CN113289029B - Monoclonal antibody-cytokine fusion protein preparation - Google Patents

Monoclonal antibody-cytokine fusion protein preparation Download PDF

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Publication number
CN113289029B
CN113289029B CN202110553145.4A CN202110553145A CN113289029B CN 113289029 B CN113289029 B CN 113289029B CN 202110553145 A CN202110553145 A CN 202110553145A CN 113289029 B CN113289029 B CN 113289029B
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monoclonal antibody
fusion protein
ser
cytokine fusion
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CN113289029A (en
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方国波
蔡则玲
吴珩
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Shanghai Celgen Biopharma Co ltd
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Shanghai Celgen Biopharma Co ltd
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Priority to PCT/CN2022/092262 priority patent/WO2022242527A1/en
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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Abstract

The application relates to a monoclonal antibody-cytokine fusion protein preparation. In particular, the application provides a monoclonal antibody-cytokine fusion protein composition comprising an anti-monoclonal antibody-cytokine fusion protein, a surfactant, a buffer and a stabilizer. The composition of the application can improve the stability of monoclonal antibody-cytokine fusion protein.

Description

Monoclonal antibody-cytokine fusion protein preparation
Technical Field
The application relates to the field of pharmaceutical preparations, in particular to a monoclonal antibody-cytokine fusion protein preparation.
Background
Because monoclonal antibody-cytokine fusion proteins have complex spatial structures, they are susceptible to various factors (e.g., temperature, shear force, shaking, freeze thawing, uv exposure, pH changes, organic solvents, microbial contamination) during production and storage, resulting in changes in the chemical and physical properties of the protein drug. Chemical changes include oxidation, deamidation, isomerization, degradation, covalent polymerization of protein molecules; physical changes include conformational changes in the protein space such as unfolding, non-covalent aggregation of monomer molecules, which are susceptible to the amino acid sequence of the protein and the environmental conditions (salt, pH and temperature) in which the protein is contained.
The protein is used as a biological medicine, and needs a long storage period of validity from the production, circulation and patient use series links. During storage, the stability of the protein is maintained, and the change of physicochemical properties is avoided, so that the safety and effectiveness of the protein are affected.
Therefore, there is a need in the art to develop a monoclonal antibody-cytokine fusion protein formulation with high stability, which improves the stability of the monoclonal antibody-cytokine fusion protein.
Disclosure of Invention
The application aims to provide a monoclonal antibody-cytokine fusion protein composition with high stability.
In a first aspect of the application, there is provided a monoclonal antibody-cytokine fusion protein composition comprising an anti-monoclonal antibody-cytokine fusion protein, a surfactant, a buffer and a stabilizer.
In another preferred embodiment, the composition is a pharmaceutical composition.
In another preferred embodiment, the monoclonal antibody-cytokine fusion protein comprises a human monoclonal antibody-cytokine fusion protein.
In another preferred embodiment, the C-terminus of the heavy chain amino acid sequence of the monoclonal antibody in the monoclonal antibody-cytokine fusion protein is linked to the N-terminus of the amino acid sequence of the cytokine.
In another preferred embodiment, the linkage is a peptide bond linkage.
In another preferred embodiment, in the monoclonal antibody-cytokine fusion protein, the monoclonal antibody has the sequence as set forth in SEQ ID No.:1 and the heavy chain amino acid sequence of SEQ ID No.:2 light chain amino acid sequence.
In another preferred embodiment, in the monoclonal antibody-cytokine fusion protein, the cytokine has the sequence as set forth in SEQ ID No.:3, and a sequence of amino acids.
In another preferred embodiment, the monoclonal antibody-cytokine fusion protein is present in an amount of 0.5-50mg/mL, preferably 0.5-40mg/mL, more preferably 0.5-30mg/mL, more preferably 0.5-20mg/mL, more preferably 1-15mg/mL, more preferably 2-10mg/mL, more preferably 3-8mg/mL, most preferably 5mg/mL.
In another preferred embodiment, the surfactant is selected from the group consisting of: a positive ionic surfactant, a negative ionic surfactant, a non-ionic surfactant, or a combination thereof.
