the browning issue, thus reducing the yield. Through continuous experimentation, the inventors found that using a chloride-type weakly basic anion exchange resin can effectively solve these problems, reducing the loss of sialyl lactose during the removal of excess acid, lactose, and small molecule salts using ion exchange technology, and improving the yield. Although ion exchange technology is a known method for purifying sialylated oligosaccharides, and weakly basic anion exchange resins have been used, existing technologies often choose weakly basic anion exchange resins because they have the advantage that counterions are not released into the eluent compared to strongly basic anion exchange resins. Their main purpose is to remove anionic impurities, which is significantly different from the purpose and function of the chloride-type weakly basic anion exchange resin used in this invention.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] This invention provides a method for purifying sialylated oligosaccharides, the method comprising: treating a solution containing sialylated oligosaccharides with an ion exchange resin; the ion exchange resin treatment comprising: treating with a chloride-type weakly basic anion exchange resin, such that the sialylated oligosaccharides are adsorbed onto the chloride-type weakly basic anion exchange resin, and after elution, collecting the eluent containing sialylated oligosaccharides.

[0009] In this invention, the chloride-type weakly basic anion exchange resin is a chloride-modified weakly basic anion exchange resin. It differs significantly in structure and properties from existing free-base weakly basic anion exchange resins and chloride-type strong anion exchange resins. Weakly basic anion exchange resins typically contain bases with lone pairs of electrons that attract protons, such as certain nitrogen-containing groups, including, for example, primary amines, secondary amines, tertiary amines (free amine groups), guanidine groups, or nitrogen-containing heteroaryl groups (such as pyridine, pyrimidine, etc.), preferably including tertiary amine groups. In the free-base form, the base is not protonated; in other words, there is no counter anion in the base. Therefore, this group can adsorb acids, i.e., acid cations (H+). + ) and acid anions (X - All of these can be removed from the feed solution without ion exchange (without releasing counterions).

[0010] Furthermore, the chloride-type weakly basic anion exchange resin exhibits significantly different binding forces for sialylated oligosaccharides and impurities such as lactose, sialic acid, and pigments. Utilizing this characteristic, impurities such as lactose, sialic acid, and pigments can be efficiently removed during the chloride-type weakly basic anion exchange resin treatment process. At the same time, it effectively reduces the dissociation and browning of sialylated oligosaccharides during the elution process, thereby reducing the loss of sialylated oligosaccharides. Moreover, it can also reduce the elution of pigment impurities (generally speaking, the decolorization step before ion exchange resin treatment cannot completely remove pigment impurities, and some pigments will be adsorbed by the anion exchange resin, so it is necessary to reduce the possibility of pigments being eluted together with sialylated oligosaccharides). During the column pass and elution process, impurities such as sialic acid and lactose are removed. First, lactose is passed directly through the chloride-type weakly basic anion exchange resin and then eluted with an eluent. Sialidized oligosaccharides are eluted off. At this time, sialic acid and pigment impurities are still bound to the chloride-type weakly basic anion exchange resin and can be removed in the resin purification and regeneration steps (for example, when purifying the anion exchange resin with sodium hydroxide, all the pigments and sialic acid are eluted off, and then converted with sodium chloride to obtain the chloride-type weakly basic anion exchange resin again).

[0011] In some embodiments of the present invention, the chloride-type weakly basic anion exchange resin is obtained by modifying a weakly basic anion exchange resin with sodium chloride.

[0012] The present invention does not impose any special restrictions on the structural framework of chlorine-type weakly basic anion exchange resins, including but not limited to styrene-based, acrylic-based, acetic acid-based, epoxy-based, vinylpyridine-based, urea-formaldehyde series, and oxyethylene-based resins.

[0013] The ion exchange resin treatment described above also includes treatment with acidic cation exchange resins. Acidic cation exchange resin treatment is beneficial for removing inorganic cation impurities as well as impurities such as organic amines, amino acids, and short peptides. The resin eluent obtained from acidic cation exchange resin treatment contains acidic sialylated oligosaccharides. The pH of this resin eluent is typically 1-2, but the material residence time in this step is very short, making it difficult for the sialylated oligosaccharides to undergo hydrolysis.

