Large-scale preparation method of sialic acid glycopeptide

Abstract

The invention relates to the technical field of separation and purification of bioactive substances, in particular to a large-scale preparation method of sialic acid glycopeptide. Comprising the following steps: taking degreased yolk powder as a raw material, adding pure water, and extracting twice at 60-70 DEG C; adding gel type cation exchange resin to adjust the pH value to 4.5 + / -0.05, filtering out the resin, and concentrating, washing and filtering by using an ultrafiltration membrane with the molecular weight of 10000; adding a sodium borate solution into the concentrated solution, adjusting the pH value to 8.6-8.8, continuing membrane separation and washing filtration, and collecting filtrate containing a boric acid-SGP complex; converting the macroporous anion exchange resin into a borate type, cooling the filtrate to 18-22 DEG C, passing through a column, washing with pure water and carrying out gradient elution with an ammonium acetate solution, and collecting an eluent with the purity of more than or equal to 95.0%; and adjusting the pH value of the eluent to 6.0 + / -0.05, carrying out nanofiltration desalination, carrying out 0.22 mu m filtration, and carrying out freeze drying to obtain the product. In this way, the purposes of large batch, low cost, high efficiency and environmental protection are achieved.

Description

A method for large-scale preparation of sialic acid glycopeptides Technical Field

[0001] This invention relates to the field of bioactive substance separation and purification technology, and in particular to a method for the large-scale preparation of sialic acid glycopeptides. Background Technology

[0002] Sialylglycopeptide (SGP) is a glycopeptide with a complete, naturally occurring complex N-glycan chain, composed of eleven monosaccharides and six amino acids. Its non-reducing terminus is typically two sialic acids, hence it is also known as a disialylglycopeptide. It is relatively abundant in egg yolks and its structure is extremely similar to human N-glycan chains, making it a highly humanized glycopeptide. The biantennary oligosaccharide structure of SGP is a representative N-glycan structure of protein glycosylation, widely used in the synthesis of functional glycopeptides and glycoproteins, and is an important raw material for synthesizing single-glycan glycopeptides and glycoproteins. Recently, SGP has been used as a substrate for enzymatic glycosylation reactions to modify antibody drugs, thereby obtaining efficacy-optimized glycoengineered antibodies and site-directed antibody-drug conjugates, becoming one of the important methods for modifying antibody-based macromolecular drugs. Therefore, sialyl glycopeptides play a vital role in the study of glycoprotein glycan structure and function, as well as in the development of biomedicine. Although various oligosaccharides can now be prepared through biosynthesis and chemical methods, current technology still cannot synthesize complete sialylated N-glycan chains on an application scale. The structural formula of SGP is as follows:

[0003] CN109824762A discloses a method for large-scale separation and purification of sialic acid glycopeptides (SGP). The method involves treating egg yolks with phenol, freeze-drying the crude SGP, separating and purifying it using a cotton-filled hydrophilic chromatography column, and finally freeze-drying the purified SGP. However, this invention still uses large amounts of phenol and acetonitrile, requires multiple freeze-drying processes for intermediate products, is cumbersome, and uses highly toxic excipients, making it unsuitable for large-scale production. CN116675734A discloses an improved industrial production method for SGP using ultrasonic degreasing and extraction. After nanofiltration, the SGP is crystallized in an alcohol solution, dissolved again in water, and then prepared using C18 liquid chromatography. The collected SGP fraction is then nanofiltered or rotary evaporated and freeze-dried to obtain the product. This invention uses multiple organic solvents such as methanol, ethanol, acetonitrile, and ethyl acetate, which require large quantities, have a complex process, a long cycle, and high packing material costs. CN117003827A discloses a method for separating and purifying sialic acid glycopeptides, which sequentially uses a mixed defatting solution composed of Bacillus saeformis and QuaraBoost lipase, followed by ethyl acetate for defatting the raw material, ethanol for extraction, and then uses a pre-packed polymer column PolyRP30-300 for separation and purification, followed by water elution and freeze-drying to obtain the pure product. This invention uses large amounts of ethyl acetate and ethanol, and the cross-use of the two solvents is not conducive to solvent recovery and reuse, while also resulting in high packing material costs.

[0004] In conclusion, it is essential to propose a large-scale preparation method for sialic acid glycopeptides that is efficient, low-cost, and environmentally friendly. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the large-scale preparation of sialic acid glycopeptides, which achieves the goals of large batch size, low cost, high efficiency, and environmental friendliness.

[0006] To achieve the above objectives, this invention employs a method for the large-scale preparation of sialic acid glycopeptides, comprising the following steps: using defatted egg yolk powder as raw material, adding pure water, and performing two extractions at 60-70℃, collecting the extract after filtration; slowly adding 001×7(H) gel-type cation exchange resin to the combined extract, stirring to adjust the pH to 4.5±0.05, filtering off the resin to obtain a solution containing the PV-SGP complex; separating and concentrating the solution using a 10000 molecular weight ultrafiltration membrane, continuously washing with pure water after concentration, and collecting the concentrate; adding sodium borate solution to the concentrate. The solution was adjusted to pH 8.6-8.8 with sodium hydroxide solution, and membrane separation was continued with continuous washing and filtration with pure water. The filtrate containing boric acid-SGP complex was collected. The D201(OH) macroporous anion exchange resin was converted to borate type. The filtrate was cooled to 18-22℃ and passed through a chromatography column. It was washed with pure water and eluted sequentially with ammonium acetate solutions of different concentration gradients. The eluent with HPLC purity ≥95.0% was collected. The pH of the eluent was adjusted to 6.0±0.05 with acetic acid. After desalting and concentrating through a nanofiltration membrane, it was filtered through a 0.22μm filter membrane and freeze-dried to obtain the sialic acid glycopeptide product.