In another preferred embodiment, the surfactant is selected from the group consisting of: polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerol fatty acid esters, polysorbates, poloxamers, or combinations thereof.
In another preferred embodiment, the polysorbate is selected from the group consisting of: polysorbate 20 (PS-20, tween-20), polysorbate 40 (PS-40, tween-40), polysorbate 60 (PS-60, tween-60), polysorbate 80 (PS-80, tween-80), or a combination thereof.
In another preferred embodiment, the surfactant comprises polysorbate 20 (PS-20, tween-20).
In another preferred embodiment, the surfactant is present in an amount of 0.001 to 8wt%, preferably 0.001 to 3wt%, more preferably 0.001 to 1wt%, more preferably 0.005 to 0.5wt%, more preferably 0.005 to 0.1wt%, more preferably 0.008 to 0.1wt%, more preferably 0.01 to 0.08wt%, more preferably 0.01 to 0.05wt%, more preferably 0.02 to 0.04wt%, most preferably 0.03wt% based on the total weight of the composition.
In another preferred embodiment, the buffer is selected from the group consisting of: citric acid-disodium hydrogen phosphate buffer, citric acid-sodium citrate buffer, phosphate-phosphate buffer, histidine buffer, or a combination thereof.
In another preferred embodiment, the medium of the buffer is water.
In another preferred embodiment, the concentration of the buffer is 3-80mM, preferably 5-60mM, more preferably 10-50mM, more preferably 20-40mM, more preferably 25-35mM, more preferably 28-32mM, most preferably 30mM.
In another preferred embodiment, the buffer comprises a citric acid-disodium hydrogen phosphate buffer.
In another preferred embodiment, the concentration of said citrate-disodium hydrogen phosphate buffer is 3-80mM, preferably 5-60mM, more preferably 10-50mM, more preferably 20-40mM, more preferably 25-35mM, more preferably 28-32mM, most preferably 30mM.
In another preferred embodiment, the stabilizer is selected from the group consisting of: amino acids, sugar alcohols, inorganic salts, or combinations thereof.
In another preferred embodiment, the amino acid is selected from the group consisting of: proline, arginine (Arginine), glycine (Glycine), histidine (Histidine), methionine (Methionine), or combinations thereof.
In another preferred embodiment, the sugar alcohol is selected from the group consisting of: sucrose (Sucrose), mannitol (Mannitol), trehalose (Trehalose), maltose (Maltose), sorbitol (Sorbitol), or combinations thereof.
In another preferred embodiment, the sugar alcohol is selected from the group consisting of: sucrose, mannitol, trehalose, sorbitol, or a combination thereof.
In another preferred embodiment, the inorganic salt is selected from the group consisting of: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or a combination thereof.
In another preferred embodiment, the stabilizer comprises sucrose.
In another preferred embodiment, the stabilizer is present in an amount of 0.5 to 50wt%, preferably 0.5 to 40wt%, more preferably 0.8 to 30wt%, more preferably 1 to 20wt%, more preferably 1 to 15wt%, more preferably 1 to 10wt%, more preferably 1 to 8wt%, more preferably 2 to 7wt%, most preferably 5wt%, based on the total weight of the composition.
In another preferred embodiment, the pH of the composition is from 5 to 8, preferably from 5.0 to 7.5, more preferably from 5.0 to 7.0, more preferably from 6.0 to 6.8, more preferably from 6.2 to 6.8, more preferably from 6.3 to 6.7, most preferably 6.5.
In another preferred embodiment, the composition further comprises a chelating agent.
In another preferred embodiment, the chelating agent comprises EDTA (ethylenediamine tetraacetic acid).
In another preferred embodiment, the composition is a liquid formulation.
In another preferred embodiment, the composition is an injectable liquid formulation.
In another preferred embodiment, the composition is a pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition is in the form of an injectable formulation.
