CN110272491B

CN110272491B - Purification process of anti-PD-1 antibody

Info

Publication number: CN110272491B
Application number: CN201910182809.3A
Authority: CN
Inventors: 刘宇鹏; 石瑞君; 刘洵
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Priority date: 2018-03-13
Filing date: 2019-03-12
Publication date: 2023-01-24
Anticipated expiration: 2039-03-12
Also published as: CN110272491A

Abstract

The invention relates to a purification process of an anti-PD-1 antibody. Specifically, the purification process includes removing contaminants using methods such as affinity chromatography, viral inactivation, anion membrane chromatography, and cation exchange chromatography. The process can reduce production cost and improve antibody yield.

Description

Purification process of anti-PD-1 antibody

Technical Field

The invention relates to the field of antibody purification, in particular to a purification process of an anti-PD-1 antibody, which comprises the steps of removing pollutants by using methods such as affinity chromatography, virus inactivation, anion membrane chromatography, cation exchange chromatography and the like.

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 pollutants 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 process for purifying the antibody comprises a plurality of steps, currently, chromatographic separation methods such as affinity chromatography, cation exchange chromatography, anion exchange chromatography and the like are commonly used, and the purification effect is influenced by different specific parameters and different filler selections in each separation method.

Affinity chromatography is a separation and purification technique established by utilizing the characteristic that most macromolecular substances in organisms have reversible binding with certain corresponding molecules in a transferability way. The method is suitable for purifying the target object from the mixture with complex components and high impurity content, can separate the active target object from the inactive target object according to the biological function of the target object, and has the characteristics of specificity, high efficiency and the like. Generally, the product after affinity chromatography can reach the purity of more than 80 percent, and simultaneously, higher recovery rate can be ensured. However, antibody drugs not only need to ensure monomer purity, but also need to minimize the residues of host proteins and DNA, and therefore, require subsequent purification steps.

Ion Exchange Chromatography (Ion Exchange Chromatography abbreviated as IEC) is a purification method commonly used in the field of biochemistry at present. The ion exchange chromatography is a chromatography method which takes an ion exchanger as a stationary phase and achieves the separation purpose by reversibly exchanging component ions in a mobile phase with counter ions on the exchanger. By ion exchange is meant a process in which one type of ion in solution is reversibly exchanged with another type of ion bound to a support, i.e. the ions in solution are bound to the support and the ions on the support are replaced. If the carrier is combined with active groups with positive charges, the carrier can exchange anions and is an anion exchanger; if a negatively charged active group is bound to the support, the cation can be exchanged, which is a cation exchanger. Ion exchange chromatography is widely applied to separation and purification of various antibodies such as rituximab, trastuzumab, adalimumab and the like.

The anti-PD-1 antibody is a class of antibodies which are currently spotlighted, a plurality of cross-country pharmaceutical enterprises are developing all over the world, nivolumab of Shi Gui Bao pharmaceutical company and pembrolizumab of Mussando company are on the market in 2014, and the sales volume breaks through billion dollars after the antibodies are on the market. WO2017054646A discloses a sequence of an anti-PD-1 antibody, the anti-PD-1 antibody is in Clinical stage III, the safety is good, and reported Clinical research results show that the anti-PD-1 antibody has a certain anti-tumor effect (J, journal of Clinical Oncology 35 (2017): e15572-e 15572).

Because the antibody has large molecular weight and complex structure, the difference between different antibodies is large, and the binding force between different antibodies and the ion exchanger is not only related to the number of charges carried by the antibodies, but also has a certain relation with the size of the molecular weight of the antibodies, the charge arrangement and the like. Therefore, it is a technical problem to be urgently solved at present to optimize a suitable ion chromatography purification process for different antibodies, reduce the production cost as much as possible and improve the antibody yield.

Disclosure of Invention

The present invention provides a method for purifying a composition comprising an antibody and a contaminant using cation chromatography comprising the steps of:

1) Loading: loading the composition onto a cation exchange material;

2) Cleaning: washing the cation exchange material with an equilibration buffer;

3) And (3) elution: eluting the target antibody from the cation exchange material with an elution buffer;

4) Diluting: collecting the eluent and diluting.

