CN110272491A

CN110272491A - A kind of purifying process of anti-PD-1 antibody

Info

Publication number: CN110272491A
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: 2019-09-24
Anticipated expiration: 2039-03-12
Also published as: CN110272491B

Abstract

The present invention relates to a kind of purifying process of anti-PD-1 antibody.Specifically, the purifying process includes using the methods of affinity chromatography, inactivation of virus, anionic membrane chromatography and cation-exchange chromatography depollution object of making a return journey.The technique can reduce production cost and improve antibody yield.

Description

A kind of purifying process of anti-PD-1 antibody

Technical field

The present invention relates to antibody purification fields, and in particular to a kind of purifying process of anti-PD-1 antibody, including use affine The methods of chromatography, inactivation of virus, anionic membrane chromatography and cation-exchange chromatography depollution object of making a return journey.

Background technique

With the continuous development of biological medicine, antibody drug shows increasingly consequence.To containing target antibody It is step essential in its production process that culture, which isolate and purify, how to pass through the purification condition of optimization antibody, into One step increases the efficiency of removal pollutant, and improving purity and yield of antibody etc. is problem important in current industrialized production.

The technique of antibody purification include multiple steps, it is currently used be affinity chromatography, cation-exchange chromatography and yin from The chromatography separating methods such as sub- displacement chromatography, in every kind of separation method design parameter it is different, it is Fillers selection it is different all can be to purifying Effect has an impact.

Affinity chromatography is that have and certain corresponding reversible knots of molecule metastatic using macromolecular substances most in organism The characteristic of conjunction and the separating and purifying technology established.Suitable for purifying target from complicated component and the big mixture of impurity content Object can also be separated active with inactive object according to its biological function, have the characteristics such as single-minded, efficient.Generally Product after affinity chromatography can achieve the purity greater than 80%, while also ensure the higher rate of recovery.But antibody Drug not only needs to guarantee monomer purity, it is also necessary to reduce the residual of host protein and DNA as far as possible, therefore also need subsequent Purification step.

Ion-exchange chromatography (Ion Exchange Chromatography is referred to as IEC) is current biochemical field In a kind of common purification process.Ion-exchange chromatography is to be separated using ion-exchanger as stationary phase according to the group in mobile phase Son reaches a kind of chromatography method for separating purpose by reversible exchange with the ion balance on exchanger.So-called ion is handed over Change, refer to the process of a certain ion in solution and another ion closed on carrier carry out it is reversible exchange, in ie in solution Ions binding to carrier on and the ion on carrier is replaced.If combining positively charged active group on carrier, Then exchangeable anions are anionite；If combining negatively charged active group on carrier, it is commutative sun from Son is cation-exchanger.Ion-exchange chromatography is widely used in various antibody such as rituximab antibody, Herceptin at present With isolating and purifying for adalimumab etc..

Anti- PD-1 antibody is a kind of antibody currently to attract attention, and the whole world has transnational medicine enterprise, more families developing, Shi Guibao The nivolumab of drugmaker and the pembrolizumab of MSD Corp. were listed in 2014, and sales volume is prominent after listing It is 1,000,000,000 dollars broken.WO2017054646A discloses a kind of sequence of anti-PD-1 antibody, which has been in third Phase, good security, the clinical study results registered have shown that it with certain antitumor action ([J] .Journal of Clinical Oncology 35(2017):e15572-e15572)。

Since antibody molecule amount is big, structure is complicated, the otherness between different antibodies is also larger, and different antibodies and ion are handed over The binding force for changing agent is not only related with the number for the charge that it is carried, and still has with the size of antibody molecule amount and charge placement etc. Certain relationship.Therefore, for different antibody, the suitable ion chromatography purifying process of optimization, as far as possible reduction production cost And antibody yield is improved, it is current the technical issues of needing urgent solution.

Summary of the invention

The present invention provides a kind of methods using cationic composition of the chromatographic purifying comprising antibody and pollutant, including Following steps:

1) loading: the composition is loaded on cation exchange material；

2) it cleans: cleaning cation exchange material with equilibration buffer；

3) it elutes: eluting lower purpose antibody from cation exchange material with elution buffer；

4) it dilutes: collecting eluent, dilution.

In one embodiment of the invention, wherein loading composition pH is 4.5-5.5, and preferably pH is 4.8-5.2, optimal Selecting pH is 5.0.

