TW202308692A - Pharmaceutical composition of pembrolizumab and use thereof - Google Patents

Abstract

The invention relates to the field of pharmacy and medicine, specifically to pharmaceutical compositions of anti-PD-1 antibody pembrolizumab, which may be aqueous or lyophilized compositions. The invention further relates to the use of said compositions for treating malignant neoplasms or infectious diseases, as well as to the use of said compositions to produce a medicinal product intended for treating malignant neoplasms or infectious diseases.

Description

Pharmaceutical composition containing pembrolizumab (PEMBROLIZUMAB) and its use

The present invention relates to the fields of pharmacy and medicine, in particular to a pharmaceutical composition of anti-PD-1 antibody pembrolizumab, which can be used to treat malignant tumors or infectious diseases.

Programmed cell death protein 1 (PD-1) is an inhibitory member of the CD28 receptor family and is located on the cell surface of T lymphocytes, B cells, monocytes, NK cells and dendritic cells (Jin H.T., Ahmed R., Okazaki T. Role of PD-1 in regulating T-cell immunity. Curr Top Microbiol Immunol. 2011; 350: 17-37). PD-1 is a transmembrane receptor from the immunoglobulin family and consists of 288 amino acids. The protein structure includes extracellular IgV domain, spacer, transmembrane domain and cytoplasmic domain. The latter includes two tyrosine-containing sequences (ITIM and ITSM) involved in signaling in cells (Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev. 2010; 236: 219- 242. doi: 10.1111/j.1600-065X. 2010. 00923. x). PD-1 has two inhibitory ligands, PD-L1 and PD-L2, which are also transmembrane receptors and play an important role in immune homeostasis. PD-L1 is expressed on T and B cells, dendritic cells, macrophages, endothelial cells, hematopoietic cells and epithelial cells. Furthermore, PD-L1 expression has been detected on cells of many types of malignancies such as melanoma, renal cell carcinoma, non-small cell lung cancer, head and neck tumors, gastrointestinal tumors, ovarian cancer, lymphoma, Leukemia (Han Y., Liu D., Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res. 2020; 10(3): 727-742). PD-L2 has limited expression on activated macrophages and dendritic cells and primarily binds to the PD-1 receptor. The main factor that increases the expression of PD-L1 and PD-L2 is the anti-inflammatory cytokine IFNγ. The PD-1 receptor and its PD-L1 ligand play an important role in the survival and progression of malignant tumors. As mentioned above, PD-L1 receptor expression is increased on the surface of many types of malignant cells. The PD-L1/PD-1 interaction stimulates the development of immunosuppression in the tumor microenvironment and thus protects tumor cells from the activity of cytotoxic CD8+ T cells. PD-1/PD-L1 system is a promising therapeutic target (Wu Y., Chen W., Xu Z.P., Gu W. PD-L1 Distribution and Perspective for Cancer Immunotherapy-Blockade, Knockdown, or Inhibition. Front Immunol. 2019 ; 10: 2022, Ju X., Zhang H., Zhou Z., Wang Q. Regulation of PD-L1 expression in cancer and clinical implications in immunotherapy. Am J Cancer Res. 2020; 10(1): 1-11) . The anti-PD-1 antibody pembrolizumab is known, which is a humanized monoclonal G4 (IgG4) antibody to the human PD-1 receptor (PCT/US2008/007463). It was generated by combining the variable sequences of a high-affinity antibody to the murine PD-1 receptor with a human IgG4 κ framework containing a stabilizing S228P mutation in the Fc fragment. It selectively blocks the binding of PD-1 receptor to its ligand and restarts anti-tumor immunity. The initiation of an immune response stimulates the elimination of tumor cells. Pembrolizumab has shown high efficacy in the treatment of various types of malignancies: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, classical Hodgkin lymphoma, urothelial carcinoma, gastric cancer, high microsatellite Unstable malignant tumors, hepatocellular carcinoma, esophageal cancer, cervical cancer, Merkel cell carcinoma, renal cell carcinoma, endometrial cancer, etc. Pembrolizumab is also known for use in the treatment of infectious diseases. Currently, pembrolizumab is being studied in the treatment of other diseases or conditions where inhibition of PD-1 activity may be desired. The prior art provides Keyread, a therapeutic product comprising pembrolizumab, sucrose, polysorbate 80 and histidine buffer (PCT/US2012/031063). Nevertheless, there remains a need for new and improved stable pharmaceutical compositions of pembrolizumab.

