CN106620691B - Recombinant fully human anti-CTLA-4 monoclonal antibody preparation and application thereof - Google Patents
Recombinant fully human anti-CTLA-4 monoclonal antibody preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant fully human anti-CTLA-4 monoclonal antibody preparation and application thereof. The invention discloses a composition for resisting cytotoxic T lymphocyte-associated antigen 4, which consists of a solute and a solvent, wherein the solute comprises a monoclonal antibody for resisting the cytotoxic T lymphocyte-associated antigen 4, a buffering agent, an osmotic pressure regulator and a chelating agent, and the solvent is water. Compared with the existing anti-CTLA-4 monoclonal antibody preparation, the anti-CTLA-4 monoclonal antibody preparation provided by the invention has high stability and can improve the safety of clinical application.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and relates to a recombinant fully human anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) monoclonal antibody preparation and application thereof.
Background
CTLA-4 and CD28 have close relationship in gene structure, chromosome location, sequence homology and gene expression, and are receptors of co-stimulatory molecule B7, mainly expressed on the surface of activated T cells. However, CTLA-4 functions in opposition to the CD28 molecule as a costimulatory signal for lymphocyte activation, and in normal cases, T cell activation relies on dual activation of a first signal (antigen-antibody complex formation) and a second signal (B7 mediated activation signal). Binding of CTLA-4 to B7 will generate inhibitory signals and inhibit T cell activation. The monoclonal antibody (CTLA-4mAb) as CTLA-4 blocker can specifically eliminate the immunological suppression of CTLA-4 to body and activate T cell, and has wide application foreground in gene therapy of tumor, parasite and other diseases.
IBI310 (ipilimumab mimic pharmaceutical) is a macromolecular monoclonal antibody drug designed and developed according to the above mechanism. The recombinant human monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 is a drug imitation of marketed drugs YERVOY (ipilimumab), and the administration route is designed to be intravenous drip, and the dosage is predicted to be 3 mg/kg. The preparation components of the ipilimumab (ipilimumab) comprise: DTPA (diethyltriaminepentaacetic acid), mannitol, sodium chloride, Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride), PS80 (polysorbate 80), pH 7.0. The preparation has low chemical stability, and the product is easy to undergo chemical degradation reaction. These changes may have an impact on the safety and effectiveness of the final product. Therefore, it is very important to establish a proper formulation to ensure the stability and safety of the product. IBI310 acts as a mimetic of ipilimumab, and due to its structural complexity, proteins are prone to aggregation and charge variants are prone to transition from basic to acidic components in non-optimized formulations.
Monoclonal antibodies have the advantages of strong specificity and long half-life, and are gradually paid attention to in the development of pharmaceutical proteins. At present, monoclonal antibody drugs are produced by fermentation, are mainly expressed in CHO cells, and are glycosylated, oxidized, deaminated, aggregated, degraded and isomerized during cell culture, purification, preparation process and storage, and almost all of the processes cause heterogeneity of surface charges of monoclonal antibodies. Changes in the heterogeneity of surface charge of monoclonal antibodies can affect the spatial conformation of monoclonal antibodies and even cause changes in biological activity and are prone to self-aggregation. These disadvantages present a significant challenge in formulating such drugs into stable, safe, effective formulations.
The problems to be solved are that the physical and chemical stability of the anti-CTLA-4 monoclonal antibody preparation is improved, the generation rate of charge isomers is slowed down, the quality uniformity and consistency of products are improved, the shelf life of the products is prolonged, and the clinical use stability of the products is improved.
Disclosure of Invention
The technical problem to be solved by the invention is to improve the stability of the anti-CTLA-4 monoclonal antibody preparation.
In order to solve the above technical problems, the present invention provides a composition against cytotoxic T lymphocyte-associated antigen 4, which comprises a solute containing a monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4, a buffer, an osmo-regulator and a chelating agent, and a solvent, which is water.
