CN113416243B

CN113416243B - Method for purifying antibody

Info

Publication number: CN113416243B
Application number: CN202110964569.XA
Authority: CN
Inventors: 陈伟峰; 徐会; 焦竞; 刘钰莹; 贺旭刚; 陈爱华; 黄黎明; 汤辰翔
Original assignee: Shanghai Maijin Biomedical Technology Co ltd; Jiangsu Hengrui Medicine Co Ltd; Shanghai Shengdi Pharmaceutical Co Ltd
Current assignee: Shanghai Maijin Biomedical Technology Co ltd; Jiangsu Hengrui Medicine Co Ltd; Shanghai Shengdi Pharmaceutical Co Ltd
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2021-12-10
Anticipated expiration: 2041-08-23
Also published as: CN113416243A

Abstract

The present disclosure relates to a method of purifying an antibody. In particular, the purification process effectively removes aggregates and undesired glycosylated impurities using affinity chromatography, complex mode chromatography. The method is based on the difference between the aggregate and the monomer molecule in the binding force of the affinity filler, and the aggregate is effectively removed through affinity chromatography; based on the difference between the non-glycosylated impurities and the target molecules in charge, hydrophobicity and the like, the non-expected glycosylated impurities are effectively removed through the composite mode chromatography. In addition, the present disclosure adds a specific concentration of amino acid component to the buffer system during chromatographic purification, stabilizing the conformation of the antibody, particularly bispecific antibody, inhibiting the formation of aggregates.

Description

Method for purifying antibody

Technical Field

The present disclosure relates to the field of methods for purifying antibodies, in particular, methods for removing antibody aggregates and undesired glycosylation impurities using affinity chromatography, complex mode chromatography.

Background

With the continuous development of biological medicines, antibody drugs show more and more important positions. The separation and purification of the culture containing the target antibody is an essential step in the production process, and how to further increase the efficiency of removing impurities and improve the purity and yield of the antibody by optimizing the purification conditions of the antibody is an important problem in the current industrial production.

The dual-specificity and multi-specificity antibodies can be simultaneously combined with different antigens or different epitopes of the same antigen, and have good application prospect. The bi-and multi-specific antibody has flexible and various structures, but has the problems of poor stability, easy generation of aggregates and degradation fragments, and difficult removal of such impurities.

With the increasing expression level of antibodies in the cell culture stage, the heterogeneity of the expression products becomes more obvious, and the antibody purification process faces more serious challenges. Glycosylation is an important post-translational modification, and has relevance to factors such as antibody sequences, expression systems, culture medium components, culture processes and the like. Glycosylation modifications affect the conformation, stability, pharmacokinetics, activity, and immunogenicity of the antibody. Undesired glycosylation impurities increase antibody heterogeneity and may adversely affect product stability, safety and effectiveness, and therefore need to be removed by suitable purification processes.

The antibody purification process comprises a plurality of steps, and currently, chromatographic separation methods such as affinity chromatography, cation exchange chromatography, anion exchange chromatography and the like are commonly used. Aggregates and unintended glycosylated impurities, which are often very close in physicochemical properties to the target antibody molecule, are difficult to remove using single mode chromatography. Therefore, the optimization of a proper purification process aiming at different antibodies and the reduction of the content of aggregates and unintended glycosylation impurities as far as possible are technical problems which need to be solved urgently at present.

Disclosure of Invention

The antibody purification method provided by the present disclosure can effectively remove antibody aggregates and glycosylation impurities.

The present disclosure provides a method of purifying an antibody comprising: utilizing a) affinity chromatography and b) complex mode chromatography, optionally further comprising the step of contacting a buffer comprising an amino acid with the affinity chromatography packing and the complex mode chromatography packing. In some embodiments, the buffer containing the amino acid is contacted with only the affinity chromatography packing, or the buffer containing the amino acid is contacted with only the composite mode chromatography packing, or the buffer containing the amino acid is contacted with both the affinity chromatography packing and the composite mode chromatography packing.

In some embodiments, the amino acid is a basic amino acid. In some embodiments, the basic amino acid is selected from one or more of arginine, histidine, lysine, for example, the amino acid may be arginine.

In some embodiments, the concentration of amino acids in step a) affinity chromatography buffer of the present disclosure is from 10 to 100mM, and the concentration of amino acids in step b) complex mode chromatography buffer is from 10 to 50 mM.

In some embodiments, the concentration of amino acids in step a) affinity chromatography buffer of the present disclosure is about 100mM, and the concentration of amino acids in step b) complex mode chromatography buffer is about 20 mM.

In some embodiments, the antibodies of the present disclosure present glycosylation sites. For example, the glycosylation site is a characteristic sequence of "Asn-X1-X2" on the antigen binding fragment, wherein X1 can be any amino acid and X2 is any one of serine, threonine, cysteine.

In some embodiments, the methods of the present disclosure are capable of removing antibody aggregates and glycosylation impurities.

In some embodiments, the present disclosure provides a method of purifying an antibody comprising the steps of:

a) affinity chromatography

1) Loading: loading the cell culture supernatant containing the bispecific antibody onto a pretreated affinity chromatography packing;

2) balancing: washing affinity chromatography packing with a balance buffer solution;

3) and (3) elution: eluting with elution buffer solution, and collecting eluate.

b) Complex mode chromatography

1) Loading: loading the adjusted affinity chromatography intermediate product on the pretreated composite mode chromatography filler;

2) balancing: cleaning the composite mode chromatographic packing by using a balance buffer solution;

3) and (3) elution: gradient eluting with elution buffer solution, and collecting eluate.

