A purification process and endotoxin removal method for recombinant mytilus galloprovincialis mucin

Abstract

This invention belongs to the field of mussel adhesive protein purification technology, specifically involving a purification process and endotoxin removal method for recombinant type 1 mussel adhesive protein. This invention achieves efficient purification and deep endotoxin removal through a seven-step synergistic design: cell disruption and biomimetic affinity chromatography under acidic conditions, utilizing catechol's specific recognition of the dopa structure to enrich the target protein and inhibit oxidative aggregation; ultrafiltration concentration followed by mixed-mode chromatography for purification, utilizing the protein's high isoelectric point binding medium to remove host proteins; and finally, hydroxyapatite chromatography as the core for endotoxin removal, where the target protein binds to phosphate groups at pH 5.5-6.5, allowing endotoxin to flow through and synergistically removing trace impurities. The entire process is acidic to near-neutral, with the addition of ascorbic acid, nitrogen protection, and lyophilization protectants to reduce protein oxidative inactivation. The final product is white and loose, with good resolubility, purity >97%, yield 55-60%, and endotoxin <1 EU / mg, demonstrating significant application value.

Description

Technical Field

[0001] This invention belongs to the field of mussel adhesive protein purification technology, specifically relating to a purification process for recombinant type 1 mussel adhesive protein and a method for removing endotoxins. Background Technology

[0002] Mussel adhesive protein is extracted from the byssal glands of marine mussels and purified using industrial chromatography to obtain a high-purity single protein. This type of protein is often referred to as mussel foot protein (Mfp) in scientific literature. The identified structures include six types, Mfp-1 to Mfp-6, with Mfp-1 being the most abundant component, composed of 70-90 repeating polypeptide motifs. It possesses functions such as promoting cell adhesion and crawling, promoting wound healing, inhibiting itching, broad-spectrum adhesion, and forming a water-resistant protective film. It can be widely used in surface chemistry, biomedicine, marine engineering, and daily chemical products. However, the source of natural mussel adhesive protein is extremely limited; only micrograms of protein can be extracted from each mussel, and the quality varies significantly between different batches, making it difficult to meet industrialization needs. This has prompted researchers to turn to recombinant expression using genetic engineering techniques.

[0003] Currently, *E. coli* is the primary expression system for recombinant mussel adhesive protein due to its clear genetic background, short culture cycle, and low cost, making it the preferred host for industrial production. However, the *E. coli* expression system also presents new technical challenges. Recombinant mussel adhesive protein undergoes oxidative cross-linking and aggregation, leading to reduced target protein activity and decreased purification yield. Endotoxins from the *E. coli* cell wall are released in large quantities during cell disruption and coexist with the target protein in the lysis buffer. The positively charged nature of mussel adhesive protein makes it prone to non-specific binding to negatively charged endotoxin molecules, increasing the difficulty of subsequent separation. Endotoxins are key pyrogens that can cause fever, shock, and even death in humans. Biomedical materials have strict limits on endotoxin content. Therefore, efficiently removing endotoxins without damaging protein activity has become the core challenge in the purification process of recombinant mussel adhesive protein.

[0004] To address the aforementioned issues, existing technologies have proposed various purification strategies. For example, patent CN114853863B employs a combination of cell disruption and impurity removal, enzymatic oxidation, and chromatographic purification to obtain recombinant mussel adhesive protein with a purity greater than 95% and an endotoxin content less than 20 EU / mg. However, this process requires additional enzymatic treatment steps, and the yield still has room for improvement. Another patent, CN115611972A, addresses the endotoxin removal problem by combining urea-sodium chloride treatment with acid-heat reaction to reduce endotoxin levels to below 5 EU / mg. However, this method involves extreme acid and alkaline conditions, which may damage protein activity. Overall, existing processes generally suffer from cumbersome steps, low yields, incomplete endotoxin removal, or significant loss of protein activity, making it difficult to simultaneously meet the industrial production requirements of high purity, low endotoxin, and high yield. Therefore, developing a simple, mild process for preparing recombinant mussel adhesive protein that can synergistically achieve efficient purification and deep removal of endotoxins is of great significance for promoting its widespread application in the field of biomedical materials. Summary of the Invention

[0005] The purpose of this invention is to address existing problems by providing a purification process for recombinant mussel adhesive protein type 1 and a method for removing endotoxins.

[0006] This invention is achieved through the following technical solution:

[0007] A purification process and endotoxin removal method for recombinant mussel adhesive protein type 1 includes the following steps:

[0008] S1. Cell disruption and clarification:

[0009] The Escherichia coli fermentation broth expressing recombinant mussel adhesive protein was centrifuged at 7000-8000 rpm to collect bacterial cells, resuspended in buffer, homogenized under high pressure, centrifuged to collect the supernatant and deep filtered to obtain a clear lysate.

[0010] S2, biomimetic polyphenol affinity chromatography capture:

[0011] The clarified lysate obtained in step S1 was subjected to affinity capture using an immobilized catechol biomimetic affinity chromatography medium. After washing, it was eluted with an acidic elution buffer containing salt to obtain an enriched mussel adhesive protein eluent.

[0012] S3, Concentration and Liquid Replacement:

[0013] The eluent obtained in step S2 was concentrated by ultrafiltration and replaced with an acidic buffer solution of pH 3.8-4.5 to obtain a high-concentration protein concentrate.

[0014] S4, Composite Mode Flow-through Purification:

[0015] After adjusting the pH of the concentrate obtained in step S3 to 4.8-5.5 online and controlling the conductivity to be stable, it is purified by a mixed-mode chromatography medium that combines cation exchange and hydrophobic interaction. Under low conductivity conditions, the target protein binds to the medium. After washing, it is eluted with elution buffer to obtain the target protein eluent.

[0016] S5. Online preparation under intermediate conditions:

[0017] The eluent obtained in step S4 is mixed online with the phosphate mother liquor to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography;

[0018] S6, hydroxyapatite endotoxin removal:

[0019] The loading intermediate obtained in step S5 is passed through a hydroxyapatite chromatography medium. Under these conditions, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin flows through to remove it. After washing, the intermediate is eluted with high phosphate buffer to recover the target protein solution.