In another preferred embodiment, the composition comprises:
monoclonal antibody-cytokine fusion proteins 1-15mg/mL
Polysorbate 20 0.01-0.08wt％
Citric acid-disodium hydrogen phosphate buffer 20-40mM
Sucrose 1-15wt%; and
pH 5.0-7.0。
in another preferred embodiment, the composition comprises:
in another preferred embodiment, the composition comprises:
monoclonal antibody-cytokine fusion proteins 3-8mg/mL
Polysorbate 20 0.02-0.04wt％
Citric acid-disodium hydrogen phosphate buffer 28-32mM
Sucrose 2-7wt%; and
pH 6.3-6.7。
in a second aspect of the application there is provided a method of preparing a monoclonal antibody-cytokine fusion protein composition according to the first aspect of the application, said method comprising the steps of:
after mixing the components of the composition according to the first aspect of the application, a monoclonal antibody-cytokine fusion protein composition is obtained.
In a third aspect of the present application, there is provided a lyophilized formulation prepared by:
the composition according to the first aspect of the application is freeze-dried to obtain a freeze-dried formulation.
In another preferred embodiment, the lyophilized formulation further comprises a lyoprotectant.
In another preferred embodiment, the lyoprotectant is selected from the group consisting of: glucose, mannitol, fructose, galactose, or a combination thereof.
In a fourth aspect of the present application there is provided the use of a composition according to the first aspect of the present application or a lyophilized formulation according to the third aspect of the present application for the manufacture of a medicament for the prevention and/or treatment of a tumour.
In another preferred embodiment, the tumor shown comprises a solid tumor.
In another preferred embodiment, the tumor shown comprises a solid tumor that is HER2 positive for expression.
In a fifth aspect of the present application, there is provided a method of preventing and/or treating a tumour, said method comprising: administering to a subject in need thereof a composition as described in the first aspect of the application or a lyophilized formulation as described in the third aspect of the application, thereby treating a tumor.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the tumor shown comprises a solid tumor.
In another preferred embodiment, the tumor shown comprises a solid tumor that is HER2 positive for expression.
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Detailed Description
The inventor researches and develops a composition which can effectively maintain the stability of the anti-monoclonal antibody-cytokine fusion protein, and the monoclonal antibody-cytokine fusion protein in the composition can maintain the stability under the conditions of high temperature, vibration and the like, so that the composition can stabilize the quality of the monoclonal antibody-cytokine fusion protein, prolong the shelf life of products and improve the safety of clinical practicality. On this basis, the present application has been completed.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, "mM" is in mmol/L units, e.g., 1mM = 1mmol/L.
In the compositions of the present application, the weight content (wt.%) of each component is based on the weight of the composition.
As used herein, the term "EDTA" is ethylenediamine tetraacetic acid.
Monoclonal antibody-cytokine fusion proteins
In the present application, the monoclonal antibody-cytokine fusion protein is a fusion protein formed by connecting a monoclonal antibody with a cytokine (peptide bond connection).
As used herein, the term "monoclonal antibody" consists of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain.
In a preferred embodiment of the present application, the C-terminus of the heavy chain amino acid sequence of the monoclonal antibody in the monoclonal antibody-cytokine fusion protein is linked to the N-terminus of the amino acid sequence of the cytokine.
In another preferred embodiment, in the monoclonal antibody-cytokine fusion protein, the monoclonal antibody has the sequence as set forth in SEQ ID No.:1 and the heavy chain amino acid sequence of SEQ ID No.:2 light chain amino acid sequence.
In another preferred embodiment, in the monoclonal antibody-cytokine fusion protein, the cytokine has the sequence as set forth in SEQ ID No.:3, and a sequence of amino acids.
Composition and method for preparing same
The application provides a monoclonal antibody-cytokine fusion protein composition, which comprises an anti-monoclonal antibody-cytokine fusion protein, a surfactant, a buffer solution and a stabilizer.
The composition according to the application is preferably a pharmaceutical composition.
The composition of the present application is preferably a liquid formulation, such as an oral liquid formulation or an injectable liquid formulation.
In a preferred embodiment of the present application, the monoclonal antibody-cytokine fusion protein comprises a human monoclonal antibody-cytokine fusion protein.