Wherein the elution buffer in step a), the equilibration buffer and the elution buffer in step b) all contain amino acids. In some embodiments, the amino acid is a basic amino acid. In some embodiments, the basic amino acid is selected from one or more of arginine, histidine, lysine, for example, the amino acid may be arginine.

In some embodiments, the concentration of amino acids in the elution buffer of step a) of the present disclosure is from 10 to 100 mM; the concentration of amino acids in the equilibration buffer and the elution buffer in the step b) is 10-50 mM.

In some embodiments, the concentration of amino acids in the elution buffer of step a) of the present disclosure is about 100 mM; the amino acid concentration in the equilibration buffer, elution buffer of step b) was about 20 mM.

In some embodiments, the affinity chromatography packing of step a) of the present disclosure is a packing in which Protein a is a ligand that is cross-linked to a matrix including, but not limited to, agarose, polyacrylic resin, polystyrene divinyl phenyl resin, polymethacrylic resin, polystyrene resin, glass.

In some embodiments, step a) of the present disclosure the affinity chromatography packing is MabSelect prism a, i.e. the Protein a ligand is cross-linked to agarose.

In some embodiments, the buffering substance in the equilibration buffer of step a) of the present disclosure is selected from phosphate, MES, citric acid or citrate.

In some embodiments, the buffer substance in step a) of the disclosure is phosphate at a concentration of 10-50mM, e.g., about 10mM, about 20mM, about 30mM, about 40mM, about 50 mM.

In some embodiments, the buffer substance in the equilibration buffer of step a) of the present disclosure is phosphate at a concentration of about 20 mM.

In some embodiments, step a) of the present disclosure the equilibration buffer has a pH of 7.1-7.5, e.g., about 7.1, about 7.2, about 7.3, about 7.4, about 7.5.

In some embodiments, the pH of the equilibration buffer of step a) of the present disclosure is 7.4 ± 0.1.

In some embodiments, the buffer substance in step a) of the present disclosure is selected from acetate, citrate, citric acid, acetic acid.

In some embodiments, the buffer substance in step a) of the disclosure is acetate at a concentration of 10-50mM, e.g., about 10mM, about 20mM, about 30mM, about 40mM, about 50 mM.

In some embodiments, the buffer substance in step a) of the present disclosure is acetate at a concentration of about 50 mM.

In some embodiments, step a) of the present disclosure further comprises an amino acid in a concentration of about 100mM in elution buffer-B.

In some embodiments, step a) of the present disclosure the elution buffer has a pH of 3.4-3.6 (e.g., 3.5 ± 0.1).

In some embodiments, step b) of the present disclosure is a composite mode chromatography packing selected from Capto MMC, Capto MMC imprints, hydroxyapatite, said composite mode chromatography packing being Capto MMC imprints.

In some embodiments, prior to loading in step b) of the present disclosure, 1M Tris is used to adjust the affinity intermediate pH to 5.5-6.5; deionized water dilution conductivity <6 mS.

In some embodiments, prior to loading in step b) of the present disclosure, 1M Tris is used to adjust the affinity intermediate pH to about 6.0; deionized water dilution conductivity <6 mS.

In some embodiments, the buffer substance in step b) equilibration buffer of the present disclosure is selected from phosphate, citrate, acetate, phosphoric acid, citric acid, acetic acid.

In some embodiments, the elution buffer of step B) of the present disclosure comprises elution buffer a and elution buffer B, and the buffer substances in step B) equilibration, elution buffer a and elution buffer B are phosphate and citric acid, all at a concentration of 10-50mM, e.g., about 10mM, about 20mM, about 30mM, about 40mM, about 50 mM.

In some embodiments, the buffer substance in step B) equilibration buffer, elution buffer-a, and elution buffer-B of the present disclosure is citric acid at a concentration of about 10mM and contains phosphate at a concentration of about 20 mM.

In some embodiments, step B) of the present disclosure comprises a pH of 5.5 to 6.5, e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5.

In some embodiments, step B) of the present disclosure has a pH of 6.0 for equilibration buffer, elution buffer-a, and elution buffer-B.

In some embodiments, step B) of the present disclosure further comprises amino acids in the equilibration buffer, elution buffer-a and elution buffer-B at a concentration of 10 to 50 mM.

In some embodiments, step B) of the present disclosure further comprises an amino acid in a concentration of about 20mM in the equilibration buffer, elution buffer-a, and elution buffer-B.

In some embodiments, step B) of the present disclosure elution buffer-B further comprises sodium chloride at a concentration of 400-600mM, e.g., about 400mM, about 450mM, about 500mM, about 550mM, about 600 mM.

In some embodiments, step B) elution buffer-B of the present disclosure further comprises sodium chloride at a concentration of about 400 mM.

In some embodiments, the buffer substance in both the equilibration buffer and the elution buffer of method steps a) and b) of the present disclosure comprises one or more of phosphate, MES, acetate, acetic acid, citric acid, or citrate.

In some embodiments, step a) of the present disclosure comprises a phosphate buffer at 10-50mM, pH 7.1-7.5; step a) the elution buffer contains 10-50mM acetate, pH 3.4-3.6; and step B) the equilibration buffer and the elution buffer both contain 10-50mM phosphate and citric acid and have a pH value of 5.5-6.5, wherein the elution buffer of step B) comprises elution buffers A and B, wherein the elution buffer B further contains 400-600mM sodium chloride.

