CN113150148B

CN113150148B - Purification method of anti-IL-17 RA monoclonal antibody

Info

Publication number: CN113150148B
Application number: CN202110189506.1A
Authority: CN
Inventors: 白义; 刘晓航; 孟艳敏
Original assignee: Beijing Dongfang Baitai Biotechnology Co Ltd
Current assignee: Beijing Dongfang Baitai Biotechnology Co Ltd
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2021-10-08
Anticipated expiration: 2040-06-19
Also published as: CN111690065A; CN111690065B; CN113150148A; CN113150149B; CN113150149A

Abstract

The invention relates to the technical field of antibody engineering, and particularly provides a purification method of an anti-IL-17 RA monoclonal antibody, which comprises affinity chromatography, cation exchange chromatography and anion exchange chromatography. The anti-IL-17 RA monoclonal antibody purified by the purification method provided by the invention has stronger affinity and good bioactivity, can be specifically combined with an IL-17RA antigen, and is an autoimmune disease treatment drug with great potential.

Description

Purification method of anti-IL-17 RA monoclonal antibody

Technical Field

The invention relates to the technical field of antibody engineering, in particular to a purification method of an anti-IL-17 RA monoclonal antibody.

Background

Autoimmune diseases refer to diseases caused by the body's immune reaction to autoantigens, which results in damage to the tissues. Many diseases are subsequently classified as autoimmune diseases, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma and the like. Among them, Psoriasis (Psoriasis) is also called Psoriasis and is characterized in that the skin has erythema, scaling plaques with different sizes and clear boundaries, and is covered with a large amount of dry silvery white scales. The histological features of psoriatic skin are epidermal keratinocyte hyperproliferation, vascular proliferation, and infiltration of dendritic cells, macrophages, neutrophils, T cells, the pathogenesis of psoriasis involving a complex inflammatory response and immune system. The prevalence rate of China is about 0.47%, and more than 600 million psoriasis patients are calculated at present in China. Psoriasis, once it develops, often suffers from lifelong, recurrent episodes, with the majority of patients presenting with a course of alternating relapses and remissions. Rheumatoid Arthritis (RA) is a systemic autoimmune disease characterized by chronic erosive arthritis, and is characterized by synovitis, and the resulting destruction of articular cartilage and bone mass, ultimately leading to joint deformity and loss of function. The worldwide morbidity is about 0.5-1.0%, the national morbidity is 0.3-0.4%, and the morbidity of women is 3 times that of men generally. Ankylosing Spondylitis (AS) is a chronic inflammatory disease with inflammation of sacroiliac joints, spinal attachment points and axial bones AS main symptoms, and fibrosis and ossification of connective tissues around the annulus fibrosus and intervertebral disc and ankylosis AS pathological features, belonging to the category of rheumatism, and the cause of the disease is unknown. It is often associated with infection, genetic factors, environmental factors (e.g., long term exposure to cold, humid environments), etc.

With the continuous and intensive research of biological antibody drugs, interleukin-17 (IL-17) is a characteristic cytokine secreted by helper T cell 17(Th17 cell), which can promote the local production of chemokines, recruit neutrophils, promote the proliferation and differentiation of cells; however, it may also bind to receptors to produce a cascade-like inflammatory effect, causing tissue damage, which has been shown to be closely related to the pathogenesis of various autoimmune diseases. Antagonizing and blocking IL-17/IL-17R signal channel is an autoimmune disease treatment target with great potential, and is expected to effectively relieve the symptoms of autoimmune diseases and prevent the progress of diseases.

The IL-17 receptor (IL-17R) family consists of 5 members, IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, all of which are type I single-transmembrane glycoproteins with conserved structural motifs, including an extracellular fibronectin III-like domain and an intracellular domain. The human IL-17RA gene is located on chromosome P22, and the human IL-17RA protein has 866 amino acids in total length, multiple disulfide bonds and glycosylation sites, two modification sites, an extracellular region, a transmembrane region and an intracellular region.

IL-17RA is a receptor for IL-17A and also a receptor for IL-17F, and IL-17RA binds IL-17A with greater affinity than IL-17F. Activation of IL-17RA reduces the expression of inflammatory factors such as CXCL1, CXCL8/IL-8 and IL-6. IL-17RA is widely expressed, especially in hematopoietic tissues at higher levels. IL-17RA comprises two distinct domains, a toll/interleukin 1 receptor-like loop domain and a C-terminal domain, proximal to SEF1R, and IL-17RA can cause the production and release of a variety of molecules, such as cytokines (IL-6, G-CSF, GM-CSF), chemokines (CCL2, CCL7, CCL20, CXCL1, CXCL5), antimicrobial peptides (beta defensin-2, S100A7, S100A8, S100A9), mucins (mucin 5B and mucin 5AC), and matrix metalloproteinases (MMP1, MMP3, MMP9, MMP12, and MMP13), and can also bind to a variety of IL-17 family members to exert biological effects.

At present, many problems are often encountered during the process development process for anti-IL-17 RA monoclonal antibody, the dosage of monoclonal antibody products is generally high, about 200 mg/time, but the administration volume of subcutaneous injection is limited, and 1.0-1.5ml is already a limit, which indicates that the concentration of anti-IL-17 RA monoclonal antibody is as high as 100-150mg/ml, which is more demanding for the process purification. brodalumab is the first and only fully humanized monoclonal antibody selectively targeting IL-17 receptor a (IL-17RA), but the purification process of brodalumab is complex, and in the conventional purification process, after each step is completed, the pH condition of the next step needs to be adapted by replacing or debugging buffer solution, so that a lot of time is wasted in the batch production process, and impurities are likely to be introduced in the debugging process, so that the intermediate operation steps are more, the recovery rate is low, and the cost is increased.

Disclosure of Invention

In order to meet the requirements of domestic markets, the invention utilizes a phage antibody library display technology and a high-throughput screening method to screen an anti-IL-17 RA monoclonal antibody with higher affinity and better activity, and meanwhile, the invention can overcome the defects existing in the purification process of the existing anti-IL-17 RA monoclonal antibody according to the characteristics of the monoclonal antibody, and finally provides a purification method suitable for the anti-IL-17 RA monoclonal antibody provided by the invention through a large number of experiments.

The specific technical scheme of the invention is as follows:

the invention provides a purification method of an anti-IL-17 RA monoclonal antibody, which comprises the following steps:

s1, affinity chromatography;

s11, firstly, using 3-5CV buffer solution A to balance the affinity chromatography column at the flow rate of 100-200cm/h, and loading the cell supernatant containing the anti-IL-17 RA monoclonal antibody at the flow rate of 100-200 cm/h;

s12, secondly, after the sample loading is finished, the buffer solution A is used for carrying out rebalancing for the first time, after the ultraviolet signal is stable, the buffer solution B is used for carrying out leaching for the second time, and the rinsing is stopped until the curve of the ultraviolet absorption value is reduced to be stable;

s13, finally, eluting the affinity chromatography column by using a buffer solution C at the flow rate of 100-200cm/h, collecting the affinity eluent when the ultraviolet absorption value rises to 100mAU, and ending the collection when the ultraviolet absorption value falls to 100mAU for later use;

s2, cation exchange chromatography;

s21, firstly, using 3-5CV buffer solution D to balance the cation exchange chromatography column at the flow rate of 100-;

s22, secondly, after the sample loading is finished, rebalancing is carried out by using the buffer solution D, after the ultraviolet absorption value is stable, the buffer solution E is used for leaching the cation exchange chromatography column at the flow rate of 100-200cm/h, and the washing volume is 5-15 CV;

s23, finally, eluting the cation exchange chromatography column by using a buffer solution F at the flow rate of 100-200cm/h, collecting cation eluent when the ultraviolet absorption value begins to rise, and finishing collection when the ultraviolet absorption value is reduced to 300mAU for later use;

s3, anion exchange chromatography;

s31, firstly, balancing the anion exchange chromatography column with a buffer solution G with the concentration of 7-10CV at the flow rate of 100-;

and S32, after the sample loading is finished, washing by using the buffer solution G again, collecting the flow-through liquid when the ultraviolet absorption value is increased to 100mAU, and finishing the collection when the ultraviolet absorption value is decreased to 200 mAU.