[0014] Preferably, the acidic cation exchange resin is H + Strong acid cation exchange resin.

[0015] For H + The structural framework of the strong acid cation exchange resin is not specifically limited in this invention, and includes, but is not limited to, styrene-type, acrylic-type, etc.

[0016] In some specific embodiments of the present invention, the ion exchange resin treatment described above includes: first treating the solution containing sialylated oligosaccharides with an acidic cation exchange resin, and then treating it with a chloride-type weakly basic anion exchange resin.

[0017] In other specific embodiments of the present invention, the ion exchange resin treatment described above includes: first treating the solution containing sialylated oligosaccharides with a strong basic anion exchange resin, then with an acidic cation exchange resin, and finally with a chloride-type weak basic anion exchange resin. Introducing the strong basic anion exchange resin treatment before the acidic cation exchange resin treatment allows acidic impurities such as sialic acid to be adsorbed onto the strong basic anion exchange resin, thereby pre-removing acidic impurities such as sialic acid. The present invention does not have any particular limitations on the structural framework of the strong basic anion exchange resin, and various known strong basic anion exchange resins for purification can be used.

[0018] Preferably, after treatment with acidic cation exchange resin, the eluent containing sialylated oligosaccharides is collected, and the eluent is directly treated with a chloride-type weakly basic anion exchange resin without pH adjustment of the eluent obtained from the acidic cation exchange resin treatment.

[0019] In the above method, the eluent used in the treatment of the chlorine-type weakly basic anion exchange resin is a neutral salt and / or a weakly acidic salt.

[0020] Preferably, the concentration of the eluent is 0.01-0.05M.

[0021] The present invention has found that when the eluent concentration is higher than 0.05M, sialic acid and sialic acid oligosaccharides will be eluted together, while when the eluent concentration is 0.01-0.05M, sialic acid oligosaccharides will be eluted first, while sialic acid will not be eluted, thus allowing the collection of an eluent containing sialic acid oligosaccharides.

[0022] In some specific embodiments of the present invention, the concentration of the eluent is 0.03-0.05M.

[0023] Preferably, the eluent is a sodium salt.

[0024] In some embodiments of the present invention, the eluent is sodium chloride and / or sodium acetate.

[0025] The above method does not include a chromatographic treatment step after treatment with a chlorinated weakly basic anion exchange resin, and especially does not include an ion exchange resin treatment step. This invention achieves effective removal of impurities such as sialic acid, lactose, and pigments through the above-described ion exchange resin treatment process, ensuring high purity of sialic acid oligosaccharides, without requiring additional chromatographic treatment steps to remove impurities such as miscellaneous sugars, as is done in existing technologies such as CN111447845B.

[0026] To remove the eluent used in the treatment of the chlorine-type weakly basic anion exchange resin, a step of removing the eluent is also included after the chlorine-type weakly basic anion exchange resin treatment.

[0027] Preferably, nanofiltration is used to remove the eluent.

[0028] After removing the eluent, the resulting sialylated oligosaccharide solution can be spray-dried, freeze-dried, vacuum-dried, or crystallized to obtain sialylated oligosaccharides in solid form; or, the resulting sialylated oligosaccharide solution can be dehydrated by distillation (e.g., vacuum distillation) or nanofiltration to prepare a concentrated aqueous solution or syrup.

[0029] In the above method, the solution containing sialylated oligosaccharides is an aqueous solution containing sialylated oligosaccharides.

[0030] Preferably, the pH of the aqueous solution containing sialylated oligosaccharides is 6-7.

[0031] Preferably, the concentration of the aqueous solution containing sialylated oligosaccharides is 30-50 g / L.

[0032] The above-mentioned solution containing sialylated oligosaccharides is obtained by pretreating the fermentation broth or enzymatic reaction broth containing sialylated oligosaccharides through one or more steps.