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

In one embodiment of the invention, wherein the buffer substance in equilibration buffer is MES, citric acid, phosphoric acid, lemon Lemon hydrochlorate or phosphate, optimization citric acid.

In one embodiment of the invention, wherein the concentration of equilibration buffer is 10-30mM, preferably 20mM；Balance is slow The pH of fliud flushing is 4.5-5.5, preferably 4.8-5.2.

In one embodiment of the invention, wherein the buffer substance in elution buffer is MES, citric acid, phosphoric acid, lemon One or more mixture in lemon hydrochlorate or phosphate, concentration 10-30mM, preferably 20mM；Also contain in elution buffer Potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate, sodium sulphate, citrate, one or more salts in phosphate are dense Degree is 100-250mM, preferably 150-200mM, most preferably 180mM.

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

In one embodiment of the invention, wherein the pH of elution buffer is 4.5-5.5, and preferably pH is 4.8-5.2, most It is preferred that pH is 5.0.

In one embodiment of the invention, wherein addition water or buffer progress are dilute in elution collection liquid when dilution It releases, electrical conductivity of solution < 10mS after dilution.

Purification process of the present invention, further comprising the steps of before cation-exchange chromatography:

A) affinity chromatography；

B) inactivation of virus；

C) anionic membrane chromatographs.

In one embodiment of the invention, wherein step a) affinity chromatography first uses Equilibration buffer wash before loading, Equilibration buffer is the mixed solution of phosphate buffer and sodium chloride, pH value 7.0-8.0.

In one embodiment of the invention, wherein the elution buffer of step a) affinity chromatography is citrate buffer solution, Concentration is 40-60nM, pH value 2.0-4.0, preferable ph 2.8-3.2.

In one embodiment of the invention, it is 3.2-4.0 that wherein step b), which adjusts the pH value of affinity chromatography eluent, excellent 3.6-3.8 is selected, is virus inactivated.

In one embodiment of the invention, wherein step c) anionic membrane chromatography is first clear with cleaning buffer solution before loading It washes, cleaning buffer solution is the mixed solution of phosphate buffer and sodium chloride, pH value 7.0-8.0.

In one embodiment of the invention, wherein the equilibration buffer of step c) anionic membrane chromatography is lemon acid buffering Liquid, concentration 10-30nM, pH value 4.0-6.0.

In one embodiment of the invention, wherein further including except virus filtration and ultrafiltration after cation-exchange chromatography Step.

In one embodiment of the invention, wherein it is anti-to be selected from anti-CD 20 antibodies, anti-vegf R antibody and anti-PD-1 for antibody Body, preferably anti-PD-1 antibody.

In one embodiment of the invention, wherein the light chain variable region of the anti-PD-1 antibody or its antigen-binding fragment Include LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively；Heavy chain can Become area and includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively.

Wherein, mentioned-above each CDR sequence is as shown in the table:

Title	Sequence	Number
			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 its antigen-binding fragment are anti-PD-1 humanized antibody.

Preferably, humanized antibody light chain's variable region sequences are the sequence as shown in SEQ ID NO:10 or its variant；Institute The variant stated preferably has the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.The source of people Changing antibody heavy chain variable region sequence is the sequence as shown in SEQ ID NO:9 or its variant；The variant is preferably in weight chain variable Area has the amino acid of 0-10 to change；The amino acid of more preferably G44R changes.

Humanized antibody above-mentioned is heavy, the variable region sequences of light chain are as follows:

Heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS

SEQID NO:9

Light chain variable region

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK

SEQID NO:10

Preferably, humanized antibody light chain's sequence is the sequence as shown in SEQ ID NO:8 or its variant；The variant It is preferred that thering is the amino acid of 0-10 to change in light chain variable region；The amino acid of more preferably A43S changes.The humanized antibody weight Chain-ordering is the sequence as shown in SEQ ID NO:7 or its variant；The variant preferably has the amino of 0-10 in heavy chain variable region Acid variation；The amino acid of more preferably G44R changes.

In one embodiment of the invention, humanized antibody light chain's sequence is the sequence as shown in SEQ ID NO:8, weight Chain-ordering is the sequence as shown in SEQ ID NO:7.

Humanized antibody above-mentioned is heavy, the sequence of light chain is as follows:

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQID NO:7

Light chain

DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQID NO:8

The present invention also provides a kind of compositions, and it includes antibody and buffer that any of the above item method obtains.