[0020] S7. Ultrafiltration Concentration and Drying:

[0021] The target protein solution obtained in step S6 is concentrated by ultrafiltration, desalted, and replaced with a solution containing a lyophilization protectant. After sterile filtration, it is freeze-dried under nitrogen or inert gas protection and sealed with nitrogen to obtain the final product.

[0022] Further, the buffer solution mentioned in step S1 is a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.8-4.2, and contains 3-8 mmol / L EDTA and 0.5-2 mmol / L ascorbic acid;

[0023] The conditions for high-pressure homogenization are: pressure 700~900 bar, number of crushing cycles 2~3, and temperature 4~8℃;

[0024] The centrifugation conditions are: rotation speed 10000~12000 rpm, time 25~35 min, temperature 4~8℃;

[0025] The deep filtration process uses 0.45μm and 0.22μm filter membranes sequentially.

[0026] Furthermore, the immobilized catechol biomimetic affinity chromatography medium in step S2 is agarose-based globule-coupled catechol, prepared by an epoxy activation method, with a ligand density of 10~30 μmol / mL.

[0027] The affinity capture specifically includes:

[0028] Equilibration: Equilibrate the chromatography column with equilibration buffer;

[0029] Sample loading: Load the clarified lysis buffer onto the sample;

[0030] Washing: Remove unbound or weakly bound impurity proteins using washing buffer;

[0031] Elution: The enriched mussel adhesive protein was specifically eluted with elution buffer. The absorbance was monitored at A280, and the peaks with absorbance values ​​≥50 mAu were collected to obtain the mussel adhesive protein eluent.

[0032] Furthermore, the preparation of the immobilized catechol biomimetic affinity chromatography medium includes the following steps:

[0033] (1) Media pretreatment: Take agarose-based balls, wash them with deionized water until the eluent is free of impurities, then soak them in 0.1 mol / L NaOH solution for 30 min, filter them, and rinse them with deionized water until the pH is neutral.

[0034] (2) Epoxy activation: Add the pretreated agarose-based spheres to the reaction vessel, add epichlorohydrin (volume ratio of 1 to 1.5 times the volume of the agarose-based spheres), and then add 0.1 mol / L NaOH solution as a catalyst. Control the temperature at 30 to 40°C and stir the reaction for 2 to 4 hours to activate the surface of the agarose-based spheres and form epoxy groups.

[0035] (3) Coupling reaction: After activation, the agarose-based spheres are quickly rinsed with deionized water until neutral, and then catechol solution (prepared with 0.1 mol / L Tris-HCl buffer at pH 8.0, concentration 50~100 mmol / L) is immediately added. The mass-to-volume ratio of catechol to agarose-based spheres is 1:10 (g / mL). The mixture is stirred at 30℃ for 12~24 h to allow the catechol to be covalently coupled to the surface of the agarose-based spheres through epoxy groups.

[0036] (4) Blocking and post-treatment: After the reaction, wash the medium with deionized water until no free catechol is found, then soak it in 1 mol / L ethanolamine solution for 2 h to block the unreacted epoxy groups, and finally wash it with deionized water and equilibration buffer (20 mmol / L citrate-sodium citrate buffer, pH 3.8~4.2) in sequence.

[0037] Furthermore, the equilibration buffer is a 20 mmol / L citrate-sodium citrate buffer with a pH of 3.8-4.2, containing 0.12-0.18 M NaCl;

[0038] The washing buffer is a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.8-4.2, containing 0.2-0.3 M NaCl;

[0039] The elution buffer is a 20 mmol / L citrate-sodium citrate buffer with a pH of 3.8-4.5, containing 0.6-1.0 M NaCl;

[0040] The loading conditions are: linear flow rate 120~180cm / h, loading volume 10~20mg protein / mL medium;

[0041] Elution linear flow rate: 80~120 cm / h.

[0042] Furthermore, the ultrafiltration system in step S3 uses a membrane module with a molecular weight cutoff of 3-5 kDa, and the membrane material is regenerated cellulose or modified polyethersulfone.

[0043] The concentration process controls the protein concentration at 5~15 mg / mL, and the entire concentration and solution exchange process maintains the protein solution pH ≤ 4.5, with nitrogen gas used for protection.

[0044] The replacement buffer is a citrate-sodium citrate buffer containing 1-5 mmol / L ascorbic acid, with a pH of 3.8-4.2 and a conductivity ≤4 mS / cm.

[0045] Furthermore, the composite mode chromatography medium mentioned in step S4 is a mixed mode medium that combines cation exchange and hydrophobic interaction, selected from Capto MMC, Nuvia cPrime or equivalent media;

[0046] The specific conditions for purity include:

[0047] Equilibration / loading buffer: 20~50mmol / L citrate-sodium citrate buffer, pH 4.8~5.5, conductivity ≤5mS / cm;

[0048] Sample loading: Adjust the pH of the concentrate obtained in step S3 to 4.8-5.5 and then load the sample; the target protein will bind to the medium.

[0049] Washing: Wash impurities with loading buffer or loading buffer containing no more than 50 mmol / L NaCl;

[0050] Elution: Elute with a pH 5.5-6.5 buffer containing low concentration of salt to obtain the target protein elution peak with conductivity ≤6mS / cm.

[0051] Further, step S5 specifically involves mixing the target protein elution buffer with the phosphate mother liquor online, adjusting the final phosphate concentration to 5-20 mmol / L, pH to 5.5-6.5, and conductivity to ≤6 mS / cm, to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography;

[0052] The phosphate mother liquor is a sodium phosphate or potassium phosphate buffer solution with a concentration of 0.5~2.0 mol / L.

[0053] Furthermore, the hydroxyapatite chromatography medium in step S6 is ceramic hydroxyapatite type I or type II;

[0054] The conditions for endotoxin removal are as follows:

[0055] Sample loading: The sample loading intermediate obtained in step S5 is directly loaded at a linear flow rate of 120~180 cm / h; at this time, the target protein is bound to the phosphate group of the medium, and the negatively charged endotoxin flows through.

[0056] Washing: Wash with 5-20 mmol / L sodium phosphate buffer, pH 5.5-6.5, containing 0.08-0.12 M NaCl.

[0057] Elution: Elute with 150-250 mmol / L sodium phosphate buffer, pH 5.5-6.5, at a linear flow rate of 100-150 cm / h, and recover the target protein solution.

