CN116375872A - A purification method of ROR1 monoclonal antibody - Google Patents

Abstract

The application provides a method for purifying ROR1 monoclonal antibody, which comprises the following steps: 1) Affinity chromatography; 2) Anion chromatography; 3) Cation chromatography; 4) Nanofiltration; and 5) ultrafiltration displacement. According to the purification method, ROR1 monoclonal antibody can be effectively purified through reasonable combination of purification steps, CHO cell host protein, affinity chromatography ligand rProtein A, host DNA, culture medium components, endotoxin, virus and the like, and related impurities of products are removed, so that a target product is obtained at high recovery rate, and linear amplification is facilitated.

Description

A method for purifying ROR1 monoclonal antibody

Technical Field

The invention belongs to the field of protein purification, and particularly relates to a method for purifying a ROR1 monoclonal antibody.

Background Art

In addition to removing impurities such as host cell proteins (HCP), residual DNA and aggregates, the downstream chromatography process also needs to remove viruses that may be introduced upstream.

The presence of impurities in antibody drugs can reduce the efficacy of the drug, and in severe cases can lead to death. For example, drugs containing endotoxins or contaminated by endotoxins can cause a variety of allergic reactions after entering the human body, such as high fever, shock, and even death.

Many antibodies, including IgG and IgM, form aggregates. The presence of aggregates can produce different efficacy from the main product and lead to the formation of drug-resistant antibodies.

Residual host cell proteins may stimulate the body to mount an immune response and produce corresponding antibodies, which may also cause harm to the human body.

Host cell proteins (HCPs) are a complex mixture of unwanted host proteins that may be present in the final product after various production processes.

Therefore, during the purification process of antibody drugs, the removal of impurities directly affects the quality of the product.

ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family and belongs to the type I receptor tyrosine kinase (RTK) family. Although ROR1 is highly expressed during embryonic and infant development, its expression level decreases significantly in children and adults. However, the expression of ROR1 is significantly increased in a variety of blood cancers and solid tumors. Blood cancers that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), and myeloid blood cancers. Among solid tumors, cancer types that express ROR1 include colon cancer, lung cancer, pancreatic cancer, ovarian cancer, and many other cancers.

The antibody drug manufacturing process has a significant impact on antibody drugs in any of the five aspects, from cell line construction in the research and development period, culture medium optimization, upstream cell culture, downstream purification to the final formulation. Among them, the downstream purification process is the most complex and tedious. The downstream process contains multiple steps. The commonly used method is the tandem use of affinity chromatography, ion chromatography and hydrophobic chromatography.

The preparation of monoclonal antibody drugs through genetic engineering technology has become an important aspect of the biopharmaceutical field. Due to the strong specificity and significant efficacy of monoclonal antibody drugs, they have become one of the hot drugs in recent years.

Summary of the invention

Based on the above technical problems existing in the prior art, the present invention proposes a method for purifying ROR1 monoclonal antibodies. The method of the present invention can effectively remove CHO cell host proteins, affinity chromatography ligand rProtein A, host DNA, culture medium components, endotoxins and viruses, as well as product-related impurities such as protein aggregates, isomers and small molecule degradation products to obtain the target product with a high recovery rate. Therefore, the method can be used for commercial production.

The present application provides a method for purifying a ROR1 monoclonal antibody, comprising the following steps:

1. Affinity chromatography

The affinity chromatography comprises: subjecting the supernatant containing the ROR1 antibody to affinity chromatography in the following order: equilibrium-loading-post-equilibrium-washing-wash 2-elution-washing-storage;

2. Anion chromatography

The anion chromatography comprises: subjecting the secondary deep-filtered feed liquid containing the ROR1 antibody to anion chromatography in the following order: pre-equilibrium-equilibrium-loading-cleaning-regeneration-storage;

3. Cationic chromatography

The cationic chromatography comprises: subjecting the anionic chromatography collection solution containing the ROR1 antibody to cationic chromatography in the following order: pre-equilibrium-equilibrium-loading-equilibrium-elution-regeneration-storage;

4. Nanofiltration

5. Ultrafiltration replacement.

Preferably, before affinity chromatography, the cell fermentation broth is filtered using a two-stage deep filtration membrane to obtain a supernatant containing the ROR1 antibody.

Preferably, the two-stage deep filtration membranes are DOHC and A1HC.