Detailed description of the invention:

Fig. 1: cationic Millipore Fractogel EMD SO₃(M) gradient chromatographs map

Fig. 2: cationic Millipore Fractogel EMD SO₃(M) gradient chromatographic fraction SEC is analyzed

Fig. 3: cationic Millipore Fractogel EMD SO₃(M) stage chromatographic fraction SEC is analyzed

Detailed description of the invention

One, term

In order to be easier to understand the present invention, certain technical and scientific terms are defined in detail below.Except obviously at this It is separately explicitly defined at it in file, otherwise all other technical and scientific term used herein all has belonging to the present invention The normally understood meaning of the those skilled in the art in field.

Term " pollutant " refers to the substance different from desired antibody products.Pollutant includes but is not limited to: Su Zhuxi Born of the same parents' substance, such as Chinese hamster ovary protein (CHOP)；The albumin A of leaching；Nucleic acid；Variant, segment, the aggregation of expectation antibody Or derivative；Other polypeptides；Endotoxin；Viral pollutant；Cell culture media component.

Term " buffer " refers to the effect by the pairs of ingredient of its Acid-Base to resist the solution of pH variation.Buffers.A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., described in Ed.Calbiochem Corporation (1975) depend on for example desired pH of buffer and it is adoptable A variety of buffers.

Term " equilibration buffer " refers in the present invention for that will include antibody interested and one or more pollutions The buffer of ion balance exchange material before the composition of object is loaded on ion exchange material.

Term " cleaning buffer solution " refer to after loading composition in the present invention and elution proteins of interest matter it Before flow through the buffer of ion exchange material.Cleaning buffer solution can be used for removing one or more pollutions from ion exchange material Object does not elute expectation antibody product substantially.

Term " conductivity " refers to the ability that aqueous solution conducts electric current between two electrodes.In the solution, electric current passes through Ion transports to flow, and as the amount of ion present in aqueous solution is more and more, solution can have higher conductivity.Conductance The basic unit of the measurement of rate is Siemens's (or ohm), ohm (mS/cm), and conductivity meter can be used to measure, all Such as the Orion conductivity meter of various models.Because electrolytic conductivity is the ability of the ion carrying electric current in solution, solution Conductivity can be changed by changing ion concentration therein.Such as can change in solution the concentration of buffer and/or The concentration of salt (such as oxidation is received, sodium acetate or potassium oxide) realizes desired conductivity.

Term " antibody " is with broadest use, clearly covering monoclonal antibody (including full length monoclonal antibodies), polyclonal Antibody, multi-specificity antibody (such as bispecific antibody) and antibody fragment, as long as to show desired combination special for they Property.

Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted Antibody), refer to the antibody variable region frame that the CDR sequence of mouse is transplanted to people, i.e., different types of human germline antibody The antibody generated in frame sequence.Chimeric antibody can be overcome due to carrying a large amount of murine protein ingredients, so that induction is strong Antibody variable antibody response.Such frame sequence can be from public DNA database or public affairs including germline antibody gene sequences The bibliography opened obtains.As people's heavy chain and the germline DNA sequence dna of light-chain variable region gene can be in " VBase " human germ line sequences Database (can get) in internet www.mrccpe.com.ac.uk/vbase, and in Kabat, E.A. et al., and 1991 Sequences of Proteins of Immunological Interest is found in the 5th edition.In one implementation of the present invention In scheme, the CDR sequence of the PD-1 humanized antibody is selected from SEQ ID NO:1, and 2,3,4,5,6.

Term " antigen-binding fragment ", refers to the Fab segment with antigen-binding activity, Fab ' segment, 2 segment of F (ab '), And the Fv segment sFv segment in conjunction with people PD-1；SEQ ID NO:1 to SEQ ID is selected from comprising antibody of the present invention One or more CDR regions in NO:6.Fv segment contains antibody heavy chain variable region and light chain variable region, but does not have constant region, and Minimum antibody fragment with whole antigen binding sites.Generally, Fv antibody also includes more between VH and VL structural domain Peptide linker, and structure needed for being capable of forming antigen binding.Two antibody variable regions can also be connected with different attachments At a polypeptide chain, referred to as single-chain antibody (single chain antibody) or scFv (sFv).Term of the invention " with PD-1 combination ", referring to can interact with people PD-1.Term " antigen binding site " of the invention refer to it is discontinuous on antigen, by The three-dimensional space site of antibody or antigen-binding fragment identification of the present invention.