[0058] Furthermore, the ultrafiltration concentration in step S7 uses a 3kDa hollow fiber ultrafiltration column or a spiral wound ultrafiltration membrane;

[0059] The desalting process employs a gradient solution exchange strategy: first, dialyze 2-4 times the volume of solution in 5-10 mmol / L sodium phosphate buffer (pH 5.5-6.5), then gradually transition to a solution containing a lyophilization protectant for dialyzing 4-6 times the volume. The solution containing the lyophilization protectant consists of water for injection, 0.8-1.2% mannitol, and 0.5-2 mmol / L ascorbic acid.

[0060] After being concentrated to a protein concentration of 10-20 mg / mL, it was aseptically filtered through a 0.22 μm filter.

[0061] The drying process is freeze drying, with the following conditions: pre-freezing at -25℃ to -15℃ for 1.5 to 2.5 hours, drying at -8℃ to -2℃ for 10 to 14 hours, drying at 20℃ to 30℃ for 4 to 8 hours, and then sealing with nitrogen.

[0062] The present invention has the following advantages over the prior art:

[0063] 1. This invention provides a purification process and endotoxin removal method for recombinant type 1 mussel adhesive protein. Through a seven-step synergistic design, it achieves efficient capture, precise purification, and deep removal of the target protein and endotoxin. First, cell disruption and biomimetic affinity chromatography are performed under acidic conditions. Utilizing the specific recognition of DOPA (dopa) structural analogs in mussel adhesive protein by the catechol group, one-step efficient enrichment of the target protein is achieved. Simultaneously, the acidic environment effectively inhibits protein oxidative aggregation. Subsequently, ultrafiltration concentration and mixed-mode chromatography are used for purification. Taking advantage of the high isoelectric point of mussel adhesive protein, it is bound to cationic / hydrophobic mixed grafts under specific pH and low conductivity conditions. A large number of host proteins with similar charge properties are effectively flowed through or washed, significantly improving protein purity and creating favorable conditions for subsequent endotoxin removal.

[0064] 2. Secondly, this invention utilizes hydroxyapatite chromatography as the core endotoxin removal step. By applying a pH of 5.5–6.5, the positively charged target protein binds to the phosphate groups of the medium, while the strongly negatively charged endotoxin molecules flow directly through, achieving highly efficient separation of the target protein and endotoxin. Simultaneously, this step further removes trace amounts of host proteins and nucleic acids, complementing the upstream biomimetic affinity and mixed-mode chromatography to jointly ensure high product purity.

[0065] 3. Finally, all steps of this invention are carried out under acidic near-neutral pH conditions. Ascorbic acid is added to the buffer solution as an antioxidant. Nitrogen gas is used for protection during ultrafiltration and chromatography. Before lyophilization, the solution is replaced with a protective agent system containing mannitol and ascorbic acid, minimizing protein oxidative cross-linking and activity loss. The final product, after lyophilization, is a white, loose powder with good resolubility, protein purity exceeding 97%, and a stable yield between 55% and 60%. The endotoxin content is below 1 EU / mg, demonstrating significant application value. Detailed Implementation

[0066] To further explain the present invention, the following specific embodiments are described.

[0067] Table 1

[0068] Agarose-based spheres (Sepharose 4B) Cytiva 17-0120-01(500mL) / 17-0120-02(100mL) Capto MMC (Mixed-Mode Chromatography Media) Cytiva (formerly GE Healthcare) 17531703(1L) / 17531702(100mL / 17531710(25mL) Nuvia cPrime (mixed-mode chromatography medium) Bio-Rad 1563401 (25 mL) / 1563402 (100 mL) Ceramic hydroxyapatite I Shanghai Yuanye Biotechnology Co., Ltd. V34700-50g Ceramic Hydroxyapatite II Shanghai Yuanye Biotechnology Co., Ltd. V34703-50g Regenerated cellulose ultrafiltration membrane (RC Membrane) Sartorius 14429-050D Polyethersulfone ultrafiltration membrane (PES Membrane) Sartorius 14629-047D

[0069] Note: Unless otherwise specified, the raw materials used in this invention are all from commercially available conventional products.

[0070] The preparation of immobilized catechol biomimetic affinity chromatography media includes the following steps:

[0071] (1) Media pretreatment: Take agarose-based balls, wash them with deionized water until the eluent is free of impurities, then soak them in 0.1 mol / L NaOH solution for 30 min, filter them, and rinse them with deionized water until the pH is neutral.

[0072] (2) Epoxy activation: Add the pretreated agarose-based spheres to the reaction vessel, add epichlorohydrin (volume ratio of 1 times the volume of the agarose-based spheres), and then add 0.1 mol / L NaOH solution as a catalyst. Control the temperature at 30℃ and stir the reaction for 2 hours to activate the surface of the agarose-based spheres and form epoxy groups.

[0073] (3) Coupling reaction: After activation, the agarose-based spheres were quickly rinsed with deionized water until neutral, and then catechol solution (prepared with 0.1 mol / L Tris-HCl buffer at pH 8.0, concentration 50 mmol / L) was immediately added. The mass-to-volume ratio of catechol to agarose-based spheres was 1:10 (g / mL). The mixture was stirred at 30℃ for 12 h to allow the catechol to be covalently coupled to the surface of the agarose-based spheres through epoxy groups.

[0074] (4) Blocking and post-treatment: After the reaction, wash the medium with deionized water until no free catechol is found, then soak it in 1 mol / L ethanolamine solution for 2 h to block the unreacted epoxy groups, and finally wash it with deionized water and equilibration buffer (20 mmol / L citrate-sodium citrate buffer, pH 3.8) in sequence.

[0075] Example 1

[0076] A purification process and endotoxin removal method for recombinant mussel adhesive protein type 1 includes the following steps:

[0077] S1. Cell disruption and clarification:

[0078] The fermentation broth of *E. coli* expressing recombinant mussel adhesive protein was centrifuged at 7000 rpm to collect bacterial cells. The cells were resuspended in 20 mmol / L, pH 3.8 citrate-sodium citrate buffer containing 3 mmol / L EDTA and 0.5 mmol / L ascorbic acid, homogenized twice by autoclaving at 700 bar at 4 °C, and centrifuged at 10000 rpm for 25 min at 4 °C. The supernatant was collected and filtered sequentially through 0.45 μm and 0.22 μm filter membranes to obtain a clear lysate.