Preferably, in step 1,

Equilibrate the affinity chromatography column with affinity chromatography equilibration buffer;

Add the deep filtrate containing ROR1 antibody to the equilibrated affinity chromatography column;

Equilibrate the affinity chromatography column with affinity chromatography equilibration buffer;

The affinity chromatography column is eluted with affinity chromatography elution buffer;

The affinity chromatography column is eluted with affinity chromatography elution buffer 2;

The affinity chromatography column was eluted with affinity chromatography elution buffer, and samples of 100 mAu-150 mAu under UV280 were collected, and pH adjustment and filtration operations were performed on the collected samples;

The chromatography medium was eluted with affinity chromatography cleaning solution, and the chromatography column was washed with 20% ethanol.

Preferably, in step 1, the affinity chromatography filler is MabSelect SuRe, and the height of the affinity chromatography column is 10-25 cm.

Preferably, in step 1, the deep filtration membrane is A1HC.

The affinity chromatography equilibrium solution is 20 mM phosphate buffer (PB) + 0.15 M NaCl, pH 7.3-7.5;

The affinity chromatography eluent is 20mM PB+1M NaCl, pH 7.3-7.5;

The affinity chromatography eluent 2 is 20 mM sodium citrate-citric acid, pH 5.4-5.6;

The affinity chromatography eluent is 100 mM glycine, pH 3.4-3.6;

The affinity chromatography elution collection range is the absorption peak of 100mAu-150mAu under UV280;

The cleaning solution for the affinity chromatography is 1 M HAc.

Preferably, the method comprises: after step 1 and before step 2, adjusting the pH of the collected sample to 3.4-3.6 with 1M HAc, maintaining at room temperature for 1 hour, adjusting the pH to 5.10-5.30 with 1M Tris, and filtering with a deep filtration membrane package, preferably, the deep filtration membrane package is 2 MA1HC01FS1.

Preferably, in step 2,

Pre-equilibrate the anion chromatography column with anion chromatography pre-equilibration buffer;

Equilibrate the anion chromatography column with anion chromatography equilibration buffer;

The secondary deep-filtered liquid containing the ROR1 antibody was added to a well-equilibrated anion chromatography column, and samples with a concentration greater than 0.125 Au under UV280 were collected;

The anion chromatography column was equilibrated with anion chromatography equilibration buffer, and samples with a value greater than 0.125 Au under UV280 were collected;

The anion chromatography column was cleaned with anion chromatography pre-equilibration buffer;

Regenerating the anion chromatography column with anion chromatography regeneration buffer;

Anion chromatography columns were preserved with 10 mM NaOH.

Preferably, in step 2, the anion chromatography filler is Q sepharose fast flow, and the height of the anion chromatography column is 10-25 cm.

Preferably, in step 2, the anion chromatography pre-equilibration buffer is 20mM PB+1M NaCl, pH 7.3-7.5, the anion chromatography equilibration buffer is 20mM sodium citrate-citric acid, pH 5.10-5.30, and the anion chromatography regeneration buffer is 20mM PB+1M NaCl.

Preferably, in step 3,

Pre-equilibrate the cation chromatography column with cation chromatography regeneration buffer;

Equilibrate the cation chromatography column with cation chromatography equilibration buffer;

The anion chromatography collection solution containing ROR1 antibody is added to the equilibrated cationic chromatography column;

Equilibrate the cation chromatography column with cation chromatography equilibration buffer;

The cation chromatography column was gradient eluted with elution buffer, and samples at 0.125Au-0.300Au under UV280 were collected.

Preferably, in step 3, in the regeneration and preservation process of the chromatography column, the chromatography column is regenerated with a cationic chromatography regeneration buffer, the chromatography medium is eluted with a cleaning solution, and the chromatography column is preserved with 10 mM NaOH.

Preferably, in step 3, the cationic chromatography filler is Capto S ImpAct.

Preferably, in step 3, the cationic chromatography equilibration buffer is 20 mM sodium citrate-citric acid, pH 4.9-5.1, the cationic chromatography elution buffer is 20 mM sodium citrate-citric acid + 150 mM NaCl, pH 4.9-5.1, and the cationic chromatography regeneration buffer is 20 mM sodium citrate-citric acid + 1 M NaCl, pH 4.9-5.1.