[0033] Sialized oligosaccharides produced through fermentation or in vitro enzymatic processes typically contain, in addition to the sialylated oligosaccharides, the fermentation broth also contains biomass from the microbial cells used in fermentation, as well as proteins, protein fragments, nucleic acids, inorganic salts, endotoxins, biogenic amines, unreacted carbohydrates (e.g., lactose), carbohydrate byproducts, sialic acid, pigments, etc. In vitro enzymatic reaction solutions typically contain, in addition to the sialylated oligosaccharides, proteins, protein fragments, inorganic salts, unreacted carbohydrate acceptors (e.g., lactose), carbohydrate byproducts, sialic acid and its precursors, etc. Therefore, it is preferable to pretreat the fermentation broth or enzymatic reaction solution containing sialylated oligosaccharides before treatment with ion exchange resin.

[0034] Preferably, the pretreatment includes one or more selected from centrifugation, ultrafiltration, concentration, desalination, and decolorization.

[0035] In the above pretreatment, ultrafiltration or centrifugation is preferably set as the first step. Ultrafiltration separates biomass and high molecular weight suspended solids from soluble components in aqueous media such as fermentation broth or enzymatic reaction solutions. The soluble components are separated into the permeate through an ultrafiltration membrane. Any conventional ultrafiltration membrane can be used, with a molecular weight cutoff (MWCO) range of approximately 1–500 kDa, such as 10–250 kDa, 50–100 kDa, 200–500 kDa, 100–250 kDa, 1–100 kDa, 1–50 kDa, 10–25 kDa, 1–5 kDa, or any other suitable subrange. The membrane material can be ceramic or made from synthetic or natural polymers, such as polysulfone, polypropylene, cellulose acetate, or polylactic acid. Ultrafiltration can be performed more than once using ultrafiltration membranes with different MWCOs, where the MWCO of the membrane used in the first ultrafiltration is higher than that used in the second. The permeate obtained from the above ultrafiltration contains sialylated oligosaccharides.

[0036] In some specific embodiments of the present invention, a 20-50 nm ceramic membrane is used to ultrafilter fermentation broth or enzymatic reaction broth containing sialylated oligosaccharides. Preferably, two ultrafiltration steps are performed, with a high-temperature inactivation and decolorization filtration step between the two ultrafiltration steps; the second ultrafiltration uses an ultrafiltration membrane with an MWCO of 2-4 kDa.

[0037] The pretreatment described above may also include a concentration step, or a concentration and desalination step. Concentration or concentration and desalination may be achieved using nanofiltration.

[0038] Nanofiltration can be performed after ultrafiltration or, optionally, decolorization. Nanofiltration can concentrate and / or remove salts (primarily monovalent ions). The molecular weight cutoff (MWCO) of the nanofiltration membrane ensures retention of the target sialidized oligosaccharide, with a lower MWCO than the ultrafiltration membrane used above, and approximately 25-50% of the target sialidized oligosaccharide molecular weight. For example, a nanofiltration membrane with an MWCO of approximately 150-300 Da is suitable for retaining sialyl lactose. Nanofiltration can be used in combination with water percolation for more effective removal of permeable molecules until the permeate conductivity indicates no salt or extremely low salt content.

[0039] The pretreatment described above may further include a decolorization step, preferably using activated carbon treatment. The decolorization process can be performed after an ultrafiltration or nanofiltration step. Activated carbon treatment helps remove or reduce pigments and water-soluble contaminants (e.g., salts).

[0040] Carbohydrates such as sialylated oligosaccharides can bind to the surface of activated carbon particles from their aqueous solutions, while pigments also adsorb onto the activated carbon. When carbohydrates and pigments are adsorbed, water-soluble materials that are not bound to the activated carbon or have a weak binding can be eluted with water. Changing the eluent from water to an ethanol-water solution allows for easy elution of the adsorbed sialylated oligosaccharides, while the pigments remain adsorbed on the activated carbon, thus achieving simultaneous decolorization and desalination. Of course, under certain conditions, sialylated oligosaccharides may not be adsorbed, or at least substantially not adsorbed, onto the activated carbon particles, while the pigments remain adsorbed.

[0041] In this invention, sialylated oligosaccharides can be synthesized through in vitro enzymatic reactions or produced by culturing microorganisms capable of synthesizing sialylated oligosaccharides.

