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CN119080928A - A method for purifying anti-IL-11 monoclonal antibody - Google Patents

A method for purifying anti-IL-11 monoclonal antibody Download PDF

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CN119080928A
CN119080928A CN202310653978.7A CN202310653978A CN119080928A CN 119080928 A CN119080928 A CN 119080928A CN 202310653978 A CN202310653978 A CN 202310653978A CN 119080928 A CN119080928 A CN 119080928A
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白义
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BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD
Beijing Dongfang Baitai Biotechnology Co ltd
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BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD
Beijing Dongfang Baitai Biotechnology Co ltd
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Abstract

The invention relates to the field of biological medicine, and in particular provides a purification method of an anti-IL-11 monoclonal antibody, which comprises the steps of performing affinity chromatography, namely, primarily purifying and concentrating target protein containing the anti-IL-11 monoclonal antibody, inactivating viruses of protein products in an acid solution to obtain a protein sample, performing anion exchange chromatography, namely, secondarily purifying the protein sample, collecting a flow-through liquid, and performing cation exchange chromatography, namely, performing precise purification on the flow-through liquid to obtain a protein solution of the anti-IL-11 monoclonal antibody. The invention provides a proper purification process for the anti-IL-11 monoclonal antibody, improves the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody, wherein the flow-through liquid collected by anion exchange chromatography directly enters cation exchange chromatography without treatment, sample treatment or solution replacement is not needed in the middle, the purification time is saved, impurities are avoided being introduced in the purification process, the purity of the purified protein is improved, and the method has higher practicability.

Description

Purification method of anti-IL-11 monoclonal antibody
Technical Field
The invention relates to the technical field of biological medicine, in particular to a purification method of an anti-IL-11 monoclonal antibody.
Background
Fibrosis (fibrosis) can occur in a variety of organs, such as liver, lung, kidney, retina, intestine, heart, skin, etc., which are the leading cause of disability and mortality in many diseases, with the primary pathology being increased fibrous connective tissue in organ tissues, reduced parenchymal cells, continued progression leading to destruction and hypofunction of organ structures, and even failure, severely threatening human health and life. Statistics on the united states show that nearly 45% of patients deaths from various diseases in this country are attributed to fibroproliferative diseases.
Fibrotic diseases include various diseases such as pulmonary fibrosis, liver cirrhosis, liver diseases, kidney fibrosis, heart fibrosis, asthma, eye diseases, etc. Wherein liver fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has liver fibrosis in the course of liver repair and healing, and if injury factors cannot be removed for a long period of time, the fibrosis process continues for a long period of time to develop cirrhosis. The etiology of liver fibrosis is many, and viral hepatitis, alcoholic liver disease, fatty liver disease, autoimmune disease and the like are mostly seen clinically. The global high nonalcoholic fatty liver disease can be worsened into nonalcoholic hepatitis if not treated timely, even liver inflammation, liver cell death and liver fibrosis can be caused, and finally liver cirrhosis and liver cancer are generated, and furthermore, the pulmonary fibrosis is the final-stage change of a large type of pulmonary diseases characterized by fibroblast proliferation, a large amount of extracellular matrix aggregation, inflammation damage and tissue structure damage, and the cause of most pulmonary fibrosis patients is unknown (idiopathic), and the group of diseases is called idiopathic interstitial pneumonia and is a large type of interstitial lung diseases. The most common type of disease with pulmonary fibrosis lesions as the main manifestation form in idiopathic interstitial pneumonia is idiopathic pulmonary fibrosis, which is a serious interstitial pulmonary disease that can cause progressive loss of pulmonary function, and the pulmonary fibrosis seriously affects respiratory function of human body, and is manifested by dry cough and progressive dyspnea, and the respiratory function of patients is continuously worsened with the aggravation of illness and pulmonary injury. The incidence and mortality of idiopathic pulmonary fibrosis increases year by year, with an average survival of only 2.8 years after diagnosis, and mortality higher than most tumors, known as a "neoplastic-like disease. At present, no particularly effective medicine is available for treating the fibrosis diseases of all organs, the treatment effect is general, and the research and development of the medicine for treating the anti-fibrosis diseases are particularly urgent.
Interleukin-11 (IL-11), a hematopoietic cytokine, is one of the cytokines of the multifunctional interleukin 6 (IL 6) family, sharing the same signaling receptor subunit (GP 130). This family plays a critical role in the development, progression and metastasis of tumors. It has been found that blocking of this pathway can be an effective treatment for a variety of neoplastic cancers, chronic fibrosis and inflammatory diseases. At present, medicines taking IL-11 as targets are not marketed globally, so that the technological development of anti-IL-11 monoclonal antibodies has important clinical significance for meeting urgent demands of patients.
At present, in the process of technological development of the anti-IL-11 monoclonal antibody, the conventional antibody purification method is used for purifying the anti-IL-11 monoclonal antibody, degradation products and target proteins are often difficult to separate, samples are unstable in the process, and the purification efficiency, purity and recovery rate are low.
Disclosure of Invention
In order to meet urgent demands of fibrotic patients as soon as possible, improve the recovery rate and purity of the drug in the drug development process, and ensure the biological activity of the anti-IL-11 monoclonal antibody in the purification process, the invention provides a purification method specially applicable to the anti-IL-11 monoclonal antibody.
The specific technical scheme of the invention is as follows:
The invention provides a method for purifying an anti-IL-11 monoclonal antibody, which comprises the following steps:
S1, performing affinity chromatography, namely primarily purifying and concentrating target protein containing an anti-IL-11 monoclonal antibody, and inactivating viruses of the collected protein product in an acid solution to obtain a crude and pure protein sample;
s2, anion exchange chromatography, namely performing secondary purification on the protein sample, and collecting a penetrating fluid;
S3, cation exchange chromatography, namely obtaining the protein solution of the high-purity anti-IL-11 monoclonal antibody after the flow-through liquid is subjected to fine purification.
Further, the anti-IL-11 monoclonal antibody comprises 3 heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and 3 light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, and is selected from any one of the following:
A-I, wherein the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 6;
A-II, wherein the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 7;
A-III, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 8, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 9, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 10, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 11, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 12, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 13;
A-IV, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 14, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 15, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 6.
The invention has the beneficial effects that firstly, the optimal conditions and chromatographic packing of the anti-IL-11 monoclonal antibody provided by the invention are screened by a continuous three-step purification method, the purification efficiency and recovery rate of the anti-IL-11 monoclonal antibody provided by the invention are effectively improved, wherein the flow-through liquid collected by anion exchange chromatography can directly enter cation exchange chromatography without treatment, sample treatment or solution replacement is not needed in the middle, the purification time is greatly saved, impurities are avoided from being introduced in the purification process, the purity of the purified protein is improved, the practicability is higher, and secondly, the anti-IL-11 monoclonal antibody provided by the invention has higher binding capacity with IL-11 antigen, can block the binding of IL-11 antigen with the receptor thereof, further effectively inhibit the pro-fibrosis effect of IL-11, inhibit or prevent the generation or proliferation of fiber cells, and can be effectively used for treating or preventing human fibrosis diseases, inflammation, cancers or autoimmune diseases.
Drawings
FIG. 1 is a plasmid map of pScFv-Disb-HS vector in example 2 of the present invention;
FIG. 2 is a graph showing comparison of affinity of gradient dilution ELISA anti-IL-11 phage monoclonal antibodies in example 3 of the present invention;
FIG. 3 is a map of the carrier pTSE in example 5 of the present invention;
FIG. 4 is a diagram showing the gel electrophoresis of a denatured polyacrylamide gel of a murine antibody molecule of example 5 of the present invention;
FIG. 5 is a graph showing the comparison of the binding capacity of murine antibody molecules of example 6 of the present invention to IL-11;
FIG. 6 is a graph showing the comparison of the experiment of competitive inhibition of murine antibody with IL-11 receptor protein IL-11RA in example 7 of the present invention;
FIG. 7 is a graph showing the comparison of the inhibition of IL-11 binding to the BaF/3-IL-11RA cell surface IL-11RA receptor by murine antibodies in example 8 of the present invention;
FIG. 8 is a graph showing the comparison of murine antibody of example 9 of the present invention inhibiting the secretion of TIMP-1 by embryonic lung fibroblast MRC-5;
FIG. 9 is a photograph showing the gel electrophoresis of a denatured polyacrylamide gel of the humanized antibody molecule of example 14 of the present invention;
FIG. 10 is a graph showing the comparison of the binding capacity of humanized antibody molecules to IL-11 in example 15 of the present invention;
FIG. 11 is a graph showing the comparison of the inhibition of IL-11 binding to the BaF/3-IL-11RA cell surface IL-11RA receptor by humanized antibody molecules of example 16 of the present invention;
FIG. 12 is a graph showing comparison of the inhibition of IL-11 binding to BaF/3-GP130 cell surface GP130 receptor by humanized antibody molecules of example 17 of the present invention;
FIG. 13 is a graph showing comparison of biological activity detection (reporter gene) of humanized antibody molecules of example 18 of the present invention;
FIG. 14 is a graph showing the comparison of the inhibition of TIMP-1 secretion by embryonic lung fibroblast MRC-5 by a humanized antibody molecule of example 19 of the present invention;
FIG. 15 is a graph showing comparison of the cross-binding experiments of humanized antibody molecules with IL-11 of different species in example 20 of the present invention;
FIG. 16 is a bar graph showing the lung to body weight ratio change in a mouse lung fibrosis model in example 21 of the present invention;
FIG. 17 is a Hematoxylin and Eosin (HE) staining and Ma Songran color charts of lung tissue sections in a mouse lung fibrosis model in example 21 of the present invention;
FIG. 18 is a bar graph showing the change in heart to body weight ratio in a mouse heart fibrosis model in example 22 of the present invention;
FIG. 19 is a Hematoxylin and Eosin (HE) staining and Ma Songran color charts of heart tissue sections in a mouse heart fibrosis model in example 22 of the present invention;
FIG. 20 is a bar graph showing the urine protein content of the kidney in the kidney fibrosis model of the mouse in example 23 of the present invention;
FIG. 21 is a Hematoxylin and Eosin (HE) staining and Ma Songran color chart of kidney tissue sections in a mouse kidney fibrosis model in example 23 of the present invention;
FIG. 22 is a bar graph showing liver weight change in a mouse liver fibrosis model in example 24 of the present invention;
FIG. 23 is a bar graph showing changes in alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels in mouse serum from a mouse liver fibrosis model in example 24 of the present invention;
FIG. 24 is a Hematoxylin and Eosin (HE) staining and Ma Songran color charts of liver tissue sections in a mouse liver fibrosis model in example 24 of the present invention;
FIG. 25 is a graph showing the evaluation of the thermostability of the anti-IL-11 monoclonal antibody HA-I-A in example 25 of the present invention.