Preferably, in step 4, the sample eluted by cationic chromatography is subjected to virus removal nanofiltration. Preferably, the virus removal filter membrane is 3VI-28-FCGFO.

Preferably, in step 5, the sample filtered out after virus removal is ultrafiltered using a preparation buffer. Preferably, the preparation buffer is 10mM sodium citrate + 0.05mM EDTA + 10g/L trehalose, pH 4.7-5.7; preferably, the pore size of the ultrafiltration membrane is 30KD.

Beneficial Effects

The affinity chromatography step of the present invention can capture the target antibody, low pH incubation and secondary deep filtration can inactivate lipid envelope viruses, effectively remove CHO cell host proteins, rProtein A, host cell DNA to reduce the load of subsequent chromatography steps; anion chromatography can effectively remove CHO cell host proteins, rProtein A, host cell DNA and viruses; cation chromatography can further improve the purity of the target antibody; subsequent nanofiltration can remove non-lipid envelope viruses; and the final ultrafiltration operation can replace the target antibody into the preparation buffer and adjust the concentration at the same time.

In summary, the present invention can effectively purify ROR1 monoclonal antibodies by rationally combining the above purification steps, remove CHO cell host proteins, affinity chromatography ligand rProtein A, host DNA, culture medium components, endotoxins and viruses, and product-related impurities such as protein aggregates, isomers and small molecule degradation products, obtain the target product with a high recovery rate, and facilitate linear amplification.

DETAILED DESCRIPTION

The technical contents of the present invention are described in detail below through specific implementation modes to make it better understood; however, the protection scope of the present invention is not limited by the following specific implementation modes.

Herein, "HAc" means acetic acid, "Tris" means tris(hydroxymethyl)aminomethane, "EDTA" means ethylenediaminetetraacetic acid, and "PB" means phosphate buffer.

The sources of the main equipment and reagents used below are shown in Tables 1 and 2. In addition, the detection methods and reagents/instruments not specifically listed herein are all commonly used detection methods and reagents/instruments in the art.

Table 1 Main equipment and consumables for stock solution production

Table 2 Raw materials and auxiliary materials involved in the purification process

Example

1. Protein A chromatography 1, column model: BPG300

Chromatography medium: MabSelect SuRe

Column height: 10.0cm Column volume: 7.1L

Column efficiency: HETP = 0.03 Symmetry: As = 1.15

Flow rate: 71L/h

2. Connect the chromatography column to the akta process and flush it with 0.1 M NaOH for 2 to 4 column volumes (CV) for 15 to 30 min.

3. Equilibrate in 20 mM PB + 0.15 M NaCl, pH 7.4 ± 0.1 for 5 CV.

4. Loading: loading capacity ≤ 50 g/L.

The sample loaded was the supernatant obtained by deep filtration of the cell culture fluid through DOHC and A1HC as follows.

CHO-K1 cells (purchased from ATCC, with a deposit number of CCL61) were cultured through the following steps: cell recovery, seed expansion, reactor seed expansion, and production-scale culture.

Specifically, the process conditions for cell recovery are as follows: the frozen cells are thawed and inoculated into basic culture medium 1 (CHO cell Growth A growth medium (BalanCD_CHO_GrowthA) 23.72 g/L, NaHCO ₃ 2.20 g/L, L-glutamine 0.73 g/L, ultrapure water to volume, pH 6.80-7.20, filtered and sterilized), and cultured at a temperature of 36.5°C, a CO ₂ concentration of 5.0%, and a shaker speed of 120 rpm. The live cell density after recovery is: 0.187×10 ⁶ cells/ml, and the viability is: 92.31%.

The process conditions for seed expansion are as follows: use basic culture medium 1 and subculture under the conditions of shaker temperature setting value of 36.5℃, rotation speed of 120rpm, _CO2 concentration of 5%, and amplitude of 50mm. After subculture, the seed viable cell density was measured to be 2.22× ¹⁰⁶ cells/ml, and the viability was 96.72%.