[0042] Preferably, the method for producing the sialylated oligosaccharide is microbial fermentation. The microorganisms used for fermentation include, but are not limited to, Escherichia coli.

[0043] In some embodiments of the present invention, the method for preparing the solution containing sialylated oligosaccharides includes: removing cells and proteins from the fermentation broth containing sialylated oligosaccharides and then performing a decolorization treatment, followed by concentrating and desalting the clear liquid obtained after decolorization.

[0044] The concentration and desalination can be performed using nanofiltration technology; the removal of bacteria and proteins can be performed using ultrafiltration technology.

[0045] In this invention, the sialylated oligosaccharide contains at least one sialic acid group.

[0046] Preferably, the sialylated oligosaccharide is sialylated human lactose oligosaccharide; more preferably, it is sialylated lactose.

[0047] The aforementioned sialylated human milk oligosaccharides are selected from 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), 3-fucosyl-3'-sialyl lactose (FSL), LSTA, fucosyl-LSTa (FLSTa), LSTb, fucosyl-LSTb (FLSTb), LSTc, fucosyl-LSTc (FLSTc), sialyl-LNH (SLNH), sialyl-lacto-N-hexasaccharide (SLNH), sialyl-lacto-N-neohexose I (SLNH-I), sialyl-lacto-N-neohexose II (SLNH-II), or disialyl-lacto-N-tetrasaccharide (DS-LNT).

[0048] In some embodiments of the present invention, the sialylated oligosaccharide is 3'-sialyllactose or 6'-sialyllactose. The purification method of the present invention is particularly suitable for purifying 3'-sialyllactose or 6'-sialyllactose produced by fermentation.

[0049] In some embodiments of the present invention, a method for purifying sialic acid lactose from a fermentation broth obtained by culturing microorganisms capable of producing sialic acid lactose is provided, the method comprising the following steps:

[0050] (1) The fermentation broth was subjected to ultrafiltration to obtain ultrafiltration permeate;

[0051] (2) The ultrafiltration permeate is subjected to a heating inactivation treatment to obtain an inactivated product;

[0052] (3) The inactivated substance was decolorized with activated carbon to obtain a decolorized solution;

[0053] (4) Ultrafiltration is performed on the decolorizing solution to obtain ultrafiltration permeate;

[0054] (5) Perform nanofiltration on the ultrafiltration permeate to obtain nanofiltration retentate;

[0055] (6) The nanofiltration retentate is first treated with an acidic cation exchange resin, and the eluent containing sialylated oligosaccharides is collected. The eluent containing sialylated oligosaccharides is then treated with a chloride-type weakly basic anion exchange resin so that the sialylated oligosaccharides are adsorbed onto the chloride-type weakly basic anion exchange resin. The eluent containing sialylated oligosaccharides is then eluted with an eluent and collected.

[0056] The beneficial effects of this invention include at least the following: the purification method for sialylated oligosaccharides provided by this invention is not only simple and easy to implement, but also reduces the dissociation and browning of sialylated oligosaccharides during the purification process, effectively reducing the loss of sialylated oligosaccharides and improving the yield of sialylated oligosaccharides; this purification method does not rely on subsequent chromatographic separation to remove impurities, and effectively removes impurities such as impurities by using ion exchange technology. The sialylated oligosaccharides obtained using this purification method can achieve high purity and yield, and have good application value in industrial production. Detailed Implementation

[0057] In this invention, the term "sialylated oligosaccharide" refers to a sugar polymer containing at least two monosaccharide units, at least one of which is a sialic acid group (N-acetylneuraminoyl) moiety. Sialinated oligosaccharides can have a linear or branched structure, comprising monosaccharide units linked together by glycosidic bonds. Sialinated oligosaccharides are sialylated human lactose oligosaccharides.