Detailed Description
The invention will be described in further detail with reference to the following examples.
Example 1
The present invention provides a method for purifying an anti-IL-11 monoclonal antibody, comprising:
S1, performing affinity chromatography, namely primarily purifying and concentrating target protein containing an anti-IL-11 monoclonal antibody, and inactivating viruses of the collected protein product in an acid solution to obtain a crude and pure protein sample;
s2, anion exchange chromatography, namely performing secondary purification on a protein sample, and collecting a flow-through liquid;
s3, cation exchange chromatography, namely, carrying out fine purification on the flow-through liquid to obtain a high-purity protein solution of the anti-IL-11 monoclonal antibody.
Wherein the anti-IL-11 monoclonal antibody comprises 3 heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and 3 light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, and the anti-IL-11 monoclonal antibody is selected from any one of the following.
The anti-IL-11 monoclonal antibody provided by the invention is used for treating or preventing human fibrosis diseases, inflammation, cancer or autoimmune diseases, wherein the fibrosis diseases comprise heart, liver, kidney, lung, gall bladder, stomach, bone marrow, penis, breast, blood vessel, eyes, pancreas, spleen, brain, intestinal tract, muscle or skin fibrosis and the like, the inflammation comprises, but is not limited to, hepatitis, myocarditis, nephritis, pneumonia, cholecystitis, cystitis, gastritis, osteomyelitis, prostatitis, mastitis, pancreatitis, enteritis, arthritis, polymyositis, dermatomyositis or dermatitis and the like, the cancer comprises, but is not limited to leukemia, lung cancer, gastric cancer, esophagus cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer or bladder cancer and the like, and the autoimmune diseases comprise, but are not limited to, psoriasis, crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus or multiple sclerosis and the like.
EXAMPLE 2 murine antibody molecular screening
The invention optimizes the immune method by immunizing mice with IL-11 antigen (IL-11 protein, IL-11-Fc antigen, IL-11-mFc ligand protein are all human IL-11 in subsequent experiments), creates phage display library, and constructs, screens and identifies the specific phage display library as follows:
step one, immunizing mice with IL-11 antigen
1. Experimental animals:
species strain BALB/c, female, mouse;
18-20g of body weight;
Experimental animals provider Beijing Fukang Biotechnology Co., ltd.
2. Immunization, namely, mice are immunized, and the immune antigen is human IL-11 (synthesized gene of Nanjing Jinsri biotechnology Co., ltd., carrier is constructed and expressed and purified by the company).
Step two, constructing phage antibody library
The method comprises the steps of taking mouse spleen cells with higher titers, extracting total RNA in the mouse spleen cells by using Trizol reagent (purchased from Ambion, cat# 15596026), obtaining cDNA by RT-PCR, and carrying out PCR amplification by using the cDNA as a template and degenerate primers (used degenerate primer reference: journal of Immunological Methods233 (2000) 167-177) so as to obtain an immune mouse antibody heavy chain variable region gene library (VH) and a light chain variable region gene library (VL). pScFv-Disb-HS vector is prepared by modifying vector pComb3 vector (purchased from China center for type culture Collection of plasmid vector strain genes) by a series of gene cloning method, which is used for constructing and expressing phage single-chain antibody library, the modified vector is named pScFv-Disb-HS vector, the plasmid map is shown in figure 1, and the mouse immune phage antibody library is constructed based on the vector. The light chain variable region gene library and the heavy chain variable region gene library are respectively subjected to double enzyme digestion and connected to the vector pScFv-Disb-HS which is subjected to enzyme digestion in the same steps, so as to construct a pScFv-Disb-HS-VH-VL gene library.
And thirdly, coating an immune tube with IL-11 serving as an antigen, wherein the antigen coating amount is 5 mug/500 mug/tube, coating at 4 ℃ overnight, and then sealing the immune tube and an immune phage antibody library respectively by using 4% skimmed milk powder/PBST for 1h at room temperature. The blocked immune phage antibody library is added into an immune tube for antigen-antibody binding, the input amount of phage is about 10 9~1012, after reaction is carried out for 1h at room temperature, unbound phage is washed off by using PBST-PBS, the phage is eluted by using 0.1M Glycine-HCl with pH of 2.2, and finally the eluted phage antibody solution is neutralized to about pH7.0 by using 1.5M Tris-HCl with pH of 8.8.
And step four, infecting 10ml of the neutralized phage to TG1 bacterial liquid growing to a logarithmic phase, standing for 30min in a 37 ℃ incubator, taking out part of bacterial liquid for gradient dilution, and coating the bacterial liquid on a 2YTAG plate for calculating phage output. The remaining bacterial liquid was centrifuged to discard the supernatant, the bacterial pellet was resuspended in a small amount of medium, aspirated and spread on a 2YTAG large plate, ready for the next round of screening.
Scraping the thallus coated with the infected plate from a large plate, inoculating the thallus to a 2YTAG liquid culture medium, shaking to a logarithmic phase, adding M13KO7 auxiliary phage to superinfect, culturing overnight at 220rpm under the condition of 28 ℃ to prepare phage, and carrying out PEG/NaCl sedimentation to purify phage for the next round of screening, thereby carrying out a round of phage library enrichment screening.
Screening IL-11 phage single-chain antibody positive clone, selecting well-separated monoclonal colony after one round of screening, inoculating to 96-well deep-well plate added with 2YTAG liquid culture medium, culturing at 37 deg.C and 220rpm to logarithmic growth phase, adding about 10 10 auxiliary phage M13KO7 into each well, and standing at 37 deg.C for infection for 30min.4000rpm, centrifugation for 15min, discarding supernatant, re-suspending the pellet with 2YTAK, and culturing overnight at 28℃and 220 rpm. Centrifuging at 4000rpm and 4 ℃ for 15min, absorbing amplified phage supernatant for ELISA identification, finally screening to obtain four murine antibody molecules with higher affinity, namely MA-I, MA-II, MA-III and MA-IV, carrying out gene sequencing on the obtained monoclonal antibodies to determine the correct antibody sequences, and sequencing the 4 monoclonal antibody sequences selected as follows:
specifically, SEQ ID No. 16 (amino acid sequence of the heavy chain variable region of MA-I, MA-II):
EVKLEESGGGLVKPGGSLKLSCAASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMTSLKSEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;
SEQ ID No. 17 (amino acid sequence of the light chain variable region of MA-I, MA-IV):
DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK;
18 (amino acid sequence of light chain variable region of MA-II):
DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSR LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK;
SEQ ID No. 19 (amino acid sequence of the heavy chain variable region of MA-III):
EVKLEQSGAEVVKPGALVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIG VIDPSDSYTTYNQKFKGKATLTVDTSSSTGYMQLSSLTSEDSAVYYCSQYGYDVN WYFDVWGAGTTVTVSS;
SEQ ID No. 20 (amino acid sequence of the light chain variable region of MA-III):
DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLI YEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGT KLEIK;
SEQ ID No. 21 (amino acid sequence of heavy chain variable region of MA-IV):
EVQLEESGGGLVKPGGSLKLSCVASGFTFSDYYMFWVRQTPEKRLEWVATI SDGGSYSYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAMYYCARDGGYISS PEAMDYWGQGTSVTVSS.
example 3 gradient dilution ELISA comparison of affinity of antibodies
The 4 murine antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in example 2 were subjected to monoclonal phage display and purification, and then to phage gradient dilution ELISA experiments to identify affinities, as follows:
IL-11 antigen was coated with carbonate buffer at pH9.6, 100 ng/well/100. Mu.L, and at 4℃overnight. The 4 phage monoclonal antibodies selected in example 2 were each diluted with five-fold gradient of PBST, washed three times with PBST, 100. Mu.l of diluted sample was added to each well, and allowed to stand at room temperature for 1 hour. ELISA plates were washed with PBST, and HRP-anti-M13 (purchased from Bio-viewshine, cat# GE 27-9421-01) diluted with 1% BSA-PBST was added to the ELISA plates and left at room temperature for 1h. TMB chromogenic kit developed (purchased from Kangji, cat# CW 0050S), developed for 10 minutes at room temperature, after termination with 2M H2SO4, the microplate reader was read at 450nm/630nm and the corresponding EC50 value was calculated as follows:
By the above data and as shown in FIG. 2, 4 different murine antibody molecules screened in example 2 were all able to bind IL-11, thus demonstrating that the monoclonal antibodies provided by the present invention have higher affinity to IL-11.
Example 4
Example 4 of the present invention further defines on the basis of example 2 that the anti-IL-11 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is one of SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25 or SEQ ID No. 26, and the amino acid sequence of the light chain constant region is one of SEQ ID No. 22, and the specific sequence is as follows:
SEQ ID No. 22 (murine C k type light chain constant region amino acid sequence):
ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC;
SEQ ID No. 23 (murine heavy chain constant region amino acid sequence of IgG1 type):
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;
SEQ ID No. 24 (murine heavy chain constant region amino acid sequence of IgG2a type):
AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;
SEQ ID No. 25 (murine heavy chain constant region amino acid sequence of IgG2b type):
AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;
SEQ ID No. 26 (murine heavy chain constant region amino acid sequence of IgG3 type):
ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.