The process conditions for reactor seed expansion are as follows: basal culture medium 1 is used for seed expansion culture in a 50L wave reactor for 5 days, the reactor temperature setting value is 36.5°C, the rotation speed is 15-25rpm, the compressed air flow rate is 0.5L/min, the _CO2 flow rate is 0.045L/min, and the pH is controlled above 6.80. In the N-1 wave reactor seed expansion, basal culture medium 2 (GrowthA118.96g/L, L-glutamine 0.73g/L, NaHCO3 2.20g/L, GlycosylationAdjust (GAL+) 10ml/L, ultrapure water, pH 6.80-7.20, filtered and sterilized) is added four times for a total of 20L. Before each addition, samples are taken from the reactor to confirm that the viable cell density is higher than 2.0× ¹⁰⁶ cells/ml and the viability is ≥90.00%.

The process conditions for production-scale culture are as follows: in a STR200 stirred tank reactor, basal medium 2 is used for scale culture for 14 days, and the cell culture fluid is harvested. The reactor temperature is set to 36.5°C, the stirring speed is 80 rpm, DO 40.0%, pH = 7.10 ± 0.20, OV ventilation is 0.5 L/min, SP ventilation is 0.5 L/min, and feed medium 3 (CHO cell feed medium Feed 4 (BalanCD CHO Feed 4) 67.31 g/L, glucose 40.00 g/L, NaHCO ₃ 2.20 g/L, Glycosylation Adjust (GAL+) 22.22 ml/L, fixed to volume with ultrapure water, pH 7.00-7.60, filtered and sterilized) is added at 3%, 3%, 7%, 7%, 7%, 3% and 3% of the initial culture volume on days 3, 5, 7, 9, 11 and 13, respectively.

5. 20 mM PB + 0.15 M NaCl, pH 7.4 ± 0.1 and re-equilibrate for 5CV.

6. Elution in 20 mM PB + 1 M NaCl, pH 7.4 ± 0.1 for 5 CV.

7, 20 mM sodium citrate-citric acid, pH 5.5±0.1, elution for 5 CV.

8, 100 mM glycine, pH 3.5±0.1, elution, UV280 absorption 0.100-0.150 Au range elution peak collection.

9. Wash with 1M HAc for 2CV.

10. Repeat the above steps to complete the purification of all supernatants.

After affinity chromatography, the purified sample was subjected to low pH virus inactivation and secondary depth filtration using A1HC. The exemplary operation is as follows:

1. The collected samples were adjusted to pH 3.5±0.1 with 1 M HAc and maintained at room temperature for 1 hour, and then adjusted to pH 5.2±0.1 with 1 M Tris.

2. Depth filter membrane package: MA1HC01FS1*2

2. Anion Exchange Chromatography

1. Chromatography column model: BPG200

Chromatography medium: Q.FF

Column height: 18.0cm Column volume: 5.7L

Column efficiency: HETP = 0.03 Symmetry: As = 0.96

Flow rate: 57L/h

2. Connect the chromatography column to the akta process and flush it with 1M NaOH for 2-4CV and maintain it for 30min.

3, 20 mM PB + 1 M NaCl, pH 7.4 ± 0.1 pre-equilibrated for 2CV.

4, 20 mM sodium citrate-citric acid, pH 5.2±0.1, equilibrated for 8CV.

5. Load the sample, loading ≤85g/L, UV280≥0.125Au, start collecting the flow-through peak. (The sample loaded is the sample that has been filtered through the secondary depth)

6, 20 mM sodium citrate-citric acid, pH 5.2±0.1, re-equilibrate to UV280≤0.125Au and stop collecting.

7, 20 mM PB + 1 M NaCl, pH 7.4 ± 0.1, wash for 2CV.

8. Wash with 1M NaOH for 2CV.

3. Cationic Chromatography

1. Chromatography column model: BPG300

Chromatography medium: Capto S ImpAct

Column height: 13.7 cm Column volume: 9.7 L

Column efficiency: HETP = 0.01 Symmetry: As = 1.09

Flow rate: 97L/h

2. Connect the chromatography column to the akta process and flush it with 1M NaOH for 2-4CV and maintain it for 30min.

3, 20 mM sodium citrate-citric acid + 1 M NaCl, pH 5.0 ± 0.1, pre-equilibration for 2CV.

4, 20 mM sodium citrate-citric acid, pH 5.0±0.1, equilibration for 3CV.

5. The pH of the anion flow-through sample was adjusted to 5.0±0.1 with 1M Hac and loaded with a loading of ≤49 g/L.

6, 20 mM sodium citrate-citric acid, pH 5.0±0.1, and re-equilibrate for 2CV.