In a preferred embodiment of the application, the monoclonal antibody-cytokine fusion protein is present in an amount of 0.5-50mg/mL, preferably 0.5-40mg/mL, more preferably 0.5-30mg/mL, more preferably 0.5-20mg/mL, more preferably 1-15mg/mL, more preferably 2-10mg/mL, more preferably 3-8mg/mL, most preferably 5mg/mL.
In a preferred embodiment of the present application, the surfactant includes (but is not limited to): a positive ionic surfactant, a negative ionic surfactant, a non-ionic surfactant, or a combination thereof.
Typically, the surfactants include (but are not limited to): polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerol fatty acid esters, polysorbates, poloxamers, or combinations thereof.
Typically, the polysorbates include (but are not limited to): polysorbate 20 (PS-20, tween-20), polysorbate 40 (PS-40, tween-40), polysorbate 60 (PS-60, tween-60), polysorbate 80 (PS-80, tween-80), or a combination thereof.
Typically, the surfactant comprises polysorbate 20 (PS-20, tween-20).
In a preferred embodiment of the present application, the surfactant is present in an amount of 0.001 to 8wt%, preferably 0.001 to 3wt%, more preferably 0.001 to 1wt%, more preferably 0.005 to 0.5wt%, more preferably 0.005 to 0.1wt%, more preferably 0.008 to 0.1wt%, more preferably 0.01 to 0.08wt%, more preferably 0.01 to 0.05wt%, more preferably 0.02 to 0.04wt%, most preferably 0.03wt% based on the total weight of the composition.
In a preferred embodiment of the present application, the buffer includes (but is not limited to): citric acid-disodium hydrogen phosphate buffer, citric acid-sodium citrate buffer, phosphate-phosphate buffer, histidine buffer, or a combination thereof.
In another preferred embodiment, the medium of the buffer is water.
In a preferred embodiment of the application, the concentration of the buffer is 3-80mM, preferably 5-60mM, more preferably 10-50mM, more preferably 20-40mM, more preferably 25-35mM, more preferably 28-32mM, most preferably 30mM.
Typically, the buffer comprises a citric acid-disodium hydrogen phosphate buffer.
In a preferred embodiment of the application, the concentration of said citrate-disodium hydrogen phosphate buffer is 3-80mM, preferably 5-60mM, more preferably 10-50mM, more preferably 20-40mM, more preferably 25-35mM, more preferably 28-32mM, most preferably 30mM.
In a preferred embodiment of the present application, the stabilizer includes (but is not limited to): amino acids, sugar alcohols, inorganic salts, or combinations thereof.
Typically, the amino acids include (but are not limited to): proline, arginine (Arginine), glycine (Glycine), histidine (Histidine), methionine (Methionine), or combinations thereof.
Representatively, the sugar alcohols include (but are not limited to): sucrose (Sucrose), mannitol (Mannitol), trehalose (Trehalose), maltose (Maltose), sorbitol (Sorbitol), or combinations thereof.
Representatively, the sugar alcohols include (but are not limited to): sucrose, mannitol, trehalose, sorbitol, or a combination thereof.
In a preferred embodiment of the present application, the inorganic salts include (but are not limited to): sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or a combination thereof.
Typically, the stabilizing agent comprises sucrose.
In a preferred embodiment of the present application, the stabilizer is present in an amount of 0.5 to 50wt%, preferably 0.5 to 40wt%, more preferably 0.8 to 30wt%, more preferably 1 to 20wt%, more preferably 1 to 15wt%, more preferably 1 to 10wt%, more preferably 1 to 8wt%, more preferably 2 to 7wt%, most preferably 5wt%, based on the total weight of the composition.
In a preferred embodiment of the application, the pH of the composition is from 5 to 8, preferably from 5.0 to 7.5, more preferably from 5.0 to 7.0, more preferably from 6.0 to 6.8, more preferably from 6.2 to 6.8, more preferably from 6.3 to 6.7, most preferably 6.5.
In a preferred embodiment of the application, the composition further comprises a chelating agent.
Typically, the chelating agent comprises EDTA (ethylenediamine tetraacetic acid).