EXAMPLE 5 preparation of murine antibody molecules
Example 5 of the present invention preferably defines, on the basis of example 4, murine antibody molecules comprising a murine heavy chain constant region of the IgG1 type (the amino acid sequence of which is shown in SEQ ID No. 23) and a murine light chain constant region of the C k type (the amino acid sequence of which is shown in SEQ ID No. 22). The preparation method of the antibody specifically comprises the following steps:
1. In cloning the coding genes of the heavy chain VH and the light chain VL of the 4 antibody molecules selected in example 2 into vectors pTSE (shown in FIG. 3) containing the heavy chain and light chain constant region genes, the preferred heavy chain constant region is a murine IgG1 type constant region (amino acid sequence shown in SEQ ID No. 23), the light chain constant region is a murine C k chain (amino acid sequence shown in SEQ ID No. 22), and the pTSE vector structure is shown in FIG. 3 (pTSE for vector preparation see page 3 of the description of CN103525868A [0019 ]).
2. HEK293 cells (purchased from basic medical institute of China medical sciences, cat# GNHu) were transiently transfected, antibody expression was performed, 4 monoclonal antibodies were obtained by protein A affinity column purification using an AKTA instrument, protein concentration was determined using a BCA kit (purchased from Beijing Hui Tian Oriental science and technology Co., ltd., cat# BCA 0020), and then protein sizes were identified by SDS-PAGE, as shown in FIG. 4, non-reduced MA-I, MA-II, MA-III and MA-IV were sequentially performed from left to right, protein molecular weight Marker1, protein molecular weight Marker2, and reduced MA-I, MA-II, MA-III and MA-IV murine anti-IL-11 monoclonal antibodies were obtained, and the molecular weight of each band was consistent with theory.
EXAMPLE 6 experiments on the binding of murine antibody molecules to IL-11
IL-11 antigen was coated with carbonate buffer at pH9.6, 100 ng/well/100. Mu.L, and at a temperature of 4℃overnight. Five washes with 300. Mu.L/well PBST, followed by 1% BSA-PBST, blocking at 37℃for 1h in 280. Mu.L/well, adding MA-I, MA-II, MA-III and MA-IV murine antibody molecules in different dilution concentrations, starting at a maximum concentration of 5. Mu.g/mL for 4 antibody molecules, respectively, with 5-fold gradient dilutions, 8 gradients per antibody total, and incubation for 1h at 37 ℃. Five washes with 300. Mu.L/well PBST were performed, and Goat Anti-Mouse IgG-HRP (purchased from solarbio, cat# SE 131) diluted with 1% BSA-PBST 1:2000 was added and incubated for 1h at 37 ℃. The TMB development kit developed, 100. Mu.L/well, developed for 8min at room temperature, and then stopped with 2M H 2SO4. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value, the specific data are as follows:
through the above data and as shown in FIG. 5, 4 different murine antibody molecules were screened for binding to IL-11 with higher affinity.
EXAMPLE 7 experiments on the competitive inhibition of murine antibodies with the IL-11 receptor protein IL-11RA
IL-11-Fc was coated with carbonate buffer at pH9.6, 200 ng/well/100. Mu.L, at 4℃overnight. Washing with 300. Mu.L/well PBST five times, adding 1% BSA-PBST, blocking 280. Mu.L/well at 37℃for 1h, adding IL-11RA-Fc (IgG 4 type) diluted to 0.5. Mu.g/mL by 1% BSA-PBST, 50. Mu.L/well, adding MA-I, MA-II, MA-III and MA-IV murine antibodies at different dilution concentrations, 50. Mu.L/well, starting the highest concentration of 5 antibodies was 100. Mu.g/mL, diluting each antibody by 2-fold gradient, diluting 13 gradients each antibody total, and incubating for 3h at 37 ℃. Five washes with 300. Mu.L/well PBST and then Anti-Human IgG4-HRP Mouse monoclonal antibody (purchased from Sigma, cat# SAB 4200770) diluted with 2% BSA-PBST 1:5000 were added and incubated for 1h at 37 ℃. The TMB development kit developed, 100. Mu.l/well, developed for 15min at room temperature, and then stopped with 2M H 2SO4. The microplate reader reads at 450nm/630nm and calculates the corresponding IC50 values as follows:
Through the data described above and as shown in FIG. 6, the 4 different murine antibodies screened out all competed with the receptor protein IL-11RA, demonstrating that they all effectively inhibited the binding of IL-11 to the receptor protein IL-11 RA.
EXAMPLE 8 murine antibody inhibits IL-11 binding to BaF/3-IL-11RA cell surface IL-11RA receptor
BaF/3-IL-11RA cell lines were counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6cells/mL, 100. Mu.L/well, and added to a 96-well plate. IL-11-mFc ligand protein was diluted in PBS and prepared to a concentration of 18. Mu.g/mL, 50. Mu.L/well, and added to the corresponding position of 96-well plates containing BaF/3-IL-11RA cells. After gentle mixing, 96-well plates were placed at 4 ℃ and incubated for 1h. 4 murine antibody molecules MA-I, MA-II, MA-III and MA-IV were diluted in PBS in a gradient of 800. Mu.g/mL initial concentration, diluted in a 3-fold gradient, 10 gradients in total, 50. Mu.L/well, and added to the corresponding positions of 96-well plates containing BaF/3-IL-11RA cells and IL-11-mFc ligand protein cocktail. After mixing well, the mixture was incubated at 4℃for 2h. After the incubation, the cells were centrifuged at 3000rpm, washed once with PBS buffer, and the cell pellet was collected. The pre-formulated coat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech under the trade designation 1030-02) was added to the cell pellet, after incubation at 4℃for 30min, centrifugation at 3000rpm, washing with PBS buffer once, re-suspension with 100. Mu. LPBS buffer, and detection on a flow cytometer was performed, and fluorescence signals in the FL1-A channel were collected. Dose-response curves were plotted and corresponding IC50 values were calculated, with the following specific data:
As can be seen from the above data and FIG. 7, 4 different murine candidate molecules were screened to be able to effectively inhibit the binding of IL-11 ligand protein to cell surface IL-11RA receptor.
EXAMPLE 9 murine antibody inhibits the secretion of TIMP-1 by embryonic lung fibroblast MRC-5
Embryonic lung fibroblasts (MRC-5) were counted after pancreatin digestion, a certain number of cells were taken, centrifuged, and the cells were resuspended in MEM complete medium (purchased from GIBCO under the trade designation 10370-021) and the cell density was adjusted to 2E+5cells/mL, 100. Mu.L/well, and added to 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium to a concentration of 16. Mu.g/mL and 50. Mu.L/well was added to the corresponding 96-well plate. MEM complete medium gradient dilution of 4 murine antibody molecules MA-I, MA-II, MA-III, MA-IV, preparation of initial concentration 40 u g/mL,2 times gradient dilution, total dilution of 8 gradients, 50 u L/hole, adding into a 96-well plate containing cell suspension and IL-11-mFc ligand protein suspension, gently mixing, and placing in a 37 DEG CCO 2 incubator for overnight incubation for about 20h. Cell culture supernatants were assayed using TIMP-1 ELISA kit (purchased from Ekesai Biotechnology Co., ltd.) under the designation EH 021-96.
Human TIMP-1 assay kit, cell supernatants and standards were added to sample wells, 100. Mu.L/well. Biotinylated antibody working solution (1:100 dilution) was immediately added, 50. Mu.L/well, covered with a plate membrane, and incubated with shaking at room temperature for 2h. After the incubation, the plates were washed 4 times, and 100. Mu.L/well of enzyme conjugate working solution (1:100 dilution) was added to the TIMP-1 assay kit. Covering a sealing plate membrane, and vibrating at room temperature for incubation for 1h. After incubation, the plates were washed 4 times with wash solution. TMB color development was added, 100. Mu.L/well incubated at room temperature for about 15 minutes in the dark, and 100. Mu.L/well Stop solution was used to terminate the reaction. The microplate reader reads at 450nm and calculates the corresponding IC50 values as follows:
From the above data and FIG. 8, it can be seen that the 4 different murine candidate molecules screened out are each capable of effectively inhibiting IL-11 ligand protein from stimulating human embryonic lung fibroblast MRC-5 to release TIMP-1.
Example 10
The invention of example 10 further defines an anti-IL-11 monoclonal antibody as a chimeric antibody molecule, the chimeric antibody molecule further comprising a human antibody constant region comprising a human antibody heavy chain constant region and a human antibody light chain constant region, the amino acid sequence of the human antibody heavy chain constant region being one of SEQ ID No. 27, SEQ ID No. 28 or SEQ ID No. 29, and the amino acid sequence of the human antibody light chain constant region being SEQ ID No. 30.
SEQ ID No. 27 (heavy chain constant region amino acid sequence of human IgG1 type):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
SEQ ID No. 28 (heavy chain constant region amino acid sequence of human IgG2 type):
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
SEQ ID No. 29 (heavy chain constant region amino acid sequence of human IgG4 type):
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;
30 (light chain constant region amino acid sequence of human C k chain):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.
EXAMPLE 11 preparation of chimeric antibody molecule antibody
Example 11 of the present invention further defines, on the basis of example 10, that the human antibody constant region includes a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown in SEQ ID No. 27) and a human C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No. 30).
The specific preparation method comprises the following steps:
The heavy chain variable region VH (SEQ ID No: 16) and the MA-I light chain variable region VL genes (SEQ ID No: 17) of the murine antibody molecule MA-I, MA-II obtained by screening the immune phage antibody library of example 2 and the light chain variable region VL genes (SEQ ID No: 18) of MA-II were kept unchanged in murine sequences and cloned into vectors pTSE (shown in FIG. 3) harboring heavy chain constant region and light chain constant region genes, respectively, the heavy chain constant region being human IgG1 type (amino acid sequence shown as SEQ ID NO: 27) and the light chain constant region being human C k type (amino acid sequence shown as SEQ ID NO: 30). HEK293E cells (purchased from basic medical institute of China medical sciences, accession number GNHu) were transiently transfected and antibody expression was performed to obtain chimeric antibody CA-I, CA-II.
