Patent Application
20240245609
The present invention relates to the field of biopharmaceutical technology, in particular to a freeze-dried composition containing anti-HER2 drug conjugate, a freeze-dried preparation prepared from the freeze-dried composition, and a preparation method thereof and use thereof.
Human epidermal growth factor receptor 2 (HER2/ErbB2) is a member of the epithelial growth factor receptor family, which is a transmembrane tyrosine kinase. The amplification of HER2 gene exists in about 30% of the primary human breast cancers, and sometimes it is also found in many other solid tumors. The amplification of HER2 gene usually leads to overexpression, resulting in up to one million HER2 protein molecules on the surface of each cancer cell. Increased HER2 protein expression and HER2 gene amplification are associated with poor clinical prognosis, including shortened relapse-free survivals and overall survivals (OSs) of breast cancer and gastric cancer, and shortened overall survivals of lung cancer, ovarian cancer, colon cancer and pancreatic cancer. Several therapeutic agents, antibodies and antibody-drug conjugates (ADCs, including T-DM1) targeting HER2 have been proved to have clinical anticancer activity.
A pharmaceutical must be marketed in the form of final preparation, and the selection of dosage form, the types and dosages of excipients, and the preparation process all have an impact on the stability of the product. A qualified product should have stable and controllable quality within its validity period, and be convenient for transportation, storage and clinical use. Pharmaceutical stability is one of the important indicators for evaluating pharmaceutical effectiveness and safety. Stability research is the main basis for determining the storage condition and shelf life of a pharmaceutical, which can also provide scientific basis for the pharmaceutical production, packaging, transportation condition and so on. For a pharmaceutical, especially a biological product, the maintenance of molecular configuration and biological activity of the active ingredient depends on various covalent and non-covalent forces, and the maintenance of these forces are closely related to the microenvironment they are in, such as temperature, light, ion concentration, and mechanical shear force.
ADC is a highly effective and specific pharmaceutical used to treat cancers and other diseases, in which the antibody part specifically binds to the antigen thereof on the target cells, allowing the drug to exert its cytotoxicity or other therapeutic effects on the target cells. However, like other protein pharmaceuticals, the antibody is prone to degradation, such as through oxidation, deamidation and fragmentation, as well as to form particles and aggregates. The purpose of developing an ADC formulation is to ensure stable administration to patients and reliable quality. However, compared with naked antibody, ADC has a more complex structure and has more unstable factors. The numerous formulation characteristics which are exhibited during the development process of a biological preparation may affect the safety, effectiveness and quality controllability of the product. Therefore, ADC poses a challenge to the preparation used for therapeutic purpose. In order to provide a stable ADC pharmaceutical during transportation and storage, careful selections of the carrier, excipient, and/or stabilizer in the pharmaceutical preparation are necessary.
The ADC quality attribute includes the aspect of free small molecule drug, and the aspect of naked antibody, as well as the aspect of antibody drug conjugate. The content of free payload may affect the safety of the ADC, while the naked antibody itself has inherent critical quality attribute of an antibody, such as chemical instability, like aggregation, fragmentation, charge heterogeneity, degradation, oxidation and so on, which may affect a variety of clinical effects such as biological activity, immunogenicity, drug efficacy and safety. The unique quality attribute of an ADC, such as chemical stability and drug-antibody ratio of the conjugate, may affect the efficacy, drug metabolism and safety of the ADC.
Based on the particularity of an ADC preparation, in order to provide a novel and stable ADC preparation, one object of the present invention is to provide a freeze-dried composition containing anti-HER2 drug conjugate.
Another object of the present invention is to provide a freeze-dried preparation containing anti-HER2 drug conjugate and a preparation method thereof.
Further object of the present invention is to provide use of the freeze-dried composition and the freeze-dried preparation.
The freeze-dried composition containing anti-HER2 drug conjugate provided by the present invention comprises ARX788 and one or more of pharmaceutically acceptable lyoprotectant, buffer salt and surfactant, wherein the pH value of the freeze-dried composition is from 5.7 to 6.3.
In some preferred embodiments, the pH value of the freeze-dried composition may be from 6.0 to 6.3, and in some most preferred embodiments, the pH value of the freeze-dried composition may be 6.0.