In one embodiment of the invention, wherein the loading composition has a pH of 4.5 to 5.5, preferably a pH of 4.8 to 5.2, most preferably a pH of 5.0.

In one embodiment of the invention, wherein the conductivity of the loading composition is < 5mS.

In one embodiment of the invention, wherein the buffer substance in the equilibration buffer is MES, citric acid, phosphoric acid, citrate or phosphate, preferably citric acid.

In one embodiment of the invention, wherein the concentration of the equilibration buffer is between 10 and 30mM, preferably 20mM; the pH of the equilibration buffer is between 4.5 and 5.5, preferably between 4.8 and 5.2.

In one embodiment of the invention, wherein the buffer substance in the elution buffer is a mixture of one or more of MES, citric acid, phosphoric acid, citrate or phosphate at a concentration of 10-30mM, preferably 20mM; the elution buffer also contains one or more salts selected from potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, and phosphate, and has a concentration of 100-250mM, preferably 150-200mM, and most preferably 180mM.

In one embodiment of the invention, wherein the conductivity of the elution buffer is between 10 and 25mS, preferably between 18 and 22mS.

In one embodiment of the invention, wherein the elution buffer has a pH of 4.5-5.5, preferably a pH of 4.8-5.2, most preferably a pH of 5.0.

In one embodiment of the invention, wherein the dilution is performed by adding water or buffer to the elution pool, the conductivity of the solution after dilution is less than 10mS.

The purification method further comprises the following steps before cation exchange chromatography:

a) Affinity chromatography;

b) Inactivating viruses;

c) Anion membrane chromatography.

In one embodiment of the invention, wherein step a) affinity chromatography is performed before loading by washing with an equilibration buffer solution, the equilibration buffer solution is a mixed solution of phosphate buffer and sodium chloride, and the pH value is 7.0-8.0.

In one embodiment of the invention, wherein the elution buffer for affinity chromatography in step a) is a citric acid buffer at a concentration of 40-60nM and a pH of 2.0-4.0, preferably a pH of 2.8-3.2.

In one embodiment of the invention, wherein step b) adjusts the pH of the elution solution of affinity chromatography to a value of 3.2-4.0, preferably 3.6-3.8, virus inactivation is performed.

In one embodiment of the present invention, the anion membrane chromatography in step c) is washed with a washing buffer solution before loading, wherein the washing buffer solution is a mixed solution of phosphate buffer solution and sodium chloride, and the pH value is 7.0-8.0.

In one embodiment of the present invention, wherein the equilibration buffer for anionic membrane chromatography in step c) is a citrate buffer, the concentration is 10-30nM and the pH is 4.0-6.0.

In one embodiment of the invention, the cation exchange chromatography is followed by virus-removing filtration and ultrafiltration.

In one embodiment of the present invention, wherein the antibody is selected from the group consisting of an anti-CD 20 antibody, an anti-VEGFR antibody and an anti-PD-1 antibody, preferably an anti-PD-1 antibody.

In one embodiment of the present invention, wherein the light chain variable region of the anti-PD-1 antibody or an antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively.

Wherein, the CDR sequences are shown in the following table:

name (R)	Sequence of	Number of
			HCDR1	SYMMS	SEQID NO：1
HCDR2	TISGGGANTYYPDSVKG	SEQID NO：2
			HCDR3	QLYYFDY	SEQID NO：3
LCDR1	LASQTIGTWLT	SEQID NO：4
			LCDR2	TATSLAD	SEQID NO：5
LCDR3	QQVYSIPWT	SEQID NO：6

Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 humanized antibody.

Preferably, the humanized antibody light chain variable region sequence is as shown in SEQ ID NO 10 or a variant thereof; the variant preferably has 0-10 amino acid changes in the light chain variable region; more preferably, the amino acid sequence of A43S is changed. The humanized antibody heavy chain variable region sequence is shown as SEQ ID NO. 9 or the variant thereof; the variant preferably has 0-10 amino acid changes in the heavy chain variable region; more preferably the amino acid of G44R.