In some embodiments, step a) of the present disclosure the equilibration buffer contains about 20mM phosphate, at a pH of about 7.4; step a) the elution buffer contains about 50mM acetate at a pH of about 3.5; and step B) both the equilibration buffer and the elution buffer containing about 20mM phosphate and about 10mM citric acid at a pH of about 6.0, said elution buffers comprising elution buffers A and B, wherein elution buffer B further contains about 400mM sodium chloride.

In some embodiments, the antibody of the present disclosure is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, Fab ', F (ab') 2, Fv, disulfide-linked Fv, scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, and a bispecific antibody.

In some embodiments, the antibodies of the present disclosure bind to any one or more of PD-1, PD-2, PD-L1, PD-L2, LAG-3, CD20, VEGFR, CTLA-4, TIM-3 immune checkpoint molecules.

In some embodiments, the antibodies of the present disclosure specifically bind to PD-1 and LAG-3.

In some embodiments, the antibody heavy chain amino acid sequence of the present disclosure is the sequence shown as SEQ ID NO. 1 and the light chain amino acid sequence is the sequence shown as SEQ ID NO. 2.

In some embodiments, the antibodies of the present disclosure specifically bind B7H3 comprising HCDR1 as shown in NYDN (SEQ ID NO: 3), HCDR2 as shown in WIFPGDGSTQYNETFKG (SEQ ID NO: 4), HCDR3 as shown in QTTGTWFAY (SEQ ID NO: 5); and, LCDR1 as shown at RASQSISKYLH (SEQ ID NO: 6), LCDR2 as shown at YANQSIR (SEQ ID NO: 7), and LCDR3 as shown at QNGHSFPLT (SEQ ID NO: 8). The full length sequence of this antibody of US20170362322a1 is incorporated herein in its entirety.

The present disclosure includes the following embodiments:

1. a method for purifying an antibody comprising the steps of using a) affinity chromatography and b) complex mode chromatography, and contacting the affinity chromatography packing and the complex mode chromatography packing with a buffer containing an amino acid.

2. The method of claim 1, wherein the amino acid is a basic amino acid.

3. The method of claim 2, wherein the basic amino acid is selected from one or more of arginine, histidine, lysine.

4. The method of item 3, wherein the basic amino acid is arginine.

5. The method according to any one of the preceding claims, wherein the concentration of amino acids in the affinity chromatography buffer of step a) is between 10 and 100mM and the concentration of amino acids in the complex mode chromatography buffer of step b) is between 10 and 50 mM.

6. The method of claim 5, wherein the concentration of amino acids in step a) affinity chromatography buffer is about 100mM and the concentration of amino acids in step b) complex mode chromatography buffer is about 20 mM.

7. The method of item 6, wherein the antibody has a glycosylation site.

8. The method of claim 7, wherein the glycosylation site is a characteristic sequence of "Asn-X1-X2" on the antigen binding fragment, wherein X1 can be any amino acid and X2 is any one of serine, threonine, and cysteine.

9. The method of any one of the preceding claims, wherein the method is capable of removing antibody aggregates and glycosylation impurities.

10. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

1) Loading: loading the cell culture clarified liquid containing the antibody on the pretreated affinity chromatography filler;

3) and (3) elution: eluting with an elution buffer solution, and collecting the eluate;

b) complex mode chromatography

1) Loading: loading the adjusted affinity chromatography elution product on a pretreated composite mode chromatography filler;

3) and (3) elution: gradient eluting with elution buffer solution, and collecting eluate;

wherein the elution buffer in step a), the equilibration buffer and the elution buffer in step b) all contain amino acids.

11. The method of item 10, wherein the amino acid is a basic amino acid.

12. The method of claim 11, wherein the basic amino acid is selected from one or more of arginine, histidine, lysine.

13. The method of claim 12, wherein the basic amino acid is arginine.

14. The method according to any one of claims 10 to 13, wherein the concentration of amino acids in the elution buffer of step a) is between 10 and 100 mM; the concentration of amino acids in the equilibration buffer and elution buffer of step b) is 10-50 mM.

15. The method of claim 14, wherein the concentration of amino acids in the elution buffer of step a) is about 100 mM; the amino acid concentration in the equilibration buffer, elution buffer of step b) was about 20 mM.

16. The method according to any of the preceding claims, wherein the affinity chromatography packing of step a) is a packing with Protein a as ligand and/or the composite mode chromatography packing of step b) is selected from Capto MMC, Capto MMC impress or hydroxyapatite.

17. The method of item 16, wherein the step a) affinity chromatography packing is MabSelect prism a and the step b) complex mode chromatography packing is Capto MMC impress.

18. The method of claim 17, wherein the buffer material in the equilibration and elution buffers of steps a) and b) of the method comprises one or more of phosphate, MES, acetate, acetic acid, citric acid, or citrate.

19. The method of claim 18, wherein:

the equilibration buffer of step a) contains 10-50mM phosphate, pH 7.1-7.5;

the elution buffer of step a) contains 10-50mM acetate, pH 3.4-3.6; and

the elution buffer of step B) comprises elution buffer A and elution buffer B,

the equilibrium buffer solution of the step B) and the elution buffer solution A and the elution buffer solution B of the step B) both contain 10-50mM phosphate and citric acid, the pH value is 5.5-6.5, and the elution buffer solution B of the step B) further contains 400-600mM sodium chloride.

