CN107619442B

CN107619442B - Recombinant anti-TNF-alpha fully human monoclonal antibody preparation

Info

Publication number: CN107619442B
Application number: CN201710077091.2A
Authority: CN
Inventors: 郭亚军; 寇庚; 钱卫珠; 郭怀祖; 徐进
Original assignee: Taizhou Mabtech Pharmaceutical Co ltd
Current assignee: Taizhou Mabtech Pharmaceutical Co ltd
Priority date: 2016-07-15
Filing date: 2016-07-15
Publication date: 2022-09-20
Anticipated expiration: 2036-07-15
Also published as: CN107619442A

Abstract

The invention discloses a stable preparation for the novel recombinant anti-TNF-alpha fully human monoclonal antibody. The invention provides a novel anti-TNF-alpha antibody, wherein a light chain of the anti-TNF-alpha antibody has the amino acid sequence shown in SEQ ID NO:1, and the heavy chain has the amino acid sequence of SEQ ID NO:3, or a pharmaceutically acceptable salt thereof. The invention also encompasses aqueous pharmaceutical formulations comprising a therapeutically effective dose of an antibody in a buffer that enhances the stability of the antibody pharmaceutical formulation.

Description

Recombinant anti-TNF-alpha fully human monoclonal antibody preparation

The present application is a divisional application of a patent application having an application number of "201610554636X" entitled "a recombinant anti-TNF- α fully human monoclonal antibody preparation".

Technical Field

The invention belongs to the technical field of biology, and particularly discloses a preparation formula of a recombinant anti-TNF-alpha fully human monoclonal antibody.

Background

Tumor necrosis factor (TNF-. alpha.) is a cytokine produced by various types of cells, including monocytes and macrophages, and was originally identified by their ability to induce necrosis of certain tumors in mice (see Old, L. (1985) Science 230: 630-. Subsequently, it was confirmed that a factor called cachexia protein associated with cachexia is the same molecule as TNF-. alpha.. TNF- α has been shown to be associated with mediating shock, and with the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft versus host disease, among others.

TNF- α is an important inflammatory cytokine that plays an important role in normal immune function and in the cascade of reactions that trigger inflammatory processes. In the synovial fluid of the joint of a patient with rheumatoid arthritis, TNF-alpha and other inflammatory factors are abundantly expressed. The role of TNF- α as a key inflammatory factor in the cascade of inflammatory processes and possible mechanisms in rheumatoid arthritis include: (1) inducing the synthesis of other inflammatory factors such as IL-1, IL-6, IL-8, TGF, GM-CSF and the like; (2) stimulating the production of inflammatory mediators such as prostaglandin E2 and leukotriene B4; (3) activating leukocytes and assisting in the exudation of leukocytes to inflammatory sites by up-regulating E-selectin, vascular cell adhesion molecules and intracellular adhesion molecules; (4) stimulates the production of collagenase and matrix metalloproteinase by neutrophils and fibroblasts. In addition, TNF- α can also induce apoptosis and produce acute phase responses. Therefore, compared with the blocking of other cytokines (such as IL-1), the blocking of the generation and the action of the TNF-alpha can more comprehensively and effectively inhibit the development of the rheumatoid arthritis.

The TNF-alpha inhibitor is the most effective treatment means for treating rheumatoid arthritis at present, has quick response and definite curative effect, and particularly is an anti-TNF-alpha monoclonal antibody. However, originally, murine anti-TNF- α monoclonal antibodies were prepared for use in the treatment of rheumatoid arthritis and for neutralizing TNF- α. However, researches find that the mouse-derived monoclonal antibody has many defects as a therapeutic drug, and the mouse-derived monoclonal antibody has strong immunogenicity, fast elimination in vivo and short half-life when being used for a human body, so that the clinical curative effect is limited and the side effect is great. The humanized monoclonal antibody technology partially overcomes the defects of the anti-TNF-alpha murine monoclonal antibody. Wherein the human murine fully human anti-TNF-alpha monoclonal antibody (Infliximab,

) The variable region of the monoclonal antibody is still obtained from a murine TNF-alpha monoclonal antibody by utilizing a genetic engineering upstream construction technology, the specificity and the affinity (Ka-1010M-1) of the monoclonal antibody combined with a soluble fragment and a transmembrane region of a tumor necrosis factor are reserved, the constant region is replaced by the constant region of human IgG1, and the half-life period in vivo is greatly prolonged.