In the composition, the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is 2.5-7.5 mg/ml, the concentration of the buffer in the composition is 1.21-3.63 mg/ml, the concentration of the osmotic pressure regulator in the composition is 2.93-8.78 mg/ml, and the concentration of the chelating agent in the composition is 0.02-0.06 mg/ml; the pH value is 5.5-6.5;
the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is preferably 5.0mg/ml, the concentration of the buffer in the composition is preferably 2.42mg/ml, the concentration of the tonicity modifier in the composition is preferably 5.85mg/ml, and the concentration of the chelating agent in the composition is preferably 0.04 mg/ml.
In the composition, the buffering agent is sodium citrate or tris (hydroxymethyl) aminomethane;
the osmotic pressure regulator is sodium chloride;
the chelating agent is disodium ethylene diamine tetraacetate.
The composition of any of the above, further comprising a protein protectant;
the concentration of the protein protective agent in the composition is 5-15 mg/ml, and preferably 10 mg/ml.
In the composition, the protein protective agent is mannitol and/or arginine.
The composition of any of the above, further comprising a nonionic surfactant;
the concentration of the non-ionic surfactant in the composition is 0.1-0.4mg/ml, preferably 0.1mg/ml or 0.4 mg/ml.
In the composition, the nonionic surfactant is polysorbate 80 and/or polysorbate 20.
In the above composition, the solute consists of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4, the buffer, the tonicity adjusting agent, the chelating agent, the protein protecting agent and the nonionic surfactant; the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is 2.5-7.5 mg/ml, the concentration of the buffer in the composition is 1.21-3.63 mg/ml, the concentration of the osmotic pressure regulator in the composition is 2.93-8.78 mg/ml, the concentration of the chelating agent in the composition is 0.02-0.06 mg/ml, the concentration of the protein protective agent in the composition is 5-15 mg/ml, and the concentration of the nonionic surfactant in the composition is 0.1-0.4 mg/ml; the pH value of the composition is 5.5-6.5.
In the composition, the buffering agent is sodium citrate or tris (hydroxymethyl) aminomethane;
the osmotic pressure regulator is sodium chloride;
the chelating agent is disodium ethylene diamine tetraacetate;
the protein protective agent is mannitol and/or arginine;
the nonionic surfactant is polysorbate 80 and/or polysorbate 20.
In any of the above compositions, the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is 5.0mg/ml, the concentration of sodium citrate in the composition is 2.42mg/ml, the concentration of mannitol in the composition is 10mg/ml, the concentration of sodium chloride in the composition is 5.85mg/ml, the concentration of disodium edetate in the composition is 0.04mg/ml, the concentration of polysorbate 80 in the composition is 0.1mg/ml, and the pH is 5.5.
In any one of the above compositions, the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is 5.0mg/ml, the concentration of the tris in the composition is 2.42mg/ml, the concentration of the arginine in the composition is 10mg/ml, the concentration of the sodium chloride in the composition is 5.85mg/ml, the concentration of the disodium edetate in the composition is 0.04mg/ml, the concentration of the polysorbate 20 in the composition is 0.4mg/ml, and the pH is 6.5.
In the composition as described in any of the above, the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of said monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 are shown in SEQ ID No.1 from position 46 to 57, 73 to 79 and 112 to 120, respectively, and the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown in SEQ ID No.2 from position 45 to 51, 70 to 76 and 118 to 126, respectively;
the amino acid sequence of the variable region of the light chain of the monoclonal antibody of the anti-cytotoxic T lymphocyte-associated antigen 4 is preferably shown as 23 rd to 130 th positions of SEQ ID No.1, and the amino acid sequence of the variable region of the heavy chain is preferably shown as 20 th to 137 th positions of SEQ ID No. 2;
the amino acid sequence of the light chain of the monoclonal antibody of the anti-cytotoxic T lymphocyte-associated antigen 4 is preferably shown as SEQ ID No.1, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No. 2.