EXAMPLE 12 humanization of murine antibody molecules
First, the sequence of the murine antibody molecule MA-I, MA-II in example 2 was selected and compared with a human antibody germline database (v-base), and human antibody light and heavy chain germlines with higher homology were searched as candidate sequences, and then the sequence of the CDR of the murine antibody molecule MA-I, MA-II was transplanted onto the human candidate sequences for homology modeling. The back mutations of the humanized antibodies were then designed by three-dimensional structure modeling to calculate key framework amino acid residues that might play an important role in maintaining the CDR loop structure. The designed light chain variable region sequence and heavy chain variable region sequence of the humanized antibody containing the reverse mutation are respectively and optimally synthesized by Nanjing Jinsri biotechnology Co., ltd, then are connected to a transient expression vector, and the humanized light chain and heavy chain combined analysis is carried out on the humanized light chain and heavy chain combined analysis, wherein MA-I obtains humanized anti-IL-11 monoclonal antibody molecules, HA-I-A, HA-I-B, HA-I-C, HA-I-D, MA-II obtains humanized antibody molecules, HA-II-A, HA-II-B, HA-II-C, HA-II-D, and 8 monoclonal antibody sequences selected by the screening are as follows:
Specifically, SEQ ID No. 31 (amino acid sequences of the heavy chain variable regions of HA-I-A, HA-I-C, HA-II-A and HA-II-B):
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVSS PEAMDYWGQGTLVTVSS;
SEQ ID No. 32 (amino acid sequence of the light chain variable region of HA-I-A):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;
SEQ ID No. 33 (amino acid sequences of the heavy chain variable regions of HA-I-B and HA-II-C):
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVST ISDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGGYVS SPEAMDYWGQGTLVTVSS;
34 (amino acid sequence of light chain variable region of HA-I-B):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;
SEQ ID No. 35 (amino acid sequence of the light chain variable region of HA-I-C):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;
SEQ ID No. 36 (amino acid sequences of the heavy chain variable regions of HA-I-D and HA-II-D):
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMFWVRQAPGKGLEWVATI SDGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARDGGYVS SPEAMDYWGQGTSVTVSS;
37 (amino acid sequence of light chain variable region of HA-I-D):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;
SEQ ID No. 38 (amino acid sequence of the light chain variable region of HA-II-A):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;
SEQ ID No. 39 (amino acid sequences of the light chain variable regions of HA-II-B and HA-II-C):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK;
SEQ ID No. 40 (amino acid sequence of the light chain variable region of HA-II-D):
DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGTVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGGGTKVEIK.
Example 13
In example 13 of the present invention, on the basis of example 12, it is further defined that the constant region of the human antibody comprises a constant region of a heavy chain of the human antibody and a constant region of a light chain of the human antibody, the constant region of the heavy chain of the human antibody has an amino acid sequence shown in SEQ ID No. 27, SEQ ID No. 28 or SEQ ID No. 29, and the constant region of the light chain of the human antibody has an amino acid sequence shown in SEQ ID No. 30.
The specific sequence of the constant region of the above-mentioned human antibody was the same as in example 10.
EXAMPLE 14 preparation of humanized antibody molecules
Example 14 of the present invention further defines, on the basis of example 13, that the constant region of the human antibody comprises a heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown in SEQ ID No. 27) and a light chain constant region of human C k type (the amino acid sequence of which is shown in SEQ ID No. 30).
The 8 humanized anti-IL-11 monoclonal antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained in example 12 were cloned into vector pTSE (shown in FIG. 3) harboring heavy chain constant region and light chain constant region genes, respectively, the heavy chain constant region being human IgG1 type (amino acid sequence shown as SEQ ID NO: 27) and the light chain constant region being C k chain (amino acid sequence shown as SEQ ID NO: 30).
The 2 chimeric antibodies CA-I and CA-II obtained in example 11 and the 8 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D obtained in example 12 were transiently transfected into HEK293 cells (purchased from basic medical institute of China medical sciences, accession number GNHu), respectively, were subjected to antibody expression, and were purified by protein A affinity column using AKTA instrument to obtain monoclonal antibodies, while protein concentration was measured using BCA kit (purchased from Beijing Hui Oriental technologies Co., ltd., accession number: BCA 0020), followed by SDS-PAGE to identify the size of the protein, and the results were shown in FIG. 9, as a result of non-reduced protein molecular weight HA-I-A, HA-I-B, HA-I-C, HA-I-D, and the reduced protein molecular weight CA-II prepared in example 11 were purified by protein A affinity column, and were identical to those of the chimeric antibodies CA-II, HA-38II-A, HA-38-II, each of which were large and 38-II, respectively.
EXAMPLE 15 humanized antibody molecules binding experiments to IL-11
IL-11 antigen was coated with carbonate buffer at pH9.6, 100 ng/well/100. Mu.L, at 4℃overnight. Five washes with 300. Mu.L/well PBST and 1% BSA-PBST 280. Mu.L/well was added and blocked at 37℃for 1h. Humanized antibody HA-I-A, HA-I-B, HA-I-C, HA-I-D, HA-II-A, HA-II-B, HA-II-C, HA-II-D and chimeric antibody CA-I, CA-II prepared in example 11 were diluted 1% BSA-PBST, the initial concentration of humanized antibody was 10 μg/mL, 8 gradients were added in 5-fold gradient dilutions, and incubated for 1h at 37 ℃. Five washes with 300. Mu.L/well PBST and then goat anti Human IgG Fab HRP (available from invitrogen, cat# 31482) diluted with 1% BSA-PBST 1:5000 were added and incubated for 1h at 37 ℃. The TMB development kit developed, 100. Mu.L/well, developed for 5min at room temperature, and then stopped with 2M H 2SO4. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value, the specific data are as follows:
As shown in FIG. 10, 8 different humanized antibody molecules can be combined with IL-11, the EC50 value of the HA-I-A, HA-I-B, HA-I-C, HA-I-D humanized antibody molecule is close to that of the chimeric antibody CA-I, and the EC50 value of the HA-II-A, HA-II-B, HA-II-C, HA-II-D humanized antibody molecule is close to that of the chimeric antibody CA-II, so that the humanized antibody molecule retains the high combination capacity of the murine parent antibody MA-I, MA-II and IL-11.
EXAMPLE 16 humanized antibody molecules inhibit IL-11 binding to BaF/3-IL-11RA cell surface IL-11RA receptor
The 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D with better protein level binding activity are selected for cell activity evaluation experiments. BaF/3-IL-11RA cell lines were counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6cells/mL, 100. Mu.L/well, and added to a 96-well plate. IL-11-mFc ligand protein was diluted in PBS and prepared to a concentration of 18. Mu.g/mL, 50. Mu.L/well, and added to the corresponding position of 96-well plates containing BaF/3-IL-11RA cells. After gentle mixing, 96-well plates were placed at 4 ℃ and incubated for 1h. 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were diluted in PBS buffer gradient, initial concentration was made 800. Mu.g/mL, 3-fold gradient dilution was performed, total 10 gradients, 50. Mu.L/well, and 96-well plates containing BaF/3-IL-11RA cells and IL-11-mFc ligand protein cocktail were added in corresponding positions. After mixing well, the mixture was incubated at 4℃for 2h. After the incubation, cells were washed once with 3000rpm centrifugation of PBS buffer and cell pellet was collected. The pre-formulated coat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech under the trade designation 1030-02) was added to the cell pellet, after incubation at 4℃for 30min, the pellet was washed once at 3000rpm by centrifugation, resuspended at 100. Mu. LPBS, and then detected by flow cytometry on-line to collect the fluorescent signal in the FL1-A channel. Dose-response curves were plotted and corresponding IC50 values were calculated, with the following specific data:
As can be seen from the above data and FIG. 11, the 4 humanized candidate molecules screened out each inhibit the binding of IL-11 ligand protein to the BaF/3-IL-11RA cell surface IL-11RA receptor.
EXAMPLE 17 humanized antibody molecules inhibit IL-11 binding to BaF/3-GP130 cell surface GP130 receptor
BaF/3-GP130 cell lines were counted, a certain number of cells were taken, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 1E+6cells/mL, 100. Mu.L/well, and added to a 96-well plate. IL-11-mFc ligand protein was diluted in PBS and prepared to a concentration of 12. Mu.g/mL, 50. Mu.L/well, and added to the corresponding position of 96-well plates containing BaF/3-GP130 cells. After gentle mixing, 96-well plates were placed at 4 ℃ and incubated for 1h. 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were diluted in PBS buffer gradient, initial concentration was prepared at 2000. Mu.g/mL, 2-fold gradient dilution was performed, 10 gradients total, 50. Mu.L/well, and 96 well plates containing BaF/3-GP130 cells and IL-11-mFc ligand protein were added at corresponding positions. After mixing well, 96-well plates were incubated at 4 ℃ for 2h. After the incubation, cells were washed once with 3000rpm centrifugation of PBS buffer and cell pellet was collected. The pre-formulated coat anti-mouse IgG Human ads-FITC antibody (purchased from SouthernBiotech under the trade designation 1030-02) was added to the cell pellet and incubated at 4℃for 30min at 100. Mu.L/well. After centrifugation of the PBS buffer at 3000rpm, 100. Mu.L/well of PBS buffer was used to resuspend the cells, and the fluorescence signal in the FL1-A channel was collected by on-line detection by a flow cytometer. Dose-response curves were plotted and corresponding IC50 values were calculated, with the following specific data:
From the above data and FIG. 12, it can be seen that each of the 4 selected humanized candidate molecules is capable of blocking the binding of IL-11 ligand protein to the BaF/3-GP130 cell surface GP130 receptor.
Example 18 detection of biological Activity of humanized antibody molecules (reporter Gene)
The BaF/3-IL-11RA-GP130-STAT3-Luc engineering cell lines were counted, the cell density was adjusted to 2E+6cells/mL by using a sample dilution (the components of which include 90% IMDM, 10% FBS, 10ng/mL mouseIL-3), and after gentle mixing, the cell solution was added to a 96-well plate at 50. Mu.L/well. 4 humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D are respectively diluted to an initial concentration of 200 mug/ml by using sample diluent, and subjected to 5-time gradient dilution, 10 gradients total, 100 mug/hole are added into corresponding positions of a 96-well plate containing engineering cell strains, and two multiple wells are arranged for each sample concentration. The IL-11 protein was prepared at a concentration of 10. Mu.g/mL and 50. Mu.L/well in a 96-well plate containing the engineered cell line and the humanized antibody molecule. The cell culture plates were gently mixed and incubated in a 37℃CCO 2 incubator for 6h. The supernatant was centrifuged off, lysate was added, 10. Mu.L/well was added to 384-well plates, an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., ltd., product No. E2610) was added, the reaction was carried out at room temperature for 5min, the fluorescence values were read under a microplate reader, and the corresponding IC50 values were calculated as follows:
through the above data and as shown in FIG. 13, the 4 humanized antibody molecules screened each blocked IL-11 binding to IL-11RA and GP130 receptors, inhibiting signaling pathway transduction.