[0007] In the step of using defatted egg yolk powder as raw material, adding pure water, and performing two extractions at 60-70℃: the defatted egg yolk powder is selected from one of the following two types: a protein byproduct obtained after extracting egg yolk lecithin using supercritical carbon dioxide degreasing + ethanol degreasing process, with a total fat content ≤5% and a particle size of 120-200 mesh; or ordinary commercially available egg yolk powder as starting material, which is degreased by high-concentration alcohol, with a total fat content ≤3% and a particle size of 120-200 mesh; the process parameters for the two extractions are: the solid-liquid ratio for the first extraction is 1kg:4-5L, the extraction temperature is 65±2℃, and the extraction time is 1-1.5h; the solid-liquid ratio for the second extraction is 1kg:3-4L, the extraction temperature is 65±2℃, and the extraction time is 0.5-1h.

[0008] In the steps of separating and concentrating the solution using a 10,000 molecular weight ultrafiltration membrane, adding pure water for continuous washing and filtration, and collecting the concentrate: the ultrafiltration membrane is a high-temperature ultrafiltration membrane, the ultrafiltration feed temperature is controlled at 60℃, the pressure is maintained at 1.2MPa, and the amount of pure water added for washing is 5 to 7 times the volume of the concentrate.

[0009] In the steps of adding sodium borate solution to the concentrate, adjusting the pH to 8.6-8.8 with sodium hydroxide solution, continuing membrane separation and continuous washing with pure water, and collecting the filtrate containing boric acid-SGP complex, the mass ratio of sodium borate to defatted egg yolk powder is 0.7-1.2 g: 1 kg, and the concentration of the sodium borate solution is 10-15 g / L.

[0010] In the steps of converting the D201(OH) macroporous anion exchange resin to borate type, cooling the filtrate to 18~22℃ and passing it through a chromatography column, rinsing with pure water, and eluting sequentially with ammonium acetate solutions of different concentration gradients, and collecting the eluent with HPLC purity ≥95.0%: the flow rate of the chromatography column is controlled at 2~4 BV / h; the concentration gradient of the ammonium acetate solution is 40mM, 80mM, 120mM, 160mM, and 200mM, and the volume of the eluent for each concentration is 2 BV.

[0011] In the step of converting D201(OH) macroporous anion exchange resin to borate type, cooling the filtrate to 18~22℃ and passing it through a chromatography column, rinsing with pure water, and eluting with ammonium acetate solutions of different concentration gradients to collect the eluent with HPLC purity ≥95.0%, the conversion method of D201(OH) macroporous anion exchange resin is as follows: boric acid solution is passed into the chromatography column packed with resin until the eluent is weakly acidic, and then rinsed with pure water.

[0012] In the step of adjusting the pH of the eluent to 6.0 with acetic acid, desalting and concentrating it through a nanofiltration membrane, filtering it through a 0.22μm filter membrane, and then freeze-drying it to obtain the sialic acid glycopeptide product: the nanofiltration membrane desalting and concentration process is as follows: after concentrating it to a volume of 1 / 5 to 1 / 6 of the initial eluent volume, add 4 to 5 times the volume of the concentrated liquid and pure water for continuous washing and filtration.

[0013] This invention discloses a method for the large-scale preparation of sialic acid glycopeptides. First, defatted egg yolk powder is used as raw material, and pure water is added. The mixture is extracted twice at 60-70°C, and the extract is collected after filtration. Then, 001×7 (H) gel-type cation exchange resin is slowly added to the combined extract, and the pH is adjusted to 4.5±0.05 by stirring. The resin is then filtered off to obtain a solution containing the PV-SGP complex. The solution is then separated and concentrated using a 10000 molecular weight ultrafiltration membrane. After concentration, pure water is added for continuous washing and filtration, and the concentrate is collected. Next, sodium borate solution is added to the concentrate, and the pH is adjusted to 8.6-8.8 using sodium hydroxide solution. Membrane separation was continued, and pure water was added for continuous washing and filtration. The filtrate containing boric acid-SGP complex was collected. The D201(OH) macroporous anion exchange resin was converted to borate type. The filtrate was cooled to 18-22℃ and passed through a chromatography column. It was washed with pure water and eluted sequentially with ammonium acetate solutions of different concentration gradients. The eluent with HPLC purity ≥95.0% was collected. Finally, the pH of the eluent was adjusted to 6.0±0.05 with acetic acid. After desalting and concentrating through a nanofiltration membrane and filtering through a 0.22μm filter membrane, the product was freeze-dried to obtain sialic acid glycopeptide. Through the above method, the goals of large-scale production, low cost, high efficiency, and green environmental protection were achieved.

[0014] The technical mechanism of this invention is as follows: Egg yolk high-phosphorus protein (PV) is one of the main protein components of egg yolk. Its molecular structure is rich in phosphate groups, which have cation exchange function. The peptide chain of SGP contains three free amino groups, Lys-1 and Lys-5. The extract of defatted egg yolk powder contains a large amount of PV. Without introducing new anions, a gel-type cation exchange resin is used to remove cations, adjusting the pH of the extract to below the isoelectric point of SGP, causing the amino groups on its peptide chain to ionize. These ionized amino groups then interact with the phosphate groups on PV through ionic bonds, forming a water-soluble PV-SGP protein complex. The bound SGP cannot permeate through a low-molecular-weight ultrafiltration membrane, while inorganic salts, amino acids, peptides, and small-molecule proteins can. SGP can effectively separate from substances with molecular weights smaller than PV. Subsequently, by adjusting the solution pH, SGP is released from the protein complex and permeates through the ultrafiltration membrane, achieving separation from PV and proteins with molecular weights larger than PV. Meanwhile, when adjusting the pH of the solution, sodium borate solution is added. Under these conditions, the cis-ortho-dihydroxy group on SGP can quickly form a complex with boric acid, and the filtrate can be directly used for the next purification step.