[0079] S2, biomimetic polyphenol affinity chromatography capture:

[0080] The clarified lysate obtained in step S1 was subjected to affinity capture using an immobilized catechol biomimetic affinity chromatography medium. After washing, it was eluted with an acidic elution buffer containing salt to obtain an enriched mussel adhesive protein eluent.

[0081] The affinity capture specifically includes:

[0082] Equilibration: Equilibrate the chromatography column with equilibration buffer (20 mmol / L citrate-sodium citrate buffer, pH 3.8, containing 0.12 M NaCl);

[0083] Sample loading: Load the clarified lysis buffer at a linear flow rate of 120 cm / h and a loading volume of 10 mg protein / mL medium.

[0084] Washing: Remove unbound or weakly bound impurity proteins with washing buffer (20 mmol / L citrate-sodium citrate buffer, pH 3.8, containing 0.2 M NaCl);

[0085] Elution: The enriched mussel adhesive protein was specifically eluted with elution buffer (20 mmol / L citrate-sodium citrate buffer, pH 3.8, containing 0.6 M NaCl) at a linear flow rate of 80 cm / h. The absorbance was monitored by A280 spectrophotometry, and the peaks with absorbance values ​​≥50 mAu were collected to obtain the mussel adhesive protein eluent.

[0086] S3, Concentration and Liquid Replacement:

[0087] The eluent obtained in step S2 was concentrated by passing it through a membrane module with a molecular weight cutoff of 3 kDa. The membrane material was regenerated cellulose, and the eluent was replaced with an acidic buffer solution of pH 3.8 (citric acid-sodium citrate buffer solution containing 1 mmol / L ascorbic acid, pH 3.8, conductivity ≤ 4 mS / cm) to obtain a high concentration protein concentrate.

[0088] The concentration process controls the protein concentration at 5 mg / mL, and the entire concentration and solution exchange process maintains the protein solution pH ≤ 4.5, with nitrogen gas used for protection.

[0089] S4, Composite Mode Flow-through Purification:

[0090] After adjusting the pH of the concentrate obtained in step S3 to 4.8 online and controlling the conductivity to be stable, it was purified by a mixed-mode chromatography medium (selected from Capto MMC) that combines cation exchange and hydrophobic interaction. Under low conductivity conditions, the target protein was bound to the medium. After washing, it was eluted with elution buffer to obtain the target protein eluent.

[0091] The specific conditions for purity include:

[0092] Equilibration / loading buffer: 20 mmol / L citrate-sodium citrate buffer, pH 4.8, conductivity ≤5 mS / cm;

[0093] Sample loading: After adjusting the pH of the concentrate obtained in step S3 to 4.8, the target protein is loaded into the medium;

[0094] Washing: Wash impurities with loading buffer or loading buffer containing no more than 50 mmol / L NaCl;

[0095] Elution: Elution was performed using a pH 5.5 buffer solution containing a low concentration of salt to obtain the target protein elution peak with a conductivity ≤6 mS / cm.

[0096] S5. Online preparation under intermediate conditions:

[0097] The target protein eluent was mixed online with 0.5 mol / L sodium phosphate, and the final phosphate concentration was adjusted to 5 mmol / L, pH to 5.5, and conductivity to ≤6 mS / cm to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography.

[0098] S6, hydroxyapatite endotoxin removal:

[0099] The loading intermediate obtained in step S5 is passed through a hydroxyapatite chromatography medium (ceramic hydroxyapatite type I). Under these conditions, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin is removed by flow-through. After washing, the solution is eluted with high phosphate buffer and the target protein solution is recovered.

[0100] The conditions for endotoxin removal are as follows:

[0101] Sample loading: The sample loading intermediate obtained in step S5 is directly loaded at a linear flow rate of 120 cm / h; at this time, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin flows through.

[0102] Washing: Wash with 5 mmol / L sodium phosphate buffer, pH 5.5, containing 0.08 M NaCl;

[0103] Elution: Elute with 150 mmol / L sodium phosphate buffer, pH 5.5, at a linear flow rate of 100 cm / h, and recover the target protein solution;

[0104] S7. Ultrafiltration Concentration and Drying:

[0105] The target protein solution obtained in step S6 is concentrated by ultrafiltration, desalted, and replaced with a solution containing a lyophilization protectant. After sterile filtration, it is freeze-dried under nitrogen or inert gas protection and sealed with nitrogen to obtain the final product.

[0106] The ultrafiltration concentration uses a 3kDa hollow fiber ultrafiltration column or a spiral wound ultrafiltration membrane.

[0107] The desalting process employs a gradient liquid exchange strategy: first, dialyze two volumes of solution in 5 mmol / L sodium phosphate buffer (pH 5.5), and then gradually transition to a solution containing a lyophilization protectant for dialyzing four volumes. The solution containing the lyophilization protectant consists of water for injection, 0.8% mannitol, and 0.5 mmol / L ascorbic acid.

[0108] After being concentrated to a protein concentration of 10 mg / mL, it was aseptically filtered through a 0.22 μm filter.

[0109] The drying process is freeze-drying, with the following conditions: pre-freezing at -25℃ for 1.5 hours, drying at -8℃ for 10 hours, drying at 20~30℃ for 4 hours, and then sealing with nitrogen.

[0110] Example 2

[0111] A purification process and endotoxin removal method for recombinant mussel adhesive protein type 1 includes the following steps:

[0112] S1. Cell disruption and clarification:

[0113] The Escherichia coli fermentation broth expressing recombinant mussel adhesive protein was centrifuged at 7500 rpm to collect bacterial cells. The cells were resuspended in 20 mmol / L, pH 4 citrate-sodium citrate buffer containing 5 mmol / L EDTA and 1 mmol / L ascorbic acid. The cells were homogenized twice by high pressure at 800 bar at 6 °C and centrifuged at 11000 rpm for 30 min at 6 °C. The supernatant was collected and filtered sequentially through 0.45 μm and 0.22 μm filter membranes to obtain a clear lysate.

[0114] S2, biomimetic polyphenol affinity chromatography capture:

[0115] The clarified lysate obtained in step S1 was subjected to affinity capture using an immobilized catechol biomimetic affinity chromatography medium. After washing, it was eluted with an acidic elution buffer containing salt to obtain an enriched mussel adhesive protein eluent.

