CN112891530B - Injection preparation of anti-IL-17 RA monoclonal antibody - Google Patents
Injection preparation of anti-IL-17 RA monoclonal antibody Download PDFInfo
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Abstract
The invention relates to the field of biological medicine, and particularly provides an injection preparation of an anti-IL-17 RA monoclonal antibody, which comprises pharmacodynamic molecules and a buffer solution, wherein the pharmacodynamic molecules are the anti-IL-17 RA monoclonal antibody, and the buffer solution comprises buffer salt, a protein protective agent, an osmotic pressure regulator and a surfactant; the pH value of the buffer solution is 5.5-6.5. The components in the buffer solution interact and cooperate with each other, so that a storage environment suitable for long-term storage is provided for the anti-IL-17 RA monoclonal antibody, the degradation, aggregation or precipitation of the antibody caused by long-term storage or transportation of a preparation can be avoided, the antibody can be stored in the environment for more than 36 months, the stability of the activity and purity of the antibody can be ensured during storage, and the efficacy of the biological medicine is ensured; the injection preparation can be used for various autoimmune diseases, including but not limited to psoriasis, rheumatoid arthritis, ankylosing spondylitis or scleroderma and the like.
Description
Technical Field
The invention relates to the technical field of biological medicine preparations, in particular to an injection preparation 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 and plaques with different sizes and clear boundaries, and a large amount of dry silvery white scales are covered on the skin, so the Psoriasis is named. 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, belongs to the category of rheumatism, has unknown etiology, and is mostly related to infection, genetic factors, environmental factors (such AS being in cold, damp and wet environments for a long time) and the like.
The traditional drugs for treating autoimmune diseases have three types: nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, and the like. In recent years, monoclonal antibodies (targeting) targeting T cells, B cells and cytokines are a new hotspot for treating autoimmune diseases, good treatment effects are achieved, and huge economic benefits are obtained at the same time.
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 5 members of the IL-17 receptor (IL-17R) family are IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, respectively. IL-17RA is widely expressed, especially in hematopoietic tissues at a high level, and can cause the production and release of various substances, 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 the like, and can also exert biological effects in combination with a plurality of IL-17 family members.
At present, many problems are often encountered in the process of developing and developing anti-IL-17 RA monoclonal antibodies, monoclonal antibody products are generally used at high dose of about 200 mg/time, but the administration volume of subcutaneous injection is limited, 1.0-1.5ml is already a limit, which indicates that the concentration of the anti-IL-17 RA monoclonal antibody is as high as 100-150mg/ml, so that the exploration of the preparation components and the process thereof has great challenges, how to avoid the degradation of the injection preparation during long-term storage and the increase of charge variants, how to avoid the generation of polymers and even the generation of precipitates caused by the aggregation of molecules under the long-term storage under the uncertain actions, and the series of problems all affect the stability of the anti-IL-17 RA monoclonal antibody during long-term storage, for this reason, the development of highly stable anti-IL-17 RA monoclonal antibody preparations at high concentrations is an urgent need to ensure its clinical stability and safety.
Disclosure of Invention
In order to solve the problems encountered in the downstream process development of the injection preparation of the anti-IL-17 RA monoclonal antibody in the prior art, the invention discloses an injection preparation capable of keeping the long-term stability of the anti-IL-17 RA monoclonal antibody.
The specific technical scheme of the invention is as follows:
the invention provides an injection preparation of an anti-IL-17 RA monoclonal antibody, which comprises a pharmacodynamic molecule and a buffer solution, wherein the pharmacodynamic molecule is the anti-IL-17 RA monoclonal antibody with the protein content of 100-150mg/ml, and the buffer solution comprises the following components in percentage by weight:
wherein the pH value of the buffer solution is 5.5-6.5.