In a preferred embodiment of the present application, the composition comprises:
monoclonal antibody-cytokine fusion proteins 1-15mg/mL
Polysorbate 20 0.01-0.08wt％
Citric acid-disodium hydrogen phosphate buffer 20-40mM
Sucrose 1-15wt%; and
pH 5.0-7.0
in a preferred embodiment of the present application, the composition comprises:
monoclonal antibody-cytokine fusion proteins 2-10mg/mL
Polysorbate 20 0.01-0.05wt％
Citric acid-disodium hydrogen phosphate buffer 25-35mM
Sucrose 1-8wt%; and
pH 6.0-6.8
in a preferred embodiment of the present application, the composition comprises:
monoclonal antibody-cytokine fusion proteins 3-8mg/mL
Polysorbate 20 0.02-0.04wt％
Citric acid-disodium hydrogen phosphate buffer 28-32mM
Sucrose 2-7wt%; and
pH 6.3-6.7
the application also provides a preparation method of the composition, which comprises the following steps:
after mixing the components of the compositions according to the application, monoclonal antibody-cytokine fusion protein compositions are obtained.
Freeze-dried preparation
The application also provides a freeze-dried preparation, which is prepared by the following method:
the composition according to the application is freeze-dried to obtain a freeze-dried preparation.
In another preferred embodiment, the lyophilized formulation further comprises a lyoprotectant.
In another preferred embodiment, the lyoprotectant includes (but is not limited to): glucose, mannitol, fructose, galactose, or a combination thereof.
Use of the same
The application also provides a use of the composition or the lyophilized preparation of the application for preparing a medicament for preventing and/or treating tumors.
The present application also provides a method for preventing and/or treating a tumor, the method comprising: administering to a subject in need thereof a composition according to the application or the lyophilized formulation, thereby treating a tumor.
In another preferred embodiment, the tumor shown comprises a solid tumor.
In another preferred embodiment, the tumor shown comprises a solid tumor that is HER2 positive for expression.
The main advantages of the application include:
the application provides a composition containing a monoclonal antibody-cytokine fusion protein, which can improve the stability of the monoclonal antibody-cytokine fusion protein.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Examples
The citrate buffer and histidine buffer in the following examples are as follows:
citric acid buffer: the citric acid-disodium hydrogen phosphate buffer solution is water as medium, and citric acid and disodium hydrogen phosphate as solute.
Histidine buffer: the medium is water, and the solute is histidine and histidine hydrochloride.
The monoclonal antibody-cytokine fusion protein in the following examples is formed by peptide bond linkage of the C-terminus of the heavy chain amino acid sequence of a monoclonal antibody with the N-terminus of the amino acid sequence of a cytokine, wherein the heavy chain amino acid sequence of the monoclonal antibody is as set forth in SEQ ID No.:1, the light chain amino acid sequence of the monoclonal antibody is as set forth in SEQ ID No.:2, the amino acid sequence of the cytokine is set forth in SEQ ID No.:3 is shown in the figure;
SEQ ID NO.:1 (N-terminal to C-terminal):
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
SEQ ID NO.:2 (N-terminal to C-terminal):
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
SEQ ID NO.:3 (N-terminal to C-terminal):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL。
the monoclonal antibody-cytokine fusion protein is used for treating HER2 positive expressed solid tumors, and the conjugated cytokine kills tumor cells targeted by the monoclonal antibody, so that the treatment effect of the monoclonal antibody is greatly enhanced.
Universal assay method
SEC-HPLC is a size exclusion chromatography method used to analyze the purity of monoclonal antibody-cytokine fusion proteins, in particular to detect the content of aggregates and small molecule fragments of the monoclonal antibody-cytokine fusion proteins resulting from degradation.
Example 1
The study samples had the same formulation composition but contained stability of the monoclonal antibody-cytokine fusion protein in a citrate-disodium hydrogen phosphate buffer system at different pH.
The different pH values are 10mM citric acid-disodium hydrogen phosphate buffer solution with pH of 4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5 and pH 8.0.