[0116] The affinity capture specifically includes:

[0117] Equilibration: Equilibrate the chromatography column with equilibration buffer (20 mmol / L citrate-sodium citrate buffer, pH 4, containing 0.15 M NaCl);

[0118] Sample loading: Load the clarified lysis buffer at a linear flow rate of 150 cm / h and a loading volume of 15 mg protein / mL medium.

[0119] Washing: Remove unbound or weakly bound impurity proteins with washing buffer (20 mmol / L citrate-sodium citrate buffer, pH 4, containing 0.25 M NaCl);

[0120] Elution: The enriched mussel adhesive protein was specifically eluted with elution buffer (20 mmol / L citrate-sodium citrate buffer, pH 4.2, containing 0.8 M NaCl) at a linear flow rate of 100 cm / h. The absorbance was monitored by A280 spectrophotometry, and the peaks with absorbance values ​​≥50 mAu were collected to obtain the mussel adhesive protein eluent.

[0121] S3, Concentration and Liquid Replacement:

[0122] The eluent obtained in step S2 was concentrated using a membrane module with a molecular weight cutoff of 4 kDa. The membrane material was regenerated cellulose, and the eluent was replaced with an acidic buffer solution of pH 4.2 (citric acid-sodium citrate buffer solution containing 3 mmol / L ascorbic acid, pH 4, conductivity ≤ 4 mS / cm) to obtain a high-concentration protein concentrate.

[0123] The concentration process controls the protein concentration at 10 mg / mL, and the entire concentration and solution exchange process maintains the protein solution pH ≤ 4.5, with nitrogen gas used for protection.

[0124] S4, Composite Mode Flow-through Purification:

[0125] After adjusting the pH of the concentrate obtained in step S3 to 5.1 online and controlling the conductivity to be stable, it was purified by a mixed-mode chromatography medium (selected from Capto MMC) that combines cation exchange and hydrophobic interaction. Under low conductivity conditions, the target protein was bound to the medium. After washing, it was eluted with elution buffer to obtain the target protein eluent.

[0126] The specific conditions for purity include:

[0127] Equilibration / loading buffer: 35 mmol / L citrate-sodium citrate buffer, pH 5.1, conductivity ≤5 mS / cm;

[0128] Sample loading: After adjusting the pH of the concentrate obtained in step S3 to 5.1, the target protein is loaded into the medium;

[0129] Washing: Wash impurities with loading buffer or loading buffer containing no more than 50 mmol / L NaCl;

[0130] Elution: Elution was performed using a pH 6 buffer solution containing a low concentration of salt to obtain the target protein elution peak with a conductivity ≤ 6 mS / cm.

[0131] S5. Online preparation under intermediate conditions:

[0132] The target protein eluent was mixed online with sodium phosphate at a concentration of 1 mol / L. The final phosphate concentration was adjusted to 10 mmol / L, pH to 6, and conductivity to ≤6 mS / cm to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography.

[0133] S6, hydroxyapatite endotoxin removal:

[0134] The loading intermediate obtained in step S5 is passed through a hydroxyapatite chromatography medium (ceramic hydroxyapatite type I). Under these conditions, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin is removed by flow-through. After washing, the solution is eluted with high phosphate buffer and the target protein solution is recovered.

[0135] The conditions for endotoxin removal are as follows:

[0136] Sample loading: The sample loading intermediate obtained in step S5 is directly loaded at a linear flow rate of 150 cm / h; at this time, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin flows through.

[0137] Washing: Wash with 10 mmol / L sodium phosphate buffer, pH 6, containing 0.1 M NaCl;

[0138] Elution: Elute with 200 mmol / L sodium phosphate buffer, pH 6, at a linear flow rate of 125 cm / h, and recover the target protein solution;

[0139] S7. Ultrafiltration Concentration and Drying:

[0140] The target protein solution obtained in step S6 is concentrated by ultrafiltration, desalted, and replaced with a solution containing a lyophilization protectant. After sterile filtration, it is freeze-dried under nitrogen or inert gas protection and sealed with nitrogen to obtain the final product.

[0141] The ultrafiltration concentration uses a 3kDa hollow fiber ultrafiltration column or a spiral wound ultrafiltration membrane.

[0142] The desalting process employs a gradient liquid exchange strategy: first, dialyze 3 times the volume of solution in 7 mmol / L sodium phosphate buffer (pH 6), and then gradually transition to dialyze 5 times the volume of solution containing a lyophilization protectant. The solution containing the lyophilization protectant consists of water for injection, 1% mannitol, and 1 mmol / L ascorbic acid.

[0143] After being concentrated to a protein concentration of 15 mg / mL, it was aseptically filtered through a 0.22 μm filter.

[0144] The drying process is freeze-drying, with the following conditions: pre-freezing at -20℃ for 2 hours, drying at -5℃ for 12 hours, drying at 25℃ for 6 hours, and then sealing with nitrogen.

[0145] Example 3

[0146] A purification process and endotoxin removal method for recombinant mussel adhesive protein type 1 includes the following steps:

[0147] S1. Cell disruption and clarification:

[0148] The Escherichia coli fermentation broth expressing recombinant mussel adhesive protein was centrifuged at 8000 rpm to collect bacterial cells. The cells were resuspended in 20 mmol / L, pH 4.2 citrate-sodium citrate buffer containing 8 mmol / L EDTA and 2 mmol / L ascorbic acid. The cells were homogenized three times by high pressure at 8°C and 900 bar, and centrifuged at 8°C and 12000 rpm for 35 min. The supernatant was filtered sequentially through 0.45 μm and 0.22 μm filter membranes to obtain a clear lysate.

[0149] S2, biomimetic polyphenol affinity chromatography capture:

[0150] The clarified lysate obtained in step S1 was subjected to affinity capture using an immobilized catechol biomimetic affinity chromatography medium. After washing, it was eluted with an acidic elution buffer containing salt to obtain an enriched mussel adhesive protein eluent.

[0151] The affinity capture specifically includes:

[0152] Equilibration: Equilibrate the chromatography column with equilibration buffer (20 mmol / L citrate-sodium citrate buffer, pH 4.2, containing 0.18 M NaCl);

[0153] Sample loading: Load the clarified lysis buffer at a linear flow rate of 180 cm / h and a loading volume of 20 mg protein / mL medium.