The sequences of the variable regions of the heavy and light chains of the humanized antibodies are shown below:

heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS

SEQID NO：9

Light chain variable region

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

SEQID NO：10

Preferably, the humanized antibody light chain sequence is as shown in SEQ ID NO. 8 or a variant thereof; the variant preferably has 0-10 amino acid changes in the light chain variable region; more preferably, the amino acid sequence of A43S is changed. The humanized antibody heavy chain sequence is shown as SEQ ID NO. 7 or a variant thereof; the variant preferably has 0-10 amino acid changes in the heavy chain variable region; more preferably the amino acid of G44R.

In one embodiment of the invention, the humanized antibody light chain sequence is as shown in SEQ ID NO. 8 and the heavy chain sequence is as shown in SEQ ID NO. 7.

The sequences of the heavy and light chains of the humanized antibodies are shown below:

heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQID NO：7

Light chain

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQID NO：8

The invention also provides a composition comprising an antibody obtained by any one of the methods above and a buffer.

Description of the drawings:

FIG. 1: cationic Millipore Fractogel EMD SO ₃ (M) gradient chromatogram

FIG. 2 is a schematic diagram: cationic Millipore Fractogel EMD SO ₃ (M) gradient chromatography component SEC analysis

FIG. 3: cationic Millipore Fractogel EMD SO ₃ (M) staged chromatography component SEC analysis

Detailed Description

1. Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise elsewhere in this document, 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 invention belongs.

The term "contaminant" refers to a substance that is different from the desired antibody product. Contaminants 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. Buffers A Guide for the Preparation and Use of Buffers in Biological Systems, gueffroy, D., ed. Calbiochem Corporation (1975) describes a variety of Buffers that may be employed depending, for example, on the desired buffer pH.

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 "wash buffer" refers herein to a buffer that flows through the ion exchange material after loading the composition and prior to eluting the protein of interest. The wash buffer can be used to remove one or more contaminants from the ion exchange material without substantially eluting the desired antibody product.

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 "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired binding specificity.

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the strong antibody variable antibody reaction induced by the chimeric antibody because of carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/VBase), and in Kabat, E.A. et al, 1991 Sequences of Proteins of Immunological Interest, 5 th edition. In one embodiment of the invention, the CDR sequences of the humanized antibody PD-1 are selected from the group consisting of SEQ ID NO 1,2,3,4,5,6.

The term "antigen-binding fragment" refers to Fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv fragments sFv fragments that bind to human PD-1, having antigen-binding activity; comprising one or more CDR regions of an antibody of the invention selected from SEQ ID NO 1 to SEQ ID NO 6. The Fv fragment contains the variable regions of the antibody heavy and light chains, but lacks the constant region, and has the smallest antibody fragment with the entire antigen-binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined together with different linkers into a single polypeptide chain, known as single chain antibodies (scFv) or single chain Fv (sFv). The term "binds to PD-1" in the context of the present invention means capable of interacting with human PD-1. The term "antigen binding site" of the present invention refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen binding fragment of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Example 1 anti-PD-1 antibody cell culture depth filtrate was captured by affinity chromatography and initially purified

The sequences of the heavy and light chains of the anti-PD-1 antibody in this example are shown in SEQ ID NO:7 and SEQ ID NO: as shown in FIG. 8, the affinity chromatography procedure was as follows:

step one, packing the affinity chromatography packing Prosep Ultra Plus (Millipore) into the column. Washed with a disinfectant (150 mM phosphoric acid) and left to stand. Washing with balance buffer solution (20 mM phosphate buffer solution +1M sodium chloride pH7.4 + -0.1), collecting column-passing solution for detecting endotoxin with limit less than 0.25EU/mL, loading sample if it is qualified, and sterilizing and balancing if it is not qualified.

And step two, sampling in 3 cycles to ensure that the loading capacity is less than or equal to 40g/L.

Step three, washing with an equilibrium buffer (20 mM phosphate buffer solution +1M sodium chloride pH7.4 + -0.1).