The monoclonal antibody-cytokine fusion protein preparation is added into the buffer solution, the protein concentration is 5mg/ml, the preparation is incubated for 3 days (T3) and 7 days (T7) at the temperature of 2-8 ℃ and 40 ℃ respectively, the purity of the monoclonal antibody-cytokine fusion protein in the preparation is analyzed by SEC-HPLC, the purity detection results of the preparation at the temperature of 2-8 ℃ are shown in table 2, and the purity detection results of the preparation at the temperature of 40 ℃ are shown in table 3.
Table 2 2-8deg.C incubated preparation purity determination results
Table 3 results of determination of purity of formulations incubated at 40 c
The results in tables 2 and 3 are shown: each preparation is relatively stable at 2-8 ℃, the variation trend of sample stability is obvious under the condition of acceleration test (40 ℃), the sample aggregation is obvious when the pH value of the preparation is 4.5, the aggregate reaches 80.3% when the pH value is T3, and the stability of the sample aggregation is not obviously different within the pH range of 5.0-8.0; the degradation fragments are relatively stable under the condition of pH 5.5-6.5, have no obvious increasing trend, and the result shows that the pH of the preparation influences the stability of the monoclonal antibody-cytokine fusion protein, and tend to aggregate under the condition of low pH, and the degradation trend is obviously increased under the condition of high pH, and the pH range of the preparation is preferably 5.5-6.5 according to the initial result of the accelerated stability test.
Example 2
The stability of the monoclonal antibody-cytokine fusion protein in histidine buffer and citrate buffer systems was examined according to the pH range determined in example 1, and suitable buffer solutions were screened. According to the characteristics of the buffer solution, the pH range of the histidine buffer solution is 5.5-7.0, the pH range of the citric acid buffer solution is 5.5-6.5, the protein concentration is 5mg/ml, the DOE design is adopted, and the formulation prescription is designed according to 2 factors of the pH and the buffer solution concentration. After the preparation samples are incubated for 3 days under the accelerated stability test condition (50 ℃), the purity of the protein is measured, the test results of the prescription of the histidine buffer system preparation are shown in table 4, and the test results of the prescription of the citric acid buffer system preparation are shown in table 5.
The results show that: the histidine buffer solution and the citric acid buffer solution are easy to aggregate under the condition of relatively low pH (5.5), and are relatively stable under the condition of pH 6.0-6.5, and in the pH range, the concentration of the buffer solution has obvious influence on the stability of a sample, the purity of the sample protein is lower than 5mM when the concentration of histidine is 30mM, and the purity of the sample protein is higher than 5mM when the concentration of citric acid is 30mM. The pH of the buffer solution is 6.0 to 6.5, and the buffer solution is preferably citric acid buffer solution for investigation.
TABLE 4 results of protein purity detection of formulations of histidine buffer systems
TABLE 5 results of protein purity detection of formulations of citrate buffer systems
Example 3
The concentration and pH of the buffer solution were further examined by selecting a citric acid buffer solution, and DOE was prescribed at 2 factors and 3 levels, and pH was selected to be 6.0, 6.5, and 7.0, and the buffer solution concentration was 10mM, 30mM, and 50mM. The preparation samples were examined under accelerated test conditions, and after incubation at 50 ℃ for 3 days, the protein purity was determined, the measurement results are shown in table 6, and the results show that: the pH of the buffer solution of the preparation is in the range of 6.0-7.0, the sample is relatively stable, wherein the buffer solution with the pH of 6.5 has relatively good stability, when the concentration of the buffer solution is increased, the aggregate tends to be reduced, and the purity of the sample tends to be increased. Based on the results of this test, a citrate buffer concentration of 30mM, pH6.5, was preferably selected as the most suitable buffer for the formulation of the protein.
TABLE 6 protein purity detection results
Example 4
Surfactant effect: and selecting an intermediate product monoclonal antibody-cytokine fusion protein affinity chromatography eluent in the production process, adding 0.03% and 0.1% of Tween 20, observing the change of insoluble particles of a sample, and observing the influence of the Tween 20 on the stability of the sample.