The invention provides a purification method special for an anti-IL-17 RA monoclonal antibody, which not only reduces the process impurities such as host protein (HCP), protein-A, DNA and the like in antibody protein on the basis of ensuring the protein quality by a three-step chromatography method, but also simplifies the operation process, does not need to independently regulate proper pH value again in the transition process from cation exchange chromatography to anion exchange chromatography, has smooth two-step connection, avoids introducing impurities in the process of regulating the pH value, shortens the purification period, improves the purification efficiency, saves time for the pilot-scale production in the later period, and effectively removes impurities related to various products such as polymers, acid and alkali isomers and the like, thereby achieving the effect of obviously improving the purity of target protein. In the invention, affinity chromatography is used for capturing target protein, anion-cation exchange chromatography is used for finely purifying protein, product impurities are further removed, and finally the anti-IL-17 RA monoclonal antibody with qualified quality is obtained; compared with the prior art, the method can obtain high-yield active protein in the shortest time, has simple operation and lower cost, and can meet the purification requirement of pilot scale amplification on the anti-IL-17 RA monoclonal antibody.

Further, in step S1 of the above method provided by the present invention, the anti-IL-17 RA monoclonal antibody is selected from any one of the following:

(mAb-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;

(mAb-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;

(mAb-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;

(mAb-9) the amino acid sequence of the heavy chain variable region is SEQ ID NO:9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;

(mAb-10) the amino acid sequence of the heavy chain variable region is SEQ ID NO:10, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;

(mAb-11) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2 and the amino acid sequence of the light chain variable region is SEQ ID NO: 13.

The 11 anti-IL-17 RA monoclonal antibodies provided by the invention have stronger affinity and good biological activity, can be specifically combined with IL-17RA antigens, block the combination of IL-17A and IL-17RA, antagonize and block IL-17/IL-17R signal pathways, are autoimmune disease treatment drugs with great potential, effectively relieve the symptoms of autoimmune diseases, prevent the progression of diseases, and the autoimmune diseases include but are not limited to the following: psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma, or the like.

Further, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is human C kappa;

preferably, the heavy chain constant region is human IgG 1.

Further, in step S1, the medium of the affinity chromatography column is MabSelect Sure, MabSelect Sure LX or Eshmuno-A. Preferably, the MabSelect Sure LX is selected as a medium in the affinity chromatography column to capture the antibody in the cell supernatant, remove most impurities and reduce the volume of the sample, and the MabSelect Sure LX has the advantages of stable base frame, long service life, less ligand shedding and the like.

Further, in step S1, the buffer solution A comprises 50mM Tris-HCl and 150-160 mM sodium chloride, the pH of the buffer solution A is 7.2-7.6, and the conductivity of the buffer solution A is 15-20 mS/cm;

the buffer solution B comprises 50mM sodium acetate, the pH value of the buffer solution B is 5.0-6.0, and the conductivity of the buffer solution B is 2-5 mS/cm;

the buffer solution C comprises sodium acetate, sodium citrate or glycine, and the pH value of the buffer solution C is 3.5-3.7.

Further, in step S1, the buffer A is composed of 50mM Tris-HCl and 150mM NaCl, and the pH of the buffer A is 7.4; the pH of the buffer solution B is 5.0; the buffer C was 50mM sodium acetate and the pH of the buffer C was 3.6.

The buffer solution A is mainly used for reducing non-specifically bound impurities, and Tris and sodium chloride contained in the buffer solution A provide a proper buffer range to ensure the stability of the protein, so that the column can be bound with the target protein to elute and remove the impurity protein. In addition, in order to adapt to the isoelectric point of the anti-IL-17 RA monoclonal antibody, a large number of experiments prove that the buffer solution A with the pH value of 7.2-7.6 can meet the purification requirement, the buffer solution has stronger buffer capacity in the pH range, and the purification efficiency can be improved by setting the conductivity to be 15-20mS/cm according to the pH value. The buffer solution B is mainly used for leaching and removing host cells, and in addition, sodium acetate contained in the buffer solution B can improve the stability of the target protein and provides a basis for protein purification.

The affinity chromatography selects low pH solution for leaching, has low electrical conductivity, is suitable for the cation exchange chromatography of the second step, avoids the process that the existing affinity chromatography selects high salt solution for leaching and then can enter the cation exchange chromatography after desalting, greatly shortens the time of the purification process and improves the purification efficiency.

Further, in step S2, the medium of the cation exchange chromatography column is Capto SP, Eshmuno CPX or Sepharose CM, and preferably, the medium of the cation exchange chromatography column is Eshmuno CPX. The cation exchange chromatography selects Eshmuno CPX as a medium, can be used for finely purifying antibody protein, further removes impurities in the antibody protein, can separate acid and alkali isomers, improves the content of a main peak, and finally obtains the anti-IL-17 RA monoclonal antibody with qualified quality.

Anion-cation exchange chromatography is used for fine purification of protein, further removal of product impurities and final obtaining of qualified anti-IL-17 RA monoclonal antibody

Further, the buffer solution D comprises 20mmol/L sodium phosphate, 20mmol/L sodium citrate or 20mmol/L MES buffer solution, the pH value of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 2-4 mS/cm;

the buffer solution E comprises 20mmol/L sodium phosphate and 10-20 mmol/L sodium chloride, the pH value of the buffer solution E is 7.2-7.4, and the conductivity of the buffer solution E is 4-6 mS/cm;

the buffer solution F comprises 20mmol/L sodium phosphate and 70-100 mmol/L sodium chloride, the pH value of the buffer solution F is 7.2-7.4, and the conductivity of the buffer solution F is 7-15 mS/cm;

preferably, the buffer D is 20mmol/L sodium phosphate.

The invention screens the buffer solution components required in the cation exchange chromatography process through a large number of experiments, the screened buffer solution D, buffer solution E and buffer solution F can meet the fine purification requirement of the anti-IL-17 RA monoclonal antibody in the cation exchange chromatography, and simultaneously, the buffer solution D, buffer solution E and buffer solution F can directly enter anion exchange chromatography through the exploration of the preparation components and the pH value without regulating the pH value again, thereby greatly shortening the purification time and saving the materials.

Further, the medium of the anion exchange chromatographic column is POROS XQ, Capto Q or Eshmuno Q;

preferably, the medium of the anion exchange chromatography column is POROS XQ.

Further, the buffer solution G comprises 20mmol/L sodium phosphate and 50-80 mmol/L sodium chloride, the pH value of the buffer solution G is 7.2-7.4, and the conductivity of the buffer solution G is 6-12 mS/cm.

The invention has the following beneficial effects: firstly, the anti-IL-17 RA monoclonal antibody used for purification in the invention has stronger affinity and good biological activity, can be specifically combined with IL-17RA antigen, can block the combination of IL-17A and IL-17RA, antagonize and block IL-17/IL-17R signal channel, is an autoimmune disease treatment drug with huge potential, effectively relieves the symptoms of autoimmune diseases, prevents the progression of the diseases, and the autoimmune diseases comprise but are not limited to the following: psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma, or the like; secondly, the method provided by the invention adopts three steps of affinity chromatography and anion-cation exchange chromatography to complete the purification of the anti-IL-17 RA monoclonal antibody, the affinity chromatography is used for capturing target protein, the cation exchange chromatography is used for removing an acid peak, the anion exchange chromatography is used for removing aggregates, and finally the anti-IL-17 RA monoclonal antibody with qualified quality is obtained, compared with the purification method provided by the prior art, the method is easy for pilot scale production, does not have the adjustment step of an intermediate sample, has smooth process connection in each step, shortens the process period, shortens the purification time of the existing anti-IL-17 RA monoclonal antibody from 3 days to 10-12 hours, greatly improves the purification efficiency, in addition, the method is suitable for continuous flow process production, the sample in the previous process step can directly enter the next process step without adjusting the product, therefore, the invention can realize the whole process in a closed system, reduce the risk of pollution and ensure the safety of the antibody.