EXAMPLE 19 humanized antibody molecules inhibit the secretion of TIMP-1 by embryonic lung fibroblast MRC-5
Embryonic lung fibroblasts (MRC-5) were counted after pancreatin digestion, a certain number of cells were taken, centrifuged, resuspended in MEM complete medium, and the cell density was adjusted to 2E+5cells/mL, 100. Mu.L/well, and plated in 96-well plates. IL-11-mFc ligand protein was diluted in MEM complete medium, prepared at a concentration of 16. Mu.g/mL, 50. Mu.L/well, and added to the corresponding 96-well plate. The four humanized antibody molecules HA-I-A, HA-I-B, HA-I-C and HA-I-D were diluted in gradient in MEM complete medium at an initial concentration of 40. Mu.g/mL, 3-fold gradient dilution, total of 8 gradients, 50. Mu.L/well, added to 96-well plates containing cell suspension and IL-11-mFc ligand protein suspension, gently mixed, incubated overnight in a 37℃CCO 2 incubator for about 20 hours, and cell culture supernatants were assayed using TIMP-1 ELISA kit (method as in example 9). The microplate reader reads at 450nm and calculates the corresponding IC50 values as follows:
From the above data and FIG. 14, the 4 humanized antibody molecules screened were each effective in inhibiting IL-11 ligand protein from stimulating human embryonic lung fibroblast MRC-5 to release TIMP-1.
EXAMPLE 20 humanized antibody molecules Cross-binding experiments with IL-11 of different species
And selecting humanized antibody molecule HA-I-A with better protein level and function detection activity to carry out cross-binding detection with IL-11 of different species. Human IL-11, mouse IL-11 (purchased from Beijing Yiqiao Shenzhou technologies Co., ltd., cat# 50117-MNCE), rat IL-11 (purchased from Kang Lang organism, cat# KL40001 Ra), cynomolgus monkey IL-11 (purchased from Yiqiao Shenzhou, cat# 90925-CNCE) were coated with 100 ng/well/100. Mu.L, respectively, overnight at a temperature of 4 ℃. Five washes with 300. Mu.L/well PBST, followed by 1% BSA-PBST, 280. Mu.L/well, were performed and blocked at 37℃for 1h. The humanized antibody HA-I-A was diluted 1% BSA-PBST at an initial concentration of 50. Mu.g/mL, 5-fold gradient dilution, 9 total gradients, two duplicate wells per gradient, 100. Mu.L/well added to 96-well plates and incubated at 37℃for 1h. Five washes with 300. Mu.L/well PBST, 1% BSA-PBST dilution goat anti Human IgG Fab HRP (purchased from invitrogen, cat# 31482), working solution concentration 1:5000, 100. Mu.L/well were added to 96-well plates and incubated for 1h at 37 ℃. The reaction was performed five times with 300. Mu.L/well PBST, developed with TMB development kit, 100. Mu.L/well, developed for 5min at room temperature in the dark, and then stopped with 2M H 2SO4. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value, the specific data are as follows:
From the above data and as shown in FIG. 15, the humanized antibody molecule HA-I-A was able to bind to human IL-11, mouse IL-11, rat IL-11, cynomolgus monkey IL-11 with high affinity.
EXAMPLE 21 therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on pulmonary fibrosis
The therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on pulmonary fibrosis was studied using bleomycin (bLF) modeling.
Animal species C57BL/6J mice (purchased from Jiangsu Ji Yikang Biotechnology Co., ltd.)
Number, sex and age of mice are 6/group, male, 6-8 weeks;
the control group was injected with physiological saline only;
the dosing group was given HA-I-a antibody molecules twice weekly for 4 weeks.
Animal body weight was measured once a week and observed for abnormalities, organ weight detection, which was performed by collecting lung organs, measuring the weight of the lung organs, calculating the lung to body weight ratio, and lung pathology detection, which was performed by slicing the lung and observing the degree of pulmonary fibrosis using Hematoxylin Eosin (HE) and masson staining.
The results are shown in FIG. 16, in which the lung to body weight ratio of mice in the administered group is significantly lower than that in the control group, and in which lung fibrosis is significantly reduced in the lung section of the administered group compared to the control group, as shown in FIG. 17, and thus it can be demonstrated that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule is effective in inhibiting the production of lung fibrosis.
EXAMPLE 22 therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on cardiac fibrosis
The therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on cardiac fibrosis was investigated using isoprenaline modeling.
Animal species C57BL/6J mice (purchased from Jiangsu Ji Yikang Biotechnology Co., ltd.)
Number, sex and age of mice are 6/group, male, 6-8 weeks;
the control group was injected with physiological saline only;
the dosing group was given HA-I-a antibody molecules twice weekly for 4 weeks.
Animal body weight was measured once a week and observed for abnormalities, organ weight detection, heart was collected, heart weight was measured, heart to body weight ratio was calculated, heart pathology detection, heart section and heart fibrosis degree was observed using Hematoxylin Eosin (HE) and masson staining.
The results are shown in FIG. 18, in which the ratio of heart to body weight of mice in the administered group is significantly smaller than that in the control group, and in which the cardiac section of the administered group shows significantly reduced cardiac fibrosis compared to the control group, thus demonstrating that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule is effective in inhibiting the production of cardiac fibrosis.
EXAMPLE 23 therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on renal fibrosis
The therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on renal fibrosis was investigated using doxorubicin (dKF) modeling.
Animal species BALB/c mice (purchased from Jiangsu Ji Yikang Biotechnology Co., ltd.)
Number, sex and age of mice are 6/group, male, 6-8 weeks;
the control group was injected with physiological saline only;
the dosing group was given HA-I-a antibody molecules twice weekly for 4 weeks.
Animal body weight was measured once a week and observed for abnormalities, organ weight measurement, heart collection, kidney weight measurement, urine protein content detection, kidney pathology detection, kidney section and kidney fibrosis degree observation using Hematoxylin Eosin (HE) and masson staining.
The results are shown in FIG. 20, in which the urine protein content in the kidneys of the mice in the administration group is significantly lower than that in the control group, and in which the kidney section in the administration group shows significantly reduced kidney fibrosis compared to the control group, as shown in FIG. 21, thus it can be demonstrated that the anti-IL-11 monoclonal antibody HA-I-A antibody molecule can effectively inhibit the generation of kidney fibrosis.
EXAMPLE 24 therapeutic efficacy experiment of anti-IL-11 monoclonal antibody on liver fibrosis
The therapeutic effect of the anti-IL-11 monoclonal antibody HA-I-A on liver fibrosis was studied using CCl 4 modeling.
Animal species C57BL/6J mice (purchased from Jiangsu Ji Yikang Biotechnology Co., ltd.)
Number, sex and age of mice are 6/group, male, 6-8 weeks;
the control group was injected with physiological saline only;
the dosing group was given HA-I-a antibody molecules twice weekly for 4 weeks.
Body weight monitoring, which is to measure animal body weight once a week and observe the presence or absence of abnormality of the animal, liver pathology detection, which is to cut liver and observe the degree of liver fibrosis by Hematoxylin Eosin (HE) and masson staining, organ weight detection, which is to collect kidneys, measure liver weight and conduct HE staining, and serum detection, which is to collect serum and detect alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels in mouse serum.
As shown in fig. 22, the liver weight of mice in the administered group was significantly lower than that in the control group, as shown in fig. 23, the ALT and AST levels in the serum of mice in the administered group were significantly lower than those in the control group, and as shown in fig. 24, liver sections of the administered group showed significantly reduced liver fibrosis compared to the control group, thus demonstrating that the anti-IL-11 monoclonal antibody HA-I-a antibody molecule was able to effectively inhibit the generation of liver fibrosis.
EXAMPLE 25 evaluation of thermal stability of anti-IL-11 monoclonal antibody HA-I-A
The thermostability of the anti-IL-11 monoclonal antibody HA-I-A was evaluated using a multifunctional protein thermostability analysis system (purchased from Unchained Labs). Protein conformational stability was assessed by monitoring protein intrinsic fluorescence over temperature (starting at 25 ℃ and increasing to 95 ℃ at a rate of 0.3 ℃ per minute) to determine protein melting temperature Tm. When the sample is aggregated, the scattered light waves interfere, the scattered light signals increase, and the colloidal stability (characterized by Tagg) of the protein is measured by static light scattering, and the results are shown in the following table and fig. 25.
The melting temperature Tm of the anti-IL-11 monoclonal antibody HA-I-A is 79.5 ℃, and the average Tagg is 75.9 ℃, so that the anti-IL-11 monoclonal antibody HA-I-A HAs better conformational stability and colloid stability.
Example 26
The anti-IL-11 monoclonal antibody HA-I-A obtained in the above example was purified by the following method:
S1, performing affinity chromatography, namely balancing an affinity chromatography column through a buffer solution A, wherein the filler of the affinity chromatography column is MabSelect Sure LX, the buffer solution A is 30mM HEPES buffer solution, the pH value of the buffer solution A is 7, the conductivity of the buffer solution A is 10mS/cm, S102, loading a target protein containing an anti-IL-11 monoclonal antibody HA-I-A onto the affinity chromatography column at the flow rate of 100cm/h, the loading capacity of the target protein is 20mg protein/ml filler, the height of the column bed of the affinity chromatography column is 18cm, S103, after loading is finished, the buffer solution A is used for re-balancing, the buffer solution B is used for at least one time of leaching until the curve of the ultraviolet absorption value is reduced to be stable, the buffer solution A is used for a second time leaching, the buffer solution B is 30mM HEPES buffer solution, the pH value of the buffer solution is 5, the buffer solution C is used for eluting the affinity chromatography column, the protein product is collected after eluting, the loading capacity of 20mM protein-glycine is used for the buffer solution C, the pH value of the buffer solution is 3, the protein is adjusted to the pH value of 3, and the pH value of the protein is adjusted to be 3, namely the pure protein is adjusted to the pH value of the solution is 5, and the crude protein is adjusted to the pH value of the solution is adjusted to be 1, and the pH value of the protein is adjusted to the pH value of the pure solution is adjusted to the pH value to be 1, and the pH value is adjusted to the pH value of the pure solution is1 to the pH solution is 1.