ARX788 described in the present invention is an anti-HER2 ADC, also known as anti-HER2 monoclonal antibody AS269 conjugate (the structural formula thereof is shown in
The pharmaceutically acceptable lyoprotectant, buffer salt and surfactant described in the present invention may be any type commonly used in pharmaceutical field, especially those commonly used in ADC preparations. In some preferred embodiments, the buffer salt includes but is not limited to acetate, phosphate, citrate, histidine salt, succinate, 2-(N-morpholinyl) ethanesulfonic acid, edetate disodium, etc: the surfactant includes but is not limited to polysorbate 20, polysorbate 80, etc: the lyoprotectant includes but is not limited to sucrose, trehalose, etc.
In some preferred embodiments, the freeze-dried composition described in the present invention comprises 10 mg/mL to 30 mg/mL of ARX788, 5 mM to 10 mM of histidine, 2.5 wt. % to 8 wt. % trehalose and 0.01 wt. % to 0.05 wt. % of polysorbate 80.
In some more preferred embodiments, the freeze-dried composition described in the present invention comprises 20 mg/mL of ARX788, 5 mM of histidine, 6 wt. % trehalose and 0.02 wt. % of polysorbate 80, and the pH of the freeze-dried composition is 6.0.
The present invention also provides a freeze-dried preparation containing anti-HER2 drug conjugate, which is prepared from the freeze-dried composition described in any one of the above technical solutions.
The present invention also provides a preparation method for the freeze-dried preparation, comprising the following steps:
In some preferred embodiments, in the preparation method provided by the present invention, the step S1 comprises: cooling down the freeze-dried composition described in any one of the above technical solutions to 2° C. to 5° C. within 0.5 hour to 1.5 hours and holding at the same temperature for 0.5 hour to 1.5 hours, then cooling down to −50° C. to −40° C. within 3 hours to 5 hours and holding at the same temperature for 3 hours to 6 hours. In some more preferred embodiments, the step S1 comprises: cooling down the freeze-dried composition described in any one of the above technical solutions to 4° C. within 1 hour and holding at the same temperature for 1 hour, then cooling down to −45° C. within 3.5 hours and holding at the same temperature for 5 hours.
In some preferred embodiments, in the preparation method provided by the present invention, the step S2 comprises: heating up the freeze-dried composition after treated in step S1 to −23° C. to −20° C. within 0.2 hour to 0.8 hour and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 35 hours to 45 hours. In some more preferred embodiments, the step S2 comprises: heating up the freeze-dried composition after treated in step S1 to −23° C. to −20° C. within 0.5 hour and holding at the same temperature and a vacuum degree of 13 Pa for 40 hours.
In some preferred embodiments, in the preparation method provided by the present invention, the step S3 comprises: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. within 2 hours to 8 hours and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 5 hours to 10 hours. In some more preferred embodiments, the step S3 comprises: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. within 5 hours and holding at the same temperature and a vacuum degree of 13 Pa for 8 hours.
The present invention also provides use of the freeze-dried composition described in any one of the above technical solutions or the freeze-dried prepatation described in any one of the above technical solutions for the preparation of a medicament for treating a cancer.
In the use of the present invention, the cancer includes but is not limited to breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, urothelial cancer, biliary tract cancer, liver cancer, brain cancer, esophageal cancer, endometrial cancer, uterine cancer, kidney cancer, bladder cancer, thyroid cancer, salivary gland cancer or pancreatic cancer.
The freeze-dried composition and the freeze-dried preparation provided by the present invention are specifically designed for the novel ADC drug ARX788. By setting specific pH value, composition formula and even freeze-drying process, the drop of the small molecule drug and the decline of CEX main peak can be effectively reduced. The resulting freeze-dried product has very excellent stability and can maintain good stability and biological activity for a long time even at high temperatures (such as 40° C.). Moreover, the freeze-dried product also has good appearance, short reconstitution time (within one minute, even up to within 30 seconds) and low moisture content. The product quality is safe and reliable, and the production efficiency is high. The preparation methods of the freeze-dried composition and the freeze-dried preparation provided by the present invention are simple and have low-cost, and they can be used for treating various cancers, and have broad application prospects.
