CN114642726A - Stable anti-PD-1/PD-L1 bispecific antibody liquid preparation - Google Patents
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- CN114642726A CN114642726A CN202011490759.4A CN202011490759A CN114642726A CN 114642726 A CN114642726 A CN 114642726A CN 202011490759 A CN202011490759 A CN 202011490759A CN 114642726 A CN114642726 A CN 114642726A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
Abstract
The invention discloses a liquid preparation of a PD1/PD-L1 bispecific antibody, which comprises a tetravalent bispecific antibody combined with human PD1 and PD-L1, a histidine-histidine hydrochloride buffer solution, trehalose and a surfactant, can be used for preparing a medicament for treating cancers and autoimmune diseases, and has good stability.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a stable anti-PD-1/PD-L1 bispecific antibody liquid preparation.
Background
Programmed death receptor 1(PD-1) is an important immunosuppressive molecule, and its ligand is programmed death receptor-ligand 1 (PD-L1). The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, promoting proliferation of antigen-specific T cells. One way for tumor cells to escape T cell destruction as antigens is to generate PD-L1 on the surface of the tumor cells, and when PD-1 on the surface of T cells of immune cells recognizes PD-L1, the tumor cells can conduct inhibition signals, and the T cells cannot find the tumor cells and send attack signals to the tumor cells.
Bispecific antibodies refer to antibody molecules that specifically bind to two antigens or two epitopes simultaneously. The anti-PD-1/PD-L1 bispecific antibody has the action mechanism of preventing the recognition process of PD-1 and PD-L1 and partially restoring T cell function, so that T cells can kill tumor cells. Bispecific antibodies are more complex in primary structure, larger in molecular weight and more demanding on the stability of the formulation than monoclonal antibodies, and therefore formulation formulations suitable for clinical use need to be developed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a novel liquid preparation of an anti-PD-1/PD-L1 bispecific antibody. The liquid preparation can be stored at 2-8 ℃ for at least 24 months, and at 25 ℃ for at least 6 months, and has good stability.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
in one aspect, the present invention provides a liquid formulation comprising a tetravalent bispecific antibody that binds to human PD1 and PD-L1, a histidine-histidine hydrochloride buffer, trehalose, and a surfactant, the pH of the liquid formulation being 5.0-5.8.
Preferably, the heavy chain of the antibody is as set forth in SEQ ID NO: 1, and the light chain is shown as SEQ ID NO: 2, respectively.
Preferably, the concentration of the bispecific antibody is 20-80mg/ml, preferably 20-50mg/ml, more preferably 40-50 mg/ml.
Preferably, the concentration of the histidine-histidine hydrochloride buffer solution is 10-20 mM.
Preferably, the concentration of the trehalose is 70-100mg/ml, and preferably, the concentration of the trehalose is 80-90 mg/ml.
Preferably, the surfactant is polysorbate 80 or polysorbate 20, and the concentration of the surfactant is 0-0.9mg/ml, preferably 0.5-0.9 mg/ml.
Preferably, the pH of the liquid preparation is in the range of 5.0-5.6.
Preferably, the liquid formulation comprises 40mg/ml bispecific antibody, 20mM histidine-histidine hydrochloride buffer, 90mg/ml trehalose, 0.5mg/ml polysorbate 80, pH 5.3.
In another aspect, the invention provides the use of said liquid formulation for the manufacture of a medicament for the treatment of cancer and autoimmune diseases.
As a preferred embodiment, the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
The invention has the beneficial effects that: the invention provides a novel anti-PD-1/PD-L1 bispecific antibody liquid preparation which can be stored at 2-8 ℃ for at least 24 months and at 25 ℃ for at least 6 months and has good stability.
Drawings
FIG. 1: SEC purity and IEC purity results for formulations at different protein concentrations
FIG. 2 is a schematic diagram: SEC purity and IEC purity results for formulations at different pH and polysorbate 80 concentrations
FIG. 3: DOE model analysis of formulations at different pH and polysorbate 80 concentrations
FIG. 4: SEC purity and IEC purity results for formulations at varying concentrations of histidine
FIG. 5: SEC purity and IEC purity results for formulations at different concentrations of trehalose
Detailed Description
The anti-PD-1/PD-L1 bispecific antibody used in the following examples was derived from PCT patent application PCT/CN2020/090442 as an anti-PD-1/PD-L1 bispecific antibody 609-Fab-PDL1-IgG4 comprising two identical heavy chains each having two heavy chain variable regions and two CH1 domains and four identical light chains each having the heavy chain amino acid sequence as set forth in SEQ ID NO: 1, and the light chain amino acid sequence is shown as SEQ ID NO: 2, respectively.