S2, anion exchange chromatography, wherein a 2CV buffer solution D is used for balancing an anion exchange chromatography column, the filler of the anion exchange chromatography is Capto Q, the buffer solution D is a citric acid buffer solution with the pH value of 6.5, S202, a crude and pure protein sample is loaded on the anion exchange chromatography column at the flow rate of 100cm/h, the loading capacity is 20mg of protein/ml filler, the height of the column bed of the anion exchange chromatography column is 18cm, S203 is used for balancing the anion exchange chromatography column again after loading is finished, and the flow-through liquid is collected for standby;
S3, cation exchange chromatography, wherein a 2CV buffer solution E is used for balancing a cation exchange chromatography column, the packing of the cation exchange chromatography column is Capto SP, the buffer solution E is 30mM citric acid buffer solution, the pH value is 5.0, the conductivity is 2mS/cm, S302, the flow through liquid after secondary purification is loaded on the cation exchange chromatography column at the flow rate of 100cm/h, the loading capacity is 20mg protein/ml packing, the column bed height of the cation exchange chromatography column is 18cm, S303, the cation exchange chromatography column is eluted by using an eluent F, and the protein solution of the high-purity anti-IL-11 monoclonal antibody is obtained after elution, wherein the buffer solution F is 30mM citric acid buffer solution and 80mM NaCl, and the pH value is 5.0.
Example 27
The anti-IL-11 monoclonal antibody HA-I-A obtained in the above example was purified by the following method:
S1, performing affinity chromatography, namely balancing an affinity chromatography column through buffer A, wherein the packing of the affinity chromatography column is AT Protein A Diamond Plus, the buffer A is 50mM Tris-HCl buffer, the Tris-HCl buffer contains 120-160mM NaCl, the preferred embodiment is 150mM NaCl, the pH value of the buffer A is 7.4, the conductivity is 18mS/cm, S102, loading a target protein containing an anti-IL-11 monoclonal antibody HA-I-A onto the affinity chromatography column at the flow rate of 150cm/h, the loading capacity is 55mg protein/ml packing, the column bed height of the affinity chromatography column is 20cm, S103, after loading is finished, the buffer A is used for rebalancing, at least one time of elution is performed through buffer B of 4CV, after the curve of the ultraviolet absorption value is reduced to be stable, the buffer A is used for a second time, the buffer B is an acetate buffer of 50mM, the pH value of the buffer is 5.5, the buffer C is used for the buffer C, the pH value of the buffer C is used for the buffer C, the pH value of the buffer is adjusted to be 3, and the pH value of the buffer C is adjusted to be 3, and the pH value of the buffer is adjusted to be 3.2, and the pH value of a product is obtained after the buffer is used for the buffer is adjusted to be the pH value of the buffer is 3, and the pH value of the pure protein is adjusted to be the pH value of the buffer is 3, and the pH value of the product is adjusted to be the pH value of the buffer is adjusted to be 2 to be the pH value of the buffer 2 and the pH value is used to be the pH 2 and the product is adjusted to be the pH value is used to be the pH 2 and the pH is used to 2 and the pH is used to 2 for the 2 is used for the 2 and is subjected to the 2 to the for the 2 to for 2.
S2, anion exchange chromatography, wherein a 4CV buffer solution D is used for balancing an anion exchange chromatography column, the filler of the anion exchange chromatography is NanoGel-50Q, the buffer solution D is phosphate buffer solution with the pH value of 7, S202, a crude and pure protein sample is loaded on the anion exchange chromatography column at the flow rate of 150cm/h, the loading capacity is 70mg protein/ml filler, the height of the column bed of the anion exchange chromatography column is 20cm, S203 is used for balancing the anion exchange chromatography column again after loading is finished, and the flow-through liquid is collected for standby;
S3, cation exchange chromatography, wherein 3CV buffer solution E is used for balancing the cation exchange chromatography column, the packing of the cation exchange chromatography column is NanoGel-50SP, the buffer solution E is 50mM acetate buffer solution, the pH value is 6, the conductivity is 2.5mS/cm, S302, the flow through liquid after secondary purification is loaded on the cation exchange chromatography column at the flow rate of 150cm/h, the loading capacity is 47mg protein/ml packing, the bed height of the cation exchange chromatography column is 20cm, S303, the eluting solution F is used for eluting the cation exchange chromatography column, and the protein solution of the high-purity anti-IL-11 monoclonal antibody is obtained after eluting, wherein the buffer solution F is 50mM acetate buffer solution and 115mM NaCl, and the pH value is 5.8.
Example 28
The anti-IL-11 monoclonal antibody HA-I-A obtained in the above example was purified by the following method:
S1, performing affinity chromatography, namely balancing an affinity chromatography column through a buffer solution A, wherein the filler of the affinity chromatography column is Eshmuno A, the buffer solution A can be replaced by NMab Pro, the buffer solution A is phosphate buffer solution with the pH value of 60mM, the conductivity of the buffer solution A is 30mS/cm, S102, loading target protein containing an anti-IL-11 monoclonal antibody onto the affinity chromatography column at the flow rate of 300cm/h, the loading amount is 60mg of protein/ml filler, the height of a column bed of the affinity chromatography column is 22cm, S103, after loading is finished, the buffer solution A is used for re-balancing, the elution is performed at least once through a buffer solution B with the pH value of 6CV until the curve of the ultraviolet absorption value is reduced to be stable, the washing is stopped, the buffer solution A is used for the second time, the buffer solution B is Tris-HCl buffer solution with the pH value of 60mM, the pH value of 6S 104 can be selected, the buffer solution C is used for eluting the affinity chromatography column, the elution is performed with the loading amount of 60mg of protein/ml filler, the column bed height is 22cm, the pH value of the protein C is adjusted to be the pH value of 4, the pH value of the pure protein is adjusted to be the pH value of 5, and the pH value of the pure protein is adjusted to be the pH value of the pure protein is 5, and the pH value is adjusted to be the pH value of the pure protein is 5, and the product is adjusted to be the pH value of the pure protein is at the pH value of the pH value is at the pH value of 5 and is at the condition of 5 and is at the conditions of 5 and is adjusted to be at the pH 1.
S2, anion exchange chromatography, wherein a 6CV buffer solution D is used for balancing an anion exchange chromatography column, the filler of the anion exchange chromatography is Capto sphere, or can be replaced by Eshmuno Q, the buffer solution D is acetate buffer solution with the pH value of 7.5, S202, a crude and pure protein sample is loaded on the anion exchange chromatography column at the flow rate of 200cm/h, the loading capacity is 120mg protein/ml filler, the height of the bed of the anion exchange chromatography column is 22cm, S203, after loading is finished, the buffer solution D is used for balancing the anion exchange chromatography column again, and the flow-through liquid is collected for standby;
S301, balancing a cation exchange chromatographic column by using a 6CV buffer solution E, wherein the buffer solution E is 60mM phosphate buffer solution with the pH value of 6.0 and the conductivity of 5mS/cm, S302, loading the secondarily purified flow through solution on the cation exchange chromatographic column at the flow rate of 200cm/h, loading 75mg of protein/ml of the buffer solution E, eluting the cation exchange chromatographic column by using an eluent F, and obtaining a protein solution of the high-purity anti-IL-11 monoclonal antibody after eluting, wherein the buffer solution F is 60mM phosphate buffer solution and 150mM NaCl, and the pH value is 6.0.
Example 29
Example 29 of the present invention in step S105, which further defines the method of purifying an anti-IL-11 monoclonal antibody based on example 27, the acid solution during the inactivation treatment is preferably acetic acid.
Examples 30 to 32
Examples 30 to 32 of the present invention further define the purification method of an anti-IL-11 monoclonal antibody based on example 29, wherein in step S105, the pH of the protein product was adjusted back to the following pH and conductivity range with 1M Tris buffer to obtain a crude and pure protein sample, and the other methods are the same as in example 27, and the specific data are as follows.
Examples PH value of Sample conductivity (mS/cm)
Example 30 6.5 5
Example 31 7.5 5
Example 32 7.0 4
Examples 33 to 35
Examples 33 to 35 of the present invention further define the eluent F of step S303 in the purification method of an anti-IL-11 monoclonal antibody on the basis of example 32, and the other methods and parameters are all the same as those of example 27, specifically as follows.
Comparative example 1
The purification method provided in comparative example 1 exchanges the anion exchange chromatography in step S2 with the cation exchange chromatography in step S3 in this order on the basis of example 35. Other methods and parameters were all the same as in example 35.
Comparative example 2
Comparative example 2 according to the present invention was based on example 35 using a classical three-step purification process of monoclonal antibodies against the IL-11 monoclonal antibody HA-I-A, all the fillers used for three-step chromatography being produced by Cytiva. The packing of the first affinity column was MabSelectSuRe LX, the packing of the second anion exchange column was Capto Q, the packing of the third cation exchange column was Capto SP ImpAct, and all other methods and parameters were the same as in example 35.
Physicochemical detection of Experimental first anti-IL-11 monoclonal antibody and detection of related impurities
The anti-IL-11 monoclonal antibody HA-I-A obtained by the purification method provided by the embodiment and the comparison example of the invention uses the gel chromatography technical means to detect the purity, analyzes the aggregate, monomer and degradation product contents of a sample in the purification process, uses the ion chromatography technical means to analyze the acid-base peak content of a charge isomer, adopts a special kit to detect the content of impurities related to the process, and simultaneously calculates the total recovery rate by the following formula, wherein the total recovery rate=affinity chromatography protein yield (%) and anion exchange chromatography protein yield (%) and cation exchange chromatography protein yield (%), and the specific data are as follows:
The experimental data show that the anti-IL-11 monoclonal antibody purified by the purification method provided by the embodiment of the invention has the SEC purity of more than 99 percent and the total protein yield of more than 75 percent, and the anti-IL-11 monoclonal antibody stock solution obtained by the purification method provided by the embodiment 35 has the highest purity and yield, the total protein recovery rate of more than 81 percent, and the recovery rate can obviously improve the yield and reduce the production cost in antibody production.