Infliximab is a human murine chimeric monoclonal antibody that specifically blocks TNF- α, which blocks the binding of TNF- α to its receptor by neutralizing the biological activity of TNF- α through high affinity binding to soluble and transmembrane forms of TNF- α. While Infliximab further kills cells expressing TNF- α by antibody-dependent cytotoxicity and complement-dependent cytotoxicity. Infliximab, however, does not neutralize the effects of the cytokine TNF β (also known as lymphotoxin), which shares the same receptor as TNF- α.

Is the trade name of Infliximab, and the product is sterile, white and freeze-dried powder injection for intravenous infusion, each bottle contains 100mg of Infliximab, 500 mg of cane sugar, 800.5 mg of polysorbate, 2.2 mg of sodium dihydrogen phosphate and 6.1 mg of disodium hydrogen phosphate, and does not contain preservatives. Before use, the mixture is dissolved in 10 ml of sterile water for injection of USP, the pH value is 7.2, and the mixture is diluted by physiological saline and then is subjected to intravenous drip.

Antibodies, like other protein therapeutics, are complex molecules and large amounts of antibodies must not be used in pharmaceutical formulations due to the therapeutically effective dose in mammals, particularly humans. Liquid formulations of protein therapeutics should retain the intact biological activity of the protein therapeutic and protect the functional groups of the protein therapeutic from degradation during production and shelf life, the degradation pathway of the protein may involve chemical or physical instability.

The antibody drug preparation should be stable for a long period of time, contain a safe and effective amount of drug preparation, and is determined by the specific structure and properties of the antibody, and the antibody drug needs an environment capable of stabilizing the antibody drug in the preparation, storage and transportation processes. For different kinds of proteins, the physicochemical properties, degradation reactions, and the like of different kinds of antibodies are different, and therefore, the formulation of the buffer composition for antibody pharmaceutical preparations is also different.

In order to solve the stability problem of antibody drugs and ensure the effectiveness of the antibody drugs in the transportation and storage processes, a plurality of antibody drug preparations are supplied to patients in the form of powder injection. However, the powder injection needs to be used in sterile water for injection or a liquid preparation prepared for dissolving the antibody, and thus the use of the powder injection causes unnecessary troubles for patients.

The Subcutaneous (SC) injection is a preferred embodiment of the monoclonal antibody for treating chronic diseases at present, and the antibody medicament is prepared into a prefilled needle type liquid preparation, so that the carrying and the use of a patient are greatly facilitated. However, factors such as the amount of drug in the formulation, the physical properties and chemical properties of the drug affect the effectiveness of the formulation, and a suitable buffer system is required to improve the stability of the high concentration antibody formulation while ensuring the therapeutic properties of the drug. Different formulation strategies have been tried to reduce the viscosity of high concentration monoclonal antibody solutions, formulated with salts, amino acids, or sugars. Sugars and polyols are non-specific protein stabilizers, with sucrose, trehalose, glycerol, mannitol, sorbitol, and the like being the most commonly used.

The commercial TNF-alpha antibody preparation Humira comprises the following preparation formula: antibody 50mg/ml, sodium chloride 6.16mg/ml, sodium dihydrogen phosphate dihydrate 0.86mg/ml, disodium hydrogen phosphate dihydrate 1.53mg/ml, sodium citrate 0.3mg/ml, citric acid monohydrate 1.3mg/ml, Tween 801 mg/ml, mannitol 12mg/ml, pH 5.2.