The detection method used in the examples of the present invention is explained as follows:
SEC purity, Polymer detection method:
mobile phase: 200mM phosphate buffer, pH 6.8. + -. 0.1. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. A chromatographic column: TSK G3000SWxl, 7.8X 300mm 5 μm, TOSOH 08541. High performance liquid chromatograph: waters Alliance e 26952489 UV/visible light Detector, Dionex Ultimate 3000VWD-3400(RS) Detector or other suitable HPLC system equipped with a UV Detector.
System applicability sample: the reference substance is diluted to 1.0mg/ml by mobile phase, centrifuged for 10min at 13000rpm, and the supernatant is transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: diluting the sample concentration to 1.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing into an HPLC sample tray. Chromatographic conditions are as follows: the column temperature is 25 +/-2 ℃; the sample temperature is 10 +/-2 ℃; detection wavelength UV280 nm; the injection volume is 20 mu L; the flow rate was 0.5ml/min and the elution time was 30 min.
Integration was performed using chromatography software and peak area normalization was used to calculate the peak area percentage of each peak. Acceptance criteria for system suitability: the separation degree of polymers and monomers of 6-needle system applicability samples is more than or equal to 1.5, the retention time RSD of a main peak is less than or equal to 1.0%, the peak area RSD of the main peak is less than or equal to 2.0%, the asymmetry of the main peak is less than or equal to 2.0, and the number of theoretical plates is more than or equal to 4000. The test article reports the results: the SEC purity of a sample is reported as the peak area percentage of the monomer main peak and the mer content as the peak area percentage of the mer peak.
The IEC purity detection method comprises the following steps:
mobile phase A: 20mM phosphate buffer, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. Mobile phase B: 20mM phosphate buffer +200mM sodium chloride, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. A chromatographic column: propac WCX-10, 4X 250mm, Thermo Dionex 054993. A high performance liquid chromatograph: waters Alliance e2695, Dionex Ultimate series 3000 or other suitable HPLC system equipped with an ultraviolet detector.
System applicability sample: the reference sample is diluted to 1.0mg/ml with ultrapure water, centrifuged at 13000rpm for 10min, and the supernatant is transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: diluting the sample to 1.0mg/ml with ultrapure water, centrifuging at 13000rpm for 10min, taking the supernatant, transferring to a sample bottle, and placing into an HPLC sample tray. Chromatographic conditions are as follows: the column temperature is 30 +/-2 ℃; the sample temperature is 10 +/-2 ℃; the detection wavelength is UV 214 nm; the injection volume is 20 mu L; the flow rate was 1.0 ml/min. The mobile phase gradient was as follows:
purity analysis: and calculating the peak area percentages of a main peak, an acid peak area and an alkali peak area on the sample map by using a peak area normalization method. The IEC purity results are reported as peak area percentages of the main peak.
The starting materials used in the examples of the present invention are commercially available unless otherwise indicated.
The examples of the present invention are further illustrative of the present invention and are not intended to limit the present invention in any way.
Example 1 investigation of antibody protein concentration
This example examines the effect of the amount of antibody protein on the formulation. The antibody protein concentration was investigated in the range of 20-80mg/ml, using histidine as buffer at a concentration of 20mM, adjusting the pH to 6.0 with histidine hydrochloride, and formulating 3 sets of formulations as given in Table 1. Samples were kept at 40 ℃ for 2 weeks and sampled at 0, 1, and 2 weeks for SEC purity and IEC purity.
TABLE 1 examination conditions for antibody protein concentration
TABLE 2 investigation results of antibody protein concentration
The results in table 2 were subjected to regression analysis and the results were analyzed by the JMP model as shown in fig. 1. As can be seen from the results in FIG. 1, there was no significant difference between the SEC purity and the IEC purity for each group of prescriptions. Therefore, the concentration of the antibody protein is preferably in the range of 20-80 mg/ml. However, since the starting point of the SEC purity of the antibody protein becomes lower at a high concentration, for example, the SEC purity at 0 point in 1-3 groups is lower than that in the other two groups, the preparation with the concentration of the antibody protein of 20-50mg/ml has better quality, and 40mg/ml is selected as the final concentration of the antibody protein.
Example 2 pH and polysorbate 80 investigation
In the embodiment, the influence of pH and the dosage of polysorbate 80 on the stability of the preparation is examined, the pH is examined to be 5.0-6.6, and the polysorbate 80 is examined to be 0-1 mg/ml, as shown in Table 3. Other components: the protein concentration was 40mg/ml, histidine/histidine hydrochloride concentration was 20mM, trehalose concentration was 90 mg/ml. The formulated samples were placed at 40 ℃ for 4 weeks and sampled at 0, 1, 2, 3, 4 weeks for SEC purity, IEC purity and insoluble particles.