[0058] The term "sialylated human milk oligosaccharide," also known as "acidic human milk oligosaccharide (acidic HMO)," refers to a complex carbohydrate found in human breast milk. It comprises a core structure with a lactose unit at the reducing end, which can be elongated by one or more β-N-acetyl-lactoamine and / or one or more β-lactose-N-disaccharide units. This core structure is partially substituted with an α-N-acetyl-neuraminoyl group (sialic acid group) and optionally partially substituted with an L-pyranofucose group. In this regard, it contains at least one sialic acid residue in its structure. Sialized human milk oligosaccharides include 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), 3-fucosyl-3'-sialyl lactose (FSL), LSTA, fucosyl-LSTa (FLSTa), LSTb, fucosyl-LSTb (FLST b), LSTc, fucosyl-LSTc (FLST c), sialic acid-LNH (SLNH), sialic acid-lact-N-hexasaccharide (SLNH), sialic acid-lact-N-neohexose I (SLNH-I), sialic acid-lact-N-neohexose II (SLNH-II), and disialialic acid-lact-N-tetrasaccharide (DS-LNT).

[0059] The term "sialic acid lactose" is preferably 3'-SL or 6'-SL.

[0060] The term "aqueous solution containing sialylated oligosaccharides" preferably refers to the raw material for ion exchange resin treatment according to the present invention, which is an optional pretreatment containing sialylated oligosaccharides. The aqueous medium containing sialylated oligosaccharides can be directly treated with the ion exchange resin of the present invention, or pretreated by one or more steps described above before ion exchange resin treatment. Through pretreatment, the aqueous medium is partially purified, and the content of impurities such as bacteria and proteins is reduced or eliminated.

[0061] In some specific embodiments of the present invention, a method for purifying sialic acid lactose from fermentation broth is provided, the technical route of which is as follows: fermentation broth → ceramic membrane filtration to remove bacteria → collection of clear liquid → protein removal → decolorization → collection of clear liquid → nanofiltration → collection of retentate → treatment with strong basic anion exchange resin to remove impurities such as sialic acid → treatment with acidic cation exchange resin → treatment with chloride-type weak basic anion exchange resin to remove impurities such as lactose and salts → elution with 0.03~0.05M sodium chloride or sodium acetate solution → collection of eluent → nanofiltration desalting → drying (freeze-drying or spray drying).

[0062] In other specific embodiments of the present invention, a method for purifying sialic acid lactose from fermentation broth is provided, the technical route of which is as follows: fermentation broth → ceramic membrane filtration to remove bacteria → collection of clear liquid → protein removal → decolorization → collection of clear liquid → nanofiltration → collection of retentate → treatment with acidic cation exchange resin → treatment with chloride-type weakly basic anion exchange resin to remove impurities such as sialic acid, lactose, and salts → elution with 0.03~0.05M sodium chloride or sodium acetate solution → collection of eluent → nanofiltration desalting → drying (freeze-drying or spray drying).

[0063] The method for purifying sialic acid lactose from fermentation broth as described above specifically includes the following steps:

[0064] (1) The fermentation broth was filtered through a ceramic membrane, and water was added to permeate the clear liquid to obtain ceramic membrane filtrate;

[0065] (2) Heat the ceramic membrane filtrate to 80-85℃ to inactivate the filtrate and precipitate the protein. Keep warm for 25-35 min to obtain the inactivated product;

[0066] (3) Cool the inactivated substance to 40~45℃, add activated carbon and stir, filter to obtain decolorized solution;

[0067] (4) Filter the decolorizing solution using a 2-4 kDa ultrafiltration membrane, add water to permeate the clear solution, and obtain the ultrafiltration membrane filtrate;

[0068] (5) Use a nanofiltration membrane to further filter the ultrafiltration membrane filtrate from step (4) to remove impurities such as salts, and obtain a nanofiltration retentate. The concentration of sialic acid lactose in the nanofiltration retentate is controlled at 30-50 g / L.

[0069] (6) The nanofiltration retentate is first treated with a strong basic anion exchange resin and the supernatant is collected; then the supernatant is treated with an acidic cation exchange resin and the supernatant is collected; then the supernatant is treated with a chloride-type weak basic anion exchange resin, where sialic acid lactose is adsorbed onto the chloride-type weak basic anion exchange resin. Sodium chloride or sodium acetate is used for elution, and the eluent is collected. The pH of the eluent is 6-7 (Cl type).