[0015] Commonly used boric acid affinity chromatography utilizes the reversible and specific binding between the boric acid groups loaded on the packing material and cis-o-dihydroxy compounds to achieve the separation and purification of target compounds from impurities. This invention also utilizes this property of boric acid, as well as the newly added anionic groups on the boric acid-SGP complex. When boric acid forms a complex with the cis-o-dihydroxy groups on SGP, the acidity is significantly enhanced, competitively displacing the borate groups loaded on the resin. 1 mol of SGP can form 4 mol of boric acid complex, and combined with its two carboxyl groups, it can form multi-site ionic bonds, significantly enhancing the adsorption capacity of SGP on the anion exchange resin. Gradient elution using ammonium acetate solutions of different concentrations can achieve good separation of SGP from monosialotyl-glycopeptides and other proteins. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 is a flowchart of the steps of the large-scale preparation method of sialic acid glycopeptide of the present invention.

[0018] Figure 2 is a process flow diagram of the large-scale preparation method of sialic acid glycopeptide of the present invention.

[0019] Figure 3 is the HPLC chromatogram of the extract in process step S100 of the present invention (SGP-RT: 7.839 min).

[0020] Figure 4 is the HPLC chromatogram of the ultrafiltration filtrate of process step S300 of the present invention (SGP-RT: 7.832 min).

[0021] Figure 5 is the HPLC chromatogram of the ultrafiltration concentrate of process step S300 of the present invention (SGP-RT: 8.008 min).

[0022] Figure 6 is the HPLC chromatogram of the product of the present invention. Figure 7 is the HPLC-ELSD-MASS chromatogram of the product of the present invention.

[0023] Figure 8 shows the product of this invention. 1 H NMR spectrum. Detailed Implementation

[0024] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0025] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0026] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0027] Please refer to Figures 1-8. This invention provides a method for the large-scale preparation of sialic acid glycopeptides, comprising the following steps: S100: Using defatted egg yolk powder as raw material, add pure water and extract twice at 60-70℃; S200: Slowly add gel-type cation exchange resin to the extract, stir to adjust the pH to 4.5±0.05, filter out the resin, and obtain a solution containing PV-SGP complex; S300: Separate and concentrate the solution using a 10000 molecular weight ultrafiltration membrane, add pure water for continuous washing after concentration, and collect the concentrate; S400: Add sodium borate solution to the concentrate. The pH was adjusted to 8.6-8.8 with sodium hydroxide solution, and membrane separation was continued with continuous washing and filtration with pure water. The filtrate containing boric acid-SGP complex was collected. S500: The macroporous anion exchange resin was converted to borate type. The filtrate was cooled to 18-22℃ and passed through a chromatography column. It was washed with pure water and eluted sequentially with ammonium acetate solutions of different concentration gradients. The eluent with HPLC purity ≥95.0% was collected. S600: The pH of the eluent was adjusted to 6.0±0.05 with acetic acid. After desalting and concentrating by nanofiltration membrane, it was filtered through a 0.22μm filter membrane and freeze-dried to obtain sialic acid glycopeptide product.

[0028] Furthermore, in the step of using defatted egg yolk powder as raw material, adding pure water, and performing two extractions at 60-70℃, followed by filtration and collection of the extract: the defatted egg yolk powder is selected from one of the following two types: a protein byproduct obtained by supercritical carbon dioxide degreasing + ethanol degreasing process after extracting egg yolk lecithin, with a total fat content ≤5% and a particle size of 120-200 mesh; or obtained by using ordinary commercially available egg yolk powder as starting material and degreasing it with high-concentration alcohol, with a total fat content ≤3% and a particle size of 120-200 mesh; the process parameters for the two extractions are: the solid-liquid ratio for the first extraction is 1kg:4-5L, the extraction temperature is 65±2℃, and the extraction time is 1-1.5h; the solid-liquid ratio for the second extraction is 1kg:3-4L, the extraction temperature is 65±2℃, and the extraction time is 0.5-1h.

[0029] Furthermore, in the steps of separating and concentrating the solution using a 10,000 molecular weight ultrafiltration membrane, continuously washing with pure water after concentration, and collecting the concentrate: the ultrafiltration membrane is a high-temperature ultrafiltration membrane, the ultrafiltration feed temperature is controlled at 60℃, the pressure is maintained at 1.2MPa, and the amount of pure water added for washing is 5 to 7 times the volume of the concentrate.

[0030] Further, in the steps of adding anhydrous sodium borate to the concentrate, adjusting the pH to 8.6-8.8 with sodium hydroxide solution, continuing membrane separation and continuously washing with pure water, and collecting the filtrate containing boric acid-SGP complex: the mass ratio of anhydrous sodium borate to defatted egg yolk powder is 0.7-1.2 g: 1 kg, and the concentration of sodium borate is 10-15 g / L.

[0031] Furthermore, in the steps of converting the D201(OH) macroporous anion exchange resin to borate type, cooling the filtrate to 18~22℃ and passing it through a chromatography column, rinsing with pure water, and eluting sequentially with ammonium acetate solutions of different concentration gradients, and collecting the eluent with HPLC purity ≥95.0%: the flow rate of the chromatography column was controlled at 2~4 BV / h; the concentration gradient of the ammonium acetate solution was 40mM, 80mM, 120mM, 160mM, and 200mM, and the volume of the eluent for each concentration was 2 BV.