Specific embodiment

With reference to embodiments for further describing the present invention, but these embodiments not limit model of the invention It encloses.

Embodiment 1, anti-PD-1 antibody cell culture in-depth filtration liquid are through affinity chromatography capture and preliminary purification

The sequence of anti-PD-1 antibody weight, light chain is affine as shown in SEQ ID NO:7 and SEQ ID NO:8 in the present embodiment Chromatographic step is as follows:

Step 1: affinity chromatography filler Prosep Ultra Plus (Millipore) is packed into column.Use thimerosal (150mM phosphoric acid) rinses, and stands.It is rinsed again with equilibration buffer (20mM phosphate buffer+1M common salt pH7.4 ± 0.1), Collection took column liquid survey endotoxin, limit be less than 0.25EU/mL, detection qualification can loading, if it is unqualified need to sterilize again and Balance.

Step 2: point 3 circulation loadings, it is ensured that carrying capacity≤40g/L.

Step 3: being rinsed with equilibration buffer (20mM phosphate buffer+1M common salt pH7.4 ± 0.1).

Step 4: being rinsed with miscellaneous buffer (20mM phosphate buffer pH7.4 ± 0.1) is washed.

Step 5: eluting destination protein with elution buffer (50mM citrate buffer solution pH3.0 ± 0.1).

Step 6: affinity chromatography collection liquid sample detection protein concentration, calculates this step rate of recovery and sample presentation QC detection SEC- HPLC purity.The result is as follows:

Table 1

Applied sample amount (mg)	Yield (mg)	The rate of recovery (%)	SEC-HPLC purity (%)
				1800	1710	95	91.6

Conclusion: cell culture fluid carries out Millipore PUP affinity chromatography according to the carrying capacity of 40mg/ml, and the rate of recovery is 95%, SEC-HPLC purity 91.6%.

Embodiment 2, low pH inactivation of virus

It collects eluent and collection liquid is divided into three groups in order to determine the pH of inactivation of virus, second and third group is passed through 1.5 hours Low pH uses 1.0M Tris tune pH to 5.0 after being incubated for, first group does not carry out the sample that low pH incubation directly adjusts pH to 5.0 and carry out SEC detection, the result is as follows:

Table 2

It can be seen that anti-PD-1 antibody SEC under the conditions of lower pH is unstable by result in table, comprehensively consider pH and processing Time to the effect of inactivation of virus, finally adjusts affinity chromatography collection liquid pH 3.6-3.8,18- with 1M Tris or 1M citric acid 90-95min is stood under the conditions of 26 DEG C to be virus inactivated.After inactivation, addition 1M Tris adjusting sample pH value to 5.0 ± 0.1, except virus results are as follows:

Table 3

Conclusion: the experimental results showed that, two kinds of viral reduction amounts (log10) are all larger than 4logs, indicate the step inactivation of viruses Effectively.

Embodiment 3, anionic membrane chromatography

Affinity chromatography collection liquid carries out anionic membrane chromatography, step is such as after low pH inactivation of virus, absorption in-depth filtration Under:

Step 1: anion chromatography film is Sartobind Q (Sai Duolisi) chromatographic film, thimerosal (1M NaOH) is rinsed.

Step 2: being rinsed with cleaning buffer solution (20mM phosphate buffer+1M common salt, pH7.4 ± 0.1).

Step 3: being rinsed with equilibration buffer (20mM citrate buffer solution, pH5.0 ± 0.1), then took in film liquid survey Toxin, limit be less than 0.25EU/mL, detection qualification can loading, if unqualified need to sterilize and balance again.

Step 4: confirmation sample solution pH5.0 ± 0.1, conductance < 5mS/cm carry out loading, flowed through according to UV280 collection Liquid.

Step 5: being washed after end of the sample with equilibration buffer top, collection is continued according to UV280 and flows through liquid, sample presentation QC detection HCP removal effect.The result is as follows:

Table 4

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

Conclusion: affinity chromatography sample purity after anionic membrane flows through mode chromatography reaches without significant change, HCP removal rate 46.7%.