The injection preparation of the anti-IL-17 RA monoclonal antibody provided by the invention is obtained through a large number of experimental verifications, a proper amount of buffer salt, a protein protective agent, an osmotic pressure regulator and a surfactant are added into a buffer solution, so that the pH value of the injection preparation can be effectively regulated, a proper environment is provided for pharmacodynamic molecules, a protective effect on pharmacodynamic molecule proteins is effectively achieved, the increase of charge variants and the degradation of small molecules caused by the influences of temperature, oxidation, enzymolysis, surface tension and the like on the pharmacodynamic molecules can be avoided, meanwhile, the aggregation and physical aggregation caused by chemical reaction or non-covalent bonds in the long-time storage process of the pharmacodynamic molecules in the buffer solution can be avoided, the aggregation of the antibody molecules into polymers and even the occurrence of precipitation and other conditions can be avoided, and the long-term stability of the high-concentration monoclonal antibody is effectively ensured.
Further, the anti-IL-17 RA monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and 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.
Further, the anti-IL-17 RA 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, the buffer salt comprises one or more of acetate buffer, phosphate buffer, citrate buffer, histidine buffer and tris buffer.
The buffer salt provided by the invention is screened in a large number of experiments according to the isoelectric point of the anti-IL-17 RA monoclonal antibody, and the buffer salt including one or more of acetate buffer solution, phosphate buffer solution, citrate buffer solution, histidine buffer solution and tris buffer solution can meet the requirements of the anti-IL-17 RA monoclonal antibody provided by the invention.
Preferably, the buffer salt is a citrate buffer solution, the pKa of the citrate buffer solution is similar to that of blood, the physiological compatibility is good, a relatively high buffer capacity can be provided under low ionic strength, and the buffer salt has a good metal chelating agent effect and can reduce the oxidation effect of trace heavy metals on antibody protein.
Preferably, the pH of the buffer solution is 6.0.
Further, the protein protective agent comprises sugar and/or amino acid, wherein the sugar comprises one or more of trehalose, mannitol and sucrose; the amino acid comprises one or more of glycine, arginine or histidine;
preferably, the protein protective agent is a combination of the trehalose, the arginine and the histidine in a content ratio of 1:1: 1.
The protein protective agent selects sugar or amino acid, can prevent the change of the secondary structure of the anti-IL-17 RA monoclonal antibody protein, has obvious inhibiting effect on the extension and aggregation of the protein, effectively plays a role in protecting the anti-IL-17 RA monoclonal antibody protein, and effectively ensures the long-term stability of the anti-IL-17 RA monoclonal antibody.
Further, the osmotic pressure regulator comprises one or more of sodium chloride, magnesium chloride, glucose and sorbitol. The osmotic pressure regulator can regulate the osmotic pressure of the preparation to be basically consistent with the physiological osmotic pressure of a human body, avoids the irritation of a high-concentration preparation to the human body, and finds that the osmotic pressure regulator can meet the requirements of the preparation by selecting one or more of sodium chloride, magnesium chloride, glucose and sorbitol through a large number of experiments according to the physicochemical properties of the anti-IL-17 RA monoclonal antibody, and the osmotic pressure can be possibly changed by changing any other components or contents.
Further, the surfactant comprises one or more of Tween 20, Tween 80 and poloxamer;
preferably, the surfactant is tween 20.
The addition of the surfactant can prevent the polymerization of the anti-IL-17 RA monoclonal antibody protein product, and effectively ensure the stability of the antibody in long-term storage.
The invention also provides application of the injection preparation of the anti-IL-17 RA monoclonal antibody in preparing a medicament for treating autoimmune diseases.
Preferably, the autoimmune disease comprises psoriasis, rheumatoid arthritis, ankylosing spondylitis, or scleroderma.