The technical solutions of the present invention will be further described in detail below in combined with specific examples.
The ADC drug ARX788 and ADC reference substance (RS) used in the examples are provided by Zhejiang Novocodex Biopharmaceuticals Co., Ltd. Other reagents or raw materials are commercially available if not particularly indicated.
The test items and test conditions used in the examples are described as follows:
The cell-based potency of the freeze-dried sample relative to RS was tested by using BT-474 cell line (ATCC, article No. HTB-20).
The specific process was as follows: BT-474 cells were cultured to obtain a sufficient quantity with ATCC Hybrid Care Medium (ATCC, article No. 46-X) containing 10% fetal bovine serum (Gibco, article No. 10099141) under the conditions of 37° C. and 5% carbon dioxide. 1.5×104 of BT-474 cells were inoculated into each well in a 96-well cell culture plate (Corning, article No. 3599), with an amount of 80 μL/well. The freeze-dried sample and RS were subjected to gradient dilution from 55.8 μg/mL to 0.2 μg/mL, with a total of 10 concentrations and 2 parallel wells for each dilution. The diluted freeze-dried sample and RS were transferred to the culture plate inoculated with BT-474 cells, with an amount of 10 μL per well, and the plate was cultured under the conditions of 37° C. and 5% carbon dioxide. 10 μL of CCK-8 (Beyotime, article No. C0043) test reagent was added into each well at day 5 for coloration under the conditions of 37° C. and 5% carbon dioxide for 6 hours. The optical density values were read by a microplate reader at 450 nm and 620 nm wavelengths. EC50 of the freeze-dried sample and RS were calculated by using SoftMax Pro software, and the relative cell-based potency of the freeze-dried sample was calculated by using the following formula: relative cell-based potency (%)=(EC50 RS)/(EC50 freeze-dried sample)×100%.
ELISA was used to test the binding potency of the freeze-dried sample relative to RS.
The specific process was as follows: HER2 antigen (R&D, article No. 1129-ER-050) was added into a 96-well plate (Corning, article No. 9018) with an amount of 50 μL/well to be coated overnight at 2° C. to 8° C. Blocking solution (Fisher Scientific, article No. 37528) was added with an amount of 200 μL/well, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. In the 96-well plate, the freeze-dried sample and RS were subjected to gradient dilution from 10 μg/mL to 0.00017 μg/mL by using the blocking solution, with a total of 11 concentrations and 2 parallel wells for each dilution, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. Second antibody (Abcam, article No. ab79115) was added with an amount of 50 μL/well, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. TMB coloring solution (Beyotime, article No. P0209) was added with an amount of 50 μL/well, and the plate was shaken at 200±10 rpm for 10 minutes at room temperature. 5% sulfuric acid was added with an amount of 50 μL/well for termination, and the light absorbance values were read at 450 nm and 620 nm respectively. EC50 of the freeze-dried sample and RS were calculated by using SoftMax Pro software, and the relative binding potency of the freeze-dried sample was calculated by using the following formula: relative binding potency (%)=(EC50 RS)/(EC50 freeze-dried sample)×100%.
Unless otherwise specified, the percentages used in the examples are all mass percentages.
Formulations 1 to 10 with different pH values were designed (see Table 1).
The samples were placed at 25° C. for a total of 2 months investigation. The investigation results are shown in Table 2, wherein TO represents the time point at which the samples began to be placed, 25° C.-1M represents the time point at which the samples were placed for one month, and 25° C.-2M represents the time point at which the samples were placed for two months.
According to the AS269 residue results (Table 2), except for formulation 3 (pH 6.0), formulation 4 (pH 6.3) and formulation 10 (pH 6.0), the growth trend of AS269 residue is observed 5 in all other formulations (pH<6.0) under the investigation at 25° C. The lower the pH is, the higher the AS269 residue is, indicating that the small molecule is prone to drop under the condition that pH is less than 6.0.
According to the CEX results, the decline of CEX main peak as well as the increase of acidic peak and basic peak are more likely to happen under the condition of pH>6.0.
It can be seen that the pH value of the freeze-dried composition is crucial for product stability. The pH value of the freeze-dried composition of the present invention is preferably between 5.7 and 6.3, more preferably around 6.0.