Drawings

FIG. 1 is a plasmid map of pScFvDisb-s in example 1 of the present invention;

FIG. 2 is a graph of Elisa experimental data for identification of monoclonal antibody phage in example 1 of the present invention;

FIG. 3 is a graph of gradient dilution Elisa experimental data of phage purified monoclonal antibody in example 2 of the present invention;

FIG. 4 is a plasmid map of pTSE in example 3 of the present invention;

FIG. 5 is a graph showing a comparison of the binding ability of anti-IL-17 RA whole antibody to IL-17RA at the molecular level in example 4 of the present invention;

FIG. 6 is a graph showing a comparison of the ability of anti-IL-17 RA whole antibody in example 5 of the present invention to competitively inhibit the binding between IL-17A and IL-17 RA;

FIG. 7 is a graph comparing the binding ability of anti-IL-17 RA whole antibody to IL-17RA at the cellular level in example 7 of the present invention;

FIG. 8 is a graph showing experimental comparison of the biological activities of the anti-IL-17 RA antibody of example 8 of the present invention in inhibiting IL-6 production in HDF cells.

Detailed Description

The present invention will be described in further detail with reference to the following examples.

EXAMPLE 1 screening of anti-IL-17 RA monoclonal antibodies

Antibody purification is the most important step in process development, and before the antibody purification method provided by the invention, an anti-IL-17 RA monoclonal antibody should be obtained first. The invention utilizes a fully synthetic ScFv single-chain phage antibody library to screen and obtain a fully human monoclonal antibody which is specifically combined with IL-17 RA. At present, the fully human antibody is the main direction of development of therapeutic antibodies, and the emergence of antibody library technology provides a good technical platform for preparation and screening of the fully human antibody. The antibody library technology bypasses the hybridoma process necessary in the previous monoclonal antibody development process, and can obtain various antibody genes and antibody molecular fragments even without an immunization process. Phage antibody libraries were the earliest and most widely used antibody libraries at present. The phage antibody library display technology is a technology firstly established by Smith, inserts a gene coding a foreign protein or polypeptide into a phage capsid protein gene, and leads the foreign protein or polypeptide and the phage capsid protein to be fused and expressed on the surface of a phage. The phage antibody library utilizes the principle to express antibodies with different specificities or functional fragments thereof (Fab, Fv and ScFv) on the surface of phage, and then carries out screening by using antigen. The phage antibody library is divided into immune library and non-immune library according to the source of antibody gene, and the non-immune library comprises natural library, semi-synthetic library and fully-synthetic library. Screening of phage antibody libraries mimics the process of antibody affinity maturation, typically by coating the antigen on a solid phase medium, adding the phage antibody library to be screened, and performing several rounds of "adsorption-washing-elution-amplification" (i.e., panning) until specific, high affinity antibodies are screened.

Specifically, the biopanning method of the anti-IL-17 RA monoclonal antibody comprises the following steps:

the method comprises the following steps: screening of phage antibody libraries

A series of gene cloning methods are adopted to modify a vector pComb3 (purchased from China plasmid vector strain cell strain gene collection center) for construction and expression of a phage single-chain antibody library. The modified vector is named as pScFvDisb-s, the plasmid map of the modified vector is shown in figure 1, and a fully synthetic phage antibody library is constructed on the basis of the vector;

IL-17RA-ECD-His is used as an antigen to coat the immune tube, the antigen coating amount is 5 mug/500 mug/tube, and the immune tube is coated overnight at 4 ℃. The immune tube and phage antibody library were blocked with PBST-4% mikk (phage input of about 109-1012) and blocked at 37 ℃ for 1h, respectively. And adding the closed phage antibody library into an immune tube for antigen-antibody combination, and reacting for 1h at 37 ℃. PBST-PBS washed away unbound or weakly bound phage, eluted by 0.1M Glycine-HCl pH 2.2, and finally the eluted phage antibody solution was neutralized to pH 7.0 with 1.5M Tris-HCl pH 8.8.

The neutralized phage was infected with 10ml of TG1 bacterial solution grown to OD of about 0.5-0.8, left to stand in an incubator at 37 ℃ for 30min, and then cultured for 1h with shaking at 150 rpm. And taking out 1% bacterial liquid, performing gradient dilution, and coating the bacterial liquid on a 2YTAG small plate for calculating the output of the phage. The remaining bacterial solution was centrifuged and the supernatant was discarded, and the pellet was resuspended in a small amount of medium, spread on a 2YTAG large plate, and cultured overnight at 37 ℃. Transferring the overnight culture to 2YTAG liquid culture medium, performing shake culture at 37 deg.C and 220rpm to logarithmic phase, adding M13K07 helper phage, performing static infection at 37 deg.C for 30min, and performing shake culture at 150rpm for 1 h. Centrifuging at 4000rpm for 15min, discarding supernatant, resuspending the mycelia with 50ml 2YTAKA medium, and performing shaking culture at 28 deg.C and 220rpm overnight to amplify the phage. The next day, the phage were purified by PEG6000-NaCl sedimentation for the next round of screening. Three rounds of enrichment and screening of phage library are performed according to the method for later use.

Step two: ELISA identification of monoclonal antibodies

After three rounds of selection, well-separated monoclonal colonies were picked, inoculated into a 96-well deep-well plate containing 1ml of 2YTAG liquid medium, cultured at 37 ℃ and 220rpm for about 5 hours to its logarithmic growth phase, supplemented with about 1010 of helper phage M13KO7 per well, and subjected to static infection at 37 ℃ for 30min, followed by shaking culture at 150rpm for 1 hour. Centrifugation was carried out at 4000rpm for 15min, the supernatant was discarded, and the mycelia were resuspended in 2YTAKA and incubated overnight at 28 ℃ and 220 rpm. The next day, centrifugation at 4000rpm and 4 ℃ for 15min, phage-containing supernatant fluid was aspirated for monoclonal ELISA identification, and as shown in FIG. 2, monoclonal antibodies with higher affinity were obtained by screening, and named mAb-1, mAb-2, mAb-3, mAb-4, mAb-5, mAb-6, mAb-7, mAb-8, mAb-9, mAb-10, and mAb-11, respectively, and the 11 monoclonal bacterial fluids were subjected to gene sequencing to determine the correct antibody sequences.