7, 20 mM sodium citrate-citric acid + 150 mM NaCl, pH 5.0±0.1 for elution, and collect the elution peak in the UV280 absorption range of 0.125-0.300 Au.

8, 20 mM sodium citrate-citric acid + 1 M NaCl, pH 5.0±0.1, wash 2CV.

9. 1M NaOH cleaning 2CV

4. Virus Removal Nanofiltration

1. 20 mM sodium citrate-citric acid, pH 5.0±0.1, clean the Akta process pipeline until the baseline is flat.

2. Virus removal pre-filter: 54A5358N9--FF

Virus removal filter: 3VI-28-FCGFO

Access to Akta Process column

3. Akta process uses pressure loading, operating pressure 2±0.2 bar, 20 mM sodium citrate-citric acid, pH 5.0±0.1, and rinsing for 15 minutes.

4. Load the sample (load capacity ≤ 617.8L/m ² ), the pressure does not exceed 2.2bar. (The sample loaded is a cation elution sample)

5. Collect the filtered product.

5. Ultrafiltration

1. Ultrafiltration membrane package: 3021445906E-SW ^0.6m2 * 3 pieces

2. Clean the ultrafiltration system with 1M NaOH for 60 min, and then clean with purified water until the pH at the permeate end is neutral.

3. Use preparation buffer to clean the ultrafiltration system until the pH of the solution at the permeate end and reflux port is the same as that at the inlet.

4. Concentrate the protein after virus removal filtration, TMP≤1.0bar, and concentrate to a protein concentration of 30-40g/L.

5. Keep the volume of concentrated protein solution unchanged, continuously add preparation buffer, change the solution by 7-10 sample volumes, close the permeate end, fully open the reflux port, detect the protein concentration, and control it at 50-55g/L.

6. Hang the inlet tube in the air to completely remove the protein in the ultrafiltration system, collect the ultrafiltration harvest liquid and retest the concentration, measure and record the pH and conductivity of the harvest liquid.

7. Clean the ultrafiltration system with 1M NaOH and store it with 20% ethanol.

As mentioned above, the intermediate deep filtration is used to effectively remove cell host proteins, affinity chromatography ligand rProteinA, and host DNA, reducing the load of the subsequent chromatography step. According to the ICH Q5a "Viral Safety Evaluation of Biotechnology Products Derived from Human or Animal Cell Lines" guidelines, the chromatography step needs to be validated for virus clearance. The anion chromatography after reducing the load can leave more operating space for the virus clearance effect, and the addition of cationic chromatography can effectively improve the product purity. In the above purification steps, the detection records of ROR1 purification samples are shown in Table 3 below.

Table 3 ROR1 purification process sample detection record

The ROR1 antibody data finally obtained after the above operation is shown in Table 4 below:

Table 4

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

1. A method for purifying ROR1 monoclonal antibody, comprising the steps of:

1) Affinity chromatography

The affinity chromatography comprises: the supernatant of the cell fermentation broth containing ROR1 antibody was subjected to affinity chromatography in the following order: balancing, loading, balancing after, leaching, eluting, cleaning and preserving;

2) Anion chromatography

The anion chromatography comprises: subjecting the ROR1 antibody-containing feed solution purified by affinity chromatography, preferably the feed solution subjected to secondary depth filtration, to anion chromatography in the following order: pre-balancing-loading-balancing-cleaning-regenerating-preserving;

3) Cationic chromatography

The cationic analysis includes: anion chromatography collections containing ROR1 antibodies were subjected to cation chromatography in the following order: pre-equilibration-loading-equilibration-elution-regeneration-preservation;

4) Nanofiltration

5) And (5) ultrafiltration displacement.

2. The method according to claim 1, wherein the cell fermentation broth is filtered using a two-stage deep filtration membrane, preferably D0HC and A1HC, prior to performing the affinity chromatography, resulting in a supernatant containing ROR1 antibodies.

3. The method according to claim 1, wherein, in step 1,

balancing the affinity chromatography column by using an affinity chromatography balancing buffer;

adding the deep filtrate containing ROR1 antibody into well-balanced affinity chromatographic column;

balancing the affinity chromatography column by using an affinity chromatography balancing buffer;

eluting the affinity chromatographic column by using an affinity chromatographic eluting buffer;

eluting the affinity chromatography column by using an affinity chromatography eluting buffer 2;

eluting the affinity chromatographic column by using an affinity chromatographic elution buffer, collecting a sample of 100mAu-150mAu under UV280, and carrying out pH adjustment and filtration operation on the collected sample;

eluting the chromatographic medium with affinity chromatography washing liquid, and washing the chromatographic column with 20% ethanol.