[0070] (7) Use a nanofiltration membrane to filter the eluent containing step (6) to remove the eluent and collect the retentate containing sialic acid lactose;

[0071] (8) Drying: The retentate containing sialic acid lactose from step (7) is dried to obtain the sialic acid lactose product.

[0072] The sialic acid lactose product obtained by the above purification method has a sialic acid lactose content of >95% and a product yield of >80%.

[0073] In this invention, non-limiting examples of weakly basic anion exchange resins include LX-360, D354-FD, Dowex™66, Amberlite™ FPA53, Lewatit S6368 A, Lewatit S4268, Lewatit S5528, Lewatit S6368A (Lanxess AG. Cologne, DE), Dowex AG 1x2 (Mesh 200-400), Dowex 1x8 (Mesh 100-200), Purolite Chromalite CGA 100x4 (Purolite GmbH, Ratingen, DE), and DowAmberlite FPA51 (DowChemicals, ML, USA), among other brands or models. Chloride-form weakly basic anion exchange resins can be obtained by modifying the above-mentioned weakly basic anion exchange resins with chloride ions.

[0074] Taking LX-360 as an example, the following method can be used for chloride ion modification: 1. Load the resin into the exchanger and backwash the resin with water until the effluent is clear. 2. Pass in an 8-10% NaCl solution twice the volume of the resin, soak the resin in the saline solution for about 24 hours, and then rinse with water until the effluent is colorless and odorless. 3. First, pass in about 4% HCl twice the volume of the resin, allowing one volume of 4% HCl to pass directly through the column, then soak the resin in another volume of 4% HCl for 4-8 hours, and wash with water until the pH is around 3. Then, pass in about 4% NaOH twice the volume of the resin, allowing one volume of 4% NaOH to pass directly through the column, then soak the resin in another volume of 4% NaOH for 4-8 hours, and wash with water until the pH is around 10. Finally, 2-3 times the resin volume of 4% sodium chloride is introduced at a rate of 2-4 BV / h to obtain the chloride-form weakly basic anion exchange resin, which is used in the following examples. This conversion method is a conventional resin modification method in the art. Those skilled in the art can make adaptive adjustments according to the specific resin conditions, and no excessive limitations are imposed.

[0075] Non-limiting examples of acidic cation exchange resins in this invention include: Amberlite™ IR100, Amberlite™ IR120, Amberlite™ FPC22Dowex™ 50WX, Finex™ CS16GC, Finex™ CS13GC, Finex™ CS12GC, Finex™ CS11GC, Lewatit™ S, Diaion™ SK, Diaion™ UBK, Amberjet™ 1000, Amberjet™ 1200, Dowex™ 88, etc.

[0076] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0077] The nanofiltration retentate containing sialic acid lactose used in the following examples was prepared by the following method:

[0078] (1) Separation of fermentation broth and clear liquid: The fermentation broth for the production of sialic acid lactose by microbial fermentation (the content of sialic acid lactose in the fermentation broth is about 30g / L) is filtered through a 30nm ceramic membrane, and process water is added to produce clear liquid to obtain ceramic membrane filtrate.

[0079] (2) Temperature inactivation: The ceramic membrane filtrate is heated to 80°C to inactivate it and precipitate the protein. The temperature is maintained for 30 minutes to obtain the inactivated product.

[0080] (3) Decolorization and filtration: Cool the inactivated substance to 40~45℃, add 4% activated carbon and stir for 30min, then filter to obtain the decolorized solution.

[0081] (4) Ultrafiltration: The decolorized liquid is filtered using a 3kDa ultrafiltration membrane, and process water is added to permeate the clear liquid to obtain the ultrafiltration membrane filtrate.

[0082] (5) Nanofiltration: The ultrafiltration membrane filtrate from step (4) is further filtered using a nanofiltration membrane to remove impurities such as salts, resulting in a nanofiltration retentate containing sialic acid lactose (i.e., an aqueous solution containing sialic acid lactose). The concentration of sialic acid lactose in the nanofiltration retentate is controlled at 30-50 g / L, and the pH is 6-7.