Definitions Unless defined otherwise herein, all technical and scientific terms used in connection with the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular. As used in this specification and the appended claims, unless the context dictates otherwise, the words "has", "comprises" and "comprises" or variations thereof such as "has", "having" , "includes", "including", "comprises" or "includes" will be understood to imply the inclusion of stated integers or groups of integers, but not the exclusion of any other integers or overall group. The term "pharmaceutical composition" refers to a composition and/or preparation comprising a therapeutically effective amount of pembrolizumab and excipients or auxiliary substances (carriers, diluents, fillers, solvents, etc.), the selection and ratio of which depends on Type and route of administration and dosage. As used herein, the term "aqueous composition" refers to a water-based composition, and the water in the composition can be: water, water for injection, physiological saline (aqueous solution of 0.9%-1.0% sodium chloride). As used herein, the term "freeze-dried" refers to a formulation that has been subjected to a process known in the art as freeze-drying, which involves freezing the formulation followed by removal of ice from the frozen contents. A pharmaceutical composition is "stable" if the active agent retains its physical and/or chemical stability and/or biological activity at storage temperatures (eg, 2-8°C) during the stated shelf life. Additionally, the active agent can retain both physical and chemical stability, as well as biological activity. Shelf life is adjusted based on the results of stability tests under accelerated or natural aging conditions. The term "long-term storage" or "long-term stability" is understood to mean that the pharmaceutical composition can be stored for three months or more, six months or more, one year or more, and the minimum stable storage period of the composition is also Can be at least two years. In general, the terms "long-term storage" and "long-term stability" further include stable storage durations compared to the stable storage periods typically required for currently available commercial formulations of the anti-PD-1 antibody pembrolizumab, It is at least equivalent or better without loss of stability making the formulation unsuitable for its intended pharmaceutical use. The term "buffer" refers to a buffer or the acid or base component (usually a weak acid or base) of a buffer solution. Buffers help to maintain the pH of a given solution at or near a predetermined value, and are typically selected to supplement the predetermined value. The buffering agent may be a single compound that produces the desired buffering effect, especially when said buffering agent is mixed with an appropriate amount (depending on the desired predetermined value) of its corresponding "acid/base conjugate" (and suitably capable of performing proton exchange). The term "buffer" or "buffer solution" refers to an acid (usually a weak acid, such as acetic acid, citric acid) and its conjugate base (such as acetate or citrate, such as sodium acetate, sodium citrate, and the Hydrates of salts such as sodium acetate trihydrate) or mixtures of bases (usually weak bases such as histidine) and their conjugate acids such as histidine hydrochloride or histidine hydrochloride monohydrate or monohydrate Aqueous solution of hydrated (m/h) mixture of L-histidine hydrochloride (h/c) or L-histidine (h/cm/h or histidine (h/cm/h). The pH of a "buffer solution" changes only slightly when a small amount of strong base or acid is added thereto, and when it is diluted or concentrated due to the "buffering effect" imparted by the "buffering agent". In this application, a "buffer system" comprises one or more buffers and/or one or more acid/base conjugates thereof, and more suitably comprises one or more buffers and one or more acid/base conjugates thereof , and most suitably contain a buffer and its acid/base conjugate. Unless otherwise stated, reference herein to any concentration of a "buffer system" (buffer concentration) may suitably refer to the combined concentration of one or more buffers and/or one or more acid/base conjugates thereof. In other words, reference herein to the concentration of a "buffer system" may refer to the combined concentration of all relevant buffer species (ie, species that are in dynamic equilibrium with each other, eg, citrate/citric acid). The overall pH of a composition comprising the relevant buffer system reflects the equilibrium concentration of each relevant buffer species (ie, the balance of one or more buffers and their one or more acid/base conjugates). The buffer solution can be, for example, acetate, phosphate, citrate, histidine, succinate, and other buffer solutions. Generally, the pH of the pharmaceutical composition is preferably in the range of 4.0-8.0. "Stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and/or chemical stability to the active agent. Stabilizers can be amino acids such as but not limited to arginine, histidine, glycine, lysine, glutamine, proline; surfactants such as but not limited to polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene glycol-polypropylene glycol and its copolymers (trade name: poloxamer, pluronic, dodecane Sodium Hydroxyl Sulfate (SDS); Antioxidants, such as but not limited to Methionine, Acetylcysteine, Ascorbic Acid, Monothioglycerol, Sulfites, etc.; Chelating Agents, such as but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc. As used herein, the terms "osmotic agent" or "tonic agent" and "osmolyte" refer to Excipients that can provide the desired osmotic pressure of the liquid antibody solution. In some embodiments, tonicity modifiers can increase the osmotic pressure of the liquid antibody formulation to an isotonic pressure such that the liquid antibody formulation is compatible with the subject's biological Physiologically compatible with the cells of body tissues. In another embodiment, tonicity modifiers can help increase the stability of the antibody. An "isotonic" formulation is one that has an osmotic pressure equal to that of human blood. The osmotic pressure of an isotonic formulation The pressure is usually about 239-376 mOsm/kg. The tonicity agent can be an enantiomer (such as the L- or D-enantiomer) or a racemic form; an isomeric form, such as α or β, including α,α; or β, β; or α, β; or β, α; free acid or free base form; salt form; hydrated form (such as monohydrate or dihydrate) or anhydrous form. Sugars (trehalose, trehalose dihydrate, sucrose, glucose), polyols (mannitol, sorbitol), amino acids (proline or L-proline, arginine, glycine) or salts ( sodium chloride, potassium chloride, magnesium chloride). As used herein, the term "solubilizer" refers to a pharmaceutically acceptable nonionic surfactant. Both a solubilizer and a combination of solubilizers can be used. Exemplary solubilizers Solvents are, but are not limited to, polysorbate 20 or polysorbate 80, poloxamer 184 or poloxamer 188, or PLURONIC®. Typically, the amino acid is an L-amino acid. For example, if using histamine acid and histidine monohydrate hydrochloride, then it is usually L-histidine acid and L-histidine monohydrate hydrochloride. For example, if proline is used, it is usually L-proline. Amino acid equivalents can also be used, for example, pharmaceutically acceptable salts of proline (e.g., proline hydrochloride). The term "drug" or "preparation" refers to tablets, capsules, solutions, ointments and intended Substances (or mixtures of substances in the form of pharmaceutical compositions) used to restore, improve or change the physiological functions of humans and animals, as well as for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetology and other ready-made forms The term "use" applies to the possibility of using the pharmaceutical composition of pembrolizumab according to the present invention to treat, alleviate the disease process, accelerate the remission of the disease or disease, and reduce the recurrence rate of the disease or disease. The term "treatment method" applies to the possibility of using the pembrolizumab pharmaceutical composition of the present invention to treat, alleviate the disease process, accelerate the remission of the disease or disease, and reduce the recurrence rate of the disease or disease. "Treating" or "treatment", "preventing" a disease, disorder or condition may include preventing or delaying the onset of clinical symptoms of a disease, disorder or condition developing in a human, inhibiting the disease, disorder or condition, That is, halting, reducing or delaying the development of the disease or its recurrence (in the case of maintenance therapy) or at least one of its clinical or subclinical symptoms, or reducing or alleviating the disease, ie causing regression of the disease, disorder or condition. The term "parenteral administration" refers to administration regimens, usually by injection (infusion), and specifically includes intravenous, intramuscular, intraarterial, intratracheal, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, Tracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection or infusion. Abbreviation IC-incoming material control FT-freeze-thaw k _D -diffusion interaction parameter Rh-hydrodynamic radius, SH-vibration T-temperature, Tons-melting onset temperature T1-first melting temperature, T2-second melting Temperature, T agr—aggregation temperature, TS—thermal stress TS50 96H—quality parameter after 96 hours of thermal stress at 50°C Δ TS50 96H—change in quality parameter after stress (96 hours at 50°C) Quality parameter after thermal stress at 50°C for 10 days, ΔTS50 - change in quality parameter after stress (thermal stress at 50°C for 10 days), Δabs - absolute change in the properties of the charged variant determined by method 21, SH800 96H-stirring (vibration) quality parameter after 96 hours, Δ SH800 96H-stress (stirring (vibration) 96 hours) after the change of quality parameter, SH800-stirring (vibration) after 14 days of quality parameter, Δ SH800-stress (stirring (vibration) Changes in quality parameters after 14 days), FT(-20)*quality parameters after 5-5 freezing cycles, FT(-20)*quality parameters after 3-3 freezing cycles, ΔFT (-20)*5-The change of quality parameters after stress (5 freeze cycles), Δ FT(-20)*3-The change of quality parameters after stress (3 freeze cycles), Acid 3.5 1H-acid hydrolysis to pH 3.5 and aging for 1 hour, Acid 4.0 - quality parameter after acid hydrolysis to pH 4.0 and aging for 10 days, Δ Acid 3.5 1H - change in quality parameter after stress (acid hydrolysis to pH 3.5 and aging for 1 hour) , Δ Acid 4.0 - changes in quality parameters after stress (acid hydrolysis to pH 4.0 and aging for 10 days), Basic 8.5 1H - quality parameters after basic hydrolysis to pH 8.5 and aging for 1 hour, Basic 8.0 - basic hydrolysis to pH 8.0 and aging Quality parameters after aging for 10 days, Changes in quality parameters after Δ Basic 8.5 1H-stress (alkaline hydrolysis to pH 8.5 and aging for 1 hour), Changes in quality parameters after Δ Basic 8.0-stress (alkaline hydrolysis to pH 8.0 and aging for 10 days) Change, Ox 0.1%-quality parameter after 4 hours of oxidation with hydrogen peroxide, ΔOx 0.1%-change of quality parameter after stress (4 hours with hydrogen peroxide oxidation), UV-light stress, after dose ICH×1 Quality parameters, Δ UV-stress (light stress, dose ICH×1) changes in quality parameters, n/a-not determined, UV-UV exposure, IE HPLC-ion-exchange high-performance liquid chromatography, CE-capillary electrophoresis, Non-red.-non-reducing condition, SW-software, Red.-reducing condition SA-specific activity (potency), SE HPLC-size exclusion high performance liquid chromatography. The invention discloses a pharmaceutical composition of anti-PD-1 antibody pembrolizumab, which can be used as a medical product for treating malignant tumors or infectious diseases. During formulation selection, we consider the purpose of the drug product, route of administration, and tolerability (eg, to reduce discomfort during administration), as well as the stability and maintenance of activity of the protein molecule within the formulation. In one aspect, the present invention relates to a pharmaceutical composition, wherein the composition comprises: (i) pembrolizumab; (ii) histidine (iii) histidine monohydrate hydrochloride; (iv) glycine; (v) trehalose and poloxamer 188, or proline; and (vi) water for injection. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) pembrolizumab; (ii) histidine (iii) histidine monohydrate hydrochloride; (iv) glycine; (v ) trehalose and poloxamer 188; and (vi) water for injection. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) pembrolizumab; (ii) histidine (iii) histidine monohydrate hydrochloride; (iv) glycine; (v ) proline; and (vi) water for injection. The concentration of pembrolizumab contained in the pharmaceutical composition of the present invention may vary depending on the desired properties of the composition as well as specific conditions, methods and purposes of use of the pharmaceutical composition. In some embodiments of the invention, pembrolizumab is present at a concentration of 5-50 mg/ml. In some embodiments of the invention, histidine is present at a concentration of 0.087-0.432 mg/ml. In some embodiments of the invention, histamine hydrochloride monohydrate is present at a concentration of 0.464-0.931 mg/ml. In some embodiments of the invention, glycine is present at a concentration of 1-2 mg/ml. In some embodiments of the invention, trehalose is present at a concentration of 70-130 mg/ml. In some embodiments of the invention, poloxamer 188 is present at a concentration of 0.8-1.2 mg/ml. In some embodiments of the invention, proline is present at a concentration of 20-34 mg/ml. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 5-50 mg/ml pembrolizumab; (ii) 0.087-0.432 mg/ml histidine; (iii) 0.464-0.931 mg/ml ml histamine hydrochloride monohydrate; (iv) 1-2 mg/ml glycine; (v) 70-130 mg/ml trehalose and 0.8-1.2 mg/ml poloxamer 188, or 20- 34 mg/ml proline; and (vi) water for injection to 1 ml. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 5-50 mg/ml pembrolizumab; (ii) 0.087-0.432 mg/ml histidine; (iii) 0.464-0.931 mg/ml ml histamine hydrochloride monohydrate; (iv) 1-2 mg/ml glycine; (v) 70-130 mg/ml trehalose and 0.8-1.2 mg/ml poloxamer 188; and (vi ) water for injection to 1 ml. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 5-50 mg/ml pembrolizumab; (ii) 0.087-0.432 mg/ml histidine; (iii) 0.464-0.931 mg/ml ml histidine hydrochloride monohydrate; (iv) 1-2 mg/ml glycine; (v) 20-34 mg/ml proline; and (vi) water for injection to 1 ml. In some embodiments of the invention, pembrolizumab is present at a concentration of 15-35 mg/ml, or 20-30 mg/ml, or 25 mg/ml. In some embodiments of the present invention, histidine is 0.200-0.319 mg/ml, or 0.200-0.250 mg/ml, or 0.210-0.240 mg/ml, or 0.210-230 mg/ml, or 0.215-0.230 mg/ml ml, or 0.215-0.225 mg/ml, or 0.220-0.225 mg/ml, or 0.221 mg/ml. In some embodiments of the invention, the histidine is L-histidine. In some embodiments of the present invention, histamine hydrochloride monohydrate is administered at 0.600-0.900 mg/ml, or 0.650-0.850 mg/ml, or 0.700-0.800 mg/ml, or 0.730-0.770 mg/ml, or present at a concentration of 0.750 mg/ml. In some embodiments of the invention, the histamine hydrochloride monohydrate is L-histamine hydrochloride monohydrate. In some embodiments of the invention, glycine is present at a concentration of 1.3-1.7 mg/ml, 1.4-1.6 mg/ml, or 1.5 mg/ml. In some embodiments of the present invention, trehalose is at a concentration of 70-100 mg/ml, or 70-90 mg/ml, or 70-85 mg/ml, or 75-85 mg/ml, or 80 mg/ml exist. In some embodiments of the invention, the trehalose is trehalose dihydrate. In some embodiments of the invention, poloxamer 188 is present at a concentration of 0.9-1.1 mg/ml, or 0.95-1.05 mg/ml, or 1.0 mg/ml. In some embodiments of the invention, proline is present at a concentration of 22-32 mg/ml, or 24-30 mg/ml, or 27 mg/ml. In some embodiments of the invention, the proline is L-proline. In some embodiments of the invention, the pH of the composition is 5.1-6.1, 5.2-6.0, 5.3-5.9, 5.4-5.8, or 5.5-5.7. In some embodiments of the invention, the pH of the composition is 5.6. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine monohydrate hydrochloride; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml poloxamer 188, or 27 mg/ml proline; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine monohydrate hydrochloride; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and the pH of the composition therein 5.5-5.7. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine monohydrate Hydrochloride; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose dihydrate and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and the composition thereof The pH is 5.5-5.7. In some embodiments of the present invention, the pharmaceutical composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine monohydrate hydrochloride; (iv) 1.5 mg/ml glycine; (v) 27 mg/ml proline; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the invention, the pH of the pharmaceutical composition is 5.6. In one aspect, the invention relates to a pharmaceutical composition of pembrolizumab provided in dry (ie powder or granule) form for reconstitution in a suitable solvent (eg water) prior to administration. Such formulations can be prepared, for example, by lyophilization (ie, a process known in the art as freeze-drying), and include freezing the product followed by removal of the solvent from the frozen material. In one aspect, the invention relates to a pharmaceutical composition of pembrolizumab produced by lyophilizing any of the pharmaceutical compositions of pembrolizumab described above. Thus, the pharmaceutical composition according to the invention may be an aqueous pharmaceutical composition or a lyophilized pharmaceutical composition (lyophilizate). The lyophilizates are used to produce other dosage forms. For example, lyophilizates for the production of injectable solutions, lyophilizates for the production of concentrates for the production of injectable solutions. The lyophilizate is reconstituted by dissolving it in a suitable solvent, most typically in water for injection. Alternatively, lyophilized compositions are first reconstituted in the required volume of solvent (most typically, water) and then further diluted in a suitable solvent (eg, 5% dextrose solution, 0.9% sodium chloride solution). In some embodiments of the invention, the pharmaceutical composition of pembrolizumab according to the invention is produced by lyophilizing the pharmaceutical composition of pembrolizumab. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab is produced by lyophilizing the pharmaceutical composition of pembrolizumab, the composition comprising: (i) 5-50 mg/ml pembrolizumab Monoclonal antibody; (ii) 0.087-0.432 mg/ml histidine; (iii) 0.464-0.931 mg/ml histidine hydrochloride monohydrate; (iv) 1-2 mg/ml glycine; (v) 70-130 mg/ml trehalose and 0.8-1.2 mg/ml poloxamer 188, or 20-34 mg/ml proline; and (vi) water for injection to 1 ml. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab is produced by lyophilizing the pharmaceutical composition of pembrolizumab, the composition comprising: (i) 25 mg/ml pembrolizumab (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab is produced by lyophilizing the pharmaceutical composition of pembrolizumab, the composition comprising: (i) 25 mg/ml pembrolizumab (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose dihydrate and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab is produced by lyophilizing the pharmaceutical composition of pembrolizumab, the composition comprising: (i) 25 mg/ml pembrolizumab (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 27 mg/ml proline; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab from which the lyophilized composition is produced has a pH of 5.6. In one aspect, the present invention relates to the use of the above-mentioned pembrolizumab pharmaceutical composition for treating malignant tumors or infectious diseases. In some embodiments, the present invention relates to the use of a pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: (i) 25 mg/ml pembrolizumab; (ii ) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml Poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments, the present invention relates to the use of a pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: (i) 25 mg/ml pembrolizumab; (ii ) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose dihydrate and 1.0 mg /ml poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments, the present invention relates to the use of a pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: (i) 25 mg/ml pembrolizumab; (ii ) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 27 mg/ml proline; (vi) water for injection to 1 ml; and the pH of the composition therein is 5.5-5.7. In some embodiments of the invention, the pH of the pharmaceutical composition of pembrolizumab is 5.6. In some embodiments, the present invention relates to the use of the pharmaceutical composition of pembrolizumab produced by lyophilizing the above pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases. In some embodiments, the present invention relates to the use of the pharmaceutical composition of pembrolizumab produced by freeze-drying the pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: ( i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments, the present invention relates to the use of the pharmaceutical composition of pembrolizumab produced by freeze-drying the pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: ( i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose dihydrate and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments, the present invention relates to the use of the pharmaceutical composition of pembrolizumab produced by freeze-drying the pharmaceutical composition of pembrolizumab for the treatment of malignant tumors or infectious diseases, the composition comprising: ( i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histidine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 27 mg/ml proline; (vi) water for injection to 1 ml; and wherein the pH of the composition is 5.5-5.7. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab from which the lyophilized composition is produced has a pH of 5.6. In one aspect, the present invention relates to the use of the above-mentioned pharmaceutical composition of pembrolizumab for the production of pharmaceutical products for treating malignant tumors or infectious diseases. In some embodiments of the invention, the malignancy is selected from the group consisting of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, high microsatellite Instability/DNA mismatch repair deficient (MMR) malignancies, hepatocellular carcinoma, cervical carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, esophageal carcinoma, squamous cell skin carcinoma, basal cell carcinoma, breast carcinoma Carcinoma, colorectal cancer, prostate cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, pancreatic cancer, ovarian cancer, gallbladder cancer, malignant brain tumors, glioblastoma, with high mutational burden of tumors. Infectious diseases can be caused by viral, bacterial or fungal infections. In some embodiments of the invention, infectious diseases may be caused, for example, by human immunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human papilloma virus, Epstein-Barr virus, human cytomegalovirus, and herpes virus. Many of the diseases can be chronic diseases. In some embodiments of the present invention, the pharmaceutical composition of pembrolizumab of the present invention is intended for parenteral administration. In some embodiments of the present invention, the pharmaceutical composition of pembrolizumab of the present invention is intended for intramuscular, intravenous or subcutaneous administration. In some embodiments of the invention, the pharmaceutical composition of pembrolizumab of the invention may be administered intravenously as an infusion. The pharmaceutical composition of pembrolizumab according to the present invention can be used after dilution. For this, the required volume of the composition is transferred from the vial to an infusion container containing sterile 0.9% sodium chloride solution or sterile 5% dextrose solution. Stir the resulting solution by gently inverting the infusion container. The therapeutically effective amount of the pharmaceutical composition of pembrolizumab according to the invention depends on the condition of the subject, the severity of the condition, previous therapy and the patient's medical history and response to the therapeutic agent. A suitable dosage can be adjusted at the discretion of the attending physician so that it can be administered to the patient in one or by several injections. In some embodiments of the invention, the subject or patient treated is a mammal, preferably a human subject. The subject can be male or female of any age. Pharmaceutical compositions according to the invention may be stored in any suitable container. Examples are glass or plastic containers, vials, ampoules, syringes, cartridges or bottles of desired volume. The container may be provided with additional means for administration, eg, dropper, auto-injector. The pharmaceutical compositions according to the present invention may be manufactured, packaged or widely sold in the form of ready-to-use formulations in the form of a single unit dose or multiple single unit doses. As used herein, the term "single unit dose" refers to a discrete quantity of a pharmaceutical composition containing a predetermined quantity of an active ingredient. The amount of active ingredient is usually equal to the dose of active ingredient to be administered to the subject, or a suitable fraction of such a dose, for example half or a third of such a dose. The pharmaceutical compositions can be administered as a single therapeutic agent or in combination with additional therapeutic agents as needed. Thus, in one embodiment, the present methods for treatment and/or prevention are used in combination with the administration of a therapeutically effective amount of another active agent. The other active agent may be administered before, during or after administration of the pharmaceutical composition according to the invention. Other active agents may be administered as part of the composition or as separate formulations.