The invention has the following beneficial effects: the anti-IL-17 RA monoclonal antibody provided by the invention has stronger affinity and good biological activity, can be specifically combined with an IL-17RA antigen, blocks the combination of IL-17A and IL-17RA, antagonizes and blocks an IL-17/IL-17R signal channel, is an autoimmune disease treatment drug with great potential, effectively relieves the symptoms of autoimmune diseases, and prevents the progress of the diseases; the injection preparation provided by the invention provides a storage environment suitable for long-term storage for the anti-IL-17 RA monoclonal antibody through the interaction and the synergistic cooperation of buffer salt, a protein protective agent, an osmotic pressure regulator and a surfactant in a buffer solution, can avoid the degradation, aggregation or precipitation of the antibody caused by long-term storage or transportation of the preparation, can be stored for at least more than 36 months in the environment, and can ensure the stability of the activity and purity of the antibody during storage, thereby ensuring the efficacy of biological medicines; the injection preparation can be used for various autoimmune diseases, including but not limited to psoriasis, rheumatoid arthritis, ankylosing spondylitis or scleroderma and the like.
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 the first experiment of the present invention;
FIG. 4 is a plasmid map of pTSE in experiment two 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 the third experiment of the present invention;
FIG. 6 is a graph comparing the ability of anti-IL-17 RA whole antibodies in competitive inhibition of IL-17A binding to IL-17RA in the fourth experiment of the present invention;
FIG. 7 is a graph showing a comparison of the binding ability of anti-IL-17 RA whole antibody to IL-17RA at the cellular level in the sixth experiment of the present invention;
FIG. 8 is a graph showing a comparison of the biological activities of the anti-IL-17 RA antibody of the seventh 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.
Examples 1 to 15
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 ℃. PBST-4% mik was used to block the immune tube and phage antibody library, respectively (phage input was about 10)9-1012) Blocking at 37 ℃ for 1 h. 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 5h to its logarithmic growth phase, and added to about 10 wells per well10The helper phage M13KO7 was cultured with shaking at 150rpm for 1 hour after 30min of static infection at 37 ℃. 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-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.
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.
Preferably, the monoclonal antibody is a fully human antibody.
The invention provides an anti-IL-17 RA monoclonal antibody capable of specifically binding IL-17RA and blocking the binding pathway with IL-17A.
The components and contents of the buffer solution in the injection preparations provided in examples 1 to 15 are shown in table 1.
TABLE 1 table of contents of respective components in buffer solutions in examples 1 to 15
Examples 16 to 19
Examples 16 to 19 of the present invention respectively provide an injection preparation of an anti-IL-17 RA monoclonal antibody, which comprises a pharmacodynamic molecule and a buffer solution, wherein the pharmacodynamic molecule is the anti-IL-17 RA monoclonal antibody with a protein content of 100mg/ml, and the amino acid sequence of the anti-IL-17 RA monoclonal antibody is the same as that of examples 1 to 15.
The monoclonal antibody also includes 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. The components and contents of the buffer solutions in the injection preparations provided in examples 16 to 19 are shown in Table 2.
TABLE 2 table of contents of respective components in buffer solutions in examples 16 to 19
Examples 20 to 22
Examples 20 to 22 of the present invention respectively provide an injection preparation of an anti-IL-17 RA monoclonal antibody, which comprises a pharmacodynamic molecule and a buffer solution, wherein the pharmacodynamic molecule is the anti-IL-17 RA monoclonal antibody with a protein content of 150mg/ml, and the amino acid sequence of the anti-IL-17 RA monoclonal antibody is the same as that of examples 1 to 15.
The monoclonal antibody also includes 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.
The components and contents of the buffer solutions in the injection preparations provided in examples 20 to 22 are shown in Table 3.
TABLE 3 table of contents of respective components in buffer solutions in examples 20 to 22
Example 23
The embodiment 23 of the invention provides application of an injection preparation of an anti-IL-17 RA monoclonal antibody in preparing a medicament for treating autoimmune diseases, wherein the autoimmune diseases comprise psoriasis, rheumatoid arthritis, ankylosing spondylitis or scleroderma.
Comparative example 1
Comparative example 1 of the present invention provides an injectable preparation based on example 1, in which the buffer salt in example 1 is replaced with PB buffer solution in an amount of 30mM, and the other components are the same.