[0083] Example 1

[0084] This embodiment provides a method for purifying sialic acid lactose, which includes the following steps:

[0085] The nanofiltration retentate containing 3'-sialyl lactose was passed through H... + The supernatant was collected using a strong acidic cation exchange resin (LX-150) and then passed through a chloride-type weak basic anion exchange resin (obtained by chloride ion modification of the weak basic anion exchange resin LX-360). Sialolactose was adsorbed onto the chloride-type weak basic anion exchange resin, while lactose flowed directly through the chloride-type weak basic anion exchange resin with the supernatant. Then, elution was performed using 0.05 mol / L sodium chloride to obtain an eluent containing sialic acid lactose.

[0086] The sialic acid-lactose content in the eluent was measured, and the percentage of sialic acid loss was calculated based on the sialic acid-lactose content in both the nanofiltration retentate and the eluent. The results showed that the percentage of sialic acid-lactose loss during ion exchange resin treatment was only 2.2%.

[0087] The eluent containing sialic acid lactose was subjected to nanofiltration and vacuum drying to obtain the sialic acid lactose product, wherein the sialic acid lactose content was 95.5% and the overall yield was 85.3%.

[0088] Example 2

[0089] This embodiment provides a method for purifying sialic acid lactose, which includes the following steps:

[0090] The nanofiltration retentate containing 3'-sialyl lactose is first passed through a strongly basic anion exchange resin (WSD260). SA in the material is adsorbed onto the resin, while SL is not adsorbed and passes directly through. The supernatant is collected. The supernatant is then treated with the strongly basic anion exchange resin and passed through H₂O₂. + The supernatant was collected using a strong acidic cation exchange resin (LX-150) and then passed through a chloride-type weak basic anion exchange resin (obtained by chloride ion modification of the weak basic anion exchange resin LX-360). Sialolactose was adsorbed onto the chloride-type weak basic anion exchange resin, while lactose flowed directly through the chloride-type weak basic anion exchange resin with the supernatant. Then, elution was performed using 0.05 mol / L sodium chloride to obtain an eluent containing sialic acid lactose.

[0091] The sialic acid-lactose content in the eluent was measured, and the percentage of sialic acid loss was calculated based on the sialic acid-lactose content in both the nanofiltration retentate and the eluent. The results showed that the percentage of sialic acid-lactose loss during ion exchange resin treatment was only 4.5%.

[0092] The eluent containing sialic acid lactose was subjected to nanofiltration and vacuum drying to obtain the sialic acid lactose product, wherein the sialic acid lactose content was 96.2% and the yield was 82.4%.

[0093] Example 3

[0094] This embodiment provides a method for purifying sialic acid lactose, which includes the following steps:

[0095] The nanofiltration retentate containing sialic acid lactose was passed through H... + The supernatant was collected using a strong acidic cation exchange resin (LX-150) and then passed through a chloride-type weak basic anion exchange resin (obtained by chloride ion modification of the weak basic anion exchange resin LX-360). Sialolactose was adsorbed onto the chloride-type weak basic anion exchange resin, while lactose flowed directly through the chloride-type weak basic anion exchange resin with the supernatant. Then, elution was performed using 0.03 mol / L sodium acetate to obtain an eluent containing sialic acid lactose.

[0096] The sialic acid-lactose content in the eluent was measured, and the percentage of sialic acid loss was calculated based on the sialic acid-lactose content in both the nanofiltration retentate and the eluent. The results showed that the percentage of sialic acid-lactose loss during ion exchange resin treatment was only 2.8%.

[0097] The eluent containing sialic acid lactose was subjected to nanofiltration and vacuum drying to obtain the sialic acid lactose product, wherein the sialic acid lactose content was 95.8% and the yield was 84.6%.

[0098] Comparative Example 1

[0099] This comparative example provides a method for purifying sialic acid lactose, which differs from the method in Example 1 only in that the chloride-type weakly basic anion exchange resin (obtained by chloride ion modification of weakly basic anion exchange resin LX-360) is replaced with LX-360 (a weakly basic anion exchange resin (OH-) that has not undergone chloride ion modification). - )).