According to the experimental results of Example 1, formulations 11-22 (Table 3) were designed to investigate the effect of different ADC concentrations and different polysorbate 80 concentrations on product stability. After placing the samples under the conditions of 25° C. and shaking at 200 rpm for 1 day and 3 days respectively, samples were taken for testing. The test results (Table 4) show that there are no significant changes in all indicators of the samples of the formulations 11-22 before and after shaking. Therefore, under the pH value of approximately 6.0, with the protein concentration of 5 mg/mL to 30 mg/ml and the polysorbate 80 concentration range of 0.01% to 0.05%, the protein stabilities are all good.
Formulations 23-34 were designed to investigate the effect of different combinations of anti-HER2 ADC concentrations and trehalose concentrations on the freeze-drying parameter (Tg′/Tc). The results of Table 5 show that the freeze-drying parameters of the formulations with different combinations of anti-HER2 ADC concentrations and trehalose concentrations exist differences. Considering the influence of the freeze-drying parameter (Tg′/Tc), freeze-drying filling volume and anti-HER2 ADC concentration on freeze-drying appearance, process time and stability, the concentration of anti-HER2 ADC may be 10 mg/mL to 30 mg/ml and the concentration of trebalose may be 2.5% to 8%. Two formulations (formulation 23 and formulation 24), three formulations (formulation 27, formulation 28 and formulation 29) and one formulation (formulation 31) with anti-HER2 ADC concentration of 10 mg/ml, 20 mg/ml and 30 mg/ml respectively were taken to carry out freeze-drying. According to the results (Table 6), the freeze-dried product with formulation 28 has the best appearance performance.
Formulation 28 of Example 3, that is, 20 mg/mL of anti-HER2 ADC (ARX788), 5 mM of histidine, 6 wt. % of trehalose and 0.02 wt. % of polysorbate 80, was used to investigate the freeze-drying process.
ADC samples and placebo (5 mM of histidine, 6 wt. % of trehalose and 0.02 wt. % of polysorbate 80) were prepared by referring to the sample preparation steps (1)-(4) in Example 1. The results are shown in Table 7. From the results of Table 7, it can be seen that the temperatures, vacuum degrees and other process parameters of primary sublimation and secondary sublimation will affect the appearance, moisture content and other properties of the obtained freeze-dried products. The appearances of the freeze-dried products obtained from process 1′ are severely unqualified, and the moisture content is high, which is not conducive to preparation stability.
Although the appearances of the freeze-dried products obtained from process 1 have been significantly improved, there are still a certain proportion of unqualified products. For processes 2-4, the processes of primary and secondary sublimations are comprehensively considered and optimized, resulting in the freeze-dried products with excellent appearances and low moisture content, thus the yield and product stability of the freeze-dried preparation can be significantly improved.
Three batches of pilot scale production were carried out according to process 4 determined in Example 4 (i.e., pilot scale batch −1 to pilot scale batch −3, with a batch size of approximately 1500 vials). The process was as follows:
Production data shows that all three batches of the production are successfully completed, and the freeze-dried products are white or off-white blocks in appearance. The yield of finished products is high, and there are no defective products like remelting or collapse. All the reconstitution time is less than 1 minute, and all the moisture contents are less than or equal to 1%.
For the sterile powder for injection produced by the method described in this example, the stability of the three batches of samples under long-term (5±3° C.) conditions for 24 months and accelerated (25±2° C.) conditions for 6 months was evaluated by using appearance, pH value, reconstitution time, moisture content, protein content, drug-antibody ratio, SEC, CEX, CE-SDS, AS269 residue, cell-based potency, binding potency, etc. as indicators. The results show that there are no significant changes in all indicators before and after the evaluation.
Meanwhile, one batch of the samples (pilot scale batch −2) was subjected to high-temperature impact factor investigation. The results show that the samples have very good stability, even after a 6-month evaluation at 40° C., the change ranges of CEX peak (
Unless otherwise particularly limited, the terms used in the present invention are commonly used meanings understood by those skilled in the art.
The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes, and improvements within the scope of the present invention. Therefore, the present invention is not limited to the aforementioned embodiments but is only limited by the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/107402 | 7/20/2021 | WO |