Disclosure of Invention

In order to solve the above problems and to obtain a stable pharmaceutical formulation of anti-TNF- α antibodies with low immunogenicity, the present invention provides a novel anti-TNF- α antibody, the light chain of which has the amino acid sequence of SEQ ID NO:1, and the heavy chain has the amino acid sequence of SEQ ID NO:3, or a pharmaceutically acceptable salt thereof. The invention also discloses a stable preparation for the novel recombinant anti-TNF-alpha fully human monoclonal antibody.

The invention also encompasses aqueous pharmaceutical formulations comprising a therapeutically effective amount of an antibody in a buffer, resulting in a formulation having a pH of about 4 to 8 and a shelf life of at least 24 months in the liquid state.

The present invention provides stable formulations that, in one embodiment, have enhanced stability, in another embodiment are stable after freeze-thaw cycling testing, and in other embodiments are stable after high temperature, shaking, light, and prolonged testing. The stable formulations of the invention comprise an anti-TNF-. alpha.antibody, wherein the anti-TNF-. alpha.antibody is CMAB 815.

The invention discloses:

a formulation of a recombinant anti-TNF- α fully human monoclonal antibody, comprising a therapeutically effective amount of the antibody in a buffer, wherein the formulation has a pH of about 4 to about 8 and a shelf life of at least 24 months.

The formulation of recombinant anti-TNF- α fully human monoclonal antibody as described above, wherein the antibody is present at a concentration of about 1mg/ml to about 100 mg/ml.

The formulation of recombinant anti-TNF- α fully human monoclonal antibody as described above, wherein the antibody is at a concentration of about 50 mg/ml.

The recombinant anti-TNF- α fully human monoclonal antibody formulation as described above, wherein the antibody is CMAB815 and the light chain has the amino acid sequence of SEQ ID NO:1, and the heavy chain has the nucleotide sequence of SEQ ID NO:3, or a pharmaceutically acceptable salt thereof.

The recombinant anti-TNF-alpha fully human monoclonal antibody preparation is characterized by comprising 1-100mg/ml of CMAB 815; 0.1-10mg/ml polysorbate 80; 5-20mg/ml mannitol; and a buffer system comprising citrate and/or phosphate, the pH being 4-8.

The preparation of the recombinant anti-TNF-alpha fully human monoclonal antibody is characterized in that the pH value of the buffer solution is 4.9-5.5.

A recombinant anti-TNF- α fully human monoclonal antibody formulation as described above, wherein the buffer ph5.2 comprises 50mg/ml CMAB 815; 1.0mg/ml polysorbate 80; 12.0mg/ml mannitol; 6.16mg/ml sodium chloride; 1.19mg/ml anhydrous citric acid; 0.3mg/ml citric acid dihydrate; 0.86mg/ml sodium dihydrogen phosphate dihydrate; 1.53mg/ml disodium phosphate dihydrate.

Use of a formulation of a recombinant fully human monoclonal antibody directed against TNF- α as defined above, for the preparation of a medicament for the treatment of a condition in which TNF- α activity is detrimental.

The application of the recombinant anti-TNF-alpha fully human monoclonal antibody preparation is characterized in that the preparation is used for preparing medicines for treating rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis and the like.

The present invention contemplates formulations of CMAB815 antibodies, in a preferred embodiment of the invention, the liquid pharmaceutical formulation comprises: CMAB815, which comprises 50 mg/ml; 1.0mg/ml polysorbate 80; 12.0mg/ml mannitol; 6.16mg/ml sodium chloride; 1.19mg/ml anhydrous citric acid; 0.3mg/ml citric acid dihydrate; 0.86mg/ml sodium dihydrogen phosphate dihydrate; 1.53mg/ml disodium phosphate dihydrate, pH 5.2.