[0100] The sialic acid-lactose content in the eluent was measured, and the percentage of sialic acid loss was calculated based on the sialic acid-lactose content in both the nanofiltration retentate and the eluent. The results showed that the percentage of sialic acid-lactose loss during the ion exchange resin treatment was 14.5%.

[0101] The eluent containing sialic acid lactose was subjected to nanofiltration and vacuum drying to obtain the sialic acid lactose product, wherein the sialic acid lactose content was 82.5% and the yield was 65.4%.

[0102] Comparative Example 2

[0103] This comparative example provides a method for purifying sialic acid lactose, which differs from the method in Example 1 only in that the chloride-type weak basic anion exchange resin (obtained by chloride ion modification of weak basic anion exchange resin LX-360) is replaced with a strong basic anion exchange resin WSD260.

[0104] The sialic acid-lactose content in the eluent was measured, and the percentage of sialic acid loss was calculated based on the sialic acid-lactose content in both the nanofiltration retentate and the eluent. The results showed that the percentage of sialic acid-lactose loss during ion exchange resin treatment was 8.3%, indicating that the adsorption capacity of strongly basic anion exchange resin for sialic acid-lactose was significantly worse than that of chloride-type weakly basic anion exchange resin.

[0105] The eluent containing sialic acid lactose was subjected to nanofiltration and vacuum drying to obtain the sialic acid lactose product, wherein the sialic acid lactose content was 81.3% and the yield was 68.3%.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying sialylated oligosaccharides, characterized in that, The method includes: treating a solution containing sialylated oligosaccharides with an ion exchange resin; The ion exchange resin treatment includes: first treating the solution containing sialylated oligosaccharides with an acidic cation exchange resin, and then treating it with a chloride-type weakly basic anion exchange resin, so that the sialylated oligosaccharides are adsorbed onto the chloride-type weakly basic anion exchange resin, and after elution, collecting the eluent containing sialylated oligosaccharides. Alternatively, the solution containing sialylated oligosaccharides can be treated first with a strong base anion exchange resin, then with an acidic cation exchange resin, and then with a chloride-type weak base anion exchange resin, so that the sialylated oligosaccharides are adsorbed onto the chloride-type weak base anion exchange resin. After elution, the eluent containing sialylated oligosaccharides can be collected. In this process, after treatment with acidic cation exchange resin, the eluent containing sialylated oligosaccharides is collected, and the eluent is directly treated with a chlorine-type weakly basic anion exchange resin without pH adjustment of the eluent obtained from the acidic cation exchange resin treatment. In the treatment process of the chloride-type weakly basic anion exchange resin, the eluent used for elution is a neutral sodium salt and / or a weakly acidic sodium salt; the concentration of the eluent is 0.01-0.05M; After treatment with a chlorine-type weakly basic anion exchange resin, the method does not include an ion exchange resin treatment step; The solution containing sialylated oligosaccharides is obtained by pretreating fermentation broth or enzymatic reaction broth containing sialylated oligosaccharides; the pretreatment includes concentration and desalting. The sialylated oligosaccharide is sialyl lactose.

2. The method according to claim 1, characterized in that, The acidic cation exchange resin is H + Strong acid cation exchange resin.

3. The method according to claim 1 or 2, characterized in that, After treatment with a chlorine-type weakly basic anion exchange resin, a step of removing the eluent is also included.

4. The method according to claim 3, characterized in that, Nanofiltration is used to remove the eluent.

5. The method according to any one of claims 1, 2, and 4, characterized in that, The solution containing sialylated oligosaccharides is an aqueous solution containing sialylated oligosaccharides.

6. The method according to claim 5, characterized in that, The pH of the aqueous solution is 6-7.

7. The method according to any one of claims 1, 2, 4, and 6, characterized in that, The pretreatment also includes one or more of centrifugation, ultrafiltration, and decolorization.

8. The method according to any one of claims 1, 2, 4, and 6, characterized in that, The method for preparing the solution containing sialylated oligosaccharides includes: removing cells and proteins from the fermentation broth containing sialylated oligosaccharides and then performing decolorization treatment; and concentrating and desalting the clear liquid obtained after decolorization.