Table 3 pH and polysorbate 80 investigation conditions
TABLE 4 pH and polysorbate 80 observations
Trending the data of Table 4 As shown in FIG. 2, both SEC and IEC purity decreased faster at pH6.6, and relatively slower at pH 5.0-5.8. Thus higher pH is not favorable for protein stabilization. Within the pH range of 5.0-5.8, lower polysorbate 80 doses were more favorable for protein stabilization, and 2-3 and 2-7 had greater rates of change compared to the other groups, indicating that high polysorbate 80 doses are unfavorable for protein stabilization. However, the effect of polysorbate 80 on the granules is obvious, for example, in groups 2-1, 2-4 and 2-8, the granules are serious under the condition of not adding polysorbate 80, and the comprehensive selection of the dosage of polysorbate 80 is about 0.5mg/ml, which is more appropriate.
The results in table 4 were subjected to slope analysis and the slope of each set was analyzed using the DOE model as shown in fig. 3. As is clear from the results in FIG. 3, the pH is preferably in the range of pH5.0 to 5.6, and polysorbate 80 is preferably in the range of 0 to 0.9 mg/ml. The comprehensive trend analysis and slope analysis show that the comparison of pH5.3 and polysorbate 800.5 mg/ml is satisfactory.
Example 3 trehalose and histidine investigation
This example examines the effect of trehalose and histidine levels on the formulation. Trehalose was investigated in a range of 70-100mg/ml and histidine was investigated in a range of 10-30mM, as shown in Table 5. Other components: the protein concentration was 40mg/ml and polysorbate 80 was added at 0.5mg/ml, pH 5.3. Samples were left at 40 ℃ for 4 weeks and sampled at 0, 1, 2, 3, 4 weeks for SEC purity and IEC purity.
TABLE 5 trehalose and histidine survey conditions
TABLE 6 trehalose and histidine observations
The results in table 6 were analyzed and shown in fig. 4 and 5. From the results of fig. 4, it can be seen that the slope of the formulation containing 30mM histidine is greater than that of the formulations containing 10mM and 20mM histidine, indicating that the degradation rate is faster, which is not favorable for protein stabilization, and the slope of the formulations containing 10mM and 20mM histidine has no significant difference, so the preferred concentration range of histidine is 10-20 mM. As can be seen from FIG. 5, the trehalose concentration has no significant effect on the protein quality, 3-2 is slightly better than that of the other groups, and the trehalose concentration range may be selected to be 70-100 mg/ml. Considering the buffer capacity of the injection and the principle of human body isosmosis, the concentration of histidine is preferably 20mM, and the concentration of trehalose is preferably 90 mg/ml.
Sequence listing
<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH
<120> a stable anti-PD-1/PD-L1 bispecific antibody liquid preparation
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Claims (10)
1. A liquid formulation comprising a tetravalent bispecific antibody that binds human PD1 and PD-L1, histidine-histidine hydrochloride buffer, trehalose, and a surfactant, the pH of the liquid formulation being 5.0-5.8.
2. The liquid formulation of claim 1, wherein the heavy chain of the antibody is as set forth in SEQ ID NO: 1, and the light chain is shown as SEQ ID NO: 2, respectively.
3. The liquid formulation according to claim 1, wherein the concentration of bispecific antibody is 20-80mg/ml, preferably 20-50mg/ml, more preferably 40-50 mg/ml.
4. The liquid formulation of claim 1, wherein the histidine-histidine hydrochloride buffer is at a concentration of 10 to 20 mM.
5. The liquid formulation of claim 1, wherein said trehalose is at a concentration of 70-100mg/ml, preferably wherein said trehalose is at a concentration of 80-90 mg/ml.
6. The liquid formulation of claim 1, wherein the surfactant is polysorbate 80 or polysorbate 20, and wherein the surfactant concentration is 0 to 0.9mg/ml, preferably 0.5 to 0.9 mg/ml.
7. The liquid formulation of claim 1, wherein the liquid formulation has a pH in the range of 5.0 to 5.6.
8. The liquid formulation of claim 1, wherein the liquid formulation comprises 40mg/ml bispecific antibody, 20mM histidine-histidine hydrochloride buffer, 90mg/ml trehalose, 0.5mg/ml polysorbate 80, pH 5.3.
9. Use of a liquid formulation according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment of cancer and autoimmune diseases.
10. The use of claim 9, wherein the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
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