Example 27 the packing and buffer compositions and amounts of the chromatography column provided in example 27 are more compatible with the purification process conditions of the anti-IL-11 monoclonal antibody HA-I-a than examples 26 and 28.
Example 29 the acid solution was acetic acid and the appearance of the inactivated sample was clear, without precipitation and with higher protein recovery than example 27.
In step S105, the pH of the protein product was adjusted back to 7.0 with 1M Tris buffer and the conductivity was 4mS/cm, as compared with examples 30-32, to significantly increase the total recovery of the anti-IL-11 monoclonal antibody HA-I-A protein.
As can be seen from a comparison of examples 33-35, the recovery of protein was higher when eluent F included 50mM acetate buffer and 100mM NaCl at pH 5.5.
Example 35 the exchange of anion exchange chromatography in step S2 with cation exchange chromatography in step S3 compared to example 1 is detrimental to the purification of the anti-IL-11 monoclonal antibody HA-I-a, and the sequence of affinity chromatography, anion exchange chromatography and cation exchange chromatography provided in example 35 is the best 3-step purification process for the purification of the anti-IL-11 monoclonal antibody HA-I-a.
Compared with example 2, the example 35 is different from the example 2 only in the packing of the chromatographic column, the purification effect of the packing provided in example 35 is obviously higher than that of the comparative example 2, the total recovery rate is high, the cost is lower, the product related impurities and the process related impurities can be effectively removed, and therefore, the purification of the anti-IL-11 monoclonal antibody HA-I-A can be obtained and is more suitable for the packing provided in example 35; the preferred affinity chromatography of the invention is AT Protein A Diamond Plus, AT Protein A Diamond Plus is taken as a filler, HAs excellent binding specificity, alkali resistance and pressure-flow velocity characteristics, lower ligand is dropped, antibody in cell supernatant can be captured, most of impurities are removed, in addition, the capacity can be obviously improved, the affinity chromatography is alkali-resistant Protein A affinity filler, more efficient cleaning and sterilization can be realized, cross contamination is avoided, the service life is prolonged, the filler of an anion exchange chromatographic column is preferably NanoGel-50Q, the filler of a cation exchange chromatographic column is preferably NanoGel-50SP, nanogel-50SP adopts a binding elution mode, namely, target Protein passes through the chromatographic column and impurities are adsorbed on the chromatographic column during sample addition, the purification purpose is effectively achieved, nanoGel-50Q and NanoGel-50SP belong to a rigid polymer matrix monodisperse matrix, the particles are uniform, the particle size is smaller, the resolution ratio is high, the separation effect is good, the combination of the fillers of different chromatographic columns can not only effectively remove HCP, DNA, protein A and other related impurities, but also can adsorb relevant impurities, the quality of the product can be remarkably improved, the quality of the product can be purified, and the product can meet the requirements of the quality of the product is obviously purified, and the quality of the product is improved.
Experimental detection of binding Activity and biological Activity of anti-IL-11 monoclonal antibodies
Aiming at the anti-IL-11 monoclonal antibody HA-I-A purified by the purification method provided by the above embodiment of the invention and comparative example 1-2, the invention adopts ELISA means to analyze the specific binding capacity of the purified anti-IL-11 monoclonal antibody HA-I-A and IL-11 so as to evaluate the activity of the purified antibody, and the detection result is as follows:
Examples Binding Activity (%) Biological Activity (%)
Example 26 86 101
Example 27 103 72
Example 28 82 99
Example 29 79 82
Example 30 84 98
Example 31 92 104
Example 32 81 79
Example 33 96 88
Example 34 105 93
Example 35 76 84
As can be seen from the table, the anti-IL-11 monoclonal antibody HA-I-A purified by the method provided by the embodiment of the invention HAs better binding activity and biological activity, and the binding activity and biological activity are within the range of 90+/-20%, which indicates that the anti-IL-11 monoclonal antibody HA-I-A purified by the method defined by the method provided by the invention HAs better biological activity under a large number of experimental condition screening.
The present application is not limited to the above-mentioned preferred embodiments, and any person who can obtain other various products under the teaching of the present application can make any changes in shape or structure, and all the technical solutions that are the same or similar to the present application fall within the scope of the present application.

Claims (11)

1.一种抗IL-11单克隆抗体的纯化方法,其特征在于,该方法包括:S1、亲和层析:初步纯化和浓缩含有抗IL-11单克隆抗体的目的蛋白,并将收集的蛋白产物在酸溶液中进行病毒灭活,得到粗纯的蛋白样品;S2、阴离子交换层析:将所述蛋白样品进行二次提纯,收集流穿液;S3、阳离子交换层析:将所述流穿液进行精纯后,即获得高纯度的抗IL-11单克隆抗体的蛋白溶液。1. A method for purifying an anti-IL-11 monoclonal antibody, characterized in that the method comprises: S1, affinity chromatography: preliminarily purifying and concentrating the target protein containing the anti-IL-11 monoclonal antibody, and inactivating the collected protein product in an acid solution to obtain a crude and pure protein sample; S2, anion exchange chromatography: performing secondary purification on the protein sample and collecting the flow-through; S3, cation exchange chromatography: after purifying the flow-through, a high-purity protein solution of the anti-IL-11 monoclonal antibody is obtained. 2.如权利要求1所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述抗IL-11单克隆抗体包括3个分别用HCDR1、HCDR2和HCDR3表示的重链互补决定区和3个分别用LCDR1、LCDR2和LCDR3表示的轻链互补决定区,所述抗IL-11单克隆抗体选自以下任意一种:2. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that the anti-IL-11 monoclonal antibody comprises three heavy chain complementary determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and three light chain complementary determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, and the anti-IL-11 monoclonal antibody is selected from any one of the following: A-I:所述重链互补决定区HCDR1的氨基酸序列如SEQ ID No:1所示,所述重链互补决定区HCDR2的氨基酸序列如SEQ ID No:2所示,所述重链互补决定区HCDR3的氨基酸序列如SEQID No:3所示,所述轻链互补决定区LCDR1的氨基酸序列如SEQ ID No:4所示,所述轻链互补决定区LCDR2的氨基酸序列如SEQ ID No:5所示,所述轻链互补决定区LCDR3的氨基酸序列如SEQ ID No:6所示;A-I: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 2, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 3, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 6; A-Ⅱ:所述重链互补决定区HCDR1的氨基酸序列如SEQ ID No:1所示,所述重链互补决定区HCDR2的氨基酸序列如SEQ ID No:2所示,所述重链互补决定区HCDR3的氨基酸序列如SEQ ID No:3所示,所述轻链互补决定区LCDR1的氨基酸序列如SEQ ID No:4所示,所述轻链互补决定区LCDR2的氨基酸序列如SEQ ID No:5所示,所述轻链互补决定区LCDR3的氨基酸序列如SEQ ID No:7所示;A-II: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 2, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 3, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 7; A-Ⅲ:所述重链互补决定区HCDR1的氨基酸序列如SEQ ID No:8所示,所述重链互补决定区HCDR2的氨基酸序列如SEQ ID No:9所示,所述重链互补决定区HCDR3的氨基酸序列如SEQ ID No:10所示,所述轻链互补决定区LCDR1的氨基酸序列如SEQ ID No:11所示,所述轻链互补决定区LCDR2的氨基酸序列如SEQ ID No:12所示,所述轻链互补决定区LCDR3的氨基酸序列如SEQ ID No:13所示;A-III: the amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 8, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 9, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 10, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 11, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 12, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 13; A-Ⅳ:所述重链互补决定区HCDR1的氨基酸序列如SEQ ID No:1所示,所述重链互补决定区HCDR2的氨基酸序列如SEQ ID No:14所示,所述重链互补决定区HCDR3的氨基酸序列如SEQ ID No:15所示,所述轻链互补决定区LCDR1的氨基酸序列如SEQ ID No:4所示,所述轻链互补决定区LCDR2的氨基酸序列如SEQ ID No:5所示,所述轻链互补决定区LCDR3的氨基酸序列如SEQ ID No:6所示。A-IV: The amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 14, the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 15, the amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No: 4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No: 6. 3.如权利要求2所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述抗IL-11单克隆抗体还包括重链可变区和轻链可变区,所述抗IL-11单克隆抗体选自以下任意一种:3. The method for purifying an anti-IL-11 monoclonal antibody according to claim 2, characterized in that the anti-IL-11 monoclonal antibody further comprises a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is selected from any one of the following: MA-Ⅰ:所述重链可变区的氨基酸序列如SEQ ID No:16所示,所述轻链可变区的氨基酸序列如SEQ ID No:17所示;MA-Ⅰ: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 17; MA-Ⅱ:所述重链可变区的氨基酸序列如SEQ ID No:16所示,所述轻链可变区的氨基酸序列如SEQ ID No:18所示;MA-Ⅱ: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 18; MA-Ⅲ:所述重链可变区的氨基酸序列如SEQ ID No:19所示,所述轻链可变区的氨基酸序列如SEQ ID No:20所示;MA-III: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 20; MA-Ⅳ:所述重链可变区的氨基酸序列如SEQ ID No:21所示,所述轻链可变区的氨基酸序列如SEQ ID No:17所示。