，其中 А ₂₈₀是在280 nm波長下的光密度值； ε為測試蛋白質的消光係數； b是樣品的總稀釋因子； l是板孔中的層厚度；對於175 µl，l=0.42 cm。 3. 通過動態光散射確定蛋白質聚集溫度。 使用DynaPro Plate Reader II確定測試蛋白質(濃度為1-5 mg/ml)的聚集點。為此，將35 µl溶液放置在具有光學透明底部的黑色聚合物板的孔中，在儀器中逐漸加熱，同時恆定測量散射光強度。 測量設置： •初始測量溫度-25℃。 •θ=158°處的散射光強度。 •每次重複的測量次數為3。 •每次測量的時間為5秒。 •加熱速率為0.15℃/分鐘。 •最終溫度為80℃。 使用Dynamics V7軟體確定溫度趨勢和聚集點。 4. 通過差示掃描螢光測定法確定蛋白質熔點。 將Sypro Orange螢光染色劑加入到蛋白質樣品中。在CFX96 C1000 Touch Thermal Cycler放大器中以實時模式分析樣品。從25℃加熱至85℃，檢測通道為ROX。使用CFX Manager (Bio-Rad)軟體來處理結果。 5. 通過動態光散射確定溶液中顆粒的流體動力學半徑 為了分析，將35 μl各濃度的樣品放置在具有光學透明底部的黑色聚合物板的孔中。使用DynaPro Plate Reader II儀器進行分析。每個孔分析10次。在Dynamics V7軟體中處理所得資料。 6. 通過動態光散射確定擴散相互作用參數(k _D) 通過逐步稀釋生產30 mg/ml至0.94 mg/ml的許多蛋白質溶液。賦形劑的適當溶液用作溶劑。 為了分析，將35 μl各濃度的樣品放置在具有光學透明底部的黑色聚合物板的孔中。使用DynaPro Plate Reader II儀器進行分析。每個孔分析10次。在Dynamics V7軟體中處理所得資料，其中繪製擴散係數對溶液中蛋白質濃度的依賴性，並確定所得依賴性的線傾角。 7. 確定在50℃熱應力(TS50)下的熱穩定性。 將測試樣品分成2個等分試樣，每個150 μl，並放置在單獨的玻璃小瓶中：將每種組成物1個小瓶儲存在5±3℃的冰箱中，將其餘小瓶放置在恆溫器中並在50℃下孵育96小時。當選擇對照點或隨後的加熱時，從恆溫器中去除小瓶，在室溫下保持約15分鐘並轉移用於分析。 8. 確定在振動(SH800)下的膠體穩定性。 將測試樣品分成2個等分試樣，每個150 μl，並放置在玻璃小瓶中，將每種製劑1個小瓶儲存在5±3℃的冰箱中，將其餘小瓶放置在熱振動器中並在800 rpm下在5±3℃下振動96小時。在選擇對照點或隨後的應力期間，從熱振動器中去除小瓶並轉移用於分析。 9. 確定在凍融(FT(-20))下的膠體穩定性。 將測試樣品分成2個等分試樣並放置在塑膠小瓶中：將每種製劑1個小瓶儲存在5±3℃的冰箱中，將其餘小瓶儲存在不高於-18℃的冷凍機中，直到完全冷凍。此後，從冷凍機中去除小瓶，保持在室溫下直到內容物完全解凍；使用渦旋混合溶液並放回到冷凍機中。重複至少三次。應力後，從冷凍機中去除小瓶，保持在室溫下直到內容物完全解凍；使用渦旋混合溶液並轉移用於分析。 10. 確定在酸水解(Acid)下的穩定性。 將測試樣品分成2個等分試樣並放置在聚合物小瓶中：將每種製劑1個小瓶儲存在5±3℃的冰箱中(對於所有研究，在儲存開始時，輸入對照可以轉移用於分析一次)，攪拌下用鹽酸溶液將其餘小瓶的pH調節至3.5±0.1，此後，將它們轉移至冰箱中用於在5±3℃下儲存。1小時後，通過加入氫氧化鈉溶液至初始pH值，在攪拌下猝滅水解。隨後轉移溶液用於分析。 11. 確定在鹼性水解(Basic)下的穩定性。 將測試樣品分成2個等分試樣並放置在聚合物小瓶中：將每種製劑1個小瓶儲存在5±3℃的冰箱中(對於所有研究，在儲存開始時，輸入對照可以轉移用於分析一次)，攪拌下用氫氧化鈉溶液將其餘小瓶的pH調節至8.5±0.1，此後，將它們轉移至冰箱中用於在5±3℃下儲存。1小時後，通過加入鹽酸溶液至初始pH值，在攪拌下猝滅水解。隨後轉移溶液用於分析。 12. 加速老化。 將測試樣品分成單獨的等分試樣(一個等分試樣用於輸入對照，對於所有研究，在儲存開始時，允許轉移用於分析一次)並放置在單獨的無菌玻璃小瓶中：將每種製劑的小瓶的一部分放置在冰箱中用於在5±3℃下儲存(輸入對照)，將其餘小瓶放置在恆溫器中並在25±2℃下孵育6個月，根據計畫定期選擇對照點。當選擇對照點和隨後的儲存時，從恆溫器中去除小瓶並轉移用於分析。 13. 通過尺寸排阻高效液相色譜法(SE HPLC)確定樣品純度。 柱：Tosoh TSK-GelG3000SWXL 7.8 mm ID×30 cm，5 μm。 預柱：TSK-Gel Guard SW _XL，6.0 mm ID×4.0 cm，7 μm，300Å。 柱溫：25℃。 流動相流速：0.5 ml/min。 注射體積：25 μl。 樣品濃度：0.5 mg/ml。 檢測器波長：214和360 nm。 洗脫時間：30分鐘。 流動相：無水磷酸氫二鈉14.2 mg/ml。氯化鈉11.7 mg/ml。 用正磷酸將流動相pH調節至6.9。 14. 在Caliper LabChip GX II儀器上評價毛細管中的帶電荷的變體特性 根據HT蛋白質電荷變體試劑盒的說明書進行分析。通過在0.5 ml Amicon Ultra 10 kDa離心篩檢程式(Millipore)中稀釋或濃縮(取決於樣品的初始濃度)將測試樣品調節至1 mg/ml的蛋白質濃度。在280 nm波長下通過UV分光光度法確定蛋白質含量。 將2 µl羧肽酶溶液加入到每個所得樣品中，並將樣品在37±2℃的溫度下孵育2小時。在規定時間後，將樣品在Amicon Ultra離心管中對水透析，並濃縮至2 mg/ml。 96孔板負載如在說明書中規定量的標記緩衝溶液、染料混合物溶液和25 μl測試樣品，將板放置在暗處10分鐘，隨後每個孔負載60 µl水並混合。 使用板離心機轉子離心具有溶液的板，並放置在Caliper LabChip GX II儀器中。分析使用特殊晶片，該晶片充滿根據說明書的pH的運行緩衝溶液。用LabChip GX軟體處理結果。 15. 通過離子交換高效液相色譜法(iE HPLC)確定電荷變體特性。 柱：ProPac WCX-10，4×250 mm，細微性：10 μm (Thermo Scientific, USA)。 預柱：ProPac WCX-10G，4×50 mm，細微性：10 μm (Thermo Scientific, USA)。 洗脫液A：20 mM 2-(N-嗎啉代)-乙磺酸，4%乙腈的溶液，pH=7.0。 洗脫液B：20 mM磷酸鈉緩衝液，95 mM NaCl溶液，4%乙腈的溶液，pH=8.0。 流速：0.6 ml/min-1.0 ml/min。 柱溫：45℃。 自動取樣器溫度：5℃。 檢測器：UV，280 nm。 參考波長：360 nm，100 nm頻寬 樣品體積：80 µl。 色譜時間：43分鐘。 將測試樣品稀釋至濃度為1.0 mg/ml，並在37±2℃的溫度下用羧肽酶B以100:1的比率處理20分鐘。 洗脫模式：洗脫液A為從100%至80%，洗脫液B為從0%至20%。 16. 在十二烷基硫酸鈉存在/不存在下通過毛細管聚丙烯醯胺凝膠電泳(CE red.和non-red.)確定純度和相關的雜質。 將樣品稀釋至4.0 mg/ml的濃度。將23 μL所得溶液放置在1.5 mL微管中；向其中加入70 μl SDS-MW樣品緩衝液、2 μl分子量為10 kDa的內標、5 μl 0.5M碘乙醯胺溶液(CE non-red.)或5 μl 2-巰基乙醇(CE red.)。將所得溶液攪拌15秒，以2800 rpm的速度離心5秒，並放置在70℃的固態恆溫器中30分鐘。將溶液冷卻至室溫。 SDS MW分離-PA 800 plus.met分析方法用於32Karat軟體。 毛細管凝膠電泳的條件： 毛細管：50 μm×30.2 cm 毛細管的有效長度：20.0 cm 極性：相反，進口在左側(-)，出口在右側(+) 毛細管溫度：25℃ 分析時間和分離電壓：35分鐘，15 kV 檢測波長：220 nm。 17. 確定相對比活性。 使用生物學測試確定比活性，該測試是針對阻斷PD-L1依賴性抑制基於T細胞的Jurkat-PD1-NFAT報導細胞系的活化的能力。使用機器人平臺液體處理臂(LiHa)處理樣品；使用具有25 mM HEPES、24 mM碳酸氫鈉、含有2 mM L-穀胺醯胺、10% FBS、50 μg/ml慶大黴素的RPMI-1640作為測定培養基(用於定量確定的培養基)。 使用測定培養基(用於定量確定的培養基)將抗體的測試樣品稀釋至1 mg/ml的濃度，並且放置在機器人平臺中。機器人平臺液體操作臂(LiHa)用於使用測定培養基製備濃度為1 000、50、10、1、0.5、0.25、0.1、0.025、0.01、0.001 μg/ml的標準樣品和測試樣品的三種獨立稀釋液。我們將稀釋液和測定培養基轉移到培養板中，加入以(1.00±0.1)×10 ⁶個細胞/ml的濃度穩定表達PDL-1的Raji-PDL1 cl.3細胞懸浮液、以(1.67±0.1)×10 ⁶個細胞/ml的濃度的Jurkat-NFAT-PD1 cl.1報導細胞系懸浮液和抗CD3/CD20的雙特異性抗體。將培養板放置在CO ₂培養箱中，在(37±1)℃的溫度下，在具有5%二氧化碳含量的加濕空氣中培養22-24小時。 孵育期之後，將培養板在室溫下保持至少15分鐘，並加入BioGlo螢光素酶底物。使用微板讀數器和Magellan 7.2軟體，以相對發光單位(RLU)測量發光水準。使用發光測量的結果，我們為每個板建立了使用Levenberg-Marquardt演算法對標準樣品和測試樣品優化的四參數曲線，確定測試樣品相對於標準樣品的相對比活性。 18. 通過疏水相互作用的高效液相色譜(疏水相互作用HPLC)確定氧化產物。 柱：TSKgel Phenyl-5PW 7.5×75 mm，細微性：10μm (Tosoh Booscience, Japan)。 洗脫液A：5 mM磷酸鈉緩衝液，2%乙腈的溶液，pH=7.0。 洗脫液B：5 mM磷酸鈉緩衝液，400 mM硫酸銨，4%乙腈的溶液，pH=6.9。 流速：0.5 ml/min。 柱溫：30℃。 自動取樣器溫度：5℃。 檢測器：UV，280 nm，頻寬：16 nm。 參考波長：360 nm，100 nm頻寬 樣品體積：25μL。 色譜時間：82分鐘。 將測試樣品稀釋至濃度為3.0 mg/ml，並在37±2℃的溫度下用羧肽酶B以100:1的比率處理20分鐘。 洗脫方式：洗脫液A為0%-100%，洗脫液B為100%-0%。 19. 確定在氧化下的穩定性。 將測試樣品分成2個等分試樣，每個150 μl，並放置在單獨的玻璃小瓶中：將每種製劑1個小瓶儲存在5±3℃的冰箱中，將過氧化氫加入到剩餘的樣品中至樣品中過氧化氫的最終濃度為0.1%，將樣品在(5±3)℃下老化4小時。通過加入等量的L-甲硫胺酸來猝滅氧化。 20. 確定光穩定性。 將測試樣品分成兩個等分試樣，並放置在單獨的玻璃小瓶中。作為深色對照，我們在第二個包裝中使用用鋁箔緊密包裹的產品。將所有樣品放置在具有光源的氣候室中，並在1,200,000 lux•h和200 W•h/m ²(劑量ICH×1)下發射光應力程式。在達到期望的應力水準後，將所有樣品從室中去除並轉移用於分析。 21. 結果的處理。 通過下式計算當在應力下時品質指標的絕對變化： Δ=(應力後的值-應力前的值) 通過下式計算電荷變體特性的絕對變化(abs)： Δ = | 應力前酸性級分含量 - 應力後酸性級分含量 | +| 應力前鹼性級分含量 - 應力後鹼性級分含量 | +| 應力前主要級分含量 - 應力後主要級分含量 |實施例1. 緩衝系統的選擇。 在本研究中，選擇2種適合腸胃外給藥的典型的緩衝體系(乙酸鹽和組胺酸緩衝體系)作為藥學組成物的基礎。在具有兩個水準和中心點的完全雙因素實驗設計中進行研究。研究pH水準(5.0-6.5)和緩沖劑的濃度(5-50 mM)作為定量因素。 為了評估緩衝系統的適用性，我們研究了緩衝溶液的性質對蛋白質的膠體和構象穩定性的作用。確定聚集溫度、熔點、擴散相互作用參數、熱應力後的純度變化和酸-鹼特性作為回應。測試藥學組成物示於表1中。