In order to solve the above technical problems, the present invention also provides the use of any one of the above compositions in the preparation of a product as shown in any one of the following (1) to (6):
(1) products against cytotoxic T lymphocyte-associated antigen 4;
(2) products for relieving the inhibition of T lymphocyte activation by cytotoxic T lymphocyte-associated antigen 4;
(3) products for relieving the suppression of the immunity of the cytotoxic T lymphocyte-associated antigen 4 to the body;
(4) products that activate T lymphocytes;
(5) products for the prevention and/or treatment of diseases in which T lymphocyte activity is inhibited;
(6) products for enhancing the immune level of the body;
the product is preferably a preparation, more preferably a preparation for intravenous drip;
the disease is preferably advanced metastatic malignant melanoma.
The recombinant human anti-CTLA-4 monoclonal antibody preparation prepared by IBI310 can be used as a preparation for intravenous drip. Moreover, the IBI310 can be replaced by other anti-CTLA-4 monoclonal antibodies (including anti-CTLA-4 monoclonal antibodies obtained by genetic engineering technology) to prepare anti-CTLA-4 monoclonal antibody preparations with the same stability.
The recombinant fully human anti-CTLA-4 monoclonal antibody preparation provided by the invention can ensure that the contained anti-CTLA-4 monoclonal antibody is stably stored under the conventional storage condition, and has improved stability even under the high-temperature acceleration condition, and the preparation has longer quality guarantee period and can improve the safety of clinical application.
Drawings
FIG. 1 shows SDS-PAGE of a Protein of interest after affinity purification of Protein A.
FIG. 2 shows the CEX-HPLC detection result of the target Protein after affinity purification of Protein A.
FIG. 3 shows the SEC-HPLC detection result of the Protein of interest after Protein A affinity purification.
FIG. 4 shows the result of affinity assay of ipilimumab.
FIG. 5 shows the result of affinity detection of the target Protein obtained by Protein A affinity purification of the harvest of 56B3-29F 6.
FIG. 6 shows the result of the splicing of the IBI310 heavy chain sequencing fragment by DNASTAR-Seqman software.
FIG. 7 shows the result of the IBI310 light chain sequencing fragment spliced by DNASTAR-Seqman software.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The reduced SDS-PAGE method used in the following examples was as follows: according to appendix IV C of pharmacopoeia of the people's republic of China (2010 edition, three parts), the purity is detected by adopting reduced SDS-PAGE gel electrophoresis, and the purity of the sample is calculated by an area normalization method.
The charge variant detection (CEX-HPLC) method used in the following examples is as follows: the determination is carried out according to appendix III B of pharmacopoeia of the people's republic of China (2010 edition, three parts), a sample is detected by using a weak cation analytical column, and the purity of acid, alkali and main components of the sample is calculated by an area normalization method.
The size exclusion chromatography (SEC-HPLC) method used in the following examples was as follows: the determination was carried out according to appendix III B of the pharmacopoeia of the people's republic of China (2010 edition, three parts), the samples were tested with a hydrophilic silica gel size exclusion chromatography column, and the purity of the samples was calculated with an area normalization method.
The primary drug for the ipilimumab prescription formulation in the following examples was YERVOY (ipilimumab) Injection, Bristol-Myers Squibb, Application No. 125377; 3/25/2011, wherein the ipilimumab adopted in the following examples is the original grinding medicine; the imitation drug is recombinant human anti-CTLA-4 monoclonal antibody injection (IBI310 for short), and the product of the Xindao biopharmaceutical company Limited has the batch number of 3D 74403.
The Chinese hamster ovary cell line subtype S (CHO-S) is Invitrogen, Cat.A 13696-1.
CD FortiCHO is Invitrogen, catalog number A11483-01.
Anti-caking Agent is available from Invitrogen under the catalog number 0010057 AE.
MTX (methotrexate) is a product from Calbiochem under the catalog number A6770.