After sequencing, the sequence of the antibody of the 11 monoclonal antibodies screened above is as follows, and the monoclonal antibody protected by the invention is selected from any one of the following antibodies:

(mAb-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2 and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3 and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4 and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5 and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;

(mAb-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7 and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;

(mAb-10) the amino acid sequence of the heavy chain variable region is SEQ ID NO:10 and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;

Specifically, SEQ ID NO:1 (amino acid sequence of heavy chain variable region in mAb-1):

QVQLVQSGAEVKKPGASVKVSCKASGYSYSNYGISWVRQAPGQGLEWMGWISTYSGNTNYSKKLKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

2 (amino acid sequence of heavy chain variable region in mAb-2 and mAb-11):

QVQLVQSGAEVKKPGASVKVSCKASGYAWTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAKKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

3 (amino acid sequence of heavy chain variable region in mAb-3):

QVQLVQSGAEVKKPGASVKVSCKASGYAFSRFGISWVRQAPGQGLEWMGWISTYSGNTNYAKHLLGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRFLKFDYWGQGTLVTVSS；

4 (amino acid sequence of heavy chain variable region in mAb-4):

QVQLVQSGAEVKKPGASVKVSCKASGYAYSNYGISWVRQAPGQGLEWMGWISTYSGNTNYAKKFIGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

SEQ ID NO:5 (amino acid sequence of heavy chain variable region in mAb-5):

QVQLVQSGAEVKKPGASVKVSCKASGFAFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYADQFIGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

SEQ ID NO:6 (amino acid sequence of heavy chain variable region in mAb-6):

QVQLVQSGAEVKKPGASVKVSCKASGYSFTRYGISWVRQAPGQGLEWMGWISTYSGNTNYAKQFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

SEQ ID NO:7 (amino acid sequence of heavy chain variable region in mAb-7):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISTYSGNTNYAKNLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

SEQ ID NO:8 (amino acid sequence of heavy chain variable region in mAb-8):

QVQLVQSGAEVKKPGASVKVSCKASGYGFASRGISWVRQAPGQGLEWMGWISTYSGNTNYAKKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

SEQ ID NO:9 (amino acid sequence of heavy chain variable region in mAb-9):

QVQLVQSGAEVKKPGASVKVSCKASGYTWTSYGISWVRQAPGQGLEWMGWISTYSGNTNYAKKFLGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

10 (amino acid sequence of heavy chain variable region in mAb-10):

QVQLVQSGAEVKKPGASVKVSCKASGYSFSSYGISWVRQAPGQGLEWMGWISTYSGNTNYAKKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRVLRLDYWGQGTLVTVSS；

11 (amino acid sequence of light chain variable region in mAb-1-5):

DIQMTQSPSSLSASVGDRVTITCRASQDIDSSLNWYQQKPGKAPKLLIYAASNRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWANMPGTFGQGTKVEIK；

12 (amino acid sequence of light chain variable region in mAb-6 and mAb-7):

EIVMTQSPATLSVSPGERATLSCRASQDISISLGWFQQKPGQAPRPLIYGWNTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQQYSDPFLTFGGGTKVEIK；

13 (amino acid sequence of light chain variable region in mAb-8 to mAb-11):

DIQMTQSPSSLSASVGDRVTITCRASQSVHTGLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFEHNPLTFGQGIKVEIK。

further, the monoclonal antibody also comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is human C kappa;

preferably, the heavy chain constant region is human IgG 1.

Example 2 gradient dilution of phage Elisa to compare the affinity of anti-IL-17 RA monoclonal antibodies

3.1 preparation of monoclonal antibody purified phage:

the bacterial liquid of the 11 monoclonal antibodies with higher affinity (mAb-1, mAb-2, mAb-3, mAb-4, mAb-5, mAb-6, mAb-7, mAb-8, mAb-9, mAb-10, mAb-11) obtained in example 1 was transferred to 2YTAG liquid medium, after shaking culture to logarithmic growth phase, M13K07 was added to assist phage infection, after centrifugation, the cells were resuspended in 2YTAKA, cultured overnight at 28 ℃ to amplify phages, and the phages were purified by PEG6000-NaCl sedimentation the next day. Purified phages against IL-17RA monoclonal antibodies (AMH14/AML14) provided in the core patent US7833527B2 of Brodalumab, a product marketed, were prepared as positive controls.

3.2 affinity comparison at phage level

IL-17RA-ECD-His was coated with 0.01M PBS buffer pH 7.2 at 100 ng/well/100. mu.l overnight at 4 ℃. The following day, the Elisa plates were washed 3 times with PBST, PBST-4% mil k (200. mu.l/well) was added, and blocked at 37 ℃ for 1 h. Meanwhile, 11 strains of the monoclonal purified phage samples obtained by screening in example 1 and the monoclonal purified phage samples prepared from the anti-IL-17 RA monoclonal antibody (AMH14/AML14) provided in patent US7833527B2 were diluted in 5-fold gradient by PBST-4% mik, and the gradient diluted phage samples were blocked at 37 ℃ for 1 hour. PBST-4% mil in an Elisa plate was discarded, and a gradient diluted phage sample (1) was added00. mu.l/well), and left standing at 37 ℃ for 1 hour. The Elisa plates were washed 5 times with PBST, and then added with PBST-4% mil k 1:5000 diluted anti-M13-HRP monoclonal antibody (100. mu.l/well) and left at 37 ℃ for 1 h. The Elisa plates were washed 5 times with PBST, developed with TMB development kit (100. mu.l/well), developed for 10min at room temperature, and developed with 2M H₂SO₄(100. mu.l/well) the color development was stopped. The microplate reader wavelength was 450nm and 630nm read. The data were analyzed and plotted using the software GraphPad Prism5Demo, with results as shown in figure 3 and data as shown in table 1.

TABLE 1 results of gradient dilution Elisa experiments at phage level

Name of antibody	EC50	Name of antibody	EC50
				Brodalumab	0.02327	mAb-6	0.01238
mAb-1	0.004287	mAb-7	0.01023
				mAb-2	0.009335	mAb-8	0.003126
mAb-3	0.004054	mAb-9	0.003962
				mAb-4	0.003761	mAb-10	0.004997
mAb-5	0.00844	mAb-11	0.006777

As shown in FIG. 3, the selected 11 different monoclonal antibodies can bind to IL-17RA, and compared with the anti-IL-17 RA monoclonal antibody provided by the patent US7833527B2, the monoclonal antibody provided by the invention has stronger binding ability to IL-17RA and higher affinity.

Example 3 preparation of anti-IL-17 RA Total antibody

The heavy chain variable region gene and the light chain variable region gene of the 11 monoclonal antibodies selected in example 1 were cloned into a vector pTSE (shown in FIG. 4) equipped with a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgG1 constant region (the amino acid sequence is shown in SEQ ID NO:14), the light chain constant region is a kappa chain constant region (the amino acid sequence is shown in SEQ ID NO:15), the vector pTSE was obtained by modifying a PTT vector, the preparation process is shown in paragraph [0019] on page 3 of the specification of CN103525868A, and the structure is shown in FIG. 4.

14 (heavy chain constant region sequence of human IgG 1):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG；

15 (light chain constant region sequence of kappa chain):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

after the constructed vector is sequenced successfully, a large amount of plasmids are extracted by using an endotoxin-free plasmid large-extraction kit, the extracted plasmids are quantified and mixed with a transfection reagent according to a certain proportion, HEK293E cells are transiently transfected for whole antibody expression, and after the cells are cultured for 4 days, the supernatant is collected. After centrifugation and filtration of the supernatant, whole antibody protein was obtained by purification using protein A affinity column of AKTA instruments. After the protein obtained by purification was concentrated by ultrafiltration, the protein was quantified using the BCA protein quantification kit, and the results are shown in table 2. And simultaneously expressing a full antibody of the Brodalumab purified on the market as a positive control.