穩定性預測指標的研究。 擴散相互作用參數(k _D)反映作為分子濃度函數的樣品擴散係數。如果擴散係數隨濃度的增加而降低(k _D＜0)，則給定溶液的多分散性增加，並且在其中形成較大的顆粒。這樣的樣品具有低溶解度並且傾向於聚集，並且不推薦使用其製劑。 聚集溫度和熔點使得可以評估蛋白質的聚集趨勢。最穩定的樣品是其中顆粒聚集在較高溫度下開始並且其中在加熱下形成較小顆粒那些。 通過方法3的聚集溫度、通過方法4的熔點和通過方法6的擴散相互作用參數的研究結果示於表2中。最佳結果的顏色色調較淺。

確定熱穩定性。 通過方法7評估熱穩定性。在熱應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法5確定流體動力學半徑。結果示於表3中。最佳結果的顏色色調較淺。

在Minitab軟體中處理所得資料，並建立模型以選擇具有最穩定化作用的製劑。只有那些受到因素(pH和緩衝溶液的品質摩爾濃度)的顯著作用的反應(品質指標)用於建立模型。對於優化模型中沒有考慮的其它反應，假定在研究的因素範圍內沒有統計學顯著差異。基於優化結果，選擇pH為5.6的5 mM組胺酸緩衝溶液，其用以下製備： 　　L-組胺酸 0.221 mg/ml 　　一水合L-組胺酸鹽酸鹽 0.750 mg/ml 當製備具有特定含量的L-組胺酸和一水合L-組胺酸鹽酸鹽的溶液時，pH可能稍微偏離期望值，並且在5.5-5.7 (pH 5.6±0.1)的pH範圍內變化。 該製劑在所有測試樣品中顯示最佳的穩定化性質。根據模型的結果，在熱暴露下的組胺酸緩衝溶液中預期單體(蛋白質)和聚集體含量的最小變化，並且還預期聚集溫度和擴散相互作用參數的高值。與可瑞達緩衝溶液相比，最佳的組成表現出在熱暴露下單體含量的較小變化，聚集物含量的較小變化，並且擴散相互作用參數增加，這指示膠體穩定性較高。 實施例2. 滲透劑的選擇。 測試製劑。 研究了適合腸胃外給藥的賦形劑用作滲透劑。測試製劑示於表4中。

穩定性預測指標的研究。 對於滲透劑的選擇，通過方法3的聚集溫度、通過方法4的熔點和通過方法6的擴散相互作用參數的研究結果示於表5中。最佳結果的顏色色調較淺。

確定熱穩定性。 通過方法7評估熱穩定性。在熱應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表6中。最佳結果的顏色色調較淺。

確定在振動下的穩定性。 通過方法8評估在振動下的穩定性。在振動之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表7中。最佳結果的顏色色調較淺。

確定在冷凍和融化期間的穩定性。 通過方法9評估在凍融下的穩定性。在應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表8中。最佳結果的顏色色調較淺。

包含二水合海藻糖和L-脯胺酸的製劑在所有測試樣品中顯示最佳的穩定化性質。包含二水合海藻糖的樣品在聚集溫度和熔點方面顯示最佳結果。此外，該樣品在熱暴露下顯示品質指標的最小變化，觀察到在振動下的微小變化。包含L-脯胺酸的樣品也顯示聚集溫度的最佳結果之一和熔點的平均結果。具有L-脯胺酸的組成物在熱暴露下顯示平均結果，在振動下品質指標的小變化，和在凍融下的最佳結果。 引入為下一步驟選擇的二水合海藻糖或L-脯胺酸減少在凍融和熱應力下聚集體的形成。而且，所選製劑在振動下表現出輕微的品質變化。 當與含有蔗糖的製劑相比時，所選的滲透劑提供增加的熱穩定性；特別地，我們觀察到熔點/聚集溫度升高，在熱暴露下聚集體的形成速率較低，單體含量的較小變化，並且我們還觀察到酸-鹼特性的較小的絕對變化。在賦形劑中含有二水合海藻糖的樣品在擴散相互作用參數方面顯示更好的結果，這指示在濃縮和滲濾期間增加的穩定性和更小的聚集傾向。與含有蔗糖的製劑相比，含有二水合海藻糖、山梨醇或L-脯胺酸的所研究的製劑在振動和冷凍下顯示明顯更小的顆粒流體動力學半徑增加，這指示形成了大的高分子顆粒。 為下一步驟選擇的賦形劑製劑： His+Tre 100 mg/ml: L-組胺酸 0.221 mg/ml    一水合L-組胺酸鹽酸鹽 0.750 mg/ml    二水合海藻糖 100 mg/ml          His+Prol 30 mg/ml: L-組胺酸 0.221 mg/ml    一水合L-組胺酸鹽酸鹽 0.750 mg/ml    L-脯胺酸 30 mg/ml 實施例3. 滲透劑和穩定劑的篩選。 為了篩選滲透劑和穩定劑，使用適於腸胃外給藥的賦形劑。測試製劑示於表9中。根據方法2製備在測試製劑中含有帕博利珠單抗的藥學組成物。

穩定性預測指標的研究。 對於滲透劑的選擇，通過方法3的聚集溫度、通過方法4的熔點和通過方法6的擴散相互作用參數的研究結果示於表10中。最佳結果的顏色色調較淺。

確定熱穩定性。 通過方法7評估熱穩定性。在熱應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表11中。最佳結果的顏色色調較淺。

確定在酸水解下的穩定性。 對於不含泊洛沙姆188的製劑，通過方法10 (其中調節至pH 3.5並老化1小時)評估在酸水解下的穩定性。在水解之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表12中。最佳結果的顏色色調較淺。

確定在鹼水解下的穩定性。 對於不含泊洛沙姆188的製劑，通過方法11 (其中調節至pH 8.5並老化1小時)評估在鹼水解下的穩定性。在水解之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表13中。最佳結果的顏色色調較淺。

確定在振動下的穩定性。 通過方法8評估在振動下的穩定性。在應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表14中。最佳結果的顏色色調較淺。

確定在冷凍和融化期間的穩定性。 通過方法9評估在凍融下的穩定性。在應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表15中。最佳結果的顏色色調較淺。

在測試樣品中，以下製劑在穩定性方面可能是卓越的： 1. His+Tre+甘胺酸100 mM 帕博利珠單抗 1.5-30 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml 二水合海藻糖 100 mg/ml 甘胺酸 7.51 mg/ml          2. His+Tre+泊洛沙姆188 0.5 mg/ml 帕博利珠單抗 1.5-30 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml 二水合海藻糖 100 mg/ml 泊洛沙姆188 0.5 mg/ml          3. His+Prol 帕博利珠單抗 1.5-30 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml L-脯胺酸 30 mg/ml          4. His+Prol+甘胺酸100 mM 帕博利珠單抗 1.5-30 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml L-脯胺酸 30 mg/ml 甘胺酸 7.51 mg/ml          5. His+Prol+甘胺酸100 mM+ 泊洛沙姆188 0.5 mg/ml 帕博利珠單抗 1.5-30 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml L-脯胺酸 30 mg/ml 甘胺酸 7.51 mg/ml 泊洛沙姆188 0.5 mg/ml 選擇含有二水合海藻糖的製劑、二水合海藻糖與甘胺酸的組合的組成物、以及具有L-脯胺酸的組成物、以及具有L-脯胺酸與甘胺酸或甲硫胺酸的組合的組成物用於進一步開發，因為它們在應力下顯示積極穩定性結果。 與具有L-脯胺酸的製劑相比，具有二水合海藻糖的所有制劑表現出更高含量的鹼性級分。在含有二水合海藻糖的製劑中，具有甘胺酸的製劑顯示最佳結果。我們觀察到甘胺酸對聚集溫度和熔點以及對擴散相互作用參數的積極影響。當在熱應力下時，向製劑中加入甘胺酸顯示輕微的改進。向製劑中加入甘胺酸顯示在應力之前和之後純度和酸-鹼特性的改進。與沒有加入表面活性劑的可瑞達賦形劑的製劑中的樣品相比，我們觀察到熔點和聚集溫度的顯著增加，以及在酸和鹼水解下純度和酸-鹼特性的顯著較小的變化。 在含有表面活性劑的製劑中，觀察到泊洛沙姆188的積極作用。這些組成物在振動和冷凍下在純度和帶電荷的變體特性方面表現出較小的變化。因此，選擇泊洛沙姆188用作下一步驟中的表面活性劑。 實施例4. 確定賦形劑製劑的關鍵定量因素。 在具有兩個水準和中心點的階乘四因數實驗設計中進行研究。蛋白質濃度(10-40 mg/ml)、pH (5.1-6.1)、滲透劑濃度(70-130 mg/ml)、L-甘胺酸濃度(1.5-15 mg/ml)和泊洛沙姆188濃度(0.10-1.0 mg/ml)作為定量因素進行研究。測試製劑列於表16中。