Drawings

FIG. 1, CMAB815 formulation thermal stability conventional map

FIG. 2, CMAB815 formulation thermostability derivatisation profiles

FIG. 3 is a graph showing pH changes in the vibration, high temperature, light irradiation and strong destruction tests

FIG. 4 is a graph showing the change in protein content in the shaking, high temperature, light irradiation and strong destruction tests

FIG. 5 is a graph showing the purity change of protein reduction electrophoresis in the oscillation, high temperature, light irradiation and strong destruction tests

FIG. 6 is a graph showing the variation of high molecular impurities in the oscillatory, high temperature, light irradiation and strong destruction tests

FIG. 7 is a graph of the variation of charge heteroplasms in the oscillation, high temperature, light irradiation and strong destruction tests

FIG. 8 is a graph showing the change in biological activity of proteins in the shaking, high-temperature, light-irradiation and strong destruction tests

FIG. 9 graph of the change in protein content in the freeze-thaw test

FIG. 10, graph of purity change of protein reduction electrophoresis in freeze-thaw test

FIG. 11 is a graph showing the variation of high molecular weight impurities in a freeze-thaw test

FIG. 12, graph of the change in biological activity of proteins in the freeze-thaw test

FIG. 13, long-term stability assay protein content profiles of different batches

FIG. 14, long term stability assay bioactivity profiles for different batches

FIG. 15 shows the detection spectra of the reduction electrophoresis purity of different batches of long-term stability tests

FIG. 16, SEC-HPLC/UPLC high molecular weight impurity detection profiles for different batches of long-term stability tests

FIG. 17 shows the charge heteroplasmon distribution detection profiles of different batches of long-term stability tests.

Detailed Description

Example 1 preparation of recombinant anti-TNF-alpha fully human monoclonal antibody CMAB815

Artificially synthesizing a nucleotide sequence of a recombinant anti-TNF-alpha fully human monoclonal antibody CMAB815, carrying out enzyme digestion on the nucleotide sequence, and then connecting the nucleotide sequence with a plasmid pcDNA3.1(+) (Invitrogen company) to construct a fully human light chain and heavy chain eukaryotic expression vector, wherein SEQ ID NO 1 and SEQ ID NO 3 respectively show a heavy chain nucleotide sequence of the fully human monoclonal antibody CMAB 815; the amino acid sequences of the fully human monoclonal antibody CMAB815 are shown in SEQ ID NO 2 and SEQ ID NO 4, respectively. 3X 10 inoculation in 3.5cm tissue culture dish ⁵ CHO-K1 cells (ATCC CRL-9618) were transfected when cultured to 90% -95% confluence.

And (3) carrying out amplification culture on the high-expression clone obtained by screening by using a serum-free culture medium: and (2) aiming at serum-free culture, repeatedly comparing and optimizing to finally determine a culture medium (CHOM-B08) and a supplement culture medium (CHOM-S08) suitable for the recombinant anti-TNF-alpha fully human monoclonal antibody CMAB815, wherein the expression amount of the engineered cells in the optimized culture medium is more than 30pg/cell.

The fully human monoclonal antibody CMAB815 was isolated and purified by Protein A affinity column (product of GE).

The CMAB815 obtained by the above preparation method has a different sugar type from the existing products.

Example 2 preparation of formulations

CMAB815 antibody was obtained according to the preparation method of CMAB815 described in example 1. In the purification step of CMAB815, CMAB815 antibody was purified and dissolved in a solution containing 1.0mg/ml polysorbate 80 using size exclusion chromatography; 0mg/ml or 12.0mg/ml mannitol; 6.16mg/ml sodium chloride; 1.19mg/ml anhydrous citric acid; 0.3mg/ml citric acid dihydrate; 0.86mg/ml sodium dihydrogen phosphate dihydrate; 1.53mg/ml disodium hydrogen phosphate dihydrate in buffer.

After the CMAB815 antibody solution is obtained, sucrose with different concentrations is added into the preparation solution, and grouping is carried out to carry out stability test detection. The grouping situation is as in table 1.

TABLE 1 grouping of buffers for different formulations

Test example 1 analysis of thermal stability test

The thermostability of the antibodies was assayed by real-time quantitative PCR using Protein Thermal Shift Dye Kit (4461146) reagents and software from Life Technologies in various formulations of buffer for thermostability. Selecting a better preparation formula.

The thermal stability test was performed according to the test procedure described in the kit:

1) mixing the test sample with Protein Thermal Shift dye

2) And running a dissolution curve test on a real-time quantitative PCR instrument, unfolding the protein when heated, and combining the dye with the exposed hydrophobic region to emit fluorescence.