Puro (puromycin) is an Invitrogen company under catalog number A11138-03.
Example 1 preparation of anti-CTLA-4 monoclonal antibody (IBI310)
The amino acid sequence of IBI310 is identical to the amino acid sequence of Ipilimumab (Ipilimumab).
First, vector construction
According to FreedomTMCHO-STMkit instructions for insertion of heavy chain gene (SEQ ID No.4) and light chain gene (SEQ ID No.3) of IBI310 into expression vector Freedom, respectivelyTMpCHO1.0(FreedomTMCHO-STMkit, GIBCO product, catalog number A13696-01) and a second gene expression cassette from the same plasmid to construct the IBI310 antibody expression plasmid pCHO1.0-IHEKR.
Second, expression plasmid transfection of host cells
According to Free StyleTMpMAX Reagent instructions, by Invitrogen chemical transfection Reagent (FreeStyle)TMpMAX Reagent, cat No.: 16447-100) the transfection was accomplished by transferring the IBI310 antibody expression plasmid pCHO1.0-IHEKR into CHO-S host cells.
Thirdly, pressure screening after transfection
6 bottles of cell suspension after 48 hours of transfection were filtered and separately dispensed into two square bottles. The cells were cultured in an incubator by static culture using a selection medium (Table 1) containing Puro and MTX, and the cell viability was measured after 7 days, and the first and second-stage pressure selection was carried out based on the cell viability value.
TABLE 1 media Components Table
Fourth, cell population yield detection
And (3) screening the high-yield cell populations by adopting a 6-well plate 5-day static culture method for the 12 cell populations obtained in the first stage and the second stage respectively. The expression level of the antibody in the supernatant was determined by Fortebio assay. The method comprises the steps of detecting standard samples with different concentrations through a Protein A sensor, establishing a standard curve to calculate the concentration of a sample to be detected, and selecting a cell population YY 310-210/100-50/1000 with the highest antibody expression quantity.
Five, positive monoclonal screening
The selected cell population YY 310-210/100-50/1000 was monocloned using limiting dilution. The screening of high-producing clones was performed in two stages, 96-well plate and 6-well plate, using cloning medium (Table 1). According to the screening result of the 6-well plate, the 12 clones with the highest antibody expression level are amplified and cultured and subjected to sugar supplement experiments, and the cell strain 56B3 with relatively high antibody expression level is obtained.
Sixth, screening of subcloned cell lines
To ensure the monoclonality of the cells used to construct the master cell bank, cell line 56B3, which had good yield and stability within the IBI310 primary clone cell bank, was subcloned. The subcloning method is also limited dilution method, and 3 high-yield subclones are screened through two stages of 96-well plate and 6-well plate: 56B3-25D11, 56B3-26D5 and 56B3-29F6, an original subclone cell bank consisting of the 3 subclones was established.
Seventhly, determining cell strains for final production
The target Protein is obtained by affinity purification of Protein A from harvest liquid of three subclones 56B3-25D11, 56B3-26D5 and 56B3-29F 6. The results of detection by SDS-PAGE using ipilimumab as a control are shown in FIG. 1.
In FIG. 1, M is protein Marker (NEB product, cat # P7703); ST is ipilimumab; 25D11, 26D5 and 29F6 represent target proteins obtained by affinity purification of Protein A from harvest liquid of 56B3-25D11, 56B3-26D5 and 56B3-29F6, respectively.
FIG. 1 shows that the subcloned cell lines 56B3-25D11, 56B3-26D5 and 56B3-29F6 express proteins with the same relative molecular weight and purity as those of ipilimumab.
Using ipilimumab as a control, charge isomerism of the target Protein obtained by Protein A affinity purification of the harvest of each subclone was detected by CEX-HPLC, and the results are shown in FIG. 2.
In FIG. 2, subclones 25D11, 26D5, and 29F6 represent proteins of interest obtained by affinity purification of Protein A from the harvest of 56B3-25D11, 56B3-26D5, and 56B3-29F6, respectively.