[0154] Washing: Remove unbound or weakly bound impurity proteins with washing buffer (20 mmol / L citrate-sodium citrate buffer, pH 4.2, containing 0.3 M NaCl);

[0155] Elution: The enriched mussel adhesive protein was specifically eluted with elution buffer (20 mmol / L citrate-sodium citrate buffer, pH 4.5, containing 1.0 M NaCl) at a linear flow rate of 120 cm / h. The absorbance was monitored by A280 spectrophotometry, and the peaks with absorbance values ​​≥50 mAu were collected to obtain the mussel adhesive protein eluent.

[0156] S3, Concentration and Liquid Replacement:

[0157] The eluent obtained in step S2 was concentrated using a membrane module with a molecular weight cutoff of 5 kDa. The membrane material was regenerated cellulose, and the eluent was replaced with an acidic buffer solution of pH 4.5 (citric acid-sodium citrate buffer solution containing 5 mmol / L ascorbic acid, pH 4.2, conductivity ≤ 4 mS / cm) to obtain a high-concentration protein concentrate.

[0158] The concentration process controls the protein concentration at 15 mg / mL, and the entire concentration and solution exchange process maintains the protein solution pH ≤ 4.5, with nitrogen gas used for protection.

[0159] S4, Composite Mode Flow-through Purification:

[0160] After adjusting the pH of the concentrate obtained in step S3 to 5.5 online and controlling the conductivity to be stable, it was purified by a mixed-mode chromatography medium (selected from Capto MMC) that combines cation exchange and hydrophobic interaction. Under low conductivity conditions, the target protein was bound to the medium. After washing, it was eluted with elution buffer to obtain the target protein eluent.

[0161] The specific conditions for purity include:

[0162] Equilibration / loading buffer: 50 mmol / L citrate-sodium citrate buffer, pH 5.5, conductivity ≤5 mS / cm;

[0163] Sample loading: After adjusting the pH of the concentrate obtained in step S3 to 5.5, the sample is loaded, and the target protein binds to the medium;

[0164] Washing: Wash impurities with loading buffer or loading buffer containing no more than 50 mmol / L NaCl;

[0165] Elution: Elution was performed using a pH 6.5 buffer solution containing a low concentration of salt to obtain the target protein elution peak with a conductivity ≤6 mS / cm.

[0166] S5. Online preparation under intermediate conditions:

[0167] The target protein eluent was mixed online with sodium phosphate at a concentration of 2.0 mol / L, and the final phosphate concentration was adjusted to 20 mmol / L, pH to 6.5, and conductivity to ≤6 mS / cm to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography.

[0168] S6, hydroxyapatite endotoxin removal:

[0169] The loading intermediate obtained in step S5 is passed through a hydroxyapatite chromatography medium (ceramic hydroxyapatite type I). Under these conditions, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin is removed by flow-through. After washing, the solution is eluted with high phosphate buffer and the target protein solution is recovered.

[0170] The conditions for endotoxin removal are as follows:

[0171] Sample loading: The sample loading intermediate obtained in step S5 is directly loaded at a linear flow rate of 180 cm / h; at this time, the target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin flows through.

[0172] Washing: Wash with 20 mmol / L sodium phosphate buffer, pH 6.5, containing 0.12 M NaCl;

[0173] Elution: Elute with 250 mmol / L sodium phosphate buffer, pH 6.5, at a linear flow rate of 150 cm / h, and recover the target protein solution;

[0174] S7. Ultrafiltration Concentration and Drying:

[0175] The target protein solution obtained in step S6 is concentrated by ultrafiltration, desalted, and replaced with a solution containing a lyophilization protectant. After sterile filtration, it is freeze-dried under nitrogen or inert gas protection and sealed with nitrogen to obtain the final product.

[0176] The ultrafiltration concentration uses a 3kDa hollow fiber ultrafiltration column or a spiral wound ultrafiltration membrane.

[0177] The desalting process employs a gradient liquid exchange strategy: first, dialyze 4 volumes of 10 mmol / L sodium phosphate buffer (pH 6.5), and then gradually transition to a solution containing a lyophilization protectant for dialyzing 6 volumes. The solution containing the lyophilization protectant consists of water for injection, 1.2% mannitol, and 2 mmol / L ascorbic acid.

[0178] After being concentrated to a protein concentration of 20 mg / mL, it was aseptically filtered through a 0.22 μm filter.

[0179] The drying process is freeze-drying, with the following conditions: pre-freezing at -15℃ for 2.5 hours, drying at -2℃ for 14 hours, drying at 30℃ for 8 hours, and then sealing with nitrogen.

[0180] Comparative Example 1

[0181] The difference between Comparative Example 1 and Example 2 is that step S6 (hydroxyapatite endotoxin removal step) is omitted. That is, after obtaining the sample loading intermediate through online preparation in S5, the sample is directly subjected to ultrafiltration concentration and drying in step S7, without treatment with the hydroxyapatite chromatography medium. All other operating parameters, buffer formulations, chromatography medium types, and equipment are consistent with those in Example 2.

[0182] Comparative Example 2

[0183] The difference between Comparative Example 2 and Example 2 is that the immobilized catechol biomimetic affinity chromatography medium in step S2 is replaced with conventional Ni-NTA agarose gel (purchased from Cytiva, catalog number 17-5318-01). The remaining operations, such as equilibration, sample loading, washing, and elution flow rates and volumes, are the same as in Example 2. However, the elution buffer needs to be adjusted to a buffer containing 250 mmol / L imidazole according to the standard Ni-NTA operation.

[0184] Comparative Example 3

[0185] The difference between Comparative Example 3 and Example 2 is that step S4 (complex mode chromatography purification) is omitted. That is, after concentration and liquid replacement in step S3, the resulting high-concentration protein concentrate is directly introduced into step S5 for online mixing to prepare the loading intermediate, followed by S6 hydroxyapatite chromatography and S7 ultrafiltration drying.

[0186] Comparative Example 4

[0187] The difference between Comparative Example 4 and Example 2 is that the loading conditions for hydroxyapatite chromatography in step S6 are changed. Specifically, the pH of the intermediate obtained in step S5 is adjusted to 7.5 (not within the range of 5.5~6.5 required by this invention), while keeping the phosphate concentration (10 mmol / L) and conductivity (≤6 mS / cm) unchanged, and then the sample is loaded onto ceramic hydroxyapatite type I medium. The subsequent washing and elution conditions are the same as in Example 2.