[0032] Furthermore, in the steps of converting the D201(OH) macroporous anion exchange resin to the borate type, cooling the filtrate to 18~22℃ and passing it through a chromatography column, rinsing with pure water, and eluting with ammonium acetate solutions of different concentration gradients to collect the eluent with HPLC purity ≥95.0%, the conversion method of the D201(OH) macroporous anion exchange resin is as follows: purging the chromatography column packed with resin into boric acid solution until the eluent is weakly acidic, and rinsing with an appropriate amount of pure water.

[0033] Furthermore, in the steps of adjusting the pH of the eluent to 6.0±0.05 with acetic acid, desalting and concentrating it through a nanofiltration membrane, filtering it through a 0.22μm filter membrane, and then freeze-drying it to obtain the sialic acid glycopeptide product: the nanofiltration membrane desalting and concentration process is as follows: after concentrating to a volume of 1 / 5 to 1 / 6 of the initial eluent volume, add 4 to 5 times the volume of the concentrated liquid and pure water for continuous washing and filtration.

[0034] In this embodiment, defatted egg yolk powder is first used as raw material. Pure water is added, and extraction is performed twice at 60-70°C. The extract is collected after filtration. Then, 001×7(H) gel-type cation exchange resin is slowly added to the combined extract, and the pH is adjusted to 4.5±0.05 by stirring. The resin is then filtered off to obtain a solution containing the PV-SGP complex. The solution is then separated and concentrated using a 10000 molecular weight ultrafiltration membrane. After concentration, pure water is added for continuous washing and filtration, and the concentrate is collected. Next, sodium borate solution is added to the concentrate, and the pH is adjusted to 8.6-8.8 with sodium hydroxide solution. Membrane filtration continues. Separate and continuously wash with pure water, collecting the filtrate containing the boric acid-SGP complex; convert the D201(OH) macroporous anion exchange resin to the borate type, cool the filtrate to 18-22℃, pass it through a chromatography column, wash with pure water, and elute sequentially with ammonium acetate solutions of different concentration gradients, collecting the eluent with HPLC purity ≥95.0%; finally, adjust the pH of the eluent to 6.0±0.05 with acetic acid, desalt and concentrate it through nanofiltration, filter it through a 0.22μm filter membrane, and freeze-dry it to obtain the sialic acid glycopeptide product; through the above method, the goals of large-scale production, low cost, high efficiency, and environmental friendliness are achieved. Example

[0035] Weigh 2 kg of defatted egg yolk powder (SGP content determined by HPLC external standard method was 0.19%), add 10 L of pure water, stir and heat to 65℃ for 1.5 h, filter to obtain extract A and filter cake. Add 8 L of water to the filter cake, stir and heat to 65℃ for 1 h, filter to obtain extract B. Combine extracts A and B, stir and slowly add 001×7 (H) gel-type cation exchange resin until pH 4.53, filter off the resin, and separate and concentrate using a 10000 molecular weight ultrafiltration membrane until the concentrate volume is about 400 mL. Add 2 L of pure water and wash continuously to obtain concentrate C. Slowly add a solution prepared with 1.4 g of anhydrous sodium borate, slowly adjust the pH to 8.74 with sodium hydroxide solution, continue membrane separation, and slowly add 2.5 L of pure water and wash continuously to obtain 2.6 L of filtrate D.

[0036] 160g of D201(OH) was packed into a glass chromatography column, and boric acid solution was passed through it until the eluent was weakly acidic. The column was then rinsed with pure water to obtain D201 (borate type) resin. The filtrate D was cooled to 20°C in an ice-water bath and passed through the chromatography column at a flow rate of 3-4 BV / h. The column was rinsed with 500mL of pure water, and 1L of a 200mM ammonium acetate stock solution (pH 5.52) was prepared. Then, 500mL each of 40, 80, 120, 160, and 200mM ammonium acetate solutions were prepared, eluting sequentially from low to high concentration at a flow rate of 3-4 BV / h, with each fraction being 0.1 BV. HPLC analysis was performed. Eluents with HPLC purity ≥99.0% and 95.0-99.0% were mixed to obtain eluents E-1 and E-2, respectively, which were stored frozen at -20°C.

[0037] After packing the cation exchange resin column, it was regenerated by sequentially rinsing with 5% sodium hydroxide solution, 5% hydrochloric acid solution, and pure water; the anion exchange resin was regenerated by sequentially rinsing with 5% hydrochloric acid solution, 5% sodium hydroxide solution, and pure water. Example

[0038] Using the resin regenerated in Example 1, and with all other conditions remaining the same as in Example 1, eluents E-1 and E-2 were obtained and stored frozen at -20°C. Example

[0039] Using the regenerated resin from Example 2, the ultrafiltration membrane was replaced with a 10,000 molecular weight high-temperature ultrafiltration membrane. Extracts A and B were ultrafiltered sequentially according to time. The feed temperature was controlled at 60°C, and all other conditions were the same as in Example 1, yielding eluents E-1 and E-2, which were then frozen and stored at -20°C. Example

[0040] The eluents E-1 obtained in Examples 1-3 were thawed and mixed. The pH was adjusted to 5.99 with acetic acid solution. The mixture was then desalted and concentrated using a nanofiltration membrane until the volume of the concentrate was approximately 400 mL. 2 L of pure water was added for continuous washing and filtration, followed by filtration through a 0.22 μm filter membrane to obtain concentrate F-1. This concentrate was freeze-dried to obtain 3.42 g of product (purity ≥99%). The eluents E-2 obtained in Examples 1-3 were thawed and mixed. The pH was adjusted to 6.01 with acetic acid solution. The mixture was then desalted and concentrated using a nanofiltration membrane until the volume of the concentrate was approximately 400 mL. 2 L of pure water was added for continuous washing and filtration, followed by filtration through a 0.22 μm filter membrane to obtain concentrate F-2. This concentrate was freeze-dried to obtain 5.72 g of product (purity 95-99%). Examples