TABLE 2 data table of total antibody protein concentration

Name of antibody	Concentration (mg/ml)	Name of antibody	Concentration (mg/ml)
				Brodalumab	14.64	mAb-6	13.4
mAb-1	14.00	mAb-7	10.55
				mAb-2	18.03	mAb-8	17.39
mAb-3	15.75	mAb-9	18.22
				mAb-4	12.30	mAb-10	16.15
mAb-5	10.90	mAb-11	16.56

Example 4 binding experiments of anti-IL-17 RA Total antibodies to IL-17RA at the molecular level

IL-17RA-ECD-His was coated with 0.01M PBS buffer pH 7.2 at 100 ng/well/100. mu.l overnight at 4 ℃. The following day, the Elisa plates were washed 3 times with PBST, followed by the addition of PBST-4% mil k (200. mu.l/well) and blocked at 37 ℃ for 1 h. The whole antibody and positive control antibody prepared in example 3 were diluted using a PBST-4% mil gradient with an initial maximum concentration of 10 μ g/mL for all antibodies, diluted in 5-fold gradients, and 8 gradients for each whole antibody. PBST-4% mil k from Elisa plates was discarded, and a sample of whole antibody (100. mu.l/well) diluted in gradient was added and incubated at 37 ℃ for 1 h. Wash the Elisa plate 5 times with PBST, then addGoat anti-human IgG-HRP diluted with PBST-4% mil k 1:5000 was added and incubated at 37 ℃ for 1 h. The Elisa plates were washed 5 times with PBST, developed with TMB development kit (100. mu.l/well), developed for 10min at room temperature, and then developed with 2M H₂SO₄(50. mu.l/well) the color development was stopped. The microplate reader wavelength was 450nm and 630nm read. The data were plotted using the software GraphPad Prism5Demo analysis and the results are shown in fig. 5 and table 3.

TABLE 3 data table of full antibody affinity experiment EC50 values

As shown in FIG. 5 and Table 3, the selected 11 different monoclonal antibodies all can bind to IL-17RA, and the EC50 value of the 11 different monoclonal antibodies provided by the present invention is significantly lower than that of the anti-IL-17 RA monoclonal antibody provided by the patent US7833527B2, which indicates that the monoclonal antibody provided by the present invention has high binding affinity with IL-17RA and good activity, and in addition, as can be seen from FIG. 5 and Table 3, the total antibody of mAb-8 in the 11 total antibodies has the lowest EC50 value, which indicates that the monoclonal antibody has the best binding ability with IL-17RA, the highest affinity and the best activity.

Example 5 full antibody Competition inhibition of IL-17A binding to IL-17RA

IL-17A-Fc was coated with 0.01M PBS pH 7.2 (100 ng/well/100. mu.l) overnight at 4 ℃. The following day, the Elisa plates were washed 3 times with PBST, followed by addition of PBST-4% mil k and blocking at 37 ℃ for 1 h. IL-17RA-ECD-His was diluted to a concentration of 2. mu.g/ml using PBST-4% mil; the whole antibody and positive control antibody of example 3 were diluted using PBST-4% mil gradients with an initial maximum concentration of 300. mu.g/mL for all antibodies, diluted in 3-fold gradients, and 8 gradients for each whole antibody. PBST-4% mik in Elisa plates was discarded, and IL-17RA-ECD-His (50. mu.l/well) and whole antibody was added in a gradient dilutionSamples (50. mu.l/well) were incubated at 37 ℃ for 2 h. The Elisa plates were washed 5 times with PBST, and 1:5000 mouse anti-His IgG-HRP diluted with PBST-4% mil k was added and incubated at 37 ℃ for 1 h. The Elisa plates were washed 5 times with PBST, developed with TMB development kit (100. mu.l/well), developed for 10min at room temperature, and developed with 2M H₂SO₄(50. mu.l/well) the color development was stopped. The microplate reader wavelength was 450nm and 630nm read. The results are plotted using the software GraphPad Prism5Demo, as shown in fig. 6 and table 4.

TABLE 4 data table of IC50 values for whole antibody competition experiment

Name of antibody	IC50(ng/ml)	Name of antibody	IC50(ng/ml)
				Brodalumab	3443	mAb-6	2905
mAb-1	2355	mAb-7	2166
				mAb-2	2452	mAb-8	1576
mAb-3	2083	mAb-9	2322
				mAb-4	2021	mAb-10	2802
mAb-5	1911	mAb-11	2666

As shown in FIG. 6 and Table 4, the selected 11 monoclonal antibodies of different strains can effectively inhibit the binding of IL-17A and IL-17RA, and the IC50 values of the 11 monoclonal antibodies of different strains provided by the invention are obviously lower than those of the positive control Brodalumab whole antibody on the market, which indicates that the monoclonal antibodies provided by the invention have stronger inhibition capability on the binding of IL-17A and IL-17RA, and in addition, as can be seen from FIG. 6 and Table 4, the IC50 value of the mAb-8 whole antibody in the 11 monoclonal antibodies is the lowest, which indicates that the monoclonal antibodies have the strongest inhibition capability on the binding of IL-17A and IL-17 RA.

Example 6 BIAcore X100 determination of the affinity of the Whole antibody

The affinity of the whole antibody is determined by a capture method: goat anti-human IgG was coupled to the surface of the CM5 chip and antibody HBS-EP buffer was diluted to a concentration of 1. mu.g/ml to ensure that it could be captured by goat anti-human IgG. The affinity of the 11 whole antibodies provided in example 3 and the Brodalumab, a commercial product of positive control, were determined by running IL-17RA-ECD-His through the stationary phase surface with a series of concentration gradients (20. mu.g/ml, 10. mu.g/ml, 5. mu.g/ml, 2.5. mu.g/ml, 1.25. mu.g/ml, 0.625. mu.g/ml, 0.3125. mu.g/ml, 0. mu.g/ml), according to the data given in the following table.

TABLE 5 full antibody affinity results data Table for Biacore assay

Name of antibody	ka(1/Ms)	kd(1/s)	KD(M)
				mAb-1	8.077E+05	8.421E-05	1.042E-10
mAb-2	7.887E+05	5.732E-05	7.267E-11
				mAb-3	1.126E+06	4.840E-05	4.298E-11
mAb-4	1.023E+05	6.571E-06	6.426E-11
				mAb-5	1.022E+06	7.439E-05	7.275E-11
mAb-6	6.358E+04	1.066E-05	1.676E-10
				mAb-7	7.517E+04	1.936E-05	2.575E-10
mAb-8	3.213E+05	4.923E-06	1.532E-11
				mAb-9	8.160E+04	1.471E-06	1.803E-11
mAb-10	5.205E+05	3.413E-05	6.356E-11
				mAb-11	1.001E+05	1.393E-05	1.392E-10
Brodalumab	1.504E+05	4.333E-05	2.882E-10

As shown in the data in Table 5, the total antibodies of the 11 monoclonal antibodies provided by the present invention all have higher affinity, and the KD (M) value is significantly lower than that of the total antibody of the commercial Brodalumab positive control, which indicates that the 11 monoclonal antibodies provided by the present invention have higher biological activity.

Example 7 analysis of binding specificity of Whole antibody to cell surface IL-17RA

The CHO cells over-expressing IL-17RA are constructed by the prior method to detect the binding condition of different anti-IL-17 RA antibodies and cell surface IL-17 RA.

The whole antibody and control antibody of example 3 were diluted in a gradient with an initial maximum concentration of 20 μ g/ml for all antibodies, diluted in 5-fold gradients with 8 gradients for each whole antibody. CHO cells were harvested by centrifugation, washed 3 times with PBS + 1% BSA and resuspended, adjusted to a cell density of 2X 10⁶Mu.l of cells per well were added, together with 50. mu.l of gradient diluted anti-IL-17 RA whole antibody, and incubated for 1 hour at room temperature. The supernatant was centrifuged at 3000rpm, the cells were washed 3 times with PBS, 50. mu.l/well of a diluted FITC-labeled goat anti-human IgG antibody solution was added to resuspend the cells, and the cells were incubated at room temperature in the dark for 1 hour. The supernatant was centrifuged at 3000rpm, washed again 3 times with PBS, resuspended in 100. mu.l PBS and the fluorescence intensity was measured by flow cytometry. The results were plotted using Graphpad Prism5Demo and are shown in FIG. 7 and Table 6.