[0188] 1. Experimental testing

[0189] Protein purity determination: High performance liquid chromatography (HPLC) was used with a C4 reverse-phase column and a gradient elution of water and acetonitrile containing 0.1% trifluoroacetic acid. The detection wavelength was 280 nm. The purity of the target protein was calculated using the area normalization method.

[0190] Endotoxin content determination: The gel electrophoresis method was performed according to the "1143 Bacterial Endotoxin Test Method" in the General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia. The endotoxin content (EU / mg protein) was determined by serially diluting the samples using a Limulus amebocyte lysate (LAL) reagent with a sensitivity of 0.03 EU / mL.

[0191] Protein yield calculation: The ratio of total protein content (mg) in the lyophilized powder to the estimated total protein content (mg) in the initial fermentation broth. The total protein content of the initial fermentation broth was calculated by determining the total protein concentration of the clarified lysate obtained from S1 using the BCA method.

[0192] 2. Test Results

[0193] The test results are shown in Table 2 below.

[0194] Table 2

[0195] Example 1 97.0±0.6 0.90±0.15 57.5±2.5 Example 2 97.2±0.5 0.85±0.12 58.3±2.1 Example 3 97.5±0.4 0.80±0.10 59.0±2.0 Comparative Example 1 89.6±0.8 35.40±4.50 61.5±1.8 Comparative Example 2 65.4±2.1 8.20±1.50 22.7±3.0 Comparative Example 3 92.5±0.6 1.20±0.30 60.1±2.3 Comparative Example 4 96.8±0.7 5.60±0.80 57.6±2.5

[0196] As shown in Table 1 above, Example 2 achieved the highest purity (97.2%), indicating that the seven-step process worked synergistically to achieve the best purification effect. Comparative Example 2 had the lowest purity (65.4%) and the lowest yield (22.7%), indicating that the non-specific Ni-NTA medium could not effectively capture the target protein, and most of the mussel adhesive protein was lost in the flow-through and washing steps, while the impurity protein residue was serious. This verifies the high selectivity of the biomimetic affinity chromatography designed in this invention for mussel adhesive protein. The purity of Comparative Example 3 (92.5%) was slightly lower than that of Example 2, indicating that even after omitting the mixed-mode chromatography (S4), some impurities with properties similar to the target protein could not be completely removed by hydroxyapatite, proving that mixed-mode chromatography is indispensable in purification. The purity of Comparative Example 4 (96.8%) was close to that of Example 2, indicating that the change in pH of hydroxyapatite loading had little effect on the final purity, but the endotoxin content increased significantly.

[0197] Example 2 showed the lowest endotoxin content (0.85 EU / mg), meeting medical material standards. Comparative Example 1 showed an extremely high endotoxin content (35.4 EU / mg), further demonstrating that hydroxyapatite chromatography is the core step for endotoxin removal; omitting this step will result in a substandard product. Comparative Example 2, while having a lower endotoxin content (8.2 EU / mg) than Comparative Example 1, still had a higher content than Example 2. This may be due to a lower target protein capture, resulting in some endotoxin removal during flow-through, but the residual endotoxin in the product still exceeded the standard. Comparative Example 3 had a slightly higher endotoxin content (1.2 EU / mg) than Example 2, but it was still acceptable, indicating that hydroxyapatite itself has a certain endotoxin removal capacity, but pre-purification using mixed-mode chromatography helps reduce its load, making endotoxin removal more thorough. Comparative Example 4 showed a significantly increased endotoxin content to 5.6 EU / mg, indicating that after the sample loading pH deviated from the optimized range of this invention (5.5~6.5), the interaction between endotoxin and the medium or target protein changed, with some endotoxin co-eluting or binding being enhanced, leading to a decrease in removal efficiency. This confirms the rationality of the pH range selected in this invention.

[0198] Example 2's yield (58.3%) was at a moderate level, demonstrating the balance between quality and yield achieved through multi-step chromatography. Comparative Examples 1 and 3 had slightly higher yields because omitting steps reduced protein loss, but decreased product quality. Comparative Example 2 had an extremely low yield, indicating that incorrect selection of the affinity medium can lead to significant product loss. Comparative Example 4 had a yield comparable to Example 2, showing that pH changes had little impact on yield, but endotoxin removal was worsened.

[0199] In summary, the seven-step process of this invention works synergistically and is indispensable. Omission of any step or deviation of any key parameter will result in the final product failing to meet the purity or endotoxin content standards, thus failing to meet the high standards required for biomedicine.

[0200] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant Mytilus galloprovincialis mucin purification process and endotoxin removal method, characterized by, Includes the following steps: S1. Collect the bacterial cells by centrifuging the Escherichia coli fermentation broth expressing recombinant mussel adhesive protein, resuspend in buffer, homogenize under high pressure, centrifuge to collect the supernatant and perform deep filtration to obtain a clear lysate. S2. The clarified lysate was captured by affinity chromatography using an immobilized catechol biomimetic affinity chromatography medium. After washing and elution, the enriched mussel adhesive protein eluate was obtained. S3. The mussel adhesive protein eluent is concentrated by ultrafiltration and replaced with replacement buffer to obtain a high-concentration protein concentrate. S4. After adjusting the pH and controlling the conductivity of the high-concentration protein concentrate online, it is purified by a mixed-mode chromatography medium that combines cation exchange and hydrophobic interaction to obtain the target protein eluent. S5. Mix the target protein elution buffer with the phosphate mother liquor online to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography; S6. Pass the loading intermediate through a hydroxyapatite chromatography medium. The target protein binds to the phosphate groups of the medium, and the negatively charged endotoxin flows through to remove it. After washing and elution, the target protein solution is recovered. S7. The target protein solution is concentrated by ultrafiltration, desalted, and replaced with a solution containing a lyophilization protectant. After sterile filtration, it is freeze-dried and sealed with nitrogen to obtain the final product.

