CN113117071B - Freeze-dried preparation of non-glycosylated anti-PD-1 monoclonal antibody - Google Patents

Freeze-dried preparation of non-glycosylated anti-PD-1 monoclonal antibody Download PDF

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CN113117071B
CN113117071B CN202010044092.9A CN202010044092A CN113117071B CN 113117071 B CN113117071 B CN 113117071B CN 202010044092 A CN202010044092 A CN 202010044092A CN 113117071 B CN113117071 B CN 113117071B
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CN113117071A (en
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赵丽丽
石曼
刘忠
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Lunan Pharmaceutical Group Corp
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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Abstract

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a freeze-dried preparation of an aglycosylated anti-PD-1 monoclonal antibody, which comprises the aglycosylated anti-PD-1 monoclonal antibody, a buffer channel system, an excipient and a solubilizer. The invention adds an annealing step in the freeze-drying process, and the freeze-dried preparation has simple formula and fewer auxiliary materials, further reduces the water content of freeze-dried powder, improves the stability of the medicine and is convenient to store and transport.

Description

Freeze-dried preparation of non-glycosylated anti-PD-1 monoclonal antibody
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a freeze-dried preparation of an aglycosylated anti-PD-1 monoclonal antibody.
Background
Programmed Death receptor-1 (PD-1) is a type I membrane protein of 288 amino acids, which is one of the consistent major immune checkpoints (Immune Checkpoint). PD-1 is used as an immunosuppressive receptor, mainly in activating T cells and B cells, is used as a T cell inhibitory receptor, is combined with ligand PD-L1, can inhibit the activity of T lymphocytes and related in-vivo cell immune response, can limit the function of T cell effector in tumor cells, and has an important role in tumor immune escape. Tumor immunotherapy, i.e. the use of the human body's own immune system to combat cancer, is a breakthrough tumor treatment, but the tumor microenvironment protects tumor cells from effective immune destruction, so how to break the tumor microenvironment is called as an anti-tumor study focus.
The prior research results have determined the role of PD-1 in tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by ifnγ in most PD-L1 negative tumor cell lines) and is presumed to be an important target for mediating immune evasion of tumors. Biopsy was assessed by immunohistochemistry and acetonitrile found PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 expression on tumor cells in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, head and neck tumor, and the like. Thus, blocking the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor-specific T cells, and help the immune system clear tumor cells, so PD-1 is a hot target for developing tumor immunotherapy drugs.
At present, anti-PD-1 monoclonal antibodies such as opdivo, keyruda and the like are developed and developed abroad and are marketed in batches. However, these antibodies have low response rate as single drugs, and the glycosylation modification of the monoclonal antibody used in the invention is knocked out, and although the process development is relatively stable, the uniformity is improved, and a better clinical effect is expected to be obtained, compared with other anti-PD-1 monoclonal antibodies, the sensitivity of the monoclonal antibody to trypsin and the like is obviously increased due to glycosylation removal, the structural flexibility is enhanced, the tendency of polymer formation is easier at low pH, and the aggregation and precipitation are easy. In order to prevent denaturation of proteins during storage prior to use, loss of activity or structural integrity due to aggregation, development of formulation processes for non-glycosylated anti-PD-1 monoclonal antibodies is necessary.
In general, proteins have very short half-lives and undergo denaturation (e.g., aggregation, dissociation, and adsorption to the surface of a container) after exposure to various factors (e.g., unsuitable temperature, water-gas interface, high pressure, physical/mechanical stress, organic solvents, and microbial contamination). Thus, denatured proteins lose intrinsic physicochemical properties and physiological activities. Denaturation of proteins is often irreversible, and thus, once proteins are denatured, their natural properties may not return to their original state.
The formulations of PD-1 in the prior art are mostly liquid formulations, however in the biopharmaceutical industry long-term storage of proteins prepared using recombinant DNA technology in aqueous formulations is often a difficult task. In order to overcome the stability problem of the protein in the aqueous preparation, the invention provides a freeze-dried preparation of the non-glycosylated anti-PD-1 monoclonal antibody.