TABLE 6 binding experiment of anti-IL-17 RA holoantibody to IL-17RA on cell surface

Name of antibody	EC50(ng/ml)	Name of antibody	EC50(ng/ml)
				Brodalumab	272.9	mAb-6	213.1
mAb-1	176.8	mAb-7	216.1
				mAb-2	130.7	mAb-8	48.45
mAb-3	78.14	mAb-9	66.14
				mAb-4	106.3	mAb-10	101.4
mAb-5	146.0	mAb-11	189.6

Based on the above data and as shown in FIG. 7, the EC50 values of the 11 full antibodies prepared by the present invention are significantly lower than those of the positive control Brodalumab full antibody marketed, which indicates that the 11 antibodies provided by the present invention can bind to cell-expressed IL-17RA, and the EC50 value of the full antibody of mAb-8 in the 11 full antibodies is the lowest, indicating that the full antibody specifically binds to IL-17RA antigen with the best binding ability, the highest affinity, and the best activity.

Example 8 biological Activity test of anti-IL-17 RA antibody for inhibiting IL-6 production by human skin fibroblasts (HDF)

HDF cells were digested with 0.25% trypsin, plated 8000 cells/well, and incubated overnight at 37 ℃. IL-17A was diluted to a concentration of 2. mu.g/ml with PBS; the whole antibody and control antibody of example 3 were diluted in a PBS gradient with an initial maximum concentration of 200 μ g/ml for the whole antibody, diluted in 5-fold gradients, with 8 gradients for each whole antibody. IL-17A diluted to a final concentration of 1. mu.g/ml in PBS and anti-IL-17 RA antibody and control antibody (anti-IL-17 RA monoclonal antibody provided in US7833527B 2) were added to each well and incubated overnight at 37 ℃. 90. mu.l of the supernatant was subjected to quantitative analysis of the secretion level of cytokine IL-6 using IL-6ELISA kit, and the results are shown in FIG. 8 and Table 7.

TABLE 7 biological Activity test of anti-IL-17 RA antibodies

The data in Table 7 and shown in FIG. 8 show that all of the 11 monoclonal antibodies provided by the present invention can reduce the secretion level of IL-6 by binding IL-17RA competitively with IL-17A, and that the IC50 values of the 11 monoclonal antibodies provided by the present invention are significantly lower than those of Brodalumab whole antibody available on the market as a positive control, which shows that the monoclonal antibodies provided by the present invention have stronger binding ability with IL-17RA than those of the positive control antibody with IL-17RA, and that the data also show that the whole antibody of mAb-8 in the 11 whole antibodies has the best activity and the strongest competition with IL-17A.

Example 9

The mAb-8 anti-IL-17 RA monoclonal antibody with the highest affinity obtained in the above examples 1-8 is prepared by a purification process, which specifically comprises the following steps:

cell culture fluid pretreatment is firstly carried out before purification: and (3) carrying out depth filtration on a cell culture solution of the mAb-8 anti-IL-17 RA monoclonal antibody produced by CHO cell culture, and storing the obtained cell supernatant at 4 ℃ for less than 3 days. The specific purification method then follows:

s1, affinity chromatography;

s11, firstly, using 3-5CV buffer A to balance the affinity chromatography column at the flow rate of 100-;

the medium of the affinity chromatography column is MabSelect Sure, MabSelect Sure LX or Eshmuno-A, and the medium of the affinity chromatography column in the embodiment 9 of the invention is MabSelect Sure LX.

The buffer solution A comprises 50mM Tris-HCl and 160mM sodium chloride, the pH value of the buffer solution A is 7.2, and the conductivity of the buffer solution A is 15 mS/cm;

s12, secondly, after the sample loading is finished, the buffer solution A is used for carrying out rebalancing for the first time, after the ultraviolet signal is stable, the buffer solution B is used for carrying out leaching for the second time, and the washing is stopped until the curve of the ultraviolet absorption value is reduced to be stable (basic line or leveling); the buffer solution B comprises 50mM sodium acetate, the pH value of the buffer solution B is 5.5, and the conductivity of the buffer solution B is 4 mS/cm;

s13, finally, eluting the affinity chromatography column by using a buffer solution C at the flow rate of 100-200cm/h, collecting the affinity eluent when the ultraviolet absorption value rises to 100mAU, and ending the collection when the ultraviolet absorption value falls to 100mAU for later use; buffer C comprised sodium acetate, sodium citrate or glycine, and buffer C had a pH of 3.5.

S2, cation exchange chromatography;

s21, firstly, using 3-5CV buffer solution D to balance the cation exchange chromatography column at the flow rate of 100-; the medium of the cation exchange chromatographic column comprises Capto SP, Eshmuno CPX or Sepharose CM;

in the preferred embodiment 9 of the present invention, the medium of the cation exchange chromatography column is Eshmuno CPX.

The buffer solution D is 20mmol/L sodium citrate, the pH value of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 2 mS/cm;

s22, secondly, after the sample loading is finished, rebalancing is carried out by using the buffer solution D, after the ultraviolet absorption value is stable, the buffer solution E is used for leaching the cation exchange chromatography column at the flow rate of 100-200cm/h, and the washing volume is 5-15 CV; the buffer solution E comprises 20mmol/L sodium phosphate and 10mmol/L sodium chloride, the pH value of the buffer solution E is 7.2, and the conductivity of the buffer solution E is 4 mS/cm;

s23, finally, eluting the cation exchange chromatography column by using a buffer solution F at the flow rate of 100-200cm/h, collecting cation eluent when the ultraviolet absorption value begins to rise, and finishing collection when the ultraviolet absorption value is reduced to 300mAU for later use; the buffer solution F comprises 20mmol/L sodium phosphate and 70mmol/L sodium chloride, the pH value of the buffer solution F is 7.2, and the conductivity of the buffer solution F is 7 mS/cm;

s3, anion exchange chromatography;

s31, firstly, balancing the anion exchange chromatography column with a buffer solution G with the concentration of 7-10CV at the flow rate of 100-; the medium of the anion exchange chromatographic column is POROS XQ, Capto Q or Eshmuno Q; in the preferred embodiment 9 of the present invention, the medium of the anion exchange chromatography column is POROS XQ.

And S32, after the sample loading is finished, washing by using the buffer solution G again, collecting the flow-through liquid when the ultraviolet absorption value is increased to 100mAU, and finishing the collection when the ultraviolet absorption value is decreased to 200 mAU. The buffer G comprised 20mmol/L sodium phosphate and 50mmol/L sodium chloride, the pH of the buffer G was 7.2, and the conductivity of the buffer G was 6 mS/cm.

Example 10

Embodiment 10 of the present invention further defines that, on the basis of embodiment 9, the buffer solution a is composed of 50mM Tris-HCl and 150mM sodium chloride, and the pH of the buffer solution a is 7.4, and the conductivity of the buffer solution a is 18 mS/cm; the pH of the buffer solution B is 5.0, and the conductivity of the buffer solution B is 2 mS/cm; buffer C was 50mM sodium acetate and buffer C had a pH of 3.6, all other methods and parameters being the same as in example 9.

Example 11

Example 11 of the present invention on the basis of example 9, it is further defined that the buffer solution A is composed of 50mM Tris-HCl and 155mM NaCl, the pH of the buffer solution A is 7.6, and the conductivity of the buffer solution A is 20 mS/cm; the pH of the buffer solution B is 6.0, and the conductivity of the buffer solution B is 5 mS/cm; buffer C was 50mM sodium acetate and buffer C had a pH of 3.7, all other methods and parameters being the same as in example 9.