4. A method according to claim 3, wherein, in step 1,

the affinity chromatography filler is MabSelect SuRe, and the height of the affinity chromatography column is 10-25cm;

the affinity chromatography balance buffer is 20mM phosphate buffer+0.15M NaCl, pH7.3-7.5;

the affinity chromatography eluting buffer solution is 20mM phosphate buffer solution and 1M NaCl, and the pH value is 7.3-7.5;

the affinity chromatography eluting buffer solution 2 is 20mM sodium citrate-citric acid, and the pH value is 5.4-5.6;

the affinity chromatography eluting buffer is 100mM glycine, and the pH value is 3.4-3.6;

the affinity chromatography cleaning liquid is 1M HAc.

5. The method according to claim 1, wherein the method comprises: after step 1 and before step 2, the collected sample is pH-adjusted with 1M HAc for 3.4-3.6, maintained at room temperature for 1 hour, pH-adjusted with 1M Tris for 5.10-5.30, and filtered using a deep filtration membrane pack, preferably MA1HC01FS1, preferably 2 said deep filtration membrane packs.

6. The method according to claim 1, wherein, in step 2,

pre-balancing the anion chromatographic column by using anion chromatography pre-balancing buffer solution;

balancing the anion chromatographic column by using anion chromatographic balancing buffer solution;

adding the feed liquid containing the ROR1 antibody after secondary depth filtration into an anion chromatographic column with good balance, and collecting a sample with the UV280 of more than 0.125 Au;

balancing the anion chromatographic column by using anion chromatographic balancing buffer solution, and collecting a sample with the UV280 of more than 0.125 Au;

washing the anion chromatographic column with anion chromatographic pre-equilibrium buffer solution;

regenerating the anion chromatographic column with anion chromatographic regeneration buffer;

the anion chromatography column was stored with 10mM NaOH.

7. The method according to claim 6, wherein in step 2, the anion chromatography packing is Q sepharose fast flow and the height of the anion chromatography column is 10-25cm; the anion chromatography pre-equilibrium buffer solution is 20mM PB+1M NaCl,pH7.3-7.5, the anion chromatography equilibrium buffer solution is 20mM sodium citrate-citric acid, the pH is 5.10-5.30, and the anion chromatography regeneration buffer solution is 20mM PB+1M NaCl,pH 7.4.

8. The method according to claim 1, wherein, in step 3,

pre-equilibrating the cation chromatography column with a cation chromatography regeneration buffer;

balancing the cation chromatography column with a cation chromatography balancing buffer;

adding the collected solution of the anion chromatography containing the ROR1 antibody to a balanced cation chromatography column;

balancing the cation chromatography column with a cation chromatography balancing buffer;

eluting the cationic chromatographic column with a cationic chromatographic elution buffer, and collecting a sample of 0.125Au to 0.300Au under UV 280;

regenerating with cation chromatography regeneration buffer solution, eluting the chromatography medium with cleaning solution, and preserving the chromatographic column with 10mM NaOH;

preferably, the cationic chromatographic packing is Capto S ImpAct;

preferably, the cation chromatography balance buffer is 20mM sodium citrate-citric acid, the pH value is 4.9-5.1, the cation elution buffer is 20mM sodium citrate-citric acid+150 mM NaCl, the pH value is 4.9-5.1, and the cation regeneration buffer is 20mM sodium citrate-citric acid+1M NaCl, the pH value is 4.9-5.1.

9. A method according to claim 1, wherein in step 4, the sample eluted from the cationic layer is subjected to virus removal nanofiltration, preferably using a virus removal filtration membrane of 3VI-28-FCGFO.

10. The method according to claim 1, wherein in step 5, the sample after virus removal filtration is subjected to ultrafiltration exchange using a formulation buffer, preferably 10mM sodium citrate+0.05 mM edta+10g/L trehalose, pH 4.7-5.7; preferably, the ultrafiltration membrane used comprises a pore size of 30KD.