在表16中，緩衝溶液是指在表17中描述的以下製劑。

穩定性預測指標的研究。 對於滲透劑的選擇，通過方法3的聚集溫度、通過方法4的熔點和通過方法6的擴散相互作用參數的研究結果示於表18中。最佳結果的顏色色調較淺。

確定熱穩定性。 通過方法7評估熱穩定性。在熱應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表19中。最佳結果的顏色色調較淺。

通過方法8評估在振動下的穩定性。在應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表20中。最佳結果的顏色色調較淺。

確定在冷凍和融化期間的穩定性。 通過方法9評估在凍融下的穩定性。在應力之前和之後，我們通過方法13通過SE HPLC確定純度，通過方法14在毛細管中確定電荷變體特性，通過方法6確定流體動力學半徑。結果示於表21中。最佳結果的顏色色調較淺。

結果用於在Minitab軟體中建立模型。L-甘胺酸/泊洛沙姆188的濃度對帕博利珠單抗的穩定性沒有統計學顯著作用。根據優化結果，L-甘胺酸的推薦濃度為15 mg/ml，泊洛沙姆188的推薦濃度為1.0 mg/ml，二水合海藻糖的推薦濃度為84.5 mg/ml。然而，為了確保組成物的生理同滲品質摩爾濃度，將L-甘胺酸的含量降低到1.5 mg/ml，並且將二水合海藻糖的含量降低到80 mg/ml。較早選擇緩衝溶液、其濃度和pH，結果如實施例1所示。本實施例證實它們對帕博利珠單抗的穩定性的統計學顯著作用。在熱應力下，表面活性劑可能引起人工高分子量雜質的形成。如果熱應力不包括在模型中，可以推斷泊洛沙姆188濃度的增加對帕博利珠單抗的穩定性具有積極作用。根據結果，泊洛沙姆188的最佳濃度為1.0 mg/ml。 因此，最終選擇的製劑如下： 帕博利珠單抗 1.5-40 mg/ml L-組胺酸 0.221 mg/ml 一水合L-組胺酸鹽酸鹽 0.750 mg/ml 二水合海藻糖 80 mg/ml 甘胺酸 1.5 mg/ml 泊洛沙姆188 1.0 mg/ml 實施例5. 確定在應力條件下最終製劑的穩定性 對於應力穩定性測試，選擇帕博利珠單抗的最終製劑和可瑞達賦形劑的製劑。測試製劑示於表22中。根據方法2製備在測試製劑中含有帕博利珠單抗的藥學組成物。

確定熱穩定性。 通過方法7評估熱穩定性持續10天。在熱應力之前和之後，我們通過SE HPLC方法(方法13)確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法5確定流體動力學半徑。結果示於表23中。最佳結果的顏色色調較淺。

確定在振動下的穩定性。 通過方法8評估在振動下的穩定性。在振動之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表24中。最佳結果的顏色色調較淺。

確定在冷凍和融化期間的穩定性。 通過方法9評估在凍融下的穩定性。在應力之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表25中。最佳結果的顏色色調較淺。

確定在酸水解下的穩定性。 通過方法9評估在酸水解下的穩定性。在應力之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表26中。最佳結果的顏色色調較淺。

確定在鹼水解下的穩定性。 通過方法9評估在鹼水解下的穩定性。在應力之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表27中。最佳結果的顏色色調較淺。

確定在氧化下的穩定性。 通過方法9評估在氧化下的穩定性。在應力之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表28中。最佳結果的顏色色調較淺。

確定光穩定性。 通過方法9評估在氧化下的穩定性。在應力之前和之後，我們通過SE HPLC方法13確定純度，通過IE HPLC方法(方法15)確定帶電荷的變體特性，通過疏水相互作用HPLC方法(方法18)確定氧化產物的含量，通過方法6確定流體動力學半徑。結果示於表29中。最佳結果的顏色色調較淺。

根據應力穩定性測試的結果，與可瑞達的賦形劑製劑相比，帕博利珠單抗的賦形劑製劑在熱應力下表現出品質指標的較小變化，特別是流體動力學半徑的較小變化、聚集體的較小增加和單體含量的變化、形成氧化形式的顯著較低的趨勢和帶電荷的變體特性的較小變化。 當在振動和冷凍下時，觀察到應力後帶電荷的變體特性的較小變化。 進而，當在酸水解下時，觀察到聚集體的較小增加和單體含量的變化，以及鹼性級分含量的較小變化。當在鹼性水解下時，帕博利珠單抗製劑內的蛋白質形成聚集體、改變單體含量和改變帶電荷的變體特性的傾向較小。 當在光應力下時，與可瑞達賦形劑的製劑相比，觀察到明顯較低的氧化傾向，以及單體含量的較小變化、聚集體的較小增加和帶電荷的變體特性的較小變化。 實施例6. 確定在37±2℃的溫度下加速老化下的穩定性。 為了預先證實所選製劑的穩定性，在37±2℃的溫度下使其經受加速老化。 通過方法1製備蛋白質濃度(25-50 mg/ml)、pH (5.1-6.1)、二水合海藻糖濃度(70-90 mg/ml)、甘胺酸濃度(1.0-2.0 mg/ml)和泊洛沙姆188濃度(0.8-1.2 mg/ml)範圍內的藥學組成物，並在37±2℃的溫度下儲存用於穩定性測試。 測試製劑示於表30中。

加速老化。 根據技術1通過滲濾製備包含濃度為25、50和37.5 mg/ml的蛋白質的藥學組成物，並根據技術8放置在37±2℃的溫度下用於加速儲存。研究結果示於表31中。

所有測試製劑均表現出可接受的品質參數變化(通過SE HPLC確定的純度，在還原和非還原條件下的CE，帶電荷的變體特性和比活性)，這通過使用MODDE軟體的設計空間證實。根據反映因素(緩衝溶液pH和賦形劑組分含量)對回應(品質參數)的作用的實驗空間的結果，在37±2℃下加速老化4周，藥學組成物在提供的濃度、pH和賦形劑範圍內是穩定的。 實施例7. 確定在長期加速老化下的穩定性。 為了證實所選製劑的穩定性，在25±2℃的溫度下使其經受加速老化。 通過方法1製備蛋白質濃度(25-50 mg/ml)、pH (5.1-6.1)、二水合海藻糖濃度(70-90 mg/ml)、甘胺酸濃度(1.0-2.0 mg/ml)和泊洛沙姆188濃度(0.8-1.2 mg/ml)範圍內的藥學組成物，並在25±2℃的溫度下儲存用於穩定性測試。此外，我們在所有組成物中選擇最關鍵的情況，具有最低和最高賦形劑含量和最高蛋白質含量，以及具有賦形劑含量在該範圍的中心點的製劑。 測試製劑示於表32中。

加速老化。 根據技術1通過滲濾製備包含濃度為25、50和37.5 mg/ml的蛋白質的藥學組成物，並根據技術8放置在25±2℃的溫度下用於加速儲存。研究結果示於表33和圖1、2、3、4、5和6中。

在加速儲存期間所有藥學組成物證明可接受水準的變化。 含有pH範圍為5.1-6.1的組胺酸緩衝溶液、70-90 mg/ml的二水合海藻糖、1.0-2.0 mg/ml的L-甘胺酸和0.8-1.2 mg/ml的泊洛沙姆188的藥學組成物證明可接受水準的聚集(在25±2℃下6個月內聚集體的增加不超過0.32%)，以及在25 mg/ml濃度和至多50 mg/ml的增加的濃度的單克隆抗體帕博利珠單抗下酸-鹼特性的小變化(鹼性級分含量變化不超過13.0%)和在加速老化下比活性的輕微變化。 加入二水合海藻糖有助於增加帕博利珠單抗的聚集溫度和熔點。我們還觀察到在熱應力下品質參數的最小變化，在振動和冷凍後品質參數的輕微變化。此外，甘胺酸對聚集溫度和熔點以及對擴散相互作用參數具有積極影響。觀察到在酸和鹼水解下甘胺酸對蛋白質品質參數的積極影響。向製劑中加入泊洛沙姆188有助於測試蛋白質在振動和冷凍下的穩定化。 根據應力穩定性測試的結果，帕博利珠單抗賦形劑的開發製劑在熱應力、酸和鹼水解下顯示優於可瑞達製劑的顯著優點；此外，我們觀察到在光應力下顯著更低的氧化傾向，其對蛋白質的結構和比活性具有顯著作用，並且具有增加的聚集傾向。 我們提供了使用帕博利珠單抗的水性藥學組成物的研究的實例。用於進一步研究的水性藥學組成物描述於表34中。

實驗揭示，可瑞達產品的製劑具有以下幾個缺點：1)抗體的膠體穩定性不足，2)在熱應力下穩定性不足，3)在機械應力和冷凍下帶電荷的變體特性的穩定性低。 在本發明的框架內，已經生產了具有足夠的膠體穩定性和熱穩定性以及在機械應力和冷凍下帶電荷的變體特性的高穩定性的藥學組成物。此外，在其製劑(例如表34中指示的製劑)中含有海藻糖和泊洛沙姆188的藥學組成物的進一步特徵在於在長期儲存下形成高分子量雜質的低傾向，以及在光應力和長期儲存下氧化、比活性損失和雜質增加的低傾向。 與藥學組成物的穩定性有關的所得資料指示賦形劑彼此之間以及與活性物質之間的相容性。 Method 1. Prepare pembrolizumab samples. Antibody samples were prepared under stress in a stirred chamber (Millipore) at a concentration of 5-50 mg/ml. To do this, the initial antibody preparation is placed in a chamber, the protein is concentrated to the desired concentration in a stream of compressed air under continuous agitation, and at least 10 volumes of a protein containing buffer, osmotic agent, and (if applicable) is gradually added to the chamber. required) an aqueous solution of the subject formulation of an additional water-soluble stabilizer. After antibody diafiltration, we proceed to concentrate to a concentration above the target concentration, unload it from the chamber, and measure the precise protein concentration by UV spectroscopy. Concentrates of poloxamer 188 and appropriate solutions of excipients are then added to the samples to prepare solutions with target concentrations of protein. Protein samples at concentrations of 20 mg/ml or higher were prepared in Pellicon cassettes (Millipore) in tangential flow mode. To do this, the initial antibody formulation is placed in a diafiltration tank, the protein is concentrated to the desired concentration, and then at least 10 volumes of the solution with the target formulation containing buffer and, if required, additional water-soluble stabilizers is supplied to the system. After diafiltration, we proceed to concentrate to a concentration above the target concentration, unload it from the system, and measure the precise protein concentration. Concentrates of appropriate solutions of poloxamer 188 and excipients are then added to the samples to prepare solutions with target concentrations of protein. When a formulation containing a solubilizing agent (eg Poloxamer 188) is obtained, the surfactant concentrate is added to the antibody after diafiltration and concentration, where the antibody is finally diluted to the target concentration with a solution of excipient. During aseptic filling into final containers (e.g., sterile glass/plastic containers, vials, or syringes), antibody solutions are filtered through a 0.22 µm sterile membrane. 2. Determination of the protein concentration in the test sample by UV spectroscopy, measuring the protein concentration in a UV transparent plate at a wavelength of 280 nm. Each sample was diluted to a concentration of approximately 0.5 mg/ml with an appropriate solution of excipient. Place 150 µl of the diluted sample into the wells of the UV spectrophotometer plate. Measure the optical density of the solution in the plate wells using a plate spectrophotometer at a wavelength of 280 nm. Appropriate solutions of excipients were used as reference solutions. Calculate the protein concentration (C) (mg/ml) using the following formula:

, where А ₂₈₀ is the optical density value at a wavelength of 280 nm; ε is the extinction coefficient of the test protein; b is the total dilution factor of the sample; l is the layer thickness in the plate well; for 175 µl, l=0.42 cm. 3. Determine protein aggregation temperature by dynamic light scattering. Aggregation points of test proteins (concentration 1-5 mg/ml) were determined using DynaPro Plate Reader II. For this, 35 µl of the solution are placed in the wells of a black polymer plate with an optically transparent bottom and gradually heated in the instrument while the scattered light intensity is measured constantly. Measurement settings: • Initial measurement temperature -25°C. • Scattered light intensity at θ=158°. • The number of measurements per repetition is 3. • The time for each measurement is 5 seconds. • The heating rate is 0.15°C/min. • The final temperature is 80°C. Temperature trends and accumulation points were determined using Dynamics V7 software. 4. Determine the protein melting point by differential scanning fluorometry. Sypro Orange fluorescent stain was added to protein samples. Samples were analyzed in real-time mode in a CFX96 C1000 Touch Thermal Cycler amplifier. Heating from 25°C to 85°C, the detection channel is ROX. Results were processed using CFX Manager (Bio-Rad) software. 5. Determination of the hydrodynamic radius of the particles in solution by dynamic light scattering For the analysis, 35 μl of the samples of each concentration were placed in wells of black polymer plates with optically transparent bottoms. Analysis was performed using a DynaPro Plate Reader II instrument. Each well was analyzed 10 times. The obtained data were processed in Dynamics V7 software. 6. Determination of the Diffusion Interaction Parameter (k _D ) by Dynamic Light Scattering A number of protein solutions ranging from 30 mg/ml to 0.94 mg/ml were produced by stepwise dilution. Appropriate solutions of excipients are used as solvents. For analysis, 35 μl of samples of each concentration were placed in wells of black polymer plates with optically clear bottoms. Analysis was performed using a DynaPro Plate Reader II instrument. Each well was analyzed 10 times. The resulting data were processed in the Dynamics V7 software, where the dependence of the diffusion coefficient on the protein concentration in solution was plotted and the line inclination of the resulting dependence was determined. 7. Determine the thermal stability under thermal stress (TS50) at 50°C. Divide the test sample into 2 aliquots of 150 μl each and place in separate glass vials: store 1 vial of each composition in a refrigerator at 5 ± 3 °C, place the rest in a thermostat and incubated at 50°C for 96 hours. When a control point or subsequent heating is selected, the vial is removed from the incubator, kept at room temperature for approximately 15 minutes and transferred for analysis. 8. Determine colloidal stability under vibration (SH800). Divide the test sample into 2 aliquots of 150 μl each and place in glass vials, store 1 vial of each formulation in a refrigerator at 5 ± 3 °C, place the rest of the vials in a thermal shaker and Shake at 5 ± 3 °C for 96 h at 800 rpm. During selection of control points or subsequent stress, vials were removed from the thermal shaker and transferred for analysis. 9. Determine colloidal stability under freeze-thaw (FT(-20)). Divide the test sample into 2 aliquots and place in plastic vials: store 1 vial of each formulation in a refrigerator at 5±3°C, store the remaining vials in a freezer not higher than -18°C, until completely frozen. Thereafter, the vial was removed from the freezer and kept at room temperature until the contents were completely thawed; the solution was mixed using a vortex and returned to the freezer. Repeat at least three times. After stress, remove the vial from the freezer and keep at room temperature until the contents are completely thawed; use a vortex to mix the solution and transfer for analysis. 10. Determine the stability under acid hydrolysis (Acid). Test samples were divided into 2 aliquots and placed in polymeric vials: 1 vial of each formulation was stored in a refrigerator at 5±3°C (for all studies, at the beginning of storage, input controls could be transferred for analysis once), the pH of the remaining vials was adjusted to 3.5±0.1 with hydrochloric acid solution under stirring, after which they were transferred to a refrigerator for storage at 5±3°C. After 1 h, the hydrolysis was quenched with stirring by adding sodium hydroxide solution to the initial pH. The solution was then transferred for analysis. 11. Determine the stability under alkaline hydrolysis (Basic). Test samples were divided into 2 aliquots and placed in polymeric vials: 1 vial of each formulation was stored in a refrigerator at 5±3°C (for all studies, at the beginning of storage, input controls could be transferred for analyzed once), the pH of the remaining vials was adjusted to 8.5±0.1 with sodium hydroxide solution under stirring, after which they were transferred to a refrigerator for storage at 5±3°C. After 1 h, the hydrolysis was quenched with stirring by adding hydrochloric acid solution to the initial pH. The solution was then transferred for analysis. 12. Accelerated aging. Divide test samples into separate aliquots (one aliquot for input control, for all studies, at the beginning of storage, allowing transfer once for analysis) and place in individual sterile glass vials: each Part of the vials of the formulation were placed in the refrigerator for storage at 5±3°C (input control), the rest of the vials were placed in a thermostat and incubated at 25±2°C for 6 months, and control points were selected periodically according to the plan . Upon selection of control points and subsequent storage, vials were removed from the incubator and transferred for analysis. 13. Determine sample purity by Size Exclusion High Performance Liquid Chromatography (SE HPLC). Column: Tosoh TSK-GelG3000SWXL 7.8 mm ID x 30 cm, 5 μm. Pre-column: TSK-Gel Guard SW _XL , 6.0 mm ID×4.0 cm, 7 μm, 300Å. Column temperature: 25°C. Mobile phase flow rate: 0.5 ml/min. Injection volume: 25 μl. Sample concentration: 0.5 mg/ml. Detector wavelengths: 214 and 360 nm. Elution time: 30 minutes. Mobile phase: anhydrous disodium hydrogen phosphate 14.2 mg/ml. Sodium chloride 11.7 mg/ml. The mobile phase pH was adjusted to 6.9 with orthophosphoric acid. 14. Evaluation of charged variant properties in capillaries on a Caliper LabChip GX II instrument Analysis was performed according to the instructions of the HT protein charge variant kit. Test samples were adjusted to a protein concentration of 1 mg/ml by dilution or concentration (depending on the initial concentration of the sample) in 0.5 ml Amicon Ultra 10 kDa centrifugal screening program (Millipore). Protein content was determined by UV spectrophotometry at a wavelength of 280 nm. Add 2 µl of carboxypeptidase solution to each of the resulting samples and incubate the samples at a temperature of 37 ± 2 °C for 2 h. After the specified time, samples were dialyzed against water in Amicon Ultra centrifuge tubes and concentrated to 2 mg/ml. A 96-well plate was loaded with labeling buffer solution, dye mixture solution and 25 μl of test sample in the amounts specified in the instructions, the plate was placed in the dark for 10 minutes, and then 60 μl of water per well was loaded and mixed. The plate with solution was centrifuged using a plate centrifuge rotor and placed in a Caliper LabChip GX II instrument. The analysis uses a special wafer filled with a running buffer solution of pH according to the specification. Results were processed with LabChip GX software. 15. Characterization of charge variants by ion exchange high performance liquid chromatography (iE HPLC). Column: ProPac WCX-10, 4×250 mm, fineness: 10 μm (Thermo Scientific, USA). Pre-column: ProPac WCX-10G, 4×50 mm, fineness: 10 μm (Thermo Scientific, USA). Eluent A: 20 mM 2-(N-morpholino)-ethanesulfonic acid, 4% acetonitrile solution, pH=7.0. Eluent B: 20 mM sodium phosphate buffer, 95 mM NaCl solution, 4% acetonitrile solution, pH=8.0. Flow rate: 0.6ml/min-1.0ml/min. Column temperature: 45°C. Autosampler temperature: 5°C. Detector: UV, 280 nm. Reference wavelength: 360 nm, 100 nm bandwidth Sample volume: 80 µl. Chromatography time: 43 minutes. Test samples were diluted to a concentration of 1.0 mg/ml and treated with carboxypeptidase B at a ratio of 100:1 for 20 minutes at a temperature of 37±2°C. Elution mode: eluent A is from 100% to 80%, and eluent B is from 0% to 20%. 16. Purity and associated impurities were determined by capillary polyacrylamide gel electrophoresis (CE red. and non-red.) in the presence/absence of sodium lauryl sulfate. Samples were diluted to a concentration of 4.0 mg/ml. 23 μL of the resulting solution was placed in a 1.5 mL microtube; 70 μl of SDS-MW sample buffer, 2 μl of an internal standard with a molecular weight of 10 kDa, 5 μl of 0.5M iodoacetamide solution (CE non-red. ) or 5 μl 2-mercaptoethanol (CE red.). The resulting solution was stirred for 15 s, centrifuged at 2800 rpm for 5 s, and placed in a solid state thermostat at 70 °C for 30 min. The solution was cooled to room temperature. SDS MW separation-PA 800 plus.met analysis method used in 32Karat software. Conditions for capillary gel electrophoresis: Capillary: 50 μm×30.2 cm Effective length of capillary: 20.0 cm Polarity: opposite, inlet on the left (-), outlet on the right (+) Capillary temperature: 25°C Analysis time and separation voltage: 35 min, 15 kV Detection wavelength: 220 nm. 17. Determine the relative specific activity. Specific activity was determined using a biological assay directed at the ability to block PD-L1-dependent inhibition of activation of the T cell-based Jurkat-PD1-NFAT reporter cell line. Samples were processed using the robotic platform liquid handling arm (LiHa); using RPMI-1640 with 25 mM HEPES, 24 mM sodium bicarbonate, containing 2 mM L-glutamine, 10% FBS, 50 μg/ml gentamicin As assay medium (medium for quantitative determination). A test sample of the antibody was diluted to a concentration of 1 mg/ml using assay medium (medium for quantitative determination) and placed in the robot platform. The robotic platform liquid handling arm (LiHa) is used to prepare three independent dilutions of standard and test samples at concentrations of 1 000, 50, 10, 1, 0.5, 0.25, 0.1, 0.025, 0.01, 0.001 μg/ml using the assay medium . We transferred the dilution and the assay medium to culture plates, and added Raji-PDL1 cl.3 cell suspension stably expressing PDL-1 at a concentration of (1.00±0.1)×10 ⁶ cells/ml, and at a concentration of (1.67±0.1 )×10 ⁶ cells/ml of Jurkat-NFAT-PD1 cl.1 reporter cell line suspension and anti-CD3/CD20 bispecific antibody. Place the culture plate in a _CO2 incubator at a temperature of (37±1) °C in humidified air with 5% carbon dioxide content for 22-24 hours. Following the incubation period, the plates were kept at room temperature for at least 15 minutes and BioGlo luciferase substrate was added. Luminescence levels were measured in relative luminescence units (RLU) using a microplate reader and Magellan 7.2 software. Using the results of the luminescence measurements, we built for each plate a four-parameter curve optimized for the standards and test samples using the Levenberg-Marquardt algorithm, determining the relative specific activity of the test samples relative to the standards. 18. Determination of oxidation products by hydrophobic interaction high performance liquid chromatography (Hydrophobic Interaction HPLC). Column: TSKgel Phenyl-5PW 7.5×75 mm, fineness: 10 μm (Tosoh Booscience, Japan). Eluent A: 5 mM sodium phosphate buffer, 2% acetonitrile solution, pH=7.0. Eluent B: 5 mM sodium phosphate buffer, 400 mM ammonium sulfate, 4% acetonitrile solution, pH=6.9. Flow rate: 0.5ml/min. Column temperature: 30°C. Autosampler temperature: 5°C. Detector: UV, 280 nm, bandwidth: 16 nm. Reference wavelength: 360 nm, 100 nm bandwidth Sample volume: 25 μL. Chromatography time: 82 minutes. Test samples were diluted to a concentration of 3.0 mg/ml and treated with carboxypeptidase B at a ratio of 100:1 for 20 minutes at a temperature of 37±2°C. Elution mode: eluent A is 0%-100%, and eluent B is 100%-0%. 19. Determine stability under oxidation. Divide the test sample into 2 aliquots of 150 μl each and place in separate glass vials: store 1 vial of each formulation in a refrigerator at 5 ± 3 °C, add hydrogen peroxide to the remaining The final concentration of hydrogen peroxide in the sample to the sample is 0.1%, and the sample is aged at (5±3)°C for 4 hours. Oxidation was quenched by adding an equal amount of L-methionine. 20. Determine photostability. The test sample was divided into two aliquots and placed in separate glass vials. As a dark control, we use the product wrapped tightly with aluminum foil in a second package. All samples were placed in a climate chamber with a light source, and a light stress program was emitted at 1,200,000 lux•h and 200 W•h/m ² (dose ICH×1). After reaching the desired stress level, all samples were removed from the chamber and transferred for analysis. 21. Disposal of Results. The absolute change in quality index when under stress is calculated by: Δ = (value after stress - value before stress) The absolute change in charge variant properties (abs) is calculated by: Δ = | acid level before stress Component Content - Acidic Fraction Content After Stress | + | Basic Fraction Content Before Stress - Basic Fraction Content After Stress | + | Main Fraction Content Before Stress - Main Fraction Content After Stress | Example 1. Buffer System s Choice. In this study, two typical buffer systems (acetate and histidine buffer systems) suitable for parenteral administration were selected as the basis of the pharmaceutical composition. The study was performed in a full two-factor experimental design with two levels and a center point. pH levels (5.0-6.5) and buffer concentrations (5-50 mM) were investigated as quantitative factors. To assess the suitability of the buffer system, we investigated the effect of the properties of the buffer solution on the colloidal and conformational stability of the protein. Aggregation temperature, melting point, diffusion interaction parameters, purity change after thermal stress, and acid-base properties were determined in response. The tested pharmaceutical compositions are shown in Table 1.