[0041] Approximately 500L of pure water was pumped into a 1000L stainless steel extraction vessel, and 100.6kg of defatted egg yolk powder (SGP content determined by HPLC external standard method was 0.19%) was added. Stirring and steam were started, and extraction was maintained at 65±2℃ for 1.5h. Steam was then turned off, and the mixture was pumped into a plate and frame filter press to obtain extract A and filter cake. The filter cake was transferred to the extraction vessel, and another 300L of pure water was pumped in. Stirring and steam were started, and extraction was maintained at 65±2℃ for 1h. Steam was then turned off, and the mixture was pumped into a plate and frame filter press to obtain extract B. After extract A was pumped into a buffer tank, stirring was started, and 001×7(H) gel-type cation exchange resin was slowly added until the pH reached 4.51. The mixture was then passed through a 0.22μm precision filter (post-pump) and pumped into a membrane separation device. Separation and concentration were performed using a 10000 molecular weight 4040 type high-temperature ultrafiltration membrane, with the pressure maintained at 1.2MPa. The treatment method for extract B is the same as that for A. Concentrate to the minimum volume (approximately 18 L), then slowly add 70 L of pure water and continuously wash and filter until the minimum volume is reached, yielding concentrate C. Adjust the pressure to 0 MPa and reduce the flow rate. Weigh 70 g of sodium borate (anhydrous) solution, add 5 L of pure water and stir until completely dissolved. Then slowly add this solution to concentrate C. Slowly adjust the pH to 8.65 with 10% sodium hydroxide solution and continue membrane separation. Add a total of approximately 90 L of pure water and continuously wash and filter, yielding approximately 92 L of filtrate D. Transfer to a refrigerator to cool.

[0042] 8 kg of D201(OH) was loaded into a PP chromatography column and converted to the borate form. Filtrate D was cooled to approximately 18°C ​​and added to the column using a peristaltic pump at a flow rate of 2-3 BV / h. The column was rinsed with 24 L of pure water. 80 L of a 200 mM ammonium acetate stock solution (pH 5.50) was prepared and diluted to 24 L each of 40, 80, 120, 160, and 200 mM ammonium acetate solutions. Elution was performed sequentially from low to high concentration at a flow rate of 3-4 BV / h, with each fraction being 0.1 BV. HPLC analysis was performed. Eluents with HPLC purity ≥99.0% were mixed, and eluents with purity between 95.0% and 99.0% were mixed to obtain eluents E-1 and E-2, respectively. These eluents were stored at -20°C and processed five times using an 1812 nanofiltration membrane. After thawing, the pH was adjusted to 6.04 with acetic acid solution. The solution was then passed through a 0.22 μm precision filter and subsequently desalted and concentrated using a nanofiltration membrane until the concentrate volume reached approximately 400 mL. 2 L of pure water was then added for continuous washing and filtration, yielding approximately concentrates F-1 and F-2. These were filtered through a 0.22 μm membrane and then freeze-dried to obtain 43.36 g of product (purity ≥99%) and 102.79 g of product (purity 95~99%). Example

[0043] Commercially available egg yolk powder was extracted by reflux with petroleum ether and 90% ethanol, respectively. The mixture was then concentrated to obtain total egg yolk fat. Solubility tests were conducted using ethanol of different concentrations at different temperatures, as shown in Table 1. 98% and anhydrous ethanol showed high solubility; however, considering the cost of reusing ethanol during production, 98% was selected. Separately, SGP lyophilized powder was added to 98% ethanol and refluxed. Small amounts were dried at 2, 4, and 6 hours, and liquid chromatography analysis showed that SGP exhibited good stability within 6 hours under reflux conditions of 98% ethanol.

[0044] Ethanol concentration 94% 94% 96% 96% 98% 98% Anhydrous Anhydrous Temperature 75℃ Boiling Point 75℃ Boiling Point 75℃ Boiling Point 75℃ Boiling Point Solubility 1.9% 6.7% 3.0% 17.1% 7.2% 25.3% 20.6% 34.1% Table 1 shows that 20 kg of commercially available egg yolk powder was weighed and placed in a 100 L glass reactor. 60 L of 98% ethanol was added, and the mixture was stirred and heated to boiling. The mixture was immediately discharged and filtered. The filter cake was re-added, and another 40 L of 98% ethanol was added. The mixture was stirred and heated to boiling, and the mixture was immediately discharged and filtered. The reactor was rinsed with about 20 L of 98% ethanol, and the filter cake was rinsed. The filter cake was dried to obtain 8.6 kg of defatted egg yolk powder. The SGP content was determined to be 0.22% by HPLC external standard method, and the total fat content was determined to be 2.6% by referring to the national standard GB5009.6-2016 (method II).

[0045] Weigh 3 kg of the above-mentioned ethanol-defatted egg yolk powder, add 15 L of pure water, stir and heat to 70 °C for 1 h, filter to obtain extract A and filter cake. Add 12 L of water to the filter cake, stir and heat to 70 °C for 0.5 h, filter to obtain extract B. Stir extracts A and B separately and slowly add 001×7 (H) gel-type cation exchange resin to pH 4.48 and 4.53 respectively. After filtering off the resin, separate and concentrate using a 10000 molecular weight high-temperature ultrafiltration membrane. After extract A is concentrated, add extract B, and finally concentrate to a volume of about 400 mL. Add 2.0 L of pure water and wash continuously to obtain concentrate C. Slowly add a solution prepared with 2.4 g of anhydrous sodium borate, slowly adjust the pH to 8.69 with sodium hydroxide solution, continue membrane separation, and slowly add 2.5 L of pure water and wash continuously to obtain 2.6 L of filtrate D.