Example 12

In the embodiment 12 of the present invention, on the basis of the embodiment 10, sodium phosphate with a buffer solution D of 20mmol/L is further defined, the pH of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 3 mS/cm; the buffer solution E comprises 20mmol/L sodium phosphate and 15mmol/L sodium chloride, the pH value of the buffer solution E is 7.3, and the conductivity of the buffer solution E is 5 mS/cm; buffer F comprised 20mmol/L sodium phosphate and 70mmol/L sodium chloride, the pH of buffer F was 7.3 and the conductivity of buffer F was 11mS/cm, all other methods and parameters being identical to those of example 10.

Example 13

In the embodiment 13 of the present invention, on the basis of the embodiment 10, a MES buffer solution with a buffer solution D of 20mmol/L is further defined, the pH of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 4 mS/cm; the buffer solution E comprises 20mmol/L sodium phosphate and 20mmol/L sodium chloride, the pH value of the buffer solution E is 7.4, and the conductivity of the buffer solution E is 6 mS/cm; buffer F comprised 20mmol/L sodium phosphate and 100mmol/L sodium chloride, the pH of buffer F was 7.4 and the conductivity of buffer F was 15mS/cm, all other methods and parameters being identical to those of example 10.

Example 14

Inventive example 14 on the basis of example 12, it is further defined that the buffer G comprises 20mmol/L sodium phosphate and 65mmol/L sodium chloride, the pH of the buffer G is 7.3, and the conductivity of the buffer G is 9mS/cm, all other methods and parameters are the same as in example 12.

Example 15

Inventive example 15 on the basis of example 12, it is further defined that the buffer G comprises 20mmol/L sodium phosphate and 80mmol/L sodium chloride, the pH of the buffer G is 7.4, and the conductivity of the buffer G is 12mS/cm, all other methods and parameters are the same as in example 12.

Comparative example 1

The comparison example 1 of the invention provides a purification method of an anti-IL-17 RA monoclonal antibody, which only removes the rinsing of buffer B on the basis of the example 9, and other method steps and step parameters are all the same.

Comparative example 2

The comparative example 2 of the invention provides a purification method of an anti-IL-17 RA monoclonal antibody, which is characterized in that on the basis of the example 9, a filler for cation exchange chromatography is replaced, the method steps and the step parameters are all the same, and specifically, the cation filler is Capto SP of GE company.

Comparative example 3

The comparison example 3 of the invention provides a purification method of an anti-IL-17 RA monoclonal antibody, which only removes the rinsing of the buffer solution E on the basis of the example 9, and other method steps and step parameters are all the same.

Experiment I, purification method purity analysis experiment of anti-IL-17 RA monoclonal antibody

The IL-17RA monoclonal antibody purified by the purification methods provided in the embodiments 9-15 and the comparative examples 1-3 of the present invention was subjected to purity detection for many times during the purification process; analyzing the contents of aggregates, monomers and degradation products in the protein liquid by using a gel chromatography technical means, simultaneously analyzing the contents of acid-base peaks by using an ion chromatography technical means, and detecting the contents of process-related impurities by using a special kit, wherein the detection result is as follows:

TABLE 8 purification analysis of anti-IL-17 RA monoclonal antibody

The above experimental results show that: as shown in Table 8, compared with comparative example 1, in example 9, after the elution of buffer B was removed in comparative example 1, it can be seen from the data that, after affinity chromatography was performed in both example 9 and comparative example 1, the contents of process impurities such as host protein (HCP), proteinA and the like in comparative example 1 were significantly higher than those in example 9, and the purity and the main peak were lower, so compared with comparative example 1, the purification method provided in examples 9-11 of the present invention can significantly reduce the contents of process impurities such as host protein (HCP), proteinA and the like in the anti-IL-17 RA monoclonal antibody protein product by adding high salt and low pH solution to the affinity chromatography step for elution, and in example 9-11, compared with comparative example 1, the components, contents and pH values of buffer A, buffer B and buffer C are further limited in example 10, so that the purity of the anti-IL-17 RA monoclonal antibody protein product can be improved, meanwhile, the main peak value is obviously higher than that of examples 9 and 11, the content of residual HCP is lower than 2000ppm, and the residual HCP of the final product meets the quality standard, so that process impurities such as host protein (HCP), proteinA A and the like are also lower than those of examples 9 and 11 after affinity chromatography in example 10 through data comparison, and therefore, the buffer solution A, the buffer solution B and the buffer solution C further defined in example 10 can improve the purification effect of the affinity chromatography.

Furthermore, through comparing example 10, example 12 and example 13, the composition, content, pH value and conductivity value of buffer D, buffer E and buffer F are further defined in example 12 of the present invention, and it is known from the data in table 8 that the protein purity of the anti-IL-17 RA monoclonal antibody is significantly improved after the cation exchange chromatography in example 12, and compared with example 10 and example 13, example 12 has a significant effect of removing residual HCP and DNA and simultaneously removing acidic isomers, so the buffer solution for the cation exchange chromatography of the present invention is preferably: the buffer solution D is 20mmol/L sodium phosphate, the pH value of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 3 mS/cm; the buffer solution E comprises 20mmol/L sodium phosphate and 15mmol/L sodium chloride, the pH value of the buffer solution E is 7.3, and the conductivity of the buffer solution E is 5 mS/cm; the buffer F comprises 20mmol/L sodium phosphate and 70mmol/L sodium chloride, the pH of the buffer F is 7.3, and the conductivity of the buffer F is 11 mS/cm.

Still further, as shown in the data in table 8, by comparing the data in examples 12, 14 and 15, example 14 further defines the composition, content and pH of buffer G in anion exchange chromatography on the basis of example 12, and can significantly improve the purity of the purified antibody protein, and the purity finally reaches 99.99%, which meets the purification requirement and is significantly higher than those in examples 12 and 15, and at the same time, has a significant removal effect on residual HCP and DNA, and finally ensures that the residual HCP and DNA in the purified antibody protein product meet the quality standards.

In addition, a new generation of affinity chromatography medium MabSelect Sure LX of GE company is used for capturing target protein in a cell culture solution in the purification process, and compared with the traditional affinity chromatography medium, the MabSelect Sure LX has the advantages of good stability, long service life, less ligand shedding and the like. Therefore, the content of the residual Protein A in the sample in the process of affinity chromatography elution not only reaches the quality standard (less than or equal to 10ppm), but also can improve the service life of the filler and reduce the cost.

Compared with the comparative example 2, the Eshmuno CPX filling material of Merck is selected for the cation exchange chromatography in the invention, so that not only can impurity HCP and DNA be removed easily, but also acid and alkali isomers can be separated, and the content of main peaks can be improved, and after the Capto SP of GE company is selected for the comparative example 2, the purity is obviously lower than that of the embodiment 9-15 after the cation exchange chromatography, so the Eshmuno CPX filling material of Merck is preferably used in the invention.

Compared with the comparative example 3, in the comparative example 3, after the elution for removing the buffer solution D, and the cation exchange chromatography, the purity of the antibody protein is obviously lower than that in the examples 9-15, so that the buffer solution D provided by the invention can improve the purification effect of the anti-IL-17 RA monoclonal antibody.

Meanwhile, examples 12 and 13 explore the separation of acidic peaks at different pH values and sodium chloride concentrations. Example 12 buffer E restriction, not only can increase the volume of washing, simultaneously can effectively remove the acidic peak and does not influence the recovery rate of protein, in the experiment washing volume to reach 8 CV.

In addition, Thermo POROS XQ filler is selected for anion exchange chromatography in embodiments 9-15 of the invention, aggregates are effectively removed, and the final purity of the sample is more than 99%. In examples 14 and 15, the parameters of pH and sodium chloride concentration were optimized, and the samples collected by cation exchange chromatography could be directly subjected to anion exchange chromatography without being treated and then subjected to anion exchange chromatography, thus greatly saving purification time, achieving good connection of anion and cation exchange chromatography, and improving purification efficiency.