20. The method of claim 19, wherein:

the equilibration buffer of step a) contains about 20mM phosphate, pH about 7.4;

the elution buffer of step a) contains about 50mM acetate at a pH of about 3.5; and

the equilibration buffer of step B) and the elution buffer a, B of step B) each contained about 20mM phosphate and about 10mM citric acid at a pH of 6.0, said elution buffer B further containing about 400mM sodium chloride.

21. The method of item 20, wherein step b) prior to loading, the elution product pH is adjusted to about 6.0 using 1M Tris for affinity chromatography and diluted with deionized water to a conductivity of <6 mS.

22. The method of any one of claims 1-21, wherein the antibody has a glycosylation site that is a characteristic sequence of "Asn-X1-X2" in the antigen-binding fragment, wherein X1 can be any amino acid and X2 is any one of serine, threonine, and cysteine.

23. The method of any one of the preceding claims, wherein the antibody is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a Fab ', a F (ab') 2, an Fv, a disulfide-linked Fv, an scFv, a single domain antibody, a bispecific antibody, and a multispecific antibody.

24. The method of claim 23, wherein the antibody is a bispecific antibody.

25. The method of any one of the preceding claims, wherein the antibody binds to any one or more of PD-1, PD-2, PD-L1, PD-L2, LAG-3, CD20, VEGFR, CTLA-4, B7H3, and TIM-3 immune checkpoint molecules.

26. The method of item 25, wherein the antibody binds PD-1 and LAG-3.

27. The method of claim 26, wherein the antibody heavy chain has the amino acid sequence set forth in SEQ ID NO. 1 and the light chain amino acid sequence has the amino acid sequence set forth in SEQ ID NO. 2.

28. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

1) Loading: loading cell culture supernatant containing bispecific antibodies shown in SEQ ID NO 1 and 2 on a pretreated Mabselect prism A affinity chromatography column, wherein the loading capacity is less than or equal to 30 g/L;

2) balancing: washing 3-5 column volumes with equilibration buffer, wherein the equilibration buffer contains about 20mM phosphate, pH7.4 + -0.1;

3) and (3) elution: eluting the target protein with an elution buffer, and collecting the eluate, wherein the elution buffer contains about 50mM acetate, about 0.1M arginine, and pH3.5 + -0.1;

b) complex mode chromatography

Adjusting the pH of the elution product of the affinity chromatography to 6.0 +/-0.1 using about 1M Tris prior to loading, and diluting the sample with deionized water selected from pure water, high purity water, ultrapure water to a conductivity of <6 mS;

1) loading: loading the adjusted affinity chromatography elution product onto a pretreated Capto MMC Impres composite mode chromatographic column, wherein the loading capacity is less than or equal to 30 g/L;

2) balancing: washing 3-5 column volumes with equilibration buffer, wherein the equilibration buffer comprises about 20mM phosphate, about 20mM arginine, about 10mM citric acid, and has a pH of about 6.0;

3) and (3) elution: eluting with elution buffer A and elution buffer B (about 20mM phosphate, about 20mM arginine, about 10mM citric acid, about 400mM sodium chloride, about pH 6.0) in gradient, and collecting the eluate; wherein elution buffer A comprises about 20mM phosphate, about 20mM arginine, about 10mM citric acid, and a pH of about 6.0; elution buffer B contained about 20mM phosphate, about 20mM arginine, about 10mM citric acid, about 400mM sodium chloride, and a pH of about 6.0.

29. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

b) complex mode chromatography

wherein the elution buffer of step a), the equilibration buffer of step b), and the elution buffer each comprise arginine, and the concentration of arginine in the elution buffer of step a) is about 100 mM; the concentration of arginine in the equilibration buffer and elution buffer of step b) is about 20mM, and the antibody has a glycosylation site, wherein the glycosylation site is a characteristic sequence of 'Asn-X1-X2' on the antigen binding fragment, X1 is any amino acid, and X2 is any one of serine, threonine and cysteine.

30. The method of claim 29, wherein the antibody heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO. 1 and the antibody light chain amino acid sequence is the amino acid sequence shown in SEQ ID NO. 2.

31. The method of claim 29, wherein:

the equilibration buffer of step a) contains about 20mM phosphate, pH 7.4. + -. 0.1;

the elution buffer of step a) contains about 50mM acetate, about 100mM arginine, pH3.5 ± 0.1;

the equilibration buffer of step b) contains about 20mM phosphate and about 10mM citric acid, and has a pH of about 6.0; and

the elution buffer of step B) comprises elution buffer a and elution buffer B, both elution buffer a and elution buffer B comprising about 20mM phosphate, about 20mM arginine and about 10mM citric acid at a pH of about 6.0, said elution buffer B further comprising 400mM sodium chloride.

32. The method of item 31, step b) prior to loading, adjusting the pH of the elution product of the affinity chromatography to about 6.0 using 1M Tris and diluting with deionized water to a conductivity of <6 mS.

33. The method of item 32, wherein step a) the affinity chromatography packing is MabSelect prism A and step b) the composite mode chromatography packing is Capto MMC Impres.

34. The method of any one of claims 30-32, wherein the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID No. 1 and the light chain comprises the amino acid sequence set forth in SEQ ID No. 2.

35. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

1) Loading: loading cell culture supernatant of bispecific antibody containing amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 on a pretreated Mabselect prism A affinity chromatography column, wherein the loading capacity is less than or equal to 30 g/L;

b) complex mode chromatography

3) and (3) elution: gradient elution is carried out by using elution buffer solution A and elution buffer solution B, and eluent is collected; wherein elution buffer A comprises about 20mM phosphate, about 20mM arginine, about 10mM citric acid, and a pH of about 6.0; elution buffer B contained about 20mM phosphate, about 20mM arginine, about 10mM citric acid, about 400mM sodium chloride, pH about 6.0,

the antibody has a glycosylation site, wherein the glycosylation site is a characteristic sequence of 'Asn-X1-X2' on the antigen binding fragment, X1 is any amino acid, and X2 is any one of serine, threonine and cysteine.

Definition of terms

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "impurity" refers to a substance that is different from the desired antibody product. Impurities include, but are not limited to: host cell material such as Chinese Hamster Ovary Protein (CHOP); leached protein a; a nucleic acid; a variant, fragment, aggregate or derivative of the desired antibody; other polypeptides; an endotoxin; viral contaminants; cell culture media components.

The term "buffer" refers to a solution that resists changes in pH by the action of its acid-base pair components. A variety of Buffers that may be employed depending on, for example, the desired buffer pH are described in buffers.A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D.Ed.Calbiochem Corporation (1975).

The term "equilibration buffer" refers herein to a buffer used to equilibrate an ion exchange material prior to loading a composition comprising an antibody of interest and one or more contaminants onto the ion exchange material.

The term "elution buffer" refers to a buffer containing a primary substance (a primary species) such as sodium citrate or sodium acetate, which is used to elute a target product, such as an antibody, a protein, etc., from a chromatography column.

The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, current flows by ion transport, and as the amount of ions present in the aqueous solution increases, the solution will have a higher conductivity. The basic units of measurement of conductivity are siemens (or ohm), ohm (mS/cm) and can be measured using a conductivity meter, such as various models of Orion conductivity meters. Because electrolytic conductivity is the ability of ions in a solution to carry current, the conductivity of a solution can be altered by changing the ion concentration therein. For example, the concentration of the buffer and/or the concentration of a salt (e.g., sodium oxide, sodium acetate, or potassium oxide) in the solution can be varied to achieve the desired conductivity.

The term "UV₂₈₀"refers to the absorbance of a sample at a wavelength of 280 nm. Protein molecules usually contain amino acids such as tyrosine, tryptophan, phenylalanine and the like, and have a maximum absorption peak at a wavelength of 280 nanometers, and the absorbance value is in direct proportion to the protein concentration.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, monospecific antibodies, bi-or multispecific antibodies, antibody fragments (e.g., Fab, modified Fab, Fab ', modified Fab ', F (ab ') 2, Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the foregoing), as well as murine, chimeric, humanized, fully human antibodies, and proteins, polypeptides, or any molecules comprising the foregoing, so long as they exhibit the desired antigen binding specificity.

The term "about" means that the numerical value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, which numerical value depends in part on how the value is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or over 1 standard deviation. Alternatively, "about" or "consisting essentially of may mean a range of up to 20%, for example between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 1%, in the present disclosure, every instance where a number or numerical range is preceded by the term" about "also includes a given number of embodiments. Furthermore, particularly for biological systems or processes, the term may mean at most an order of magnitude or at most 5 times the value. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value.

The term "gradient elution" refers to a method of gradually changing the ratio (concentration ratio, composition, ionic strength, solution polarity, etc.) or pH of an eluent according to a certain program along with the change of time in the same analysis period so as to elute different components on a chromatographic column. Therefore, the components with larger property (polarity) difference in a complex sample can achieve good separation according to the respective proper capacity factor k. Gradient elution is generally divided into binary gradient and quaternary gradient, and the method adopts binary gradient elution, namely two kinds of eluents (elution buffer A and elution buffer B) are used for gradient elution of an antibody product.

Drawings

FIG. 1 is an affinity Mabselect prism A chromatography elution profile.

FIG. 2 is a graph showing the SEC-HPLC purity results of the elution fraction of affinity Mabselect prism A chromatography.

FIG. 3 is a graph showing the results of mass spectrometric detection of eluted fractions from affinity Mabselect prism A chromatography.

FIGS. 4A and 4B are the gradient elution profiles of the fine purification step chromatography, wherein FIG. 4A is Capto MMC impress and FIG. 4B is NanoGel SP HP.

Fig. 5A, 5B and 5C are graphs of the detection results of chromatography gradient elution peak mass spectrometry of Capto MMC imprints in complex mode, and fig. 5A, 5B and 5C are graphs of peak 1, peak 2 and peak 3, respectively.

FIGS. 6A and 6B are graphs showing the results of mass spectrometry of elution peaks by cation exchange chromatography NanoGel SP HP chromatography gradient, and FIGS. 6A and 6B are

graphs showing peaks

4 and 5, respectively.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure.

Example 1 cell culture supernatant Capture by affinity chromatography with Primary purification and Complex mode chromatography

The affinity chromatography steps were as follows:

step one, filling affinity chromatography packing Mabselect prism A into a column.

Step two, rinse 3 column volumes with antiseptic solution (0.5M sodium hydroxide).

Step three, wash 3 column volumes with pre-equilibration buffer (20 mM phosphate, 1M sodium chloride, pH 7.4. + -. 0.1).

And step four, flushing 3-5 column volumes with an equilibrium solution (20 mM phosphate, pH7.4 +/-0.1) until the pH and conductivity of the effluent are consistent with those of the equilibrium solution.