[0046] 300g of D201(OH) was packed into a glass chromatography column and converted to the borate form. The filtrate D was cooled to 20°C in an ice-water bath and passed through the column at a flow rate of 2-3 BV / h. It was washed with 1000mL of pure water, and 3L of a 200mM ammonium acetate stock solution (pH 5.50) was prepared. Then, 920mL each of 40, 80, 120, 160, and 200mM ammonium acetate solutions were prepared, eluting sequentially from lowest to highest concentration at a flow rate of 3-4 BV / h, with each fraction being 0.1 BV. The HPLC analysis was performed. Mixing solutions with a purity ≥99.0% and 95.0~99.0% yields eluents E-1 and E-2, respectively. The pH is adjusted to 5.0±0.05 with acetic acid solution, and the solutions are desalted and concentrated using nanofiltration membranes until the volume of the concentrate is approximately 400 mL. Then, 2 L of pure water is added for continuous washing and filtration to obtain approximately concentrates F-1 and F-2. After filtration through a 0.22 μm filter membrane, the solutions are lyophilized to obtain 1.17 g of product (purity ≥99%) and 3.95 g of product (purity 95~99%).

[0047] Further details are shown in Table 2 below: | Name | Raw Material Content / % | Purity (≥99%) / % | Purity (95~99%) / % | Yield / % | |---|---|---|---|---|---| | Example 4 | 0.192 | 99.49 | 6.77 | 7.55 | | Example 5 | 0.187 | 99.29 | 6.37 | 5.94 | | Example 6 | 0.229 | 99.49 | 6.17 | 2.18 | Table 2. Additionally, the SGP high-performance liquid chromatography (HPLC) method includes the following solution preparation: Buffer salt solution: an aqueous solution containing 0.1% (V / V) TFA; Preparation of reference solution: Take an appropriate amount of SGP reference standard, transfer it to a volumetric flask, add pure water to dissolve and dilute to the mark, shake well, and filter to obtain a reference solution with a concentration of approximately 0.3 mg / mL; Preparation of sample solution: Take an appropriate amount of product (lyophilized powder), transfer it to a volumetric flask, add pure water to dissolve and dilute to the mark, shake well, and filter to obtain a sample solution with a concentration of approximately 0.4-1.5 mg / mL. Central control sample solution: obtained by filtration; Preparation of raw material content determination solution: Weigh 10g of defatted egg yolk powder, add 50mL of pure water, stir in a water bath at 65℃ for 2h, filter, add 40mL of pure water to the filter cake, stir in a water bath at 65℃ for 1h, filter, rinse the filter cake with 20mL of pure water, collect the filtrate, make up to 100mL, and filter to obtain the solution; HPLC conditions: mobile phase: A-mobile phase (TFA 0.1%V / V), B-acetonitrile, flow rate: 1mL / min, wavelength: 210nm, column temperature: 35℃.

[0048] Column: SHIMADZU Wondasil C18-WR 5μm 4.6*150mm.

[0049] Linear Elution Gradient Table: Time / min, Mobile Phase A / %, Mobile Phase B / % 0.991 15.85 15.1885 15.20 9.91 26.991 Table 3. Determination method: Accurately measure 10 μL and 20 μL of the reference solution and 20 μL of the sample solution, inject them into the liquid chromatograph, and inject each in parallel twice. Record the chromatogram and calculate the result by peak area according to the external standard method.

[0050] Comparative Example 1: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65±2℃ for 1 h, filter to obtain extract A and filter cake. Add another 3 L of water to the filter cake, stir and heat to 65±2℃ for 0.5 h, filter to obtain extract B. Combine extracts A and B, and measure the pH to be 5.65. Without adjusting the pH, separate and concentrate using a 6000 molecular weight ultrafiltration membrane until the concentrated liquid volume is approximately 400 mL. Determine the SGP content in the extract and filtrate using HPLC external standard method, and calculate the permeate.

[0051] Permeability = Absolute mass of SGP in filtrate / Absolute mass of SGP in extract before membrane separation * 100%.

[0052] Comparative Example 2: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65 ± 2 °C for 1 h, filter to obtain extract A and filter cake. Add another 3 L of water to the filter cake, stir and heat to 65 ± 2 °C for 0.5 h, filter to obtain extract B. Combine extracts A and B, adjust the pH to 4.51 with dilute hydrochloric acid, and separate and concentrate using a 6000 molecular weight ultrafiltration membrane until the concentrated liquid volume is approximately 400 mL. Determine the SGP content in the extract and filtrate by HPLC external standard method, and calculate the permeate.

[0053] Comparative Example 3: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65 ± 2 °C for 1 h, filter to obtain extract A and filter cake. Add another 3 L of water to the filter cake, stir and heat to 65 ± 2 °C for 0.5 h, filter to obtain extract B. Combine extracts A and B, adjust the pH to 4.52 with acetic acid, and separate and concentrate using a 6000 molecular weight ultrafiltration membrane until the concentrated liquid volume is approximately 400 mL. Determine the SGP content in the extract and filtrate by HPLC external standard method, and calculate the permeate.

[0054] Comparative Example 4: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65±2℃ for 1 h, filter to obtain extract A and filter cake. Add 3 L of water to the filter cake, stir and heat to 65±2℃ for 0.5 h, filter to obtain extract B. Combine extracts A and B, stir continuously, and slowly add D001 macroporous cation exchange resin until pH 4.49. Filter to remove the resin and determine the retention rate of SGP in the extract before and after pH adjustment. Separate and concentrate using a 6000 molecular weight ultrafiltration membrane to a concentrate volume of approximately 400 mL. Determine the SGP content in the extract and filtrate using HPLC external standard method and calculate the permeation rate. Freeze-dry the concentrate, determine the SGP content in the freeze-dried powder using HPLC external standard method, and calculate the SGP recovery rate.

[0055] SGP retention rate = SGP peak area of ​​the extract after adjustment / SGP peak area of ​​the extract before adjustment * 100%.