MA-IV: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:17. 4.如权利要求3所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述抗IL-11单克隆抗体还包括重链恒定区和轻链恒定区,所述重链恒定区的氨基酸序列为SEQ ID No:23、SEQ ID No:24、SEQ ID No:25或SEQ ID No:26所示中的一种;所述轻链恒定区氨基酸序列如SEQ IDNo:22所示。4. The method for purifying an anti-IL-11 monoclonal antibody according to claim 3, characterized in that the anti-IL-11 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is one of SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25 or SEQ ID No: 26; the amino acid sequence of the light chain constant region is as shown in SEQ ID No: 22. 5.如权利要求2所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述抗IL-11单克隆抗体还包括重链可变区和轻链可变区,所述抗IL-11单克隆抗体选自以下任意一种:5. The method for purifying an anti-IL-11 monoclonal antibody according to claim 2, characterized in that the anti-IL-11 monoclonal antibody further comprises a heavy chain variable region and a light chain variable region, and the anti-IL-11 monoclonal antibody is selected from any one of the following: HA-I-A:所述重链可变区的氨基酸序列如SEQ ID No:31所示,所述轻链可变区的氨基酸序列如SEQ ID No:32所示;HA-I-A: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 32; HA-I-B:所述重链可变区的氨基酸序列如SEQ ID No:33所示,所述轻链可变区的氨基酸序列如SEQ ID No:34所示;HA-I-B: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 34; HA-I-C:所述重链可变区的氨基酸序列如SEQ ID No:31所示,所述轻链可变区的氨基酸序列如SEQ ID No:35所示;HA-I-C: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 35; HA-I-D:所述重链可变区的氨基酸序列如SEQ ID No:36所示,所述轻链可变区的氨基酸序列如SEQ ID No:37所示;HA-I-D: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 37; HA-II-A:所述重链可变区的氨基酸序列如SEQ ID No:31所示,所述轻链可变区的氨基酸序列如SEQ ID No:38所示;HA-II-A: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 38; HA-II-B:所述重链可变区的氨基酸序列如SEQ ID No:31所示,所述轻链可变区的氨基酸序列如SEQ ID No:39所示;HA-II-B: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 39; HA-II-C:所述重链可变区的氨基酸序列如SEQ ID No:33所示,所述轻链可变区的氨基酸序列如SEQ ID No:39所示;HA-II-C: the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 39; HA-II-D:所述重链可变区的氨基酸序列如SEQ ID No:36所示,所述轻链可变区的氨基酸序列如SEQ ID No:40所示。HA-II-D: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:36, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:40. 6.如权利要求5所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述抗IL-11单克隆抗体还包括人源抗体重链恒定区和人源抗体轻链恒定区,所述人源抗体重链恒定区的氨基酸序列为SEQ ID No:27、SEQ ID No:28或SEQ ID No:29所示中的一种;所述人源抗体轻链恒定区的氨基酸序列如SEQ ID No:30所示。6. The method for purifying an anti-IL-11 monoclonal antibody according to claim 5, characterized in that the anti-IL-11 monoclonal antibody further comprises a human antibody heavy chain constant region and a human antibody light chain constant region, and the amino acid sequence of the human antibody heavy chain constant region is one of SEQ ID No: 27, SEQ ID No: 28 or SEQ ID No: 29; the amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID No: 30. 7.如权利要求1所述的抗IL-11单克隆抗体的纯化方法,其特征在于,步骤S1中,所述亲和层析包括以下步骤:7. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, wherein in step S1, the affinity chromatography comprises the following steps: S101、通过缓冲液A对亲和层析柱进行平衡;S101, balancing the affinity chromatography column with buffer A; S102、将含有所述抗IL-11单克隆抗体的目的蛋白以100-300cm/h的流速上样至所述亲和层析柱上,上样载量为20-60mg蛋白/ml填料,所述亲和层析柱的柱床高度为18-22cm;S102, loading the target protein containing the anti-IL-11 monoclonal antibody onto the affinity chromatography column at a flow rate of 100-300 cm/h, with a loading capacity of 20-60 mg protein/ml filler, and the column bed height of the affinity chromatography column is 18-22 cm; S103、上样结束后,使用所述缓冲液A进行再平衡,再通过2-6CV的缓冲液B进行至少一次的淋洗洗脱,直至紫外吸收值的曲线降至平稳后,停止冲洗,再使用所述缓冲液A进行第二次淋洗;S103, after the loading is completed, the buffer A is used for re-equilibrium, and then eluted at least once with 2-6CV of buffer B until the curve of the ultraviolet absorption value drops to a stable state, then the rinsing is stopped, and the buffer A is used for a second elution; S104、使用缓冲液C对所述亲和层析柱进行洗脱,洗脱后收集所述蛋白产物备用;S104, eluting the affinity chromatography column using buffer C, and collecting the protein product after elution for later use; S105、使用所述酸溶液将收集的所述蛋白产物的pH值调节至3-4,并在此pH条件下室温孵育0.5-3h,最后用1M的Tris缓冲液将所述蛋白产物的pH回调至5-8即得到粗纯的所述蛋白样品;S105, adjusting the pH value of the collected protein product to 3-4 using the acid solution, and incubating at room temperature for 0.5-3h under this pH condition, and finally adjusting the pH of the protein product back to 5-8 using 1M Tris buffer to obtain a crude and pure protein sample; 优选的,所述亲和层析柱的填料选自MabSelect Sure LX、Eshmuno A、NMab Pro或ATProtein A Diamond Plus。Preferably, the filler of the affinity chromatography column is selected from MabSelect Sure LX, Eshmuno A, NMab Pro or ATProtein A Diamond Plus. 8.如权利要求7所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述缓冲液A选自Tris-HCl缓冲液、HEPES缓冲液或磷酸盐缓冲液,所述缓冲液A的浓度为30-60mM,且其pH值为7-8,其电导率为10-30mS/cm;所述缓冲液B选自Tris-HCl缓冲液、HEPES缓冲液、磷酸盐缓冲液、柠檬酸盐缓冲液或醋酸盐缓冲液,所述缓冲液B的浓度为30-60mM,且其pH值为5-6;所述缓冲液C选自甘氨酸-盐酸盐、柠檬酸盐或醋酸盐缓冲液,所述缓冲液C的浓度为20-200mM,且其pH为3-4。8. The method for purifying an anti-IL-11 monoclonal antibody according to claim 7, characterized in that the buffer A is selected from Tris-HCl buffer, HEPES buffer or phosphate buffer, the concentration of the buffer A is 30-60 mM, the pH value is 7-8, and the conductivity is 10-30 mS/cm; the buffer B is selected from Tris-HCl buffer, HEPES buffer, phosphate buffer, citrate buffer or acetate buffer, the concentration of the buffer B is 30-60 mM, and the pH value is 5-6; the buffer C is selected from glycine-hydrochloride, citrate or acetate buffer, the concentration of the buffer C is 20-200 mM, and the pH value is 3-4. 9.如权利要求1所述的抗IL-11单克隆抗体的纯化方法,其特征在于,步骤S2中,所述阴离子交换层析包括以下步骤:9. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, characterized in that in step S2, the anion exchange chromatography comprises the following steps: S201、使用2-6CV的缓冲液D对阴离子交换层析柱进行平衡;S201, equilibrate the anion exchange chromatography column using 2-6 CV of buffer D; S202、将粗纯得到的所述蛋白样品以100-200cm/h的流速上样至所述阴离子交换层析柱上,上样载量为20-120mg蛋白/ml填料,所述阴离子交换层析柱的柱床高度为18-22cm;S202, loading the crudely purified protein sample onto the anion exchange chromatography column at a flow rate of 100-200 cm/h, with a loading capacity of 20-120 mg protein/ml filler, and a column bed height of the anion exchange chromatography column of 18-22 cm; S203、上样结束后,再次使用所述缓冲液D对所述阴离子交换层析柱进行平衡,收集所述流穿液,备用;S203, after the sample loading is completed, the anion exchange chromatography column is equilibrated again with the buffer D, and the flow-through liquid is collected for standby use; 优选的,所述阴离子交换层析的填料选自Capto Q、Capto adhere、Eshmuno Q或NanoGel-50Q。Preferably, the filler of the anion exchange chromatography is selected from Capto Q, Capto adhere, Eshmuno Q or NanoGel-50Q. 10.如权利要求9所述的抗IL-11单克隆抗体的纯化方法,其特征在于,所述缓冲液D选自柠檬酸缓冲液、醋酸盐缓冲液或磷酸盐缓冲液,其pH值为6.5-7.5。10 . The method for purifying an anti-IL-11 monoclonal antibody according to claim 9 , wherein the buffer D is selected from a citrate buffer, an acetate buffer or a phosphate buffer, and has a pH value of 6.5-7.5. 11.如权利要求1所述的抗IL-11单克隆抗体的纯化方法,其特征在于,步骤S3中,所述阳离子交换层析包括以下步骤:11. The method for purifying an anti-IL-11 monoclonal antibody according to claim 1, wherein in step S3, the cation exchange chromatography comprises the following steps: S301、使用2-6CV的缓冲液E,对阳离子交换层析柱进行平衡;S301, using 2-6 CV of buffer E to balance the cation exchange chromatography column; S302、将二次提纯后的所述流穿液以100-200cm/h的流速上样至所述阳离子交换层析柱上,上样载量为20-75mg蛋白/ml填料,所述阳离子交换层析柱的柱床高度为18-22cm;S302, loading the flow-through liquid after secondary purification onto the cation exchange chromatography column at a flow rate of 100-200 cm/h, with a loading capacity of 20-75 mg protein/ml filler, and the column bed height of the cation exchange chromatography column is 18-22 cm; S303、使用洗脱液F对所述阳离子交换层析柱进行洗脱,洗脱后即得到高纯度的抗IL-11单克隆抗体的蛋白溶液;S303, eluting the cation exchange chromatography column with an eluent F, to obtain a high-purity anti-IL-11 monoclonal antibody protein solution after elution; 优选的,所述阳离子交换层析柱的填料为Capto SP、Capto MMC、Eshmuno S或NanoGel-50SP;Preferably, the filler of the cation exchange chromatography column is Capto SP, Capto MMC, Eshmuno S or NanoGel-50SP; 优选的,所述缓冲液E选自柠檬酸缓冲液、醋酸盐缓冲液或磷酸盐缓冲液,所述缓冲液E的浓度为30-60mM,其pH值为5.0-6.0,其电导率为2-5mS/cm;所述缓冲液F包括缓冲液E和80-150mM的添加剂,所述添加剂为NaCl。Preferably, the buffer E is selected from a citric acid buffer, an acetate buffer or a phosphate buffer, the concentration of the buffer E is 30-60 mM, the pH value is 5.0-6.0, and the conductivity is 2-5 mS/cm; the buffer F comprises buffer E and 80-150 mM of an additive, and the additive is NaCl.
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