A study of stability predictors. The diffusion interaction parameter ( _kD ) reflects the sample diffusion coefficient as a function of molecular concentration. If the diffusion coefficient decreases with increasing concentration (k _D <0), then the polydispersity of a given solution increases and larger particles form within it. Such samples have low solubility and tend to aggregate, and their formulation is not recommended. Aggregation temperature and melting point allow assessment of the protein's tendency to aggregate. The most stable samples were those where particle aggregation started at higher temperatures and where smaller particles formed under heating. The results of investigation of aggregation temperature by method 3, melting point by method 4 and diffusion interaction parameters by method 6 are shown in Table 2. Best results are in lighter shades of color.

Determine thermal stability. Thermal stability was assessed by method 7. Before and after thermal stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 5. The results are shown in Table 3. Best results are in lighter shades of color.

The resulting data were processed in Minitab software and a model was built to select the formulation with the most stabilizing effect. Only those responses (quality indices) that were significantly affected by the factors (pH and mass molarity of the buffer solution) were used for modeling. For other responses not considered in the optimization model, no statistically significant differences were assumed within the range of factors studied. Based on the optimization results, a 5 mM histidine buffer solution with a pH of 5.6 was selected, which was prepared with: L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml When preparing a solution with a specific content of L-histidine and L-histidine monohydrate hydrochloride, the pH may deviate slightly from the desired value and vary within the pH range of 5.5-5.7 (pH 5.6±0.1). This formulation showed the best stabilization properties among all tested samples. From the results of the model, minimal changes in monomer (protein) and aggregate content were expected in histidine buffered solutions under heat exposure, and also high values of aggregation temperature and diffusion interaction parameters were expected. The optimal composition exhibited less change in monomer content, less change in aggregate content, and an increase in the diffusion interaction parameter upon heat exposure, indicating higher colloidal stability, compared to Keytar buffer solution. Example 2. Selection of penetrant. Test formulations. Excipients suitable for parenteral administration were investigated for use as osmotic agents. The test formulations are shown in Table 4.

A study of stability predictors. For the selection of penetrants, the results of the study of aggregation temperature by method 3, melting point by method 4 and diffusion interaction parameters by method 6 are shown in Table 5. Best results are in lighter shades of color.

Determine thermal stability. Thermal stability was assessed by Method 7. Before and after thermal stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 6. Best results are in lighter shades of color.

Determine stability under vibration. Stability under vibration was assessed by Method 8. Before and after shaking, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 7. Best results are in lighter shades of color.

Determine stability during freezing and thawing. Stability under freeze-thaw was assessed by Method 9. Before and after stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 8. Best results are in lighter shades of color.

The formulation comprising trehalose dihydrate and L-proline showed the best stabilization properties among all tested samples. The samples containing trehalose dihydrate showed the best results in terms of aggregation temperature and melting point. In addition, the sample showed minimal changes in quality indicators under heat exposure, and minor changes were observed under vibration. The samples containing L-proline also showed one of the best results for aggregation temperature and average results for melting point. Compositions with L-proline showed average results under heat exposure, small changes in quality indicators under vibration, and best results under freeze-thaw. Introducing trehalose dihydrate or L-proline selected for the next step reduces the formation of aggregates under freeze-thaw and heat stress. Also, selected formulations exhibited slight quality changes under vibration. The selected osmotic agents provided increased thermal stability when compared to formulations containing sucrose; in particular, we observed increased melting points/aggregation temperatures, lower rates of aggregate formation upon thermal exposure, and monomeric content , and we also observe small absolute changes in the acid-base properties. Samples containing trehalose dihydrate in the excipient showed better results in terms of diffusion interaction parameters, indicating increased stability and less tendency to aggregate during concentration and diafiltration. The investigated formulations containing trehalose dihydrate, sorbitol, or L-proline showed significantly smaller increases in particle hydrodynamic radius under shaking and freezing compared to formulations containing sucrose, indicating the formation of large polymer particles. Excipient formulations selected for the next step: His+Tre 100 mg/ml: L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml trehalose dihydrate 100mg/ml His+Prol 30 mg/ml: L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml L-proline 30mg/ml Example 3. Screening of penetrants and stabilizers. For screening penetrants and stabilizers, excipients suitable for parenteral administration are used. The test formulations are shown in Table 9. A pharmaceutical composition containing pembrolizumab in a test formulation was prepared according to Method 2.

A study of stability predictors. For the selection of penetrants, the results of the study of aggregation temperature by method 3, melting point by method 4 and diffusion interaction parameters by method 6 are shown in Table 10. Best results are in lighter shades of color.

Determine thermal stability. Thermal stability was assessed by method 7. Before and after thermal stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 11. Best results are in lighter shades of color.

Stability under acid hydrolysis was determined. For formulations without poloxamer 188, stability under acid hydrolysis was assessed by Method 10, where pH was adjusted to 3.5 and aged for 1 hour. Before and after hydrolysis, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 12. Best results are in lighter shades of color.

Stability under alkaline hydrolysis was determined. For formulations without poloxamer 188, stability under alkaline hydrolysis was assessed by method 11, where pH was adjusted to 8.5 and aged for 1 hour. Before and after hydrolysis, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 13. Best results are in lighter shades of color.

Determine stability under vibration. Stability under vibration was assessed by Method 8. Before and after stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 14. Best results are in lighter shades of color.

Determine stability during freezing and thawing. Stability under freeze-thaw was assessed by Method 9. Before and after stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 15. Best results are in lighter shades of color.

Among the tested samples, the following formulations were likely to be superior in terms of stability: 1. His+Tre+Glycine 100 mM pembrolizumab 1.5-30 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml trehalose dihydrate 100mg/ml Glycine 7.51mg/ml 2. His+Tre+poloxamer 188 0.5 mg/ml pembrolizumab 1.5-30 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml trehalose dihydrate 100mg/ml Poloxamer 188 0.5mg/ml 3. His+Prol pembrolizumab 1.5-30 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml L-proline 30mg/ml 4. His+Prol+Glycine 100 mM pembrolizumab 1.5-30 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml L-proline 30mg/ml Glycine 7.51 mg/ml 5. His+Prol+Glycine 100 mM+ Poloxamer 188 0.5 mg/ml pembrolizumab 1.5-30 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml L-proline 30mg/ml Glycine 7.51mg/ml Poloxamer 188 0.5mg/ml Selection of formulations containing trehalose dihydrate, compositions of combinations of trehalose dihydrate and glycine, and compositions with L-proline, and compositions with L-proline and glycine or methionine Compositions of the combination were used for further development as they showed positive stability results under stress. All formulations with trehalose dihydrate showed a higher content of alkaline fraction compared to the formulation with L-proline. Among the formulations containing trehalose dihydrate, the formulation with glycine showed the best results. We observed positive effects of glycine on aggregation temperature and melting point as well as on diffusion interaction parameters. Addition of glycine to the formulation showed a slight improvement when under thermal stress. Addition of glycine to the formulations showed improvements in purity and acid-base properties before and after stress. We observed a significant increase in melting point and aggregation temperature, as well as a significantly smaller change in purity and acid-base properties under acid and base hydrolysis, compared to samples in formulations with no surfactant-incorporated Keyradar excipients. Variety. In formulations containing surfactants, a positive effect of poloxamer 188 was observed. These compositions exhibit minor changes in purity and charged variant properties under shaking and freezing. Therefore, Poloxamer 188 was chosen to be used as the surfactant in the next step. Example 4. Determination of key quantitative factors for excipient formulations. The study was conducted in a factorial four-factor experimental design with two levels and a center point. Protein concentration (10-40 mg/ml), pH (5.1-6.1), osmolyte concentration (70-130 mg/ml), L-glycine concentration (1.5-15 mg/ml) and Poloxamer 188 concentration (0.10-1.0 mg/ml) was studied as a quantitative factor. The formulations tested are listed in Table 16.

In Table 16, buffer solutions refer to the following formulations described in Table 17.

A study of stability predictors. For the choice of penetrant, the results of the study of aggregation temperature by method 3, melting point by method 4 and diffusion interaction parameters by method 6 are shown in Table 18. Best results are in lighter shades of color.

Determine thermal stability. Thermal stability was assessed by Method 7. Before and after thermal stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 19. Best results are in lighter shades of color.

Stability under vibration was assessed by Method 8. Before and after stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 20. Best results are in lighter shades of color.

Determine stability during freezing and thawing. Stability under freeze-thaw was assessed by Method 9. Before and after stress, we determined purity by SE HPLC by method 13, charge variant properties in capillary by method 14, and hydrodynamic radius by method 6. The results are shown in Table 21. Best results are in lighter shades of color.

The results were used to build the model in Minitab software. The concentration of L-glycine/poloxamer 188 had no statistically significant effect on the stability of pembrolizumab. According to the optimization results, the recommended concentration of L-glycine is 15 mg/ml, the recommended concentration of poloxamer 188 is 1.0 mg/ml, and the recommended concentration of trehalose dihydrate is 84.5 mg/ml. However, in order to ensure the physiological osmolarity of the composition, the content of L-glycine was reduced to 1.5 mg/ml, and the content of trehalose dihydrate was reduced to 80 mg/ml. The buffer solution, its concentration and pH were selected earlier, and the results are shown in Example 1. This example demonstrates their statistically significant effect on the stability of pembrolizumab. Under thermal stress, surfactants may cause the formation of artificially high molecular weight impurities. If heat stress was not included in the model, it could be inferred that increasing concentrations of poloxamer 188 had a positive effect on the stability of pembrolizumab. According to the results, the optimal concentration of poloxamer 188 was 1.0 mg/ml. Therefore, the formulations finally selected were as follows: pembrolizumab 1.5-40 mg/ml L-histidine 0.221mg/ml L-histamine hydrochloride monohydrate 0.750mg/ml trehalose dihydrate 80mg/ml Glycine 1.5mg/ml Poloxamer 188 1.0mg/ml Example 5. Determining the Stability of Final Formulations Under Stress Conditions For stress stability testing, final formulations of pembrolizumab and formulations of Keyradar excipients were selected. The test formulations are shown in Table 22. A pharmaceutical composition containing pembrolizumab in a test formulation was prepared according to Method 2.

Determine thermal stability. Thermal stability was assessed by method 7 for 10 days. Before and after thermal stress, we determined the purity by the SE HPLC method (Method 13), the charged variant identity by the IE HPLC method (Method 15), and the content of oxidation products by the hydrophobic interaction HPLC method (Method 18) , to determine the hydrodynamic radius by method 5. The results are shown in Table 23. Best results are in lighter shades of color.

Determine stability under vibration. Stability under vibration was assessed by Method 8. Before and after shaking, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 24. Best results are in lighter shades of color.

Determine stability during freezing and thawing. Stability under freeze-thaw was assessed by Method 9. Before and after stress, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 25. Best results are in lighter shades of color.

Stability under acid hydrolysis was determined. Stability under acid hydrolysis was assessed by method 9. Before and after stress, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 26. Best results are in lighter shades of color.

Stability under alkaline hydrolysis was determined. Stability under alkaline hydrolysis was assessed by Method 9. Before and after stress, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 27. Best results are in lighter shades of color.

Determine stability under oxidation. Stability under oxidation was assessed by Method 9. Before and after stress, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 28. Best results are in lighter shades of color.

Determine photostability. Stability under oxidation was assessed by Method 9. Before and after stress, we determined the purity by SE HPLC method 13, the charged variant identity by IE HPLC method (Method 15), the content of oxidation products by hydrophobic interaction HPLC method (Method 18), and the content of oxidation products by Method 6 Determine the hydrodynamic radius. The results are shown in Table 29. Best results are in lighter shades of color.