[0056] SGP yield = (absolute mass of SGP in freeze-dried powder / absolute mass of SGP in extract) * 100%.

[0057] Comparative Example 5: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65±2℃ for 1 h, filter to obtain extract A and filter cake. Add 3 L of water to the filter cake, stir and heat to 65±2℃ for 0.5 h, filter to obtain extract B. Combine extracts A and B, stir continuously, and slowly add 001×7 (H) gel-type cation exchange resin until pH 4.52. Filter out the resin and determine the SGP retention rate in the extract before and after pH adjustment. Separate and concentrate using a 6000 molecular weight ultrafiltration membrane to a concentrate volume of approximately 400 mL. Determine the SGP content in the extract and filtrate using HPLC external standard method and calculate the permeation rate. Freeze-dry the concentrate, determine the SGP content in the freeze-dried powder using HPLC external standard method, and calculate the SGP recovery rate.

[0058] Comparative Example 6: Weigh 1 kg of defatted egg yolk powder, add 4 L of pure water, stir and heat to 65±2℃ for 1 h, filter to obtain extract A and filter cake. Add 3 L of water to the filter cake, stir and heat to 65±2℃ for 0.5 h, filter to obtain extract B. Combine extracts A and B, stir continuously, and slowly add 001×7 (H) gel-type cation exchange resin until pH 4.51. Filter out the resin and determine the retention rate of SGP in the extract before and after pH adjustment. Separate and concentrate using a 10000 molecular weight ultrafiltration membrane to a concentrate volume of approximately 400 mL. Determine the SGP content in the extract and filtrate by HPLC external standard method and calculate the permeation rate. Freeze-dry the concentrate, determine the SGP content in the freeze-dried powder by HPLC external standard method, and calculate the SGP recovery rate.

[0059] Comparative Example 7: Take the lyophilized powder from Comparative Example 6, dissolve it in 500 mL of water, add 0.6 g of anhydrous sodium borate, stir to dissolve, adjust the pH to 8.6 with sodium hydroxide solution, separate and concentrate using a 10000 molecular weight ultrafiltration membrane, slowly add 2.5 L of pure water for continuous washing, collect the filtrate, desalinate and concentrate using a nanofiltration membrane to obtain about 400 mL of concentrate, lyophilize, determine the SGP content of the lyophilized powder by HPLC external standard method, and calculate the SGP recovery rate.

[0060] SGP yield = Absolute mass of freeze-dried SGP in Comparative Example 7 / Absolute mass of freeze-dried SGP in Comparative Example 6 * 100%.

[0061] Further details are shown in Table 4 below: Name | Retention Rate / % | Permeability / % | Lyophilized Powder | SGP Content / % | Yield / % | Extraction Solution / / 4.9% / | Comparative Example 1 / 8 | 9.5% / | Comparative Example 2 / 5 | 5.7% / | Comparative Example 3 / 4 | 2.9% / | Comparative Example 4 | 46.3 | 1.2 | 22.8 | 45.5 | Comparative Example 5 | 98.9 | 1.9 | 25.6 | 96.4 | Comparative Example 6 | 99.1 | 3.4 | 29.7 | 94.7 | Comparative Example 7 / / 5 | 1.0 | 92.4 Tables 4 (Comparative Examples 1-3) show that the 6000 molecular weight ultrafiltration membrane has a weak retention effect on SGP in the untreated extract. While adjusting the pH with strong or weak acids has some retention effect, it is not ideal. This may be because the endogenous cations in the egg yolk powder and the high concentration of added anions are unfavorable to the ionic bonding between SGP and PV. Comparative Examples 4-6, using cation exchange resin to adjust the pH, effectively form the PV-SGP complex and achieve better retention. However, the macroporous resin used in Comparative Example 4 causes significant SGP loss. Compared to the 6000 molecular weight ultrafiltration membrane, the 10000 molecular weight ultrafiltration membrane has a slightly higher SGP loss, but the SGP content in the lyophilized powder is significantly increased. Comparative Example 7 and Figures 3-5 show that the PV-SGP reversible protein complex ultrafiltration method effectively removes impurities and significantly increases the SGP content from 4.9% in the extract to 51.0%.

[0062] Furthermore, the proton NMR data in Figure 8 are as follows: 1H NMR (600 MHz, Deuterium Oxide) δ 5.08 (s, 1H), 5.00 (d,J= 9.7Hz, 1H), 4.91 – 4.89 (m, 1H), 4.63 (dd,J= 7.7, 5.6 Hz, 1H), 4.56 (dd,J=7.9, 4.8 Hz, 3H), 4.40 (dd,J= 7.9, 2.6 Hz, 2H), 4.36 (dd,J= 8.0, 6.3 Hz,1H), 4.26 (q,J= 7.1 Hz, 1H), 4.21 (s, 1H), 4.18 (td,J= 6.4, 4.2 Hz, 1H),4.16 – 4.13 (m, 1H), 4.11 – 4.08 (m, 2H), 4.08 – 4.06 (m, 1H), 3.99 – 3.41(m, 74H), 2.96 (t,J= 7.6 Hz, 4H), 2.81 (dd,J= 16.3, 5.4 Hz, 1H), 2.71(dd,J= 16.3, 7.8 Hz, 1H), 2.62 (dt,J= 12.3, 4.4 Hz, 2H), 2.04 (s, 3H),2.02 (s, 3H), 2.02 (s, 3H), 1.98 (s, 6H), 1.96 (s, 3H), 1.84 (tdt,J= 13.5,9.1, 4.4 Hz, 1H), 1.80 – 1.71 (m, 3H), 1.71 – 1.61 (m, 7H), 1.44 – 1.35 (m,4H), 1.34 (d,J= 7.2 Hz, 3H), 1.12 (d,J= 6.4 Hz, 3H), 0.92 (d,J= 6.7 Hz,6H)。