Comparative example 2
Comparative example 2 of the present invention provides an injectable preparation based on example 4, in which the pH of the buffer solution in example 4 was replaced with 6.8 and the other components were all the same.
Comparative example 3
Comparative example 3 of the present invention provides an injectable preparation based on example 16, in which the protein protecting agent of example 16 was replaced with proline in an amount of 9% (w/v), and the other components were all the same.
Comparative example 4
Comparative example 4 of the present invention provides an injectable preparation based on example 20, in which the surfactant in example 20 was replaced with TritonX100 in an amount of 0.015% (w/v), and the other ingredients were all the same.
Experiment I, gradient dilution of phage Elisa to compare affinity of anti-IL-17 RA monoclonal antibody
1.1 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 and 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 next day, PEG6000-NaCl was sedimented to purify phages. 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.
1.2 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 l/well) was added, and blocked at 37 ℃ for 1 h. Meanwhile, 11 strains of the single clone purified phage samples obtained from the screening in example 1 and single clone purified phage samples prepared from anti-IL-17 RA monoclonal antibody (AMH14/AML14, Brodalumab) provided in patent US7833527B2 were diluted in 5-fold gradient using PBST-4% mik, respectively, for 8 gradients, and the gradient diluted phage samples were blocked at 37 ℃ for 1 hour. PBST-4% mil k from Elisa plates was discarded, and a gradient diluted phage sample (100. mu.l/well) was added and allowed to stand at 37 ℃ for 1 h. 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) stopAnd (4) developing color. The microplate reader wavelength was 450nm and 630nm read. The data were analyzed and plotted using the software GraphPad Prism 5Demo, with results as shown in figure 3 and data as shown in table 4.
TABLE 4 results of gradient dilution Elisa experiments at phage level
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.
Experiment II, preparation of anti-IL-17 RA holoantibody
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 5. And simultaneously expressing a full antibody of the Brodalumab purified on the market as a positive control.
TABLE 5 Total antibody protein concentration data Table
| 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 |
Experiment III, anti-IL-17 RA full antibody and IL-17RA binding experiment on 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 three 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 experiment two 4 were diluted using PBST-4% mil gradients with an initial maximum concentration of 10 μ g/mL for all antibodies, 5-fold dilutions, 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. The Elisa plates were washed five times with PBST, and goat 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. Microplate reader wavelength450nm and 630nm readings. The data were plotted using the software GraphPad Prism 5Demo analysis and the results are shown in fig. 5 and table 6.
TABLE 6 data table of full antibody affinity experiment EC50 values
| Name of antibody | EC50(ng/ml) | Name of antibody | EC50(ng/ml) |
| Brodalumab | 108 | mAb-6 | 46.7 |
| mAb-1 | 42.97 | mAb-7 | 61.15 |
| mAb-2 | 23.34 | mAb-8 | 14.38 |
| mAb-3 | 25.15 | mAb-9 | 25.46 |
| mAb-4 | 26.24 | mAb-10 | 35.58 |
| mAb-5 | 25.1 | mAb-11 | 30.65 |
As shown in FIG. 5 and Table 6, the selected 11 different monoclonal antibodies all can bind to IL-17RA, and the EC50 values of the 11 different monoclonal antibodies provided by the present invention are all significantly lower than those 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 6, 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.
Experiment four, experiment for inhibiting IL-17A and IL-17RA by full antibody competition
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 4 were diluted in different dilutions using PBST-4% mil gradients, with the initial highest concentration of all antibodies being 300 μ g/mL, diluted in 3-fold gradients, and 8 gradients for each whole antibody. PBST-4% mil in an Elisa plate was discarded, IL-17RA-ECD-His (50. mu.l/well) and a whole antibody sample (50. mu.l/well) diluted in a gradient were added, respectively, and incubated at 37 ℃ for 2 h. PBST washing Elisa plate 5 times, adding PBST-4% mill k dilution 1:5000 mouse anti His IgG-HRP secondary antibody, at 37 degrees C temperature conditions were incubated 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) stopAnd (4) developing color. The microplate reader wavelength was 450nm and 630nm read. The results of the experiments are shown in fig. 6 and table 7 using the software GraphPad Prism 5Demo mapping.