The insoluble particles of the sample were measured by a photoresist method, the results are shown in Table 7, and the results show that: after tween 20 was added to the sample solution, the number of insoluble particles was significantly reduced. According to the results, the addition of 0.03% tween 20 to the sample controlled the amount of insoluble particles in the sample solution.
TABLE 7 insoluble microparticle measurement results
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Remarks: wt% is weight percent based on the weight of the formulation;
sugar, salt and amino acid effects: the buffer system of the monoclonal antibody-cytokine fusion protein preparation is selected as a citric acid buffer solution, the concentration is 30mM, the pH is 6.5, and the concentration is 0.03 percent of Tween 20, and the influence of auxiliary materials of sugar, salt and amino acid on the stability of the monoclonal antibody-cytokine fusion protein is further examined.
The auxiliary materials comprise sucrose, sodium chloride and arginine, the protein concentration is 5mg/ml, the sucrose concentration is set to 0%, 5% and 10%, the sodium chloride concentration is set to 0mM, 30mM and 60mM, the arginine concentration is set to 0mM, 25mM and 50mM, and the design is carried out according to 3 factors and 3 levels. The preparation samples were examined under accelerated test conditions, and after incubation at 50 ℃ for 3 days, the protein purity was determined, the measurement results are shown in table 8, and the results show that: the preparation is incubated for 3 days under the accelerated test condition, the aggregate is obviously increased, and compared with the preparation without the auxiliary materials, the preparation with the auxiliary materials of sucrose, sodium chloride and arginine has no obvious improvement on the stability under the accelerated test condition of the sample.
Table 8 results of purity measurements of formulations containing sugar, salt and amino acid adjuvants
Remarks: wt% is weight percent based on the weight of the formulation; "-" means none.
Antioxidant and amino acid effects: the buffer system of the monoclonal antibody-cytokine fusion protein preparation is selected as a citric acid buffer solution, the concentration is 30mM, the pH is 6.5, and the concentration is 0.03 percent of Tween 20, and the influence of EDTA (ethylenediamine tetraacetic acid), histidine and glycine on the stability of the monoclonal antibody-cytokine fusion protein is further examined.
The preparation samples were examined under accelerated test conditions, and after incubation at 50 ℃ for 3 days, the protein purity was determined, the measurement results are shown in table 9, and the results show that: EDTA, histidine and glycine are added to inhibit degradation of the monoclonal antibody-cytokine fusion protein.
TABLE 9 protein purity detection results
Remarks: wt% is weight percent based on the weight of the formulation; "-" means none.
Example 5
Protein aggregation and degradation generally have a high correlation with storage temperature, examples 1 to 4 all employ accelerated stability tests incubated at 50℃for 3 days under relatively severe conditions, and according to the above-described case test studies, a preliminary preparation of a crude intended monoclonal antibody-cytokine fusion protein was formulated with a concentration of 5mg/ml monoclonal antibody-cytokine fusion protein, containing 30mM citrate buffer, 0.03wt% polysorbate 20, and pH of 6.5. The pH application range and whether the formulation can be used as an effective formulation prescription for long-term storage are confirmed for the prescription drop acceleration test conditions.
The pH range is selected to be 5.0-7.0, and the accelerated test condition is that the incubation is carried out for 2 days at 50 ℃. The test results are shown in Table 10, and the results show that: under the condition that the pH value is less than or equal to 6.0, the sample has obvious aggregation phenomenon, the pH value is in the range of 6.0-7.0, the protein is relatively stable, and the purity of the protein can be maintained to be about 95.0% after incubation for 2 days at 50 ℃. According to the test result, the preparation can be stored at 2-8 ℃ to maintain good stability, and long-term stability test is needed to confirm the preparation later, so that the pH range of the preparation is 6.0-7.0, and the optimal pH is 6.5.