[0063] Technical Advantages: 1. In the extraction and purification process, water is used as the sole solvent, eliminating the use of any organic solvents and fundamentally eliminating the environmental, health, and safety issues associated with organic solvents. Production costs are also effectively controlled. 2. During the separation process, the ionic properties of endogenous egg yolk proteins PV and SGP are utilized. By controlling the binding and dissociation of the PV-SGP protein complex, selective retention and permeation of SGP are achieved under the same conditions, thereby removing most impurities and increasing the SGP content of intermediate products to over 50% in one step. 3. The ion exchange chromatography used employs commercially available strong-basic macroporous ion exchange resin as the packing material. Compared to packing materials such as dextran gel and reverse-bonded silica gel, it is not only cheaper and reusable but also has a higher SGP loading capacity, reaching 2-4% of the wet resin weight, making it very suitable for the purification of large-scale products. 4. The process of this invention is simple, with good inter-process connectivity, facilitating large-scale production. The entire production process takes only 3 days (including freeze-drying), achieving a purity of over 97% for single batches of 100 grams, with some reaching 99%.

[0064] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein.

[0065] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for large-scale preparation of sialic acid glycopeptides, characterized in that, The procedure includes the following steps: using defatted egg yolk powder as raw material, adding pure water, and extracting twice at 60-70℃, collecting the extract after filtration; slowly adding 001×7 (H) gel-type cation exchange resin to the extract, stirring to adjust the pH to 4.5±0.05, filtering off the resin to obtain a solution containing the PV-SGP complex; separating and concentrating the solution using a 10000 molecular weight ultrafiltration membrane, continuously washing with pure water after concentration, and collecting the concentrate; adding sodium borate solution to the concentrate, and adjusting the pH to 8.6 with sodium hydroxide solution. At ~8.8, membrane separation was continued with continuous washing and filtration using pure water. The filtrate containing the boric acid-SGP complex was collected. The D201(OH) macroporous anion exchange resin was converted to a borate type. The filtrate was cooled to 18~22℃ and passed through a chromatography column. It was washed with pure water and eluted sequentially with ammonium acetate solutions of different concentration gradients. The eluent with HPLC purity ≥95.0% was collected. The pH of the eluent was adjusted to 6.0±0.05 with acetic acid. After desalting and concentrating through a nanofiltration membrane and filtering through a 0.22μm filter membrane, the product was freeze-dried to obtain the sialic acid glycopeptide product.

2. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the process of extracting defatted egg yolk powder twice with pure water at 60-70℃, the defatted egg yolk powder is selected from one of the following two types: a protein byproduct obtained by supercritical carbon dioxide degreasing + ethanol degreasing process after extracting lecithin from egg yolk, with a total fat content ≤5% and a particle size of 120-200 mesh; or a product obtained by degreasing commercially available egg yolk powder with high-concentration alcohol, with a total fat content ≤3% and a particle size of 120-200 mesh. The process parameters for the two extractions are: for the first extraction, the solid-liquid ratio is 1kg:4-5L, the extraction temperature is 65±2℃, and the extraction time is 1-1.5h; for the second extraction, the solid-liquid ratio is 1kg:3-4L, the extraction temperature is 65±2℃, and the extraction time is 0.5-1h.

3. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the steps of separating and concentrating the solution using a 10,000 molecular weight ultrafiltration membrane, continuously washing with pure water after concentration, and collecting the concentrate: the ultrafiltration membrane is a high-temperature ultrafiltration membrane, the ultrafiltration feed temperature is controlled at 60℃, the pressure is maintained at 1.2MPa, and the amount of pure water added for washing is 5 to 7 times the volume of the concentrate.

4. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the steps of adding sodium borate solution to the concentrate, adjusting the pH to 8.6-8.8 with sodium hydroxide solution, continuing membrane separation and continuously washing with pure water, and collecting the filtrate containing boric acid-SGP complex: the mass ratio of sodium borate to defatted egg yolk powder is 0.7-1.2 g: 1 kg, and the concentration of the sodium borate solution is 10-15 g / L.

5. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the steps of converting D201(OH) macroporous anion exchange resin to borate type, cooling the filtrate to 18~22℃, passing it through a chromatography column, rinsing with pure water, and eluting sequentially with ammonium acetate solutions of different concentration gradients, and collecting the eluent with HPLC purity ≥95.0%: the flow rate of the chromatography column was controlled at 2~4 BV / h; the concentration gradient of the ammonium acetate solution was 40mM, 80mM, 120mM, 160mM, and 200mM, and the volume of the eluent for each concentration was 2 BV.

6. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the steps of converting D201(OH) macroporous anion exchange resin to borate type, cooling the filtrate to 18~22℃, passing it through a chromatography column, rinsing with pure water, and eluting sequentially with ammonium acetate solutions of different concentration gradients, and collecting the eluent with HPLC purity ≥95.0%, the conversion method of D201(OH) macroporous anion exchange resin is as follows: purging boric acid solution into the resin-packed chromatography column until the eluent is weakly acidic, and then rinsing with pure water.

7. The method for large-scale preparation of sialic acid glycopeptides as described in claim 1, characterized in that, In the steps of adjusting the pH of the eluent to 6.0±0.05 with acetic acid, desalting and concentrating it through a nanofiltration membrane, filtering it through a 0.22μm filter membrane, and then freeze-drying it to obtain the sialic acid glycopeptide product: the nanofiltration membrane desalting and concentration process is as follows: after concentrating to a volume of 1 / 5 to 1 / 6 of the initial eluent volume, add 4 to 5 times the volume of the concentrated liquid and pure water for continuous washing and filtration.

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