Disclosure of Invention
The invention aims to provide a stable freeze-dried preparation of an aglycosylated anti-PD-1 monoclonal antibody, which has the advantages of simple formulation, fewer auxiliary materials, good stability and convenience in storage and transportation.
The specific technical scheme of the invention is as follows:
a lyophilized preparation of an aglycosylated anti-PD-1 monoclonal antibody comprises the aglycosylated anti-PD-1 monoclonal antibody, a buffer system, an excipient and a solubilizing agent.
Wherein the buffer system is selected from one of acetic acid/sodium acetate, citric acid/sodium citrate, histidine/histidine hydrochloride.
Wherein the excipient is selected from one or more of sucrose, trehalose, mannitol and sorbitol.
Wherein the solubilising agent is selected from polysorbate 20 or polysorbate 80.
The pH is regulated by a buffer system, and the pH is preferably 5.5-6.0.
Preferably, the lyophilized formulation of the stabilized aglycosylated anti-PD-1 monoclonal antibody comprises the following components:
further preferably, the lyophilized formulation of the stabilized aglycosylated anti-PD-1 monoclonal antibody comprises the following components:
according to the invention, an amino acid stabilizer is not required to be added, and the excipient can be used as a protective agent to surround the protein, so that the movement of macromolecular substances is blocked, the spatial structure of protein molecules is maintained, and the aggregation of the protein molecules is prevented, thereby increasing the stability of the anti-PD-1 monoclonal antibody. The invention uses polysorbate 20 or polysorbate 80 as solubilizer, which is a nonionic surfactant, and can reduce the aggregation of antibodies in the preparation, the formation and adsorption of particles in the preparation and the like in a certain dosage range.
In a preferred embodiment, a stable lyophilized formulation of an aglycosylated anti-PD-1 monoclonal antibody comprises the following components:
the invention also provides a preparation method of the antibody drug freeze-dried preparation, which comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, and filling and freeze-drying after detecting that endotoxin is qualified.
Preferably, the freeze-drying process comprises three steps of freezing control, primary drying and analytical drying, in particular the following steps:
(1) Freezing control: firstly, placing a penicillin bottle filled with antibody drug solution on a partition board of a freeze dryer, pre-freezing at 5 ℃, maintaining for 15-60 min, cooling to-45 to-40 ℃, and maintaining for 2-6 h for freezing;
(2) Primary drying: heating the temperature of the partition board system to-20 to-15 ℃, maintaining for 40-50 h, heating to 0-5 ℃ and maintaining for 8-10 h, and finishing primary drying;
(3) And (5) analysis and drying: and heating the separator system to 20-30 ℃, maintaining for 8-10 h for resolving and drying to finish the freeze-drying process.
Further preferably, the lyophilization process comprises three steps of freeze control, primary drying and analytical drying, in particular the following steps:
(1) Freezing control: placing a penicillin bottle filled with antibody drug solution on a baffle plate, setting the temperature at 5 ℃ for 15-60 min, setting the temperature of the baffle plate at-45 to-40 ℃ for 40-60 min to reach the preset temperature, maintaining for 2-6 h, setting the temperature of the baffle plate at-30 to-15 ℃ for 30-40 min to reach the preset temperature, maintaining for 1-3 h, finally, setting the temperature of the baffle plate at-45 to-40 ℃ for 30-40 min to reach the preset temperature, maintaining for 2-3 h, and ending the freezing control step;
(2) Primary drying: carrying out primary drying on the system under the condition of extreme vacuum, setting the temperature of the partition plate to be-20 to-15 ℃ and the setting time to be 40-60 min to reach the preset temperature, maintaining for 40-50 h, then setting the temperature of the partition plate to be 0-5 ℃ and the setting time to be 30-40 min to reach the preset temperature and maintaining for 8-10 h, and completing primary drying;
(3) And (5) analysis and drying: setting the temperature of the partition plate to be 20-30 ℃ and setting the time to be 40-60 min to reach the set temperature, and analyzing and drying the partition plate for 8-10 h under the condition that the pressure of the box body is 0.1-0.2 mbar to finish the freeze-drying process.
The antibody drug of the invention is an anti-PD-1 monoclonal antibody, including but not limited to glycosylated or non-glycosylated anti-PD-1 monoclonal antibodies.
In one embodiment, a lyophilization process for a lyophilized formulation of an aglycosylated anti-PD-1 monoclonal antibody is as follows:
in another embodiment, a lyophilization process for a lyophilized formulation of an aglycosylated anti-PD-1 monoclonal antibody is as follows:
in a preferred embodiment, a lyophilization process for a lyophilized formulation of an aglycosylated anti-PD-1 monoclonal antibody is as follows:
the invention optimizes the formulation of the preparation and simultaneously further improves the freeze-drying process to obtain the freeze-dried preparation of the non-glycosylated anti-PD-1 monoclonal antibody, which has the advantages of simple formulation, low water content of the freeze-dried preparation and good stability.
The amino acid sequence of the aglycosylated anti-PD-1 monoclonal antibody has patent protection, and can be referred to patent CN106519034A. Experiments prove that the preparation can also be applied to other glycosylated and non-glycosylated anti-PD-1 monoclonal antibodies.
The glycosylation-free anti-PD-1 monoclonal antibody freeze-dried preparation has few auxiliary materials, further reduces the potential safety hazard of clinical medication, has low moisture content, good stability, full appearance, uniform texture and quick re-dissolution, and has multiple indexes of pH, clarity, color, purity, sterility and the like which all meet the quality standard and are superior to the prior art. The preparation process of the non-glycosylated anti-PD-1 monoclonal antibody freeze-dried powder preparation is simple, is suitable for amplification, can ensure stable and controllable quality, has good drying effect, low water content of the product and is convenient to store and transport.
Detailed Description
The invention will now be further illustrated by way of examples which are only part of a formulation screening assay and are not intended to limit the invention to the examples described. The test methods in the following examples, for which specific conditions are not noted, were selected according to conventional methods and conditions, or according to the commercial specifications. In the examples, stability tests and related biological tests were carried out according to the specifications of the chinese pharmacopoeia, and the formulations described in the examples were all of pharmaceutical grade and were all commercially available.
Example 1
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 2
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 3
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 4
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 5
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 6
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 7
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, stabilizer arginine, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 8
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Example 9
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Comparative example 1
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Comparative example 2
Prescription:
the preparation process comprises the following steps:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Comparative example 3
Prescription:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Comparative example 4
Prescription:
weighing a prescribed amount of buffer salt, dissolving with a proper amount of water for injection, adjusting pH, sampling and detecting pH and endotoxin, accurately weighing a prescribed amount of excipient, solubilizer and non-glycosylated anti-PD-1 monoclonal antibody, adding into a buffer solution, and uniformly mixing to obtain a semi-finished product liquid; and (3) performing sterile filtration on the semi-finished product liquid by using a 0.22 mu m filter membrane, filling after detecting that endotoxin is qualified, and freeze-drying according to the freeze-drying process of the following table.
Verification embodiment
Stability test
Samples were prepared according to examples 1 to 9 and comparative examples 1 to 4, respectively, 200 bottles were taken, and storage stability was examined by using an accelerated stability test and a long-term test. The acceleration test was carried out at 25.+ -. 2 ℃ for 12 months. The equipment can control the temperature to +/-2 ℃ and monitor the actual temperature. Samples were taken at the end of the test period at month 0, month 3, month 6, month 12, and project detection was focused on stability. The long-term test is carried out at the temperature of 2-8 ℃ and is respectively detected at the end of 0 month, 3 months, 6 months, 12 months and 24 months according to the stability key investigation project; (wherein purity check is determined according to the rule 0514 molecular row chromatography and rule 0542 capillary electrophoresis of the Chinese pharmacopoeia of 2015 edition), and activity is determined according to a reporter gene method based on bioluminescence); the moisture content was measured by a Coulomb method defined in the third general rule of the Chinese pharmacopoeia of 2015 edition using a Metlele-zaidoDL 37 Karl Fischer titrator. The results are shown in tables 1 and 2.
Table 1 2-8deg.C long-term stability test results
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TABLE 2 results of accelerated stability test at 25.+ -. 2 ℃
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Claims (3)