And step four, washing with a washing mixed buffer (20 mM phosphate buffer solution, pH7.4 +/-0.1).

And step five, eluting the target protein by using an elution buffer (50 mM citric acid buffer solution pH3.0 +/-0.1).

And sixthly, sampling the affinity chromatography collection liquid to detect the protein concentration, calculating the recovery rate in the step, and sending the sample to QC (quality control) for detecting the SEC-HPLC purity. The results are given in the following table:

TABLE 1

Sample loading amount (mg)	Recovery volume (mg)	Recovery (%)	SEC-HPLC purity (%)
				1800	1710	95	91.6

And (4) conclusion: the cell culture broth was subjected to Millipore PUP affinity chromatography at 40mg/ml loading with 95% recovery and SEC-HPLC purity 91.6%.

Example 2 Low pH viral inactivation

Collecting eluate, dividing the collected solution into three groups for determining virus inactivation pH, adjusting pH to 5.0 with 1.0M Tris after 1.5 hr low pH incubation in the second and third groups, and performing SEC detection on the sample with pH adjusted to 5.0 without low pH incubation in the first group, wherein the results are as follows:

TABLE 2

The results in the table show that the SEC of the anti-PD-1 antibody is unstable under the condition of lower pH, the effect of pH and treatment time on virus inactivation is comprehensively considered, and finally, 1M Tris or 1M citric acid is used for adjusting the pH of the affinity chromatography collection liquid to be 3.6-3.8, and the virus inactivation is carried out by standing for 90-95min under the condition of 18-26 ℃. After inactivation, 1M Tris was added to adjust the pH of the sample to 5.0. + -. 0.1, and the results of virus removal are shown in the following table:

TABLE 3

And (4) conclusion: the experimental results show that the reduction (log 10) of both viruses is more than 4logs, which indicates that the virus inactivation is effective in the step.

Example 3 anion Membrane chromatography

After low pH virus inactivation and adsorption deep filtration, the affinity chromatography collection liquid is subjected to anion membrane chromatography, and the method comprises the following steps:

step one, the anion chromatographic membrane is a Sartobind Q (Sadolis) chromatographic membrane, and is washed by disinfectant (1M NaOH).

Step two, washing with a washing buffer (20 mM phosphate buffer +1M sodium chloride, pH7.4 + -0.1).

And step three, washing with an equilibrium buffer solution (20 mM citric acid buffer solution, pH5.0 +/-0.1), taking a membrane-passing solution to detect endotoxin, wherein the limit is less than 0.25EU/mL, and loading the qualified solution if the endotoxin is detected, and re-disinfecting and balancing if the endotoxin is not detected.

And step four, confirming that the pH value of the sample loading solution is 5.0 +/-0.1, the conductance is less than 5mS/cm, carrying out sample loading, and collecting the flow-through solution according to UV 280.

And step five, after the sample loading is finished, top washing is carried out by using an equilibrium buffer solution, the flow-through solution is continuously collected according to UV280, and the flow-through solution is sent to QC (quasi-cyclic) for detecting the removal effect of HCP. The results are given in the following table:

TABLE 4

	Concentration (mg/ml)	SEC(％)	HCP(ppm)	HCP removal (%)
					Before loading	12.2	90.3	1247	N/A
Flow-through liquid	12.1	90.3	664	46.7

And (4) conclusion: the purity of the affinity chromatography sample is not obviously changed after the anion membrane flow-through mode chromatography, and the HCP removal rate reaches 46.7 percent.

Example 4 cationic chromatography

After anion membrane chromatography, the collected liquid is further purified by cation chromatography, and the steps are as follows:

step one, adding cation chromatography filler Fractogel EMD SO ₃ ^- (M) (Millipore) was packed into the column. Rinsing with disinfectant (1M NaOH), and standing. Washing with balance buffer solution (20 mM citric acid buffer solution, pH5.0 + -0.1), collecting column-passing solution for detecting endotoxin with limit less than 0.25EU/mL, loading sample when the detection is qualified, and sterilizing and balancing if the detection is not qualified.