FIG. 2 shows that the target proteins expressed by the subcloned cell strains 56B3-25D11, 56B3-26D5 and 56B3-29F6 have consistent main peaks compared with ipilimumab.
The SEC-HPLC method with ipilimumab as a control was used to detect the purity of the target Protein obtained by Protein A affinity purification of the harvest of each subclone, and the results are shown in FIG. 3.
In FIG. 3, subclones 25D11, 26D5, and 29F6 represent proteins of interest obtained by affinity purification of Protein A from the harvest of 56B3-25D11, 56B3-26D5, and 56B3-29F6, respectively.
FIG. 3 shows that the retention time of the target protein expressed by the subcloned cell strains 56B3-25D11, 56B3-26D5 and 56B3-29F6 is consistent with that of ipilimumab, and the purity is higher.
The results of affinity detection of the target Protein obtained by Protein A affinity purification of the harvest solution of 56B3-29F6 using ipilimumab as a control are shown in FIGS. 4 and 5.
FIGS. 4 and 5 show that subclone 56B3-29F6 expresses a protein of interest with an affinity similar to that of ipilimumab.
The stability test of 3 subcloned cell strains 56B3-25D11, 56B3-26D5 and 56B3-29F6 for 60 days shows that: the antibody yield of the 56B3-29F6 cell line is not reduced, and the antibody yield of the two cell lines of 56B3-25D11 and 56B3-26D5 is reduced. Finally 56B3-29F6 was selected as the IBI310 subclone.
Eighth, identification of original cell lines
Sequencing the target gene in the genome of the original cell strain 56B3-29F6, identifying the expression product, and determining the molecular weight by mass spectrum. The method of sequencing the PCR products was used for both heavy and light chains.
After the IBI310 heavy chain sequencing fragment is spliced by DNASTAR-Seqman software (shown in figure 6), a gene sequence HC Seqman is obtained, and the sequence is subjected to Blast comparison with a theoretical sequence of an ipilimumab heavy chain target gene, and the result shows that the two sequences are completely consistent.
After the IBI310 light chain sequencing fragment is spliced by DNASTAR-Seqman software (shown in FIG. 7), a gene sequence LC Seqman is obtained, and the sequence is subjected to Blast comparison with a theoretical sequence of an ipilimumab light chain target gene, and the result shows that the sequence and the theoretical sequence are completely consistent.
Ninth, identification of cell expression products
Cell line 56B3-29F6 was fed-batch cultured in a shake flask for 14 days. After the harvest liquid is subjected to Protein A affinity purification, the molecular weight of a sample is detected by liquid chromatography-mass spectrometry, and the molecular weight of IBI310 is completely the same as that of ipilimumab, as shown in Table 2.
TABLE 2 molecular weight comparison of ipilimumab to IBI310
The anti-CTLA-4 monoclonal antibody (IBI310) consists of 2 light chains and 2 heavy chains, wherein one light chain and one heavy chain are connected through a disulfide bond, and the heavy chain are connected through a disulfide bond; the amino acid sequence of the light chain is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 2; the amino acid sequence of the light chain variable region is shown as 23 rd to 130 th positions of SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as 20 th to 137 th positions of SEQ ID No. 2; the CDR1, CDR2 and CDR3 sequences of the light chain variable region are shown in the 46 th to 57 th, 73 th to 79 th and 112 th to 120 th positions of SEQ ID No.1, respectively, and the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region are shown in the 45 th to 51 th, 70 th to 76 th and 118 th to 126 th positions of SEQ ID No.2, respectively.
The coding gene sequence of the light chain of the monoclonal antibody is shown as SEQ ID No.3, and the coding gene sequence of the heavy chain is shown as SEQ ID No. 4; the encoding gene sequence of the light chain variable region is shown as 67 th to 390 th sites of SEQ ID No.3, and the encoding gene sequence of the heavy chain variable region is shown as 58 th to 411 th sites of SEQ ID No. 4; the encoding gene sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as positions 136 to 171, 217 to 237 and 334 to 360 of SEQ ID No.3, respectively, and the encoding gene sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as positions 133 to 153, 208 to 228 and 352 to 378 of SEQ ID No.4, respectively.