TABLE 7 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 7, 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 7, 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.
Experiment five, BIAcore X100 determination of the affinity of the Total 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 the antibody was diluted with HBS-EP buffer to a concentration of 1. mu.g/ml each to ensure that it could be captured by goat anti-human IgG. The affinity of the 11 whole antibodies provided in experiment two and the whole antibody of Brodalumab, a commercial product of positive control, was 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) set up and the data are shown in the following table.
TABLE 8 data table of full antibody affinity results determined by Biacore
| 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 8, 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.
Experiment six, analysis of binding specificity of full antibody and 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 4 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 106Each well was incubated with 50. mu.l of cells and 50. mu.l of whole anti-IL-17 RA antibody diluted in a gradient 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 Prism 5Demo and are shown in FIG. 7 and Table 9.
TABLE 9 binding experiments of anti-IL-17 RA holoantibodies to cell surface IL-17RA
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.
Experiment seven, experiment of bioactivity 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 4 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 10.
TABLE 10 biological Activity test of anti-IL-17 RA antibodies
| Name of antibody | IC50(ng/ml) | Name of antibody | IC50(ng/ml) |
| Brodalumab | 116.8 | mAb-6 | 33.41 |
| mAb-1 | 28.37 | mAb-7 | 29.69 |
| mAb-2 | 22.72 | mAb-8 | 13.54 |
| mAb-3 | 33.42 | mAb-9 | 24.76 |
| mAb-4 | 35.77 | mAb-10 | 36.35 |
| mAb-5 | 22.92 | mAb-11 | 60.8 |
As shown in the data in Table 10 and FIG. 8, it is demonstrated 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 the positive control Brodalumab whole antibody marketed as a product, indicating 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 show that the activity of the mAb-8 whole antibody is the best among the 11 whole antibodies, and the competition with IL-17A is the strongest.
Experiment eight, accelerated stability experiment
The preparation method of the antibody preparation comprises the following steps: firstly, selecting the anti-IL-17 RA monoclonal antibody mAb-8 with the highest binding capacity and the best activity with IL-17RA from experiments one to seven as a pharmacodynamic molecule for standby, then mixing a buffer solution, a protein protective agent and an osmotic pressure regulator according to different contents in examples 1-22 and a control example 5 to prepare a buffer solution, then concentrating and replacing the anti-IL-17 RA monoclonal antibody (mAb-8) into the mixed buffer solution through an ultrafiltration tube, adjusting the concentration of the antibody protein to be 100-150mg/ml, and finally adding a surfactant to prepare the injection preparation of 26 anti-IL-17 RA monoclonal antibodies.
The injection preparations of 26 anti-IL-17 RA monoclonal antibodies prepared above were simultaneously placed at a high temperature of 37 ℃ for 8 weeks (w), and samples were taken at 0w, 2w, 4w, 6w, and 8w for SEC-HPLC purity and charge isomer examination.
(1) Effect of different buffer salts on formulation stability
Examination of anti-IL-17 RA monoclonal antibody injection preparations prepared in examples 1-15 and comparative examples 1-2 described above by SEC-HPLC method after accelerated storage at a high temperature of 37 ℃ for 8 weeks (w), purity and main peak content were examined by CEX-HPLC method, and the reduction was calculated; the stability data are summarized in table 11 below.
TABLE 11 stability data tables for injectable formulations provided in examples 1-15 and comparative examples 1-2 at various pH values
The data results in table 11 show that, during the accelerated test, the injection formulations provided in examples 1 to 15 of the present invention all showed a decreasing trend from the SEC-HPLC purity and the main peak content data, but the decreasing amplitude was significantly lower than that of comparative examples 1 to 2, which indicates that the injection formulations provided in the present invention can ensure the long-term stability of the anti-IL-17 RA monoclonal antibody.