And fifthly, loading, wherein the loading capacity is ensured to be less than or equal to 30 g/L.

Step six, washing 3-5 column volumes with an equilibration solution (20 mM phosphate, pH7.4 + -0.1) to UV₂₈₀Stable and the pH and conductivity of the effluent are consistent with those of the equilibrium liquid.

And seventhly, eluting the target protein by using an eluent (50 mM acetate, 0.1M arginine, pH3.5 +/-0.1) and collecting the target protein in sections.

And step eight, collecting an affinity chromatography elution sample, and detecting the SEC-HPLC purity.

The above method was validated using a bispecific antibody of a specific sequence. The full-length sequences of the heavy chain and the light chain of the double antibody are shown as SEQ ID NO:1 and SEQ ID NO:2, respectively.

The results are shown in Table 1, and the monomers are well separated after SEC-HPLC purity is detected; another affinity chromatography elution sample is taken for mass spectrum detection, the result is shown in figure 3, and the elution sample contains unexpected glycosylation impurities.

TABLE 1 affinity Mabselect prism A chromatography collection fraction SEC-HPLC purity

And (3) taking an affinity chromatography elution sample, adjusting the pH to 6.0 +/-0.1 by using 1mol/L Tris, and diluting the combined sample to the conductivity of less than 6 mS/cm by using ultrapure water.

The complex mode chromatography procedure was as follows:

step one, filling a composite mode chromatography filler Capto MMC Impres into a column.

And step four, flushing 3-5 column volumes with an equilibrium solution (20 mM phosphate, 20mM arginine, 10mM citric acid, pH 6.0) until the effluent pH and conductivity are consistent with those of the equilibrium solution.

Step six, wash 3-5 column volumes with equilibration solution (20 mM phosphate, 20mM arginine, 10mM citric acid, pH 6.0) to UV₂₈₀Stable and the pH and conductivity of the effluent are consistent with those of the equilibrium liquid.

Seventhly, placing an elution buffer solution A (20 mM phosphate, 20mM arginine, 10mM citric acid and pH 6.0) in a system A pump, placing an elution buffer solution B (20 mM phosphate, 20mM arginine, 10mM citric acid, 400mM sodium chloride and pH 6.0) in a system B pump, carrying out gradient elution on the target protein by 0-100% B/15CV, maintaining 3CV by 100% B after the gradient elution is finished, and collecting an elution sample.

And step eight, carrying out gradient elution by composite mode chromatography, and taking an elution peak for mass spectrum detection.

Validation of the above described complex mode chromatography method was performed using affinity chromatography elution samples of the double antibody of SEQ ID NO 1 and 2 of example 1. The spectrum is shown in fig. 4A, and the results of mass spectrometric detection of

elution peaks

1, 2, and 3 are shown in fig. 5A, 5B, and 5C, which indicates that peaks 1 and 2 both contain undesired glycosylation impurities, but no impurity is detected in peak 3. Therefore, the target product is better separated through purification. The recovery of the target product was 49.1% and the SEC purity was >99% (both greater than 99% collected in tubes).

Example 2 cation exchange chromatography

And (3) taking an affinity chromatography elution sample, adjusting the pH to 5.0 +/-0.1 by using 1mol/L Tris, and diluting the combined sample to the conductivity of less than 6 mS/cm by using ultrapure water.

The cation exchange chromatography steps were as follows:

step one, filling cation exchange chromatography filler NanoGel SP HP into a column.

Step three, wash 3 column volumes with pre-equilibration buffer (50 mM acetate, 1M sodium chloride, ph5.0 ± 0.1).

And step four, flushing 3-5 column volumes with an equilibrium solution (50 mM acetate, pH5.0 +/-0.1) until the pH and conductivity of the effluent are consistent with those of the equilibrium solution.

Step six, washing 3-5 column volumes with equilibration solution (50 mM acetate, pH5.0 + -0.1) to UV₂₈₀Stable and the pH and conductivity of the effluent are consistent with those of the equilibrium liquid.

Seventhly, placing an elution buffer solution-A (50 mM acetate, pH5.0 +/-0.1) in a system A pump, placing an elution buffer solution-B (50 mM acetate, 400mM sodium chloride, pH5.0 +/-0.1) in a system B pump, eluting the target protein by a 0-100% B/15CV gradient, maintaining 3CV by 100% B, and collecting an elution sample.

And step eight, performing gradient elution by cation exchange chromatography, and taking an elution peak for mass spectrum detection.

Validation of the cation exchange chromatography method described above was performed using affinity chromatography elution samples of the diabodies A of SEQ ID NO 1 and 2 of example 1. The spectrum is shown in fig. 4B, and the results are shown in fig. 6A and 6B, indicating that both peak 4 and peak 5 contain undesired glycosylation impurities. It is known that cation exchange chromatography cannot effectively remove undesired glycosylation impurities, and thus a target product cannot be obtained, and the recovery rate is 0.

Also, we used the method to determine the heavy and light chains as SEQ ID NO: 9 and SEQ ID NO: 10 was verified.