And step two, confirming that the pH value of the sample liquid is 5.0 +/-0.1 and the conductance is less than 5mS, and carrying out sample loading.

And step three, washing by using an equilibrium buffer solution (20 mM citric acid buffer solution, pH5.0 +/-0.1).

Step four, eluting the target protein by using an elution buffer solution of 20mM citric acid buffer solution, pH5.0 and 20mM citric acid buffer solution +200mM NaCl and pH5.0 in a gradient manner, collecting the sample in tubes for SEC detection, wherein the chromatogram is shown in figure 1, and the SEC result of each component of the collected sample is shown in figure 2.

It can be seen from the figure that in the 20mM citrate buffer, pH5.0 system, the SEC of the anti-PD-1 antibody reached a maximum of 97.5% at a salt concentration of 180mM NaCl, while impurities were contained before the peak UV280 rose to 1000mAU, which was discarded technically. The fixed elution buffer was 20mM citrate buffer +180mM NaCl, pH 5.0. The SEC results for each component of the collected sample are shown in fig. 3.

Example 5 stability of anti-PD-1 antibodies

Packing material Fractogel EMD SO by cationic chromatography ₃ ^- The purity of the anti-PD-1 antibody subjected to (M) (Millipore) chromatography is obviously improved. The purified sample is in a system with pH of 5.0 and conductance of about 20ms/cm, fibrous precipitates are visually formed in a short time, and the precipitates are still separated after 0.22 mu m filtration.

Because the PD-1 antibody obtained after cation chromatography can generate fibrous precipitates, an experiment is designed to detect the salt stability of the anti-PD-1 antibody, and the following operations are carried out: the affinity elution sample, ph5.0, was titrated with 2M NaCl, conductance was measured with a conductivity meter and recorded, and the sample clarity was checked by light, with the results given in the following table:

TABLE 5

Sample (I)	Conductance (ms/cm)	Clarity of reaction
			Initial sample	3.81	Clarification
1	7.05	A small amount of fibrous precipitate
			2	9.62	Fibrous precipitate
3	12.35	Fibrous precipitate
			4	15.23	Fibrous precipitate
5	19.50	Fibrous precipitate
			6	24.6	Fibrous precipitate
7	28.8	Fibrous precipitate

As can be seen from Table 5, the anti-PD-1 antibody has better stability under the condition of low salt (3.81 ms/cm of electric conductance) of an initial sample at pH5.0, and as the salt concentration increases, the electric conductance gradually increases, and fibrous precipitation does not appear gradually, and as the salt concentration increases, the phenomenon of fibrous precipitation redissolution is not shown, thus proving that the salt stability of the anti-PD-1 antibody is weaker. The elution sample in the cation chromatography process is directly connected into prepared water for injection for dilution, the salt concentration in the system is reduced, and the effect of controlling fibrous precipitation is achieved, the data of the sample diluted by the water for injection according to different proportions is shown in a table 6, and the sample basically does not produce precipitation when the conductivity is less than or equal to 10 ms/cm.

TABLE 6

Sample numbering	Water for injection (ml)	Sample (ml)	Conductance (ms/cm)	Clarity of the product
					A1	1	1	9.65	A small amount of fibrous precipitate
A2	2	1	6.48	A small amount of fibrous precipitate
					A3	3	1	4.77	Clarification
A4	4	1	4.08	Clarification
					A5	5	1	3.23	Clarification

Sequence listing

<110> Hengrui pharmaceutical Co., ltd of Jiangsu

Shanghai Hengrui pharmaceutical Co., ltd

Suzhou Shengdiya biomedical Co Ltd

<120> purification process of anti-PD-1 antibody

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

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

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

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

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Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

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

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

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

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

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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

<210> 9

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(116)

<223> heavy chain variable region

<400> 9

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 Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> light chain variable region

<400> 10

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

1 5 10 15

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

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

Claims

1. A method for purifying a composition comprising an antibody and a contaminant using cation chromatography, comprising the steps of:

1) Sampling: loading the composition onto a cation exchange material;

2) Cleaning: washing the cation exchange material with an equilibration buffer;

3) And (3) elution: eluting the target antibody from the cation exchange material with an elution buffer, wherein the conductivity of the elution buffer is 18-22mS/cm;

4) Diluting: collecting the eluent, diluting with water for injection, and making the diluted solution have a conductivity less than 10mS/cm;

wherein the antibody is an anti-PD-1 humanized antibody, and the light chain sequence thereof is the sequence shown as SEQ ID NO. 8, and the heavy chain sequence thereof is the sequence shown as SEQ ID NO. 7.