Example 2 anti-CTLA-4 monoclonal antibody preparation A
The composition of the anti-CTLA-4 monoclonal antibody preparation A is as follows: the preparation consists of solute and solvent, wherein the solute is anti-CTLA-4 monoclonal antibody (IBI310) prepared in example 1, sodium citrate, mannitol, sodium chloride, EDTA-2Na (disodium edetate) and polysorbate 80, and the solvent is water for injection; the concentration of the anti-CTLA-4 monoclonal antibody (IBI310) in the anti-CTLA-4 monoclonal antibody preparation A is 5mg/ml, the concentration of sodium citrate in the anti-CTLA-4 monoclonal antibody preparation A is 2.42mg/ml, the concentration of mannitol in the anti-CTLA-4 monoclonal antibody preparation A is 10mg/ml, the concentration of sodium chloride in the anti-CTLA-4 monoclonal antibody preparation A is 5.85mg/ml, the concentration of EDTA-2Na in the anti-CTLA-4 monoclonal antibody preparation A is 0.04mg/ml, and the concentration of polysorbate 80 in the anti-CTLA-4 monoclonal antibody preparation A is 0.1 mg/ml; the pH was 5.5.
When the anti-CTLA-4 monoclonal antibody preparation A is prepared, the non-ionic surfactant polysorbate 80 in the solute is added at last and then volume fixing is carried out, and the addition of other solutes is not in sequence.
And (3) aseptically subpackaging the anti-CTLA-4 monoclonal antibody preparation A into penicillin bottles, and covering rubber plugs and aluminum plastic covers to obtain finished products of the preparation.
Example 3 anti-CTLA-4 monoclonal antibody preparation B
The composition of anti-CTLA-4 monoclonal antibody preparation B was as follows: the preparation consists of solutes and a solvent, wherein the solutes are the anti-CTLA-4 monoclonal antibody (IBI310) prepared in example 1, Tris (hydroxymethyl) aminomethane (Tris), arginine, sodium chloride, EDTA-2Na (disodium edetate) and polysorbate 20, and the solvent is water for injection; the concentration of the anti-CTLA-4 monoclonal antibody (IBI310) in the anti-CTLA-4 monoclonal antibody preparation B is 5mg/ml, the concentration of tris (hydroxymethyl) aminomethane in the anti-CTLA-4 monoclonal antibody preparation B is 2.42mg/ml, the concentration of arginine in the anti-CTLA-4 monoclonal antibody preparation B is 10mg/ml, the concentration of sodium chloride in the anti-CTLA-4 monoclonal antibody preparation B is 5.85mg/ml, the concentration of EDTA-2Na in the anti-CTLA-4 monoclonal antibody preparation B is 0.04mg/ml, and the concentration of polysorbate 20 in the anti-CTLA-4 monoclonal antibody preparation B is 0.4 mg/ml; the pH was 6.5.
When the anti-CTLA-4 monoclonal antibody preparation B is prepared, the non-ionic surfactant polysorbate 20 in the solute is added at last and then volume fixing is carried out, and the addition of other solutes is not in sequence.
And (3) aseptically subpackaging the anti-CTLA-4 monoclonal antibody preparation B into penicillin bottles, and covering rubber plugs and aluminum plastic covers to obtain a finished product of the preparation.
Example 4 accelerated stability test of anti-CTLA-4 monoclonal antibody preparation A and anti-CTLA-4 monoclonal antibody preparation B
Accelerated stability experiments were performed on the anti-CTLA-4 monoclonal antibody preparations prepared in examples 2 and 3, and chemical stability of the anti-CTLA-4 monoclonal antibody in the preparations was characterized by charge variation (CEX-HPLC assay) and protein purity (SDS-PAGE assay), respectively, using the change in the percentage content of the acidic component, the basic component, and the main component and the main peak content of the protein purity as determination means.