The sequence of the present disclosure:

the full-length amino acid sequence of a heavy chain of a double antibody A:

EVQLVESGGGLVQPGGSLRLSCAASTYTFKNKCMGWFRQAPGKEREGVSVVDRFGGTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGSYTSANSCQPDALWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQRLEWMGVINPYNGDTAYNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDDGYYDYYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：1

full-length amino acid sequence of > diabody a light chain:

DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWYQQKPGKAPKRLIYATSTLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQLASSPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：2

>HCDR1

NYDIN

SEQ ID NO：3

>HCDR2

WIFPGDGSTQYNETFKG

SEQ ID NO：4

>HCDR3

QTTGTWFAY

SEQ ID NO：5

>LCDR1

RASQSISKYLH

SEQ ID NO：6

>LCDR2

YANQSIR

SEQ ID NO：7

>LCDR3

QNGHSFPLT

SEQ ID NO：8

full-length amino acid sequence of antibody B heavy chain:

QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIFPGDGSTQYNETFKGRATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：9

full-length amino acid sequence of antibody B light chain:

DIVLTQSPATLSLSPGERATLSCRASQSISKYLHWYQQKPGQAPRLLIKYANQSIRGIPARFSGSGSGSDFTLTISSLEPEDFAVYYCQNGHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：10

example 3 biological functional verification of diabody A

The dual anti-A is a bispecific antibody targeting PD-1 and LAG-3.

1. Affinity identification

Dissociation constants of diabody A and human PD-1 protein or human LAG-3 protein were determined using a Biacore T200 (GE Healthcare) instrument. Covalently coupling protein A (elegant RSPA 05) to a CM 5S series chip, capturing an antibody to be detected to the surface of the chip through affinity, flowing human PD-1 protein (Sino Biological Cat. 10377-H08H) or human LAG-3 protein with different concentrations on the surface of the chip, detecting a reaction signal in real time to obtain a binding dissociation curve, and obtaining a binding dissociation curve through fittingThe binding force constant is obtained. The solution used for the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chips were washed clean and regenerated with 10mM Glycein, pH =1.5 (GE, BR-1003-54). Through detection, the affinity K of the double-antibody A and the human PD-1 protein_DA value of about 1.5E-07M, affinity K for human LAG-3 protein_DThe value is about 1.3E-07M.

2. The double-antibody A blocks the binding of PD-1 and PD-L1 on cells, blocks the binding of LAG-3 and MHCII high-expression A735

Cloning the human PD-1 full-length gene onto a mammalian cell expression vector pTargeT by PCR, taking a linear plasmid to electrically transfect CHO-S cells (preset CHO cell parameters carried by an electrotransfer), screening for 2 weeks by 1mg/mL G418, carrying out limited dilution for 2 times, detecting the PD-1 gene on the cell surface by a flow cytometry analyzer, and selecting a monoclonal cell strain high expression human PD-1. Was named CHO-PD-1.

Collecting stable high expression PD-1 cell line CHO-PD-1, each well at 5X 10⁵A cell. The gradient dilution of double antibody A is 33.33 ug/mL, 11.11 ug/mL, 3.7 ug/mL, 1.23 ug/mL, 0.41 ug/mL, 0.13ug/mL, each well is added with 50uL, and the mixture is incubated with CHO-PD-1 in the dark for 1 hour. For detection, cells were resuspended, and each tube was incubated with 1. mu.g/mL ice of PD-L1-mIgG2a protein for 1 hour, and washed again with PBS. After washing, each tube was incubated with PE anti-mouse IgG2a (1:300) on ice for 1 hour, rinsed once with PBS, resuspended in 100uL of PBS per tube, and subjected to fluorescence detection on a BD C6 Plus flow cytometer. The mean fluorescence intensities from each dose treatment of the antibody were curve fitted and plotted using Graphpad Prism9 software to quantify PD-1 binding to PD-L1 on the diabody a-blocked cells. The results show that the double anti-A can block the binding of the PD-L1 protein to CHO-PD-1 cells, and the antibody dose dependence is shown. IC of double-antibody A for blocking PD-L1 protein and cell surface antigen PD-1₅₀About 1.8 ug/mL.

Collecting stable and high expression LAG-3 cell line CHO-LAG-3, adjusting cell density to 6 × 10 with PBS⁶Per mL, 50uL was added per well. The concentration of the double antibody A in the dilution gradient was 111.1 nM, 37.04 nM, 12.3 nM, 4.12 nM, 1.37 nM, 0.46 nM, 50 per welluL. CHO-LAG-3 cells were incubated on ice for 1 hour. For detection, cells were resuspended, washed once with PBS, and FITC anti-human IgG (1:100) was added to each tube and incubated for 1 hour on ice in the absence of light. After one more PBS rinse, 100uL PBS per tube was resuspended and fluorescence detected on a BD C6 Plus flow cytometer. The mean fluorescence intensities from each dose treatment of the antibody were curve fitted and plotted using Graphpad Prism9 software to quantify the binding of diabody a to CHO-LAG-3 cells. The results show that the binding strength of the diabody A to CHO-LAG-3 cells is antibody dose-dependent and the negative control used is NC. IC of dual-antibody blocking binding of LAG-3 protein and MHCII on cell surface₅₀Approximately 110 nM.