Embodiment 4, cation chromatography

In-depth filtration collection liquid is adsorbed after anionic membrane chromatographs, the further purification of cation chromatography need to be carried out, step is such as Under:

Step 1: by cationic chromatographic stuffing Fractogel EMD SO₃ ^-(M) (Millipore) is packed into column.With disinfection Liquid (1M NaOH) rinses, and stands.It is rinsed again with equilibration buffer (20mM citrate buffer solution, pH5.0 ± 0.1), collection took Column liquid survey endotoxin, limit be less than 0.25EU/mL, detection qualification can loading, if unqualified need to sterilize and balance again.

Step 2: confirmation sample solution pH5.0 ± 0.1, conductance < 5mS carry out loading.

Step 3: being rinsed with equilibration buffer (20mM citrate buffer solution, pH5.0 ± 0.1).

Step 4: with elution buffer 20mM citrate buffer solution, pH 5.0 and 20mM citrate buffer solution+200mM NaCl, pH5.0 gradient elution destination protein are in charge of and collect sample progress SEC detection, chromatographs map such as Fig. 1, collect sample each group Divide SEC result such as Fig. 2.

As can be seen from Figure under 20mM citrate buffer solution, the system of pH5.0, when salinity reaches 180mM NaCl The SEC of anti-PD-1 antibody reaches highest 97.5%, while containing impurity before appearance UV280 rises to 1000mAU, adopts in technique It accepts or rejects and abandons the part.Fixed elution buffer is 20mM citrate buffer solution+180mMNaCl, pH5.0's.Collect sample each group Divide SEC result such as Fig. 3.

The stability of embodiment 5, anti-PD-1 antibody

Pass through cationic chromatographic stuffing Fractogel EMD SO₃ ^-(M) the anti-PD-1 antibody after (Millipore) chromatography, Purity is significantly improved.Sample is in pH5.0 after purification, and in conductance 20ms/cm or so system, range estimation is formed in a short time Threadiness precipitating, still has the phenomenon that Precipitation after 0.22 μm of filtering.

Since the PD-1 antibody obtained after cation chromatography can generate fibrous precipitating, contrived experiment detects anti-PD-1 antibody Salt-stable, proceed as follows: by affinity elution sample, pH5.0 is titrated with 2M NaCl, detects conductance with conductivity meter And record, as a result lamp inspection sample clarity see the table below:

Table 5

Sample	Conductance (ms/cm)	Clarity
			Initial sample	3.81	Clarification
1	7.05	A small amount of threadiness precipitating
			2	9.62	Threadiness precipitating
3	12.35	Threadiness precipitating
			4	15.23	Threadiness precipitating
5	19.50	Threadiness precipitating
			6	24.6	Threadiness precipitating
7	28.8	Threadiness precipitating

As can be seen from Table 5, anti-PD-1 antibody is stablized under the conditions of pH5.0, initial sample less salt (conductance 3.81ms/cm) Property it is preferable, with the increase of salinity, conductance is increased, and gradually appears fibrous precipitating, and increase not table with salinity Reveal the phenomenon that threadiness precipitating is redissolved, it was demonstrated that the salt-stable of anti-PD-1 antibody is weaker.It takes cationic chromatography process Middle elution samples are directly accessed in pre-prepd water for injection and are diluted, and it is fine to reach control in turn for salinity in reduction system Tie up shape precipitating generate effect, water for injection press different proportion dilute sample data such as table 6, in table it can be seen from conductance When rate≤10ms/cm, sample does not generate precipitating substantially.

Table 6

Sample number into spectrum	Water for injection (ml)	Sample (ml)	Conductance (ms/cm)	Clarity
					A1	1	1	9.65	A small amount of threadiness precipitating
A2	2	1	6.48	A small amount of threadiness precipitating
					A3	3	1	4.77	Clarification
A4	4	1	4.08	Clarification
					A5	5	1	3.23	Clarification

Sequence table

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Hengrui Pharmaceutical Co., Ltd., Shanghai

Suzhou Sheng Diya biological medicine Co., Ltd

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<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 kind of includes the method for the composition of antibody and pollutant using cationic chromatographic purifying, which is characterized in that including with Lower step:

1) loading: the composition is loaded on cation exchange material；

2) it cleans: cleaning cation exchange material with equilibration buffer；

4) it dilutes: collecting eluent, dilution.

2. preferably pH is 4.8-5.2, optimal according to the method described in claim 1, wherein loading composition pH is 4.5-5.5 Selecting pH is 5.0.

3. according to the method described in claim 1, the wherein conductivity < 5mS of loading composition.

4. according to the method described in claim 1, wherein the buffer substance in equilibration buffer is MES, citric acid, phosphoric acid, lemon Lemon hydrochlorate or phosphate, optimization citric acid.