The anti-CTLA-4 monoclonal antibody preparation a prepared in example 2, the anti-CTLA-4 monoclonal antibody preparation B prepared in example 3, and ipilimumab were analyzed for changes in protein charge variant content and changes in protein purity under the high-temperature acceleration condition of 37 ± 2 ℃ for 0h and 1 month by the charge variant assay (CEX-HPLC) method and the reduced SDS-PAGE method, and the results are shown in table 3 and table 4, respectively.
TABLE 337 + -2 ℃ protein Charge variant comparison
TABLE 437. + -. 2 ℃ comparison of protein purities
The IBI310 is structurally complex, resulting in unstable chemical properties, proteins are prone to aggregation and charge variants are prone to conversion from basic to acidic components. The results in tables 3 and 4 show that the charge variant content and the decrease in protein purity of the anti-CTLA-4 monoclonal antibody preparations a and B are significantly slower than those of the ipilimumab formulation.
Meanwhile, other stability indexes of the anti-CTLA-4 monoclonal antibody preparations A and B, such as appearance, protein concentration, turbidity and the like, are equivalent to those of an ipilimumab prescription preparation under the acceleration condition through observation.
The above results indicate that the anti-CTLA-4 monoclonal antibody preparations a and B prepared in examples 2 and 3 of the present invention, respectively, can reduce the chemical degradation rate of the recombinant fully human anti-CTLA-4 monoclonal antibody contained therein, and increase the physicochemical stability of the recombinant fully human anti-CTLA-4 monoclonal antibody, so that the monoclonal antibody can stably exist in the formulation. Compared with the existing anti-CTLA-4 monoclonal antibody preparation, the anti-CTLA-4 monoclonal antibody preparation prepared by the invention has the advantages of improved uniformity and consistency of product quality, prolonged product shelf life, high stability and increased clinical safety.
Claims (3)
1. A composition against cytotoxic T lymphocyte-associated antigen 4, the composition comprising a solute and a solvent, the solvent being water, the solute comprising a monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4, a buffer, an osmolality adjusting agent, a chelating agent, a protein protecting agent, and a non-ionic surfactant;
the concentration of the monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 in the composition is 5mg/ml, the concentration of the buffer in the composition is 2.42mg/ml, the concentration of the osmolality adjusting agent in the composition is 5.85mg/ml, the concentration of the chelating agent in the composition is 0.04mg/ml, the concentration of the protein protecting agent in the composition is 10mg/ml, and the concentration of the nonionic surfactant in the composition is 0.1 mg/ml; the pH of the composition was 5.5;
the buffering agent is sodium citrate;
the osmotic pressure regulator is sodium chloride;
the chelating agent is disodium ethylene diamine tetraacetate;
the protein protective agent is mannitol;
the nonionic surfactant is polysorbate 80;
the monoclonal antibody for resisting the cytotoxic T lymphocyte-associated antigen 4 is ipilimumab.
2. A process for preparing the composition of claim 1, wherein the composition is formulated by adding the remaining solute to water, adding the non-ionic surfactant, and metering.
3. Use of the composition of claim 1 or the composition prepared by the process of claim 2 for the preparation of a product as shown in any one of (1) to (6) below:
(1) products against cytotoxic T lymphocyte-associated antigen 4;
(2) products for relieving the inhibition of T lymphocyte activation by cytotoxic T lymphocyte-associated antigen 4;
(3) products for relieving the suppression of the immunity of the cytotoxic T lymphocyte-associated antigen 4 to the body;
(4) products that activate T lymphocytes;
(5) products for the prevention and/or treatment of diseases in which T lymphocyte activity is inhibited;
(6) a product for improving immunity is provided.
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