TABLE 10 protein purity assay results
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Example 6
According to the planned dosage and injection mode of the monoclonal antibody-cytokine fusion protein preparation, the osmotic pressure of the preparation is regulated to the osmotic pressure when the physiological condition is reached under the condition of the stock solution preparation, and 5% of sucrose is selected to be added into the stock solution preparation by considering the source of auxiliary materials in combination with the research of auxiliary materials in example 4. The preparation of the monoclonal antibody-cytokine fusion protein stock solution is preliminarily prepared, wherein the concentration of the monoclonal antibody-cytokine fusion protein is 5mg/ml,30mM of citric acid buffer solution, 0.03wt% of polysorbate 20 and 5wt% of sucrose, and the pH is 6.5.
The formulated formulation was validated and incubated at 50 ℃ for 3 days, after which the protein purity was determined by SEC-HPLC and the formulation stability was assessed, the results are shown in table 11, the results show: the preparation is incubated for 3 days at 50 ℃ and the purity is maintained at 94.0% with only obvious increase of degradation. According to the test result and the relevance of the combination degradation and the temperature, the preparation is considered to be stored at the temperature of 2-8 ℃ and still maintain good stability, and the preparation can meet the requirement of the storage validity period of the protein medicine.
TABLE 11 protein purity detection results
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Shanghai Seiki biological medicine Co., ltd
<120> preparation of monoclonal antibody-cytokine fusion protein
<130> P2020-2469
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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
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Claims (14)

1. A monoclonal antibody-cytokine fusion protein composition, said composition comprising:
monoclonal antibody-cytokine fusion protein 3-8mg/mL
Polysorbate 20.01-0.1 wt%, based on the total weight of the composition
10-60mM citric acid-disodium hydrogen phosphate buffer, and
sucrose 1-10wt%, based on the total weight of the composition
Wherein the pH of the composition is 6.0-7.0, the medium of the buffer solution is water,
the C end of the heavy chain amino acid sequence of the monoclonal antibody in the monoclonal antibody-cytokine fusion protein is connected with the N end of the amino acid sequence of the cytokine;
in the monoclonal antibody-cytokine fusion protein, the monoclonal antibody has the sequence shown in SEQ ID NO:1 and the heavy chain amino acid sequence shown in SEQ ID NO:2, the cytokine has the amino acid sequence of the light chain shown in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
2. The composition of claim 1, wherein the monoclonal antibody-cytokine fusion protein is present in an amount of 5mg/mL;
the polysorbate 20 is present in an amount of 0.03 to 0.1wt%, based on the total weight of the composition;
the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 10-50mM; or (b)
The sucrose content is 1-8wt%, based on the total weight of the composition.
3. The composition of claim 1, wherein the polysorbate 20 is present in an amount of 0.03 to 0.08wt%, based on the total weight of the composition;
the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 20-40mM; or (b)
The sucrose content is 2-7wt%, based on the total weight of the composition.
4. The composition of claim 1, wherein the polysorbate 20 is present in an amount of 0.03 to 0.05wt%, based on the total weight of the composition;
the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 25-35mM; or (b)
The sucrose content is 5wt%, based on the total weight of the composition.
5. The composition of claim 1, wherein the polysorbate 20 is present in an amount of 0.03 to 0.04wt%, based on the total weight of the composition;
the concentration of the citric acid-disodium hydrogen phosphate buffer is 28-32mM.
6. The composition of claim 1, wherein the polysorbate 20 is present in an amount of 0.03wt%, based on the total weight of the composition; or (b)
The concentration of the citric acid-disodium hydrogen phosphate buffer was 30mM.
7. The composition of claim 1, wherein the pH of the composition is from 6.0 to 6.8.
8. The composition of claim 1, wherein the pH of the composition is from 6.2 to 6.8.
9. The composition of claim 1, wherein the pH of the composition is from 6.3 to 6.7.
10. The composition of claim 1, wherein the pH of the composition is 6.5.
11. The composition of claim 1, wherein in the composition:
and the pH of the composition is from 6.3 to 6.7.
12. The composition of claim 1, wherein in the composition:
and the pH of the composition was 6.5.
13. The composition of claim 1, wherein the composition is a liquid formulation.
14. Use of a composition according to claim 1 for the preparation of a medicament for the prevention or treatment of a solid tumor of HER2 positive expression.
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