3) The results of the Thermal stability analysis were obtained by the Protein Thermal Shift software, and are shown in fig. 1, fig. 2 and table 2.

Table 2, test results for different formulations of buffer are as follows:

wherein the maximum temperature of 70.91 ℃ and the minimum temperature of 70.32 ℃ are within a range of 0.59 ℃, and the significant difference index of 1 ℃ specified by the kit is not reached;

therefore, there was no significant difference in all the above formulations from the TmB perspective.

According to various test results, a preparation formula of pH5.2, sucrose 20.0g/L, polysorbate 1.0g/L, sodium chloride 6.16g/L, anhydrous citric acid 1.19g/L, sodium citrate dihydrate 0.3g/L, sodium dihydrogen phosphate dihydrate 0.86g/L and disodium hydrogen phosphate dihydrate 1.53g/L is selected.

Test example 2 stability test

A preparation formula of pH5.2, sucrose 20.0g/L, polysorbate 1.0g/L, sodium chloride 6.16g/L, anhydrous citric acid 1.19g/L, sodium citrate dihydrate 0.3g/L, sodium dihydrogen phosphate dihydrate 0.86g/L and disodium hydrogen phosphate dihydrate 1.53g/L is selected and selected, and an oscillation test, a high-temperature test, a lighting test, a freeze-thaw test and a strong destruction test are carried out to further verify the stability of the preparation. The experimental design is shown in tables 3 and 4.

TABLE 3 influence factor test and Placement conditions

TABLE 4 investigation time points and investigation items of the raw liquid influence factor test

The research result is as follows:

the results of a brute force destruction test show that the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution has a tendency of increasing high molecular weight impurities along with the standing time under the conditions of 40 ℃ and 60% relative humidity, and the high molecular weight impurities exceed the requirement of the stock solution quality standard limit (1.0%) by 10 days; the proportion of the charge heteroplasmon (K0+ K1+ K2) is in a descending trend along with the standing time, and the quality standard is still met at the end of the observation period.

The oscillation test result shows that the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution slightly increases high molecular weight impurities along with the standing time under the conditions of 2-8 ℃ and 300rpm, but the change is not obvious, and the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution still meets the quality standard until the end of the observation period.

The high-temperature test result shows that the high molecular weight impurities of the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution slightly increase along with the standing time under the conditions of 25 ℃ and 60% relative humidity, but the change is not obvious, and the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution still meets the quality standard by the end of the observation period.

The illumination test result shows that the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution is laterally placed at the temperature of 2-8 ℃ and the illumination intensity of 4500Lx, the high molecular weight impurities are in a growth trend along with the placing time, and by the 3 rd day, the high molecular weight impurities exceed the quality standard limit requirement (1.0%) of the stock solution, and the high molecular weight impurities reach 1.7% at the end of the observation period; the proportion of the charge heteroplasmon (K0+ K1+ K2) is reduced along with the standing time, and the proportion at the end of the observation period is 63.9 percent and exceeds the mass standard.

The freeze-thaw test result shows that the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution undergoes repeated freeze-thaw at-80-25 ℃, repeated freeze-thaw at 10 cycles or repeated freeze-thaw at-20-25 ℃ and repeated freeze-thaw at 10 cycles, and no significant influence is observed in all quality indexes.

The results indicate that the recombinant anti-TNF alpha fully human monoclonal antibody stock solution should be stored at low temperature in the dark to avoid oscillation. When the technological process is needed, freezing and thawing can be carried out within a limited range, and the quality of the stock solution is not influenced.

The pH change map in the oscillation, high temperature, illumination and strong destruction tests is shown in figure 3, the protein content change map is shown in figure 4, the protein reduction electrophoresis purity change map is shown in figure 5, the polymer impurity change map is shown in figure 6, the charge heteroplasmon change map is shown in figure 7, and the protein bioactivity change map is shown in figure 8. The protein content variation pattern in the freeze-thaw test is shown in figure 9, the protein reduction electrophoresis purity variation pattern is shown in figure 10, the high molecular weight impurity variation pattern is shown in figure 11, and the protein bioactivity variation pattern is shown in figure 12.