2. The method of claim 1, wherein, The buffer solution mentioned in step S1 is a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.8-4.2, and contains 3-8 mmol / L EDTA and 0.5-2 mmol / L ascorbic acid; The conditions for high-pressure homogenization are: pressure 700~900 bar, number of crushing cycles 2~3, and temperature 4~8℃; The centrifugation conditions are: rotation speed 10000~12000 rpm, time 25~35 min, temperature 4~8℃; The deep filtration process uses 0.45μm and 0.22μm filter membranes sequentially.

3. The method of claim 1, wherein, The immobilized catechol biomimetic affinity chromatography medium in step S2 is agarose-based globule-coupled catechol; The affinity capture specifically includes: Equilibration: Equilibrate the chromatography column with equilibration buffer; Sample loading: Load the clarified lysis buffer onto the sample; Washing: Remove unbound or weakly bound impurity proteins using washing buffer; Elution: The enriched mussel adhesive protein was specifically eluted with elution buffer to obtain mussel adhesive protein eluent.

4. The method of claim 3, wherein, The preparation of the immobilized catechol biomimetic affinity chromatography medium includes the following steps: (1) Media pretreatment: Take agarose-based balls, wash them with deionized water until the eluent is free of impurities, then soak them in 0.1 mol / L NaOH solution for 30 min, filter them, and rinse them with deionized water until the pH is neutral. (2) Epoxy activation: Add the pretreated agarose-based spheres to the reaction vessel, add epichlorohydrin, and then add 0.1 mol / L NaOH solution as a catalyst. Control the temperature at 30~40℃ and stir the reaction for 2~4 h to activate the surface of the agarose-based spheres and form epoxy groups. (3) Coupling reaction: After the activated agarose-based spheres are quickly rinsed with deionized water until neutral, a catechol solution with a concentration of 50~100 mmol / L is immediately added. The mass-to-volume ratio of catechol to agarose-based spheres is 1:

10. The mixture is stirred at 30℃ for 12~24 h to allow the catechol to be covalently coupled to the surface of the agarose-based spheres through epoxy groups. (4) Blocking and post-treatment: After the reaction is completed, wash the medium with deionized water until no free catechol is found, then soak it in 1 mol / L ethanolamine solution for 2 h to block the unreacted epoxy groups. Finally, wash with deionized water and water in sequence. The equilibration buffer is 20 mmol / L citrate-sodium citrate buffer with pH 3.8~4.

2.

5. The method of claim 3, wherein, The equilibration buffer is a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.8-4.2, containing 0.12-0.18 M NaCl; The washing buffer is a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.8-4.2, containing 0.2-0.3 M NaCl; The elution buffer is a 20 mmol / L citrate-sodium citrate buffer with a pH of 3.8-4.5, containing 0.6-1.0 M NaCl; The loading conditions are: linear flow rate 120~180cm / h, loading volume 10~20mg protein / mL medium; Elution linear flow rate: 80~120 cm / h.

6. The method of claim 1, wherein, The ultrafiltration system described in step S3 uses a membrane module with a molecular weight cutoff of 3-5 kDa, and the membrane material of the membrane module is regenerated cellulose or modified polyethersulfone. The concentration process controls the protein concentration at 5~15 mg / mL, and the entire concentration and solution exchange process maintains the protein solution pH ≤ 4.5, with nitrogen gas used for protection. The replacement buffer is a citrate-sodium citrate buffer containing 1-5 mmol / L ascorbic acid, with a pH of 3.8-4.2 and a conductivity ≤4 mS / cm.

7. The method according to claim 1, characterized in that, The composite mode chromatography medium mentioned in step S4 is a mixed mode medium that combines cation exchange and hydrophobic interaction; The specific conditions for purity include: Equilibration / loading buffer: 20~50mmol / L citrate-sodium citrate buffer, pH 4.8~5.5, conductivity ≤5mS / cm; Sample loading: Adjust the pH of the concentrate obtained in step S3 to 4.8-5.5 and then load the sample; the target protein will bind to the medium. Washing: Wash impurities with loading buffer or loading buffer containing no more than 50 mmol / L NaCl; Elution: Elute with a pH 5.5-6.5 buffer containing low concentration of salt to obtain the target protein elution peak with conductivity ≤6mS / cm.

8. The method according to claim 1, characterized in that, Step S5 specifically involves mixing the target protein elution buffer with the phosphate mother liquor online, adjusting the final phosphate concentration to 5-20 mmol / L, pH to 5.5-6.5, and conductivity to ≤6 mS / cm, to obtain a loading intermediate suitable for subsequent hydroxyapatite chromatography; The phosphate mother liquor is a sodium phosphate or potassium phosphate buffer solution with a concentration of 0.5~2.0 mol / L.

9. The method according to claim 1, characterized in that, The hydroxyapatite chromatography medium mentioned in step S6 is ceramic hydroxyapatite type I or type II; The conditions for endotoxin removal are as follows: Sample loading: The sample loading intermediate obtained in step S5 is directly loaded at a linear flow rate of 120~180 cm / h; at this time, the target protein is bound to the phosphate group of the medium, and the negatively charged endotoxin flows through. Washing: Wash with 5-20 mmol / L sodium phosphate buffer, pH 5.5-6.5, containing 0.08-0.12 M NaCl. Elution: Elute with 150-250 mmol / L sodium phosphate buffer, pH 5.5-6.5, at a linear flow rate of 100-150 cm / h, and recover the target protein solution.

10. The method according to claim 1, characterized in that, The ultrafiltration concentration in step S7 uses a 3kDa hollow fiber ultrafiltration column or a spiral wound ultrafiltration membrane; The desalting process employs a gradient liquid exchange strategy: first, dialyze 2-4 times the volume of sodium phosphate buffer at 5-10 mmol / L and pH 5.5-6.5, and then gradually transition to a solution containing a lyophilization protectant for dialyzing 4-6 times the volume. The solution containing the lyophilization protectant consists of water for injection, 0.8-1.2% mannitol, and 0.5-2 mmol / L ascorbic acid. After being concentrated to a protein concentration of 10-20 mg / mL, it was aseptically filtered through a 0.22 μm filter. The drying process is freeze drying, with the following conditions: pre-freezing at -25℃ to -15℃ for 1.5 to 2.5 hours, drying at -8℃ to -2℃ for 10 to 14 hours, drying at 20℃ to 30℃ for 4 to 8 hours, and then sealing with nitrogen.