2. The method of claim 1, wherein the loading composition has a pH of 4.5 to 5.5.

3. The method of claim 2, wherein the loading composition has a pH of 4.8-5.2.

4. The method of claim 2, wherein the loading composition has a pH of 5.0.

5. The method of claim 1, wherein the conductivity of the loading composition is < 5mS/cm.

6. The method of claim 1, wherein the buffer substance in the equilibration buffer is MES, citric acid, phosphoric acid, citrate, or phosphate.

7. The method of claim 6, wherein the buffer substance in the equilibration buffer is citric acid.

8. The method according to claim 1, wherein the concentration of the equilibration buffer is between 10 and 30mM and the pH of the equilibration buffer is between 4.5 and 5.5.

9. The method of claim 8, wherein the concentration of equilibration buffer is 20mM; the pH of the equilibration buffer is 4.8-5.2.

10. The method of claim 1, wherein the buffer substance in the elution buffer is a mixture of one or more of MES, citric acid, phosphoric acid, citrate or phosphate at a concentration of 10-30mM; the elution buffer solution also contains one or more salts of potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate and sodium sulfate, and the concentration is 100-250mM.

11. The method of claim 10, wherein the buffer substance in the elution buffer is a mixture of one or more of MES, citric acid, phosphoric acid, citrate or phosphate at a concentration of 20mM; the elution buffer solution also contains one or more salts of potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate and sodium sulfate, and the concentration is 150-200mM.

12. The method of claim 10, wherein the buffer substance in the elution buffer is a mixture of one or more of MES, citric acid, phosphoric acid, citrate or phosphate at a concentration of 20mM; the elution buffer solution also contains one or more salts of potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate and sodium sulfate, and the concentration is 180mM.

13. The method according to any one of claims 10-12, wherein the pH of the elution buffer is between 4.5 and 5.5.

14. The method of claim 13, wherein the pH of the elution buffer is 4.8-5.2.

15. The method of claim 13, wherein the elution buffer has a pH of 5.0.

16. The method of claim 1, further comprising, prior to cation exchange chromatography, the steps of:

a) Affinity chromatography;

b) Inactivating viruses;

c) Anion membrane chromatography.

17. The method of claim 16, wherein the affinity chromatography of step a) is performed by washing with an equilibration buffer solution before loading, wherein the equilibration buffer solution is a mixture of phosphate buffer and sodium chloride, and the pH value is 7.0-8.0.

18. The method according to claim 16, wherein the elution buffer for affinity chromatography in step a) is a citrate buffer at a concentration of 40-60nM and a pH of 2.0-4.0.

19. The method according to claim 18, wherein the elution buffer for affinity chromatography in step a) is a citrate buffer at a concentration of 40-60nM and a pH of 2.8-3.2.

20. The method of claim 16, wherein step b) adjusts the pH of the affinity chromatography eluate to 3.2-4.0 for virus inactivation.

21. The method of claim 20, wherein step b) adjusts the pH of the affinity chromatography eluate to 3.6-3.8 for virus inactivation.

22. The method according to claim 16, wherein the anionic membrane chromatography of step c) is performed by washing with a washing buffer solution before loading, wherein the washing buffer solution is a mixed solution of phosphate buffer solution and sodium chloride, and the pH value is 7.0-8.0.

23. The method according to claim 16, wherein the equilibration buffer for anionic membrane chromatography in step c) is a citrate buffer at a concentration of 10-30nM and a pH of 4.0-6.0.

24. The method of claim 1, further comprising the steps of virus removal filtration and ultrafiltration after the cation exchange chromatography.