Meanwhile, compared with examples 2, 5, 11 and 14, the injection preparation provided in example 8 has a small decrease in protein purity and main peak content, so it can be shown that the citrate buffer solution, which is preferred as the buffer salt provided in example 8, can effectively ensure the stability of the injection preparation and avoid aggregation of antibodies.
Example 8 compared with example 7, the protein purity and the main peak content decreased by a small amount, which indicates that the optimal pH value of the buffer solution provided by the present invention is 6.0.
In addition, compared with the examples 1 to 15, the antibody protein purity and the main peak content of the comparative example 1 are obviously reduced after accelerated placement for 8 weeks, which shows that the combination of one or more of the buffer salt selection acetate buffer solution, the phosphate buffer solution, the citrate buffer solution, the histidine buffer solution and the tris buffer solution provided by the invention can effectively ensure the long-term stability of the anti-IL-17 RA monoclonal antibody, and the replacement of any one of the buffer salt selection acetate buffer solution, the phosphate buffer solution, the citrate buffer solution, the histidine buffer solution and the tris buffer solution can possibly cause the aggregation or precipitation of the antibody protein, thereby causing the reduction of the stability.
Compared with the comparative example 4, the purity of the antibody protein and the content of the main peak are obviously reduced after the pH value is changed in the comparative example 2, which shows that the pH value of the buffer solution provided by the invention is 5.5-6.5, and the stability of the antibody is reduced when the pH value is more than or less than the range.
(2) Effect of different protein protective Agents on formulation stability
Examination by SEC-HPLC method of the anti-IL-17 RA monoclonal antibody (mAb-8) injection preparations prepared in the above examples 16 to 19 and comparative example 3, after accelerated storage at a high temperature of 37 ℃ for 8 weeks (w), purity and main peak content were examined by CEX-HPLC method, and the reduction was calculated; the stability data are summarized in table 12 below.
Table 12 stability data for injectable formulations provided in examples 16-19 and comparative example 3 with different protein protectants
The data in table 12 show that after the anti-IL-17 RA monoclonal antibody injection preparations provided in examples 16 to 19 of the present invention are placed for 8 weeks at an accelerated speed, the antibody protein purity and the main peak content decrease significantly less than those in comparative example 3, which indicates that the protein protective agent added to the injection preparations provided by the present invention can improve the long-term stability of the preparations, and the reduction of the preparation stability can be caused by any replacement or substitution of one of the protein protective agents;
in addition, the injection preparation provided by the embodiment 19 of the present invention has lower reduction of purity and main peak content compared with the injection preparations of the embodiments 16 to 18, which shows that the combination of the trehalose, the arginine and the histidine, which are preferably used in a content ratio of 1:1:1, in the protein protective agent provided by the embodiment 19 of the present invention, can make the injection preparation more stable for long-term storage.
(3) Effect of different surfactants on formulation stability
Examination by SEC-HPLC method of the anti-IL-17 RA monoclonal antibody (mAb-8) injection preparations prepared in the above examples 20-22 and comparative example 4, after accelerated storage at a high temperature of 37 ℃ for 8 weeks (w), purity and main peak content were examined by CEX-HPLC method, and the reduction was calculated; the stability data are summarized in table 13 below.
Table 13 stability data for injectable formulations provided in examples 20-22 and comparative example 4 with different surfactants
The data in table 13 show that after the anti-IL-17 RA monoclonal antibody injection preparations provided in examples 20 to 22 of the present invention are placed for 8 weeks at an accelerated speed, the decrease in the antibody protein purity and the main peak content is significantly less than that in comparative example 4, which indicates that one or more combinations of tween 20, tween 80 and poloxamer selected by the surfactants added to the injection preparations provided by the present invention can improve the long-term stability of the preparations, and the reduction in the stability of the preparations can be caused by the arbitrary replacement or substitution of one of the surfactants;
in addition, compared with examples 21 and 22, the injection preparation provided in example 20 of the present invention has a lower reduction in purity and main peak content, which indicates that the protein protectant, preferably tween 20, provided in the injection preparation provided in example 20 of the present invention can make the injection preparation more stable for long-term storage.