1. A lyophilized formulation of an aglycosylated anti-PD-1 monoclonal antibody comprising the following components in amounts:
2. a method for preparing a freeze-dried preparation according to claim 1, which is characterized in that a prescription amount of citric acid/sodium citrate buffer salt is weighed and dissolved by a proper amount of water for injection, the pH is adjusted to 5.5, after the sampling and the detection of the pH and endotoxin are qualified, a prescription amount of excipient sucrose or trehalose, a solubilizer polysorbate 80 and an aglycosylated anti-PD-1 monoclonal antibody are weighed and added into the citric acid/sodium citrate buffer body fluid, and the mixture is uniformly mixed to obtain a semi-finished product liquid; sterile filtering the semi-finished product liquid with a 0.22 μm filter membrane, and filling and freeze-drying after detecting endotoxin;
the freeze-drying process comprises three stages of freeze-drying, primary drying and analytical drying, and specifically comprises the following steps:
(1) Freezing control: firstly, placing a penicillin bottle filled with antibody drug solution on a partition board of a freeze dryer, pre-freezing at 5 ℃, maintaining for 15-60 min, cooling to-45 to-40 ℃, and maintaining for 2-6 h for freezing;
(2) Primary drying: heating the temperature of the partition board system to-20 to-15 ℃, maintaining for 40-50 h, heating to 0-5 ℃ and maintaining for 8-10 h, and finishing primary drying;
(3) And (5) analysis and drying: and heating the separator system to 20-30 ℃, maintaining for 8-10 h for resolving and drying to finish the freeze-drying process.
3. The method of manufacturing according to claim 2, comprising the steps of:
(1) Freezing control: placing a penicillin bottle filled with antibody drug solution on a baffle plate, setting the temperature at 5 ℃ for 15-60 min, setting the temperature of the baffle plate at-45 to-40 ℃ for 40-60 min to reach the preset temperature, maintaining for 2-6 h, setting the temperature of the baffle plate at-30 to-15 ℃ for 30-40 min to reach the preset temperature, maintaining for 1-3 h, finally, setting the temperature of the baffle plate at-45 to-40 ℃ for 30-40 min to reach the preset temperature, maintaining for 2-3 h, and ending the freezing control step;
(2) Primary drying: carrying out primary drying on the system under the condition of extreme vacuum, setting the temperature of the partition plate to be-20 to-15 ℃ and the setting time to be 40-60 min to reach the preset temperature, maintaining for 40-50 h, then setting the temperature of the partition plate to be 0-5 ℃ and the setting time to be 30-40 min to reach the preset temperature and maintaining for 8-10 h, and completing primary drying;
(3) And (5) analysis and drying: setting the temperature of the partition plate to be 20-30 ℃ and setting the time to be 40-60 min to reach the set temperature, and analyzing and drying the partition plate for 8-10 h under the condition that the pressure of the box body is 0.1-0.2 mbar to finish the freeze-drying process.
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WO2022268887A1 (en) * 2021-06-23 2022-12-29 Formycon Ag Formulations of anti-pd1 antibodies
CN117825688B (en) * 2024-01-19 2024-05-31 武汉鹰达生物科技有限公司 Animal IgG antibody freeze-dried powder preparation and preparation process thereof

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CN105708811A (en) * 2014-12-01 2016-06-29 西藏海思科药业集团股份有限公司 Stable lyophilized preparation of recombinant human anti-CD20 monoclonal antibody
CN110354073A (en) * 2018-04-09 2019-10-22 鲁南制药集团股份有限公司 A kind of liquid preparation of immunosuppressor monoclonal antibody

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CN105708811A (en) * 2014-12-01 2016-06-29 西藏海思科药业集团股份有限公司 Stable lyophilized preparation of recombinant human anti-CD20 monoclonal antibody
CN110354073A (en) * 2018-04-09 2019-10-22 鲁南制药集团股份有限公司 A kind of liquid preparation of immunosuppressor monoclonal antibody

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