3. Dual anti-A inhibition of tumor growth in humanized animal models

NOG mice (female, 6-8 weeks old, purchased from Beijing Wittingle laboratory animals technologies, Inc.) were acclimatized for one week. The test day was numbered and weighed, and the human melanoma cell line A375 in logarithmic growth phase was collected and inoculated at 6X 10 per animal⁶The dose per cell (100uL) was inoculated subcutaneously in the right flank of the mouse. Resuscitating human PBMC on the same day at 37 deg.C with 5% CO₂After culturing in the incubator for 1 hour, the cells were rinsed with PBS and adjusted to a cell density of 1X 10⁷mL, 5X 10 inoculate per animal⁶The tail vein injection was performed at a dose per cell (500 uL). 5-7 days after inoculation, when the tumor grows to 50-90mm³Thereafter, body weight, over-or under-sized tumors were removed, randomly grouped, and the corresponding antibodies were intraperitoneally injected twice a week for 6 total administrations. Mouse body weight and tumor volume were measured 2 times per week. Tumor volume calculation formula is TV = L_{Long and long}× L_{Short length} ²/2. Tumor volumes of each group were represented by mean. + -. standard deviation, statistical analysis was performed using Two-way ANOVA, and tumor inhibition ratio (% TGI) was calculated as% TGI = [1- (T-T0)/(C-C0)]X 100%. At the end of the experiment, blood was taken, erythrocytes (BD 555899) were lysed, and after incubation for 40 minutes in a mixture with monoclonal antibodies targeting human and murine CD45 (eBioscience ™ 11-0451-82, 12-9459-42), human CD45 positive cells in the mouse blood were detected as all lymphocytes by a BD C6 Plus flow cytometerAs a percentage of human PBMC reconstitution levels in this mouse model. The results are shown in Table 2.

TABLE 2 PD-1/LAG-3 double antibody dosing regimen and mouse model tumor suppression effect

(Note: significant difference in tumor size at day 31 compared to the PBS group in Two-way ANOVA statistical analysis, with P <0.0001, marked as:.)

SEQUENCE LISTING

<110> Shanghai Shengdi pharmaceutical Co., Ltd

Shanghai Mijin biological medicine science and technology Limited

JIANGSU HENGRUI MEDICINE Co.,Ltd.

<120> a method for purifying an antibody

<130> P21015378CF

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<170> PatentIn version 3.5

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Tyr Thr Phe Lys Asn Lys

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Val Val Asp Arg Phe Gly Gly Thr Ile Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Gly Ser Tyr Thr Ser Ala Asn Ser Cys Gln Pro Asp Ala Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

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Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

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Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met

165 170 175

Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Val

180 185 190

Ile Asn Pro Tyr Asn Gly Asp Thr Ala Tyr Asn Gln Lys Phe Lys Gly

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Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu

210 215 220

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Asp Asp Gly Tyr Tyr Asp Tyr Tyr Phe Asp Val Trp Gly Gln Gly Thr

245 250 255

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

260 265 270

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

275 280 285

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

290 295 300

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

305 310 315 320

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

325 330 335

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

340 345 350

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

355 360 365

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

370 375 380

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

385 390 395 400

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

405 410 415

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

420 425 430

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

435 440 445

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

450 455 460

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

465 470 475 480

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

485 490 495

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

500 505 510

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

515 520 525

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

530 535 540

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

545 550 555 560

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

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His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg

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Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

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Tyr Ala Thr Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Leu Ala Ser Ser Pro Pro

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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

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Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

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Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

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Phe Asn Arg Gly Glu Cys

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Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Thr Phe Lys

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Gly

<210> 5

<211> 9

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Gln Thr Thr Gly Thr Trp Phe Ala Tyr

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<210> 6

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Arg Ala Ser Gln Ser Ile Ser Lys Tyr Leu His

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Tyr Ala Asn Gln Ser Ile Arg

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Gln Asn Gly His Ser Phe Pro Leu Thr

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

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Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Thr Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gln Thr Thr Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

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Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Lys Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Asn Gln Ser Ile Arg Gly Ile Pro Ala Arg Phe Ser Gly

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Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

b) complex mode chromatography

wherein the elution buffer of step a), the equilibration buffer of step b), and the elution buffer each comprise arginine, and the concentration of arginine in the elution buffer of step a) is about 100 mM; the concentration of arginine in the equilibration buffer and elution buffer of step b) is about 20mM, and the antibody has a glycosylation site which is a characteristic sequence of "Asn-X1-X2" on the antigen binding fragment, wherein X1 is any amino acid, and X2 is any one of serine, threonine and cysteine; the heavy chain amino acid sequence of the antibody is the amino acid sequence shown as SEQ ID NO. 1, and the light chain amino acid sequence is the amino acid sequence shown as SEQ ID NO. 2; the "about" indicates a margin of error of at most 20%.

2. The method of claim 1, wherein:

the elution buffer of step B) comprises an elution buffer A and an elution buffer B, wherein the elution buffer A and the elution buffer B both contain about 20mM phosphate, about 20mM arginine and about 10mM citric acid, the pH value is about 6.0, and the elution buffer B contains 400mM sodium chloride; the "about" indicates a margin of error of at most 20%.

3. The method of claim 2, step b) adjusting the pH of the elution product of the affinity chromatography to about 6.0 using 1M Tris and diluting with deionized water to a conductivity <6mS before loading; the "about" indicates a margin of error of at most 20%.

4. The method of claim 3, wherein step a) the affinity chromatography packing is MabSelect prism A and step b) the composite mode chromatography packing is Capto MMC Impres.

5. A method of purifying an antibody comprising the steps of:

a) affinity chromatography

b) complex mode chromatography

the antibody has a glycosylation site, wherein the glycosylation site is a characteristic sequence of 'Asn-X1-X2' on the antigen binding fragment, X1 is any amino acid, and X2 is any one of serine, threonine and cysteine; the "about" indicates a margin of error of at most 20%.