According to the results of the stress stability test, the excipient formulation of pembrolizumab showed smaller changes in quality indicators under thermal stress, especially the hydrodynamic radius, compared with the excipient formulation of Keyradar. Smaller changes, smaller increases in aggregates and changes in monomer content, significantly lower tendency to form oxidized forms and smaller changes in the identity of charged variants. Smaller changes in the properties of the charged variant after stress were observed when under vibration and freezing. Furthermore, when under acid hydrolysis, a small increase in aggregates and a change in monomer content was observed, as well as a small change in the content of the basic fraction. When under alkaline hydrolysis, proteins within pembrolizumab formulations had less tendency to form aggregates, alter monomer content, and alter the identity of charged variants. When under light stress, a significantly lower tendency to oxidize was observed, as well as a smaller change in monomer content, a smaller increase in aggregates, and a charged variant character, compared to formulations with Keyradar excipients minor changes. Example 6. Determination of stability under accelerated aging at a temperature of 37±2°C. To pre-confirm the stability of selected formulations, they were subjected to accelerated aging at a temperature of 37±2°C. Prepare protein concentration (25-50 mg/ml), pH (5.1-6.1), trehalose dihydrate concentration (70-90 mg/ml), glycine concentration (1.0-2.0 mg/ml) and porol concentration by method 1 Pharmaceutical compositions within the concentration range of Sham 188 (0.8-1.2 mg/ml), and stored at a temperature of 37±2°C for stability testing. The test formulations are shown in Table 30.

Accelerated aging. Pharmaceutical compositions containing proteins at concentrations of 25, 50 and 37.5 mg/ml were prepared by diafiltration according to technique 1 and placed at a temperature of 37±2° C. for accelerated storage according to technique 8. The results of the study are shown in Table 31.

All tested formulations exhibited acceptable variation in quality parameters (purity by SE HPLC, CE under reducing and non-reducing conditions, charged variant identity and specific activity), which was confirmed by using the design space of MODDE software . According to the results of the experimental space reflecting the effect of factors (buffer solution pH and excipient component content) on the response (quality parameters), accelerated aging at 37 ± 2 ° C for 4 weeks, pharmaceutical composition at the provided concentration, pH and It is stable within the range of excipients. Example 7. Determination of stability under long-term accelerated aging. To demonstrate the stability of selected formulations, they were subjected to accelerated aging at a temperature of 25±2°C. Prepare protein concentration (25-50 mg/ml), pH (5.1-6.1), trehalose dihydrate concentration (70-90 mg/ml), glycine concentration (1.0-2.0 mg/ml) and porol concentration by method 1 Pharmaceutical compositions within the concentration range of Sharm 188 (0.8-1.2 mg/ml), and stored at a temperature of 25±2°C for stability testing. In addition, we selected the most critical cases among all compositions, with the lowest and highest excipient content and highest protein content, and formulations with excipient content in the midpoint of the range. The formulations tested are shown in Table 32.

Accelerated aging. Pharmaceutical compositions containing proteins at concentrations of 25, 50 and 37.5 mg/ml were prepared by diafiltration according to technique 1 and placed at a temperature of 25±2°C for accelerated storage according to technique 8. The results of the study are shown in Table 33 and Figures 1, 2, 3, 4, 5 and 6.

All pharmaceutical compositions demonstrate acceptable levels of change during accelerated storage. Contains a histidine buffer solution with a pH range of 5.1-6.1, trehalose dihydrate at 70-90 mg/ml, L-glycine at 1.0-2.0 mg/ml and poloxamer at 0.8-1.2 mg/ml The pharmaceutical composition of 188 demonstrated acceptable levels of aggregation (no more than 0.32% increase in aggregates over 6 months at 25 ± 2°C), and increased concentration at concentrations of 25 mg/ml and up to 50 mg/ml Small changes in the acid-base properties of the monoclonal antibody pembrolizumab (the content of the basic fraction did not change by more than 13.0%) and slight changes in specific activity under accelerated aging. The addition of trehalose dihydrate helps to increase the aggregation temperature and melting point of pembrolizumab. We also observed minimal changes in quality parameters under thermal stress, and slight changes in quality parameters after shaking and freezing. Furthermore, glycine has a positive influence on aggregation temperature and melting point as well as on diffusion interaction parameters. Positive effects of glycine on protein quality parameters under acid and base hydrolysis were observed. The addition of poloxamer 188 to the formulation was helpful in testing the stability of the protein under shaking and freezing. According to the results of the stress stability test, the developed formulation of pembrolizumab excipient showed significant advantages over the Keyread formulation under thermal stress, acid and alkaline hydrolysis; moreover, we observed significantly more Low oxidation tendency, which has a significant effect on protein structure and specific activity, and has increased aggregation tendency. We provide an example of a study using an aqueous pharmaceutical composition of pembrolizumab. The aqueous pharmaceutical compositions used for further studies are described in Table 34.

Experiments revealed that the preparation of Keyruida products has the following disadvantages: 1) Insufficient colloidal stability of antibodies, 2) Insufficient stability under thermal stress, 3) Stability of charged variant properties under mechanical stress and freezing Sex is low. Within the framework of the present invention, pharmaceutical compositions of high stability with sufficient colloidal and thermal stability as well as charged variant properties under mechanical stress and freezing have been produced. In addition, pharmaceutical compositions containing trehalose and poloxamer 188 in their formulations (such as those indicated in Table 34) are further characterized by a low tendency to form high molecular weight impurities under long-term storage, and under light stress and long-term storage Low tendency to oxidation, loss of specific activity and increase of impurities. The data obtained concerning the stability of pharmaceutical compositions indicates the compatibility of excipients with each other and with the active substances.

[ Fig. 1 ] is a graph of aggregate content (%) determined by SE HPLC with respect to storage time at a temperature of 25±2°C for the monoclonal antibody pembrolizumab in the test preparation. [ Fig. 2 ] is a graph of monomer content (%) determined by SE HPLC with respect to storage time at a temperature of 25±2°C for the monoclonal antibody pembrolizumab in the test preparation. [ Fig. 3 ] is a graph of the basic fraction content (%) determined by IE HPLC with respect to the storage time at a temperature of 25±2°C for the monoclonal antibody pembrolizumab in the test preparation. [ FIG. 4 ] is a graph of monomer content (%) determined by CE under non-reducing conditions with respect to storage time at a temperature of 25±2° C. for the monoclonal antibody pembrolizumab in a test preparation . [ Fig. 5 ] is the content (%) of the sum of the heavy and light chains determined by CE under reducing conditions for the monoclonal antibody pembrolizumab in the test preparation relative to the temperature at 25±2°C A graph of the storage time. [ Fig. 6 ] is a graph of relative specific activity (%) with respect to storage time at a temperature of 25±2°C for the monoclonal antibody pembrolizumab in the test preparation.

Claims (34)

A pharmaceutical composition of pembrolizumab, wherein the composition comprises: (i) pembrolizumab; (ii) histidine; (iii) Histamine hydrochloride monohydrate; (iv) Glycine; (v) trehalose and poloxamer 188, or proline; and (vi) Water for injection. The pharmaceutical composition according to claim 1, wherein pembrolizumab exists at a concentration of 5-50 mg/ml. The pharmaceutical composition according to claim 1, wherein the histidine exists at a concentration of 0.087-0.432 mg/ml. The pharmaceutical composition according to claim 1, wherein the monohydrate of histamine hydrochloride exists at a concentration of 0.464-0.931 mg/ml. The pharmaceutical composition according to claim 1, wherein glycine exists at a concentration of 1-2 mg/ml. The pharmaceutical composition according to claim 1, wherein trehalose exists at a concentration of 70-130 mg/ml. The pharmaceutical composition according to claim 1, wherein the poloxamer 188 exists at a concentration of 0.8-1.2 mg/ml. The pharmaceutical composition according to claim 1, wherein proline exists at a concentration of 20-34 mg/ml. The pharmaceutical composition according to claim 1, wherein the composition comprises: (i) 5-50 mg/ml pembrolizumab; (ii) 0.087-0.432 mg/ml histidine; (iii) 0.464-0.931 mg/ml histamine hydrochloride monohydrate; (iv) 1-2 mg/ml glycine; (v) 70-130 mg/ml trehalose and 0.8-1.2 mg/ml poloxamer 188, or 20-34 mg/ml proline; and (vi) Water for injection to 1 ml. The pharmaceutical composition according to claim 9, wherein pembrolizumab exists at a concentration of 20-30 mg/ml. The pharmaceutical composition according to claim 10, wherein pembrolizumab exists at a concentration of 25 mg/ml. The pharmaceutical composition according to claim 9, wherein the histidine exists at a concentration of 0.200-0.319 mg/ml. The pharmaceutical composition according to claim 12, wherein histidine exists at a concentration of 0.221 mg/ml. According to the pharmaceutical composition of claim 9, wherein the monohydrate of histamine hydrochloride exists at a concentration of 0.650-0.850 mg/ml. The pharmaceutical composition according to claim 14, wherein histamine monohydrate hydrochloride exists at a concentration of 0.750 mg/ml. The pharmaceutical composition according to claim 9, wherein glycine exists at a concentration of 1.5 mg/ml. The pharmaceutical composition according to claim 9, wherein trehalose exists at a concentration of 70-100 mg/ml. The pharmaceutical composition according to claim 17, wherein trehalose exists at a concentration of 75-85 mg/ml. The pharmaceutical composition according to claim 18, wherein trehalose exists at a concentration of 80 mg/ml. The pharmaceutical composition according to claim 1, wherein the trehalose is trehalose dihydrate. The pharmaceutical composition according to claim 9, wherein the poloxamer 188 exists at a concentration of 1.0 mg/ml. The pharmaceutical composition according to claim 9, wherein proline exists at a concentration of 24-30 mg/ml. The pharmaceutical composition according to claim 22, wherein proline exists at a concentration of 27 mg/ml. The pharmaceutical composition according to claim 1, wherein the pH of the composition is 5.1-6.1. The pharmaceutical composition according to claim 24, wherein the pH of the composition is 5.6. The pharmaceutical composition according to claim 1, wherein the composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histamine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and Wherein the pH of the composition is 5.5-5.7. The pharmaceutical composition according to claim 20, wherein the composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histamine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 80 mg/ml trehalose dihydrate and 1.0 mg/ml poloxamer 188; (vi) water for injection to 1 ml; and Wherein the pH of the composition is 5.5-5.7. The pharmaceutical composition according to claim 1, wherein the composition comprises: (i) 25 mg/ml pembrolizumab; (ii) 0.221 mg/ml histidine; (iii) 0.750 mg/ml histamine hydrochloride monohydrate; (iv) 1.5 mg/ml glycine; (v) 27 mg/ml proline; (vi) water for injection to 1 ml; and Wherein the pH of the composition is 5.5-5.7. The pharmaceutical composition according to any one of claims 26-28, wherein the pH of the composition is 5.6. A pharmaceutical composition of pembrolizumab, which is produced by freeze-drying the pharmaceutical composition of pembrolizumab according to any one of claims 1-29. A use of the pharmaceutical composition of pembrolizumab according to any one of claims 1-30 for treating malignant tumors or infectious diseases. According to the use of claim 31, wherein the malignant tumor is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, high microsatellite Instability/DNA mismatch repair deficient (MMR) malignancies, hepatocellular carcinoma, cervical carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, esophageal carcinoma, squamous cell skin carcinoma, basal cell carcinoma, breast carcinoma Carcinoma, colorectal cancer, prostate cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, pancreatic cancer, ovarian cancer, gallbladder cancer, malignant brain tumors, glioblastoma, with high mutational burden of tumors. A use of the pharmaceutical composition of pembrolizumab according to any one of Claims 1-30 in the production of pharmaceutical products intended to treat malignant tumors or infectious diseases. According to the use of claim 33, wherein the malignant tumor is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, high microsatellite Instability/DNA mismatch repair deficient (MMR) malignancies, hepatocellular carcinoma, cervical carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, esophageal carcinoma, squamous cell skin carcinoma, basal cell carcinoma, breast carcinoma Carcinoma, colorectal cancer, prostate cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, pancreatic cancer, ovarian cancer, gallbladder cancer, malignant brain tumors, glioblastoma, with high mutational burden of tumors.

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