Experiment II, protein activity purification and recovery rate analysis experiment of purification method of anti-IL-17 RA monoclonal antibody

The anti-IL-17 RA monoclonal antibody was purified by the methods provided in examples 9 to 15 of the present invention and comparative examples 1 to 3, the total amount (g) of antibody protein in the cell supernatant was counted before purification, and the protein recovery rate was calculated by the following formula:

recovery rate ═ 100% total antibody protein after purification (g)/total antibody before initial purification (g);

and simultaneously detecting the biological activity of the anti-IL-17 RA monoclonal antibody in the finally obtained protein solution. The invention uses ELISA means to analyze the specific binding capacity of the purified anti-IL-17 RA monoclonal antibody and IL-17RA so as to evaluate the activity of the antibody after the antibody is combined and purified. The specific test results for anti-IL-17 RA monoclonal antibodies are as follows. The detection results are as follows:

TABLE 9 analysis of recovery of anti-IL-17 RA monoclonal antibody

Examples	Recovery (%)	Biological Activity (%)
			Example 9	72.0	83.2
Example 10	76.9	90.2
			Example 11	70.4	85.4
Example 12	81.6	105.2
			Example 13	78.1	112.9
Example 14	85.7	101.4
			Example 15	80.6	107.4
Comparative example 1	42.3	130.7
			Comparative example 2	42.8	68.7
Comparative example 3	38.9	60.7

As shown in table 9, in examples 9-15 of the present invention, the protein product of the anti-IL-17 RA monoclonal antibody obtained by the purification method provided in example 14 has the highest purification recovery rate and the best biological activity, so that the purification recovery rate of the anti-IL-17 RA monoclonal antibody can be improved by the purification method provided in example 14 of the present invention on the basis of ensuring the biological activity of the antibody, and the recovery rate may be reduced by changing any other parameter, pH or conductivity.

In addition, in examples 9-15, compared with comparative example 1, the recovery rate of the anti-IL-17 RA monoclonal antibody is reduced significantly and the activity is poor after the elution of the removal buffer B in comparative example 1, and therefore, the buffer B provided by the invention is essential for the purification of the anti-IL-17 RA monoclonal antibody.

Examples 9-15 comparative example 2 changed the cation exchange chromatography medium, which resulted in a decrease in the purification recovery and the life activity of the antibody product, compared to comparative example 2, and for this reason, the present invention preferably uses Merck's Eshmuno CPX packing to effectively ensure the biological activity of the anti-IL-17 RA monoclonal antibody while increasing the recovery.

Examples 9-15 compared with comparative example 3, the elution of the removal buffer E in comparative example 3 resulted in not only a decrease in the recovery rate of the anti-IL-17 RA monoclonal antibody, but also a decrease in the product activity and a decrease in the content of the main peak, and thus it can be demonstrated that the buffer E provided by the present invention is an essential buffer for the anti-IL-17 RA monoclonal antibody in the cation exchange chromatography process.

The purification method provided by the invention is easy for pilot scale production, has no intermediate sample adjustment step, is smooth in process connection, can realize the whole process in a closed system, and reduces the risk of pollution. The purification process is suitable for continuous flow process production, samples in the previous process step can directly enter the next process step without adjusting products, and therefore, the purification time of the existing anti-IL-17 RA monoclonal antibody is shortened from 3 days to 10-12 hours, the process flow is greatly saved, and the purification efficiency is improved.

The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, are within the protection scope of the present invention.

Sequence listing

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

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

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

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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Leu Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile

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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Thr Phe Gly Gln Gly Ile Lys Val Glu Ile Lys

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

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

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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

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

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

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

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

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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

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100 105

Claims

1. A method for purifying an anti-IL-17 RA monoclonal antibody, comprising the steps of:

s1, affinity chromatography;

the buffer solution A comprises 50mM Tris-HCl and 150-160 mM sodium chloride, the pH value of the buffer solution A is 7.2-7.6, and the conductivity of the buffer solution A is 15-20 mS/cm; the buffer solution B comprises 50mM sodium acetate, the pH value of the buffer solution B is 5.0-6.0, and the conductivity of the buffer solution B is 2-5 mS/cm; the buffer solution C comprises sodium acetate, sodium citrate or glycine, and the pH value of the buffer solution C is 3.5-3.7;

s2, cation exchange chromatography;

the buffer solution D comprises 20mmol/L sodium phosphate, 20mmol/L sodium citrate or 20mmol/L MES buffer solution, the pH value of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 2-4 mS/cm; the buffer solution E comprises 20mmol/L sodium phosphate and 10-20 mmol/L sodium chloride, the pH value of the buffer solution E is 7.2-7.4, and the conductivity of the buffer solution E is 4-6 mS/cm; the buffer solution F comprises 20mmol/L sodium phosphate and 70-100 mmol/L sodium chloride, the pH value of the buffer solution F is 7.2-7.4, and the conductivity of the buffer solution F is 7-15 mS/cm;

s3, anion exchange chromatography;

s32, after the sample loading is finished, washing by using the buffer solution G again, collecting the flow-through liquid when the ultraviolet absorption value is increased to 100mAU, and finishing the collection when the ultraviolet absorption value is decreased to 200 mAU;

the buffer solution G comprises 20mmol/L sodium phosphate and 50-80 mmol/L sodium chloride, the pH value of the buffer solution G is 7.2-7.4, and the conductivity of the buffer solution G is 6-12 mS/cm;

in step S1, the anti-IL-17 RA monoclonal antibody is selected from any one of the following:

mAb-1, whose heavy chain variable region amino acid sequence is SEQ ID NO 1, and its light chain variable region amino acid sequence is SEQ ID NO 11;

mAb-2, whose heavy chain variable region amino acid sequence is SEQ ID NO. 2, and its light chain variable region amino acid sequence is SEQ ID NO. 11;

mAb-3, whose heavy chain variable region amino acid sequence is SEQ ID NO. 3, and its light chain variable region amino acid sequence is SEQ ID NO. 11;

mAb-4 whose heavy chain variable region has the amino acid sequence of SEQ ID NO. 4 and whose light chain variable region has the amino acid sequence of SEQ ID NO. 11;

mAb-5 whose heavy chain variable region has the amino acid sequence of SEQ ID NO. 5 and whose light chain variable region has the amino acid sequence of SEQ ID NO. 11.

2. The method of purifying an anti-IL-17 RA monoclonal antibody of claim 1, further comprising a heavy chain constant region that is one of human IgG1, IgG2, IgG3, IgG4 and a light chain constant region that is human ck.

3. The method of claim 2, wherein the heavy chain constant region is human IgG 1.

4. The method for purifying an anti-IL-17 RA monoclonal antibody of claim 1, wherein in step S1, the medium of the affinity chromatography column is MabSelect SurE, MabSelect Sure LX or Eshmuno-A.

5. The method for purifying an anti-IL-17 RA monoclonal antibody as claimed in claim 1, characterized in that in step S1, buffer a consists of 50mM Tris-HCl and 150mM sodium chloride, and the pH of buffer a is 7.4; the pH of the buffer solution B is 5.0; the buffer C was 50mM sodium acetate and the pH of the buffer C was 3.6.

6. The method of claim 1, wherein in step S2, the medium of the cation exchange chromatography column comprises Capto SP, Eshmuno CPX, or SepHarose DEAE.

7. The method of claim 6, wherein the medium of the cation exchange chromatography column is Eshmuno CPX.

8. The method of claim 1, wherein the buffer D is 20mmol/L sodium phosphate.

9. The method for purifying an anti-IL-17 RA monoclonal antibody as claimed in claim 1, wherein the medium of the anion exchange chromatography column is POROS XQ, Capto Q or eshumno Q.

10. The method of claim 9, wherein the medium of the anion exchange chromatography column is POROS XQ.