5. according to the method described in claim 1, wherein the concentration of equilibration buffer is 10-30mM, preferably 20mM；Equalizing and buffering The pH of liquid is 4.5-5.5, preferably 4.8-5.2.

6. according to the method described in claim 1, wherein the buffer substance in elution buffer is MES, citric acid, phosphoric acid, lemon One or more mixture in lemon hydrochlorate or phosphate, concentration 10-30mM, preferably 20mM；Also contain in elution buffer Potassium chloride, sodium chloride, potassium carbonate, sodium acetate, potassium sulfate, sodium sulphate, citrate, one or more salts in phosphate are dense Degree is 100-250mM, preferably 150-200mM, most preferably 180mM.

7. according to the method described in claim 6, wherein the conductivity of elution buffer is 10-25mS, preferably 18-22mS.

8. preferably pH is 4.8-5.2, most according to the method described in claim 6, wherein the pH of elution buffer is 4.5-5.5 It is preferred that pH is 5.0.

9. according to the method described in claim 1, electrical conductivity of solution < 10mS after wherein diluting.

10. the method according to claim 1, wherein further comprising the steps of before cation-exchange chromatography:

A) affinity chromatography；

B) inactivation of virus；

C) anionic membrane chromatographs.

11. method according to claim 10, wherein step a) affinity chromatography first uses Equilibration buffer wash before loading, put down Weigh the mixed solution that buffer is phosphate buffer and sodium chloride, pH value 7.0-8.0.

12. according to the method described in claim 10, wherein the elution buffer of step a) affinity chromatography be citrate buffer solution, Concentration is 40-60nM, pH value 2.0-4.0, preferable ph 2.8-3.2.

13. according to the method described in claim 10, wherein step b) adjust affinity chromatography eluent pH value be 3.2-4.0, It is preferred that 3.6-3.8, is virus inactivated.

14. according to the method described in claim 10, wherein step c) anionic membrane chromatography first uses cleaning buffer solution before loading Cleaning, cleaning buffer solution are the mixed solution of phosphate buffer and sodium chloride, pH value 7.0-8.0.

15. according to the method described in claim 10, wherein the equilibration buffer of step c) anionic membrane chromatography is slow for citric acid Fliud flushing, concentration 10-30nM, pH value 4.0-6.0.

16. the method according to claim 1, wherein further including except virus filtration after cation-exchange chromatography And the step of ultrafiltration.

17. according to the method described in claim 1, wherein antibody be selected from anti-CD 20 antibodies, anti-vegf R antibody, anti-PD-1 antibody, It is preferred that anti-PD-1 antibody.

18. -17 described in any item methods according to claim 1, wherein the anti-PD-1 antibody or its antigen-binding fragment Light chain variable region include respectively LCDR1, LCDR2 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 and LCDR3；Heavy chain variable region include respectively the HCDR1 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, HCDR2 and HCDR3.

19. according to the method for claim 18, wherein the anti-PD-1 antibody or its antigen-binding fragment are selected from anti-PD-1 Humanized antibody.

20. according to the method for claim 19, wherein the light-chain variable sequence of the anti-PD-1 humanized antibody is such as Sequence shown in SEQ ID NO:10 or its variant, the variant preferably have the amino acid of 0-10 to change in light chain variable region, The amino acid of more preferably A43S changes；Weight chain variabl area sequence be the sequence as shown in SEQ ID NO:9 or its variant, it is described Variant preferably has the amino acid of 0-10 to change in heavy chain variable region, the amino acid variation of more preferably G44R.

21. according to the method for claim 20, wherein the anti-PD-1 humanized antibody light chain sequence is such as SEQ ID Sequence shown in NO:8 or its variant, the variant preferably have the amino acid of 0-10 to change in light chain variable region, more preferably The amino acid of A43S changes；Sequence of heavy chain is the sequence as shown in SEQ ID NO:7 or its variant, and the variant is preferably in heavy chain Variable region has the amino acid of 0-10 to change, the amino acid variation of more preferably G44R.

22. according to the method for claim 21, wherein the anti-PD-1 humanized antibody light chain sequence is such as SEQ ID Sequence shown in NO:8, sequence of heavy chain are the sequence as shown in SEQ ID NO:7.

23. a kind of composition, it includes the antibody and buffer that are obtained by any one of claim 1-22 the method.