Experiment nine, prescription verification experiment
The prescription verification test comprises a long-term stability test, a freeze-thaw stability test and a shaking stability test.
1) Long-term stability experiments:
the anti-IL-17 RA monoclonal antibody (mAb-8) provided in examples 8, 19 and 20 was concentrated to the desired concentration, subjected to a freeze-thaw stability experiment, and the above preparation samples were placed in a 2-8 ℃ refrigerator for long-term stability study, with detection indexes including appearance, SEC-HPLC purity and main peak content, and with detection time points of 0, 3, 6, 9, 12, 18, 24 and 36 months.
TABLE 14 summary of long term stability results for antibody formulations
2) Freeze-thaw stability experiment:
the anti-IL-17 RA monoclonal antibody (mAb-8) replacement solutions provided in examples 8, 19, and 20 were concentrated to the desired concentration and subjected to freeze-thaw stability experiments for 1, 2, and 3 times of freeze-thawing, respectively. The protein purity and the charge isomer main peak content were examined with emphasis and the results are summarized in Table 15.
TABLE 15 summary of antibody formulation freeze thaw stability results
3) Shaking stability test:
the replacement solutions of the anti-IL-17 RA monoclonal antibody (mAb-8) provided in examples 8, 19 and 20 were concentrated to the desired concentrations and subjected to shaking stability experiments, with horizontal shaking (80-120rpm) and circumferential shaking (30rpm) at room temperature for 3 days, respectively. We focused on protein purity and charge isomer main peak content and the results are summarized in table 16.
TABLE 16 summary of the results of the shaking stability experiments for the antibody formulations
From tables 14-16, it can be seen that from the investigation of protein purity and charge isomer main peak content, the purity of the anti-IL-17 RA monoclonal antibody provided in the embodiment of the present invention can reach more than 97% after long-term stability experiments, freeze-thaw experiments and shaking experiments, and the concentration of the drug-effective molecules does not change much, which indicates that the preparation provided by the present invention has good stability.
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
<110> Beijing Oriental Baitai Biotechnology GmbH
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Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys His Leu
50 55 60
Leu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Phe Leu Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 4
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Tyr Ser Asn Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Lys Phe
50 55 60
Ile Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 5
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Arg Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Asp Gln Phe
50 55 60
Ile Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 6
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Arg Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Gln Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 7
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Asn Leu
50 55 60
Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 8
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Gly Phe Ala Ser Arg
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Lys Phe
50 55 60
Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 9
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Trp Thr Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Lys Phe
50 55 60
Leu Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 10
<211> 116
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Lys Lys Phe
50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Val Leu Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 11
<211> 107
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp Ser Ser
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Asn Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ala Asn Met Pro Gly
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 12
<211> 107
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Ile Ser
20 25 30
Leu Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile
35 40 45
Tyr Gly Trp Asn Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Phe Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 13
<211> 107
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val His Thr Gly
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu His Asn Pro Leu
85 90 95
Thr Phe Gly Gln Gly Ile Lys Val Glu Ile Lys
100 105
<210> 14
<211> 329
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 14
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 15
<211> 107
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 15
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
Claims (12)
1. An injection preparation of an anti-IL-17 RA monoclonal antibody, which is characterized in that the preparation comprises a pharmacodynamic molecule and a buffer solution, wherein the pharmacodynamic molecule is the anti-IL-17 RA monoclonal antibody with the protein content of 100-150mg/ml, and the buffer solution comprises the following components:
wherein the pH value of the buffer solution is 5.5-6.5;
the anti-IL-17 RA monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and 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.
2. The injectable formulation of 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 injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 2, wherein the heavy chain constant region is human IgG 1.
4. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 1, wherein the buffering salt comprises one or more combinations of acetate buffer, phosphate buffer, citrate buffer, histidine buffer, tris buffer.
5. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 4, wherein the buffer salt is a citrate buffer.
6. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 4, wherein the buffer solution has a pH of 6.0.
7. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 1, wherein the protein protectant comprises a sugar and/or amino acid, the sugar comprising one or more combinations of trehalose, mannitol, sucrose; the amino acid comprises one or more of glycine, arginine or histidine.
8. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 7, wherein the protein protectant is a combination of the trehalose, the arginine, and the histidine in a 1:1:1 ratio.
9. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 1, wherein the osmolality adjusting agent comprises one or more of sodium chloride, magnesium chloride, glucose, sorbitol.
10. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 1, wherein the surfactant comprises one or more of tween 20, tween 80, and a poloxamer in combination.
11. The injectable formulation of an anti-IL-17 RA monoclonal antibody of claim 10, wherein the surfactant is tween 20.
12. Use of an injectable formulation of an anti-IL-17 RA monoclonal antibody of any one of claims 1-3 in the manufacture of a medicament for the treatment of an autoimmune disease; the autoimmune disease comprises psoriasis, rheumatoid arthritis, ankylosing spondylitis, or scleroderma.
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| CN202010562715.1A CN111840217B (en) | 2020-06-19 | 2020-06-19 | Injection preparation of anti-IL-17 RA monoclonal antibody |
| CN202110109289.0A CN112891530B (en) | 2020-06-19 | 2020-06-19 | Injection preparation of anti-IL-17 RA monoclonal antibody |
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| WO2022113105A1 (en) * | 2020-11-25 | 2022-06-02 | Dr. Reddy's Laboratories Limited | Stable therapeutic protein formulation and methods of making the same |
| WO2022166918A1 (en) * | 2021-02-05 | 2022-08-11 | 百奥泰生物制药股份有限公司 | Anti-il-5 antibody formulation, preparation method therefor and use thereof |
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| CN101541833B (en) * | 2006-10-02 | 2014-01-29 | 麒麟-安姆根有限公司 | Il-17 receptor a antigen binding proteins |
| LT2523688T (en) * | 2010-01-15 | 2018-03-12 | Kirin-Amgen, Inc. | Antibody formulation and therapeutic regimens |
| AR083546A1 (en) * | 2010-10-25 | 2013-03-06 | Genentech Inc | TREATMENT OF GASTROINTESTINAL INFLAMMATION, SORIASIS AND ASTHMA |
| WO2013016220A1 (en) * | 2011-07-22 | 2013-01-31 | Amgen Inc. | Il-17 receptor a is required for il-17c biology |
| CN103936854B (en) * | 2014-04-30 | 2016-08-17 | 北京精益泰翔技术发展有限公司 | Anti-IL-17A monoclonal antibody and preparation and application thereof |
| CN109745559A (en) * | 2017-11-01 | 2019-05-14 | 三生国健药业（上海）股份有限公司 | The liquid preparation of the monoclonal antibody of anti-human IL-17A |
| CN108586613B (en) * | 2018-05-08 | 2021-06-22 | 济南泰和医药科技有限公司 | Human antibody targeting CD19 and preparation and application thereof |
| CN110585430B (en) * | 2019-09-29 | 2023-09-08 | 华博生物医药技术（上海）有限公司 | Pharmaceutical composition of humanized anti-human IL-17A monoclonal antibody |
| CN110840830B (en) * | 2019-10-25 | 2020-07-24 | 北京东方百泰生物科技有限公司 | Injection preparation of anti-CT L A-4 monoclonal antibody |
| CN110974958B (en) * | 2019-12-25 | 2020-08-21 | 北京东方百泰生物科技有限公司 | Injection preparation of anti-PD-L1 monoclonal antibody |
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| CN111840217A (en) | 2020-10-30 |
| CN111840217B (en) | 2021-04-02 |
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