According to the above experimental results, the formulation of pH5.2, sucrose 20.0g/L, polysorbate 1.0g/L, sodium chloride 6.16g/L, citric acid anhydrous 1.19g/L, sodium citrate dihydrate 0.3g/L, sodium dihydrogen phosphate dihydrate 0.86g/L, and disodium hydrogen phosphate dihydrate 1.53g/L is preferred.

Test example 3 Long-term stability test

Selecting a preparation formula of pH5.2, sucrose 20.0g/L, polysorbate 1.0g/L, sodium chloride 6.16g/L, anhydrous citric acid 1.19g/L, sodium citrate dihydrate 0.3g/L, sodium dihydrogen phosphate dihydrate 0.86g/L and disodium hydrogen phosphate dihydrate 1.53g/L for long-term stability test of the preparation formula.

The antibody drug preparation is placed below minus 18 ℃, 1402, 1403 and 1404 batches of antibody drug preparations are taken, and samples are taken periodically to carry out stability investigation index determination. The time and items of the long-term test investigation are shown in Table 5.

TABLE 5 Long term test investigation time points and items

Long-term stability studies were conducted on 1402, 1403 and 1404 batches of antibody pharmaceutical preparations, where long-term stability test results for 1402 batches of pharmaceutical preparations are shown in table 6, long-term stability test results for 1403 batches of pharmaceutical preparations are shown in table 7, and long-term stability test results for 1404 batches of pharmaceutical preparations are shown in table 8.

TABLE 6, 1402 batches Long term test results

TABLE 7, 1403 batch Long term test results

TABLE 8, 1404 batches of Long term test results

The long-term stability test result shows that the recombinant anti-TNF alpha fully-humanized monoclonal antibody stock solution is placed below 18 ℃ below zero, and the analysis result shows that the quality indexes such as protein content, biological activity, reduction electrophoresis purity, SEC-HPLC/UPLC high molecular weight impurities, charge heteroplasmon distribution and the like are not obviously changed. The protein content test result map is shown in FIG. 13, the bioactivity test result is shown in FIG. 14, the reduction electrophoresis purity test result is shown in FIG. 15, the SEC-HPLC/UPLC high molecular weight impurity test result is shown in FIG. 16, and the charge heteroplasmon distribution test result is shown in FIG. 17.

The results indicate that the stock solution of the recombinant anti-TNF alpha fully-humanized monoclonal antibody is preserved at the temperature below 18 ℃ below zero, and the quality of the stock solution cannot be influenced within a certain time range.

The stock long term stability test will continue to be observed for 36 months.

SEQUENCE LISTING

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Claims

1. A recombinant anti-TNF-alpha fully human monoclonal antibody preparation, wherein the TNF-alpha fully human monoclonal antibody is CMAB815, and the coding nucleotide sequence of a light chain of the TNF-alpha fully human monoclonal antibody is SEQ ID NO:1, the coding nucleotide sequence of the heavy chain is SEQ ID NO:3, characterized by consisting of the following components:

a)50mg/ml of CMAB815,

b)1.0mg/ml of polysorbate 80,

c)20.0mg/ml of sucrose in the presence of sucrose,

d)6.16mg/ml of sodium chloride,

e)1.19mg/ml anhydrous citric acid,

f)0.3mg/ml sodium citrate dihydrate,

g)0.86mg/ml sodium dihydrogen phosphate dihydrate,

h)1.53mg/ml disodium phosphate dihydrate;

wherein the pH value of the buffer solution formed by the components b-h is 5.2.

2. The use of an antibody preparation according to claim 1,

the antibody preparation is used for preparing a medicament for treating diseases caused by TNF-alpha.

3. Use of an antibody preparation according to claim 2,

the antibody preparation is used for preparing medicaments for treating rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and plaque psoriasis.