CN114392352A

CN114392352A - Stable anti-PD-1 antibody pharmaceutical preparation

Info

Publication number: CN114392352A
Application number: CN202111130853.3A
Authority: CN
Inventors: 冯翠娟; 洪娟; 郑松林; 张珈珲; 李文华; 杨彬; 张旭辉; 李文佳
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Sunshine Lake Pharma Co Ltd
Priority date: 2020-09-27
Filing date: 2021-09-26
Publication date: 2022-04-26

Abstract

The invention relates to the field of antibody preparations, in particular to a stable anti-PD-1 antibody pharmaceutical preparation which comprises an anti-PD-1 antibody, a buffer solution and a stabilizer, and the pharmaceutical preparation does not contain sodium chloride. The invention adopts phosphate as a buffer system, sucrose as a stabilizer and sodium chloride as an isotonic agent is not added in the prescription, so that the thermal stability, the physical stability, the purity and the like of the preparation can be obviously improved, the storage time is effectively prolonged, and the preparation has wide market application prospect.

Description

Stable anti-PD-1 antibody pharmaceutical preparation

Technical Field

The invention relates to the field of antibody preparations, in particular to a stable anti-PD-1 antibody pharmaceutical preparation.

Background

PD-1 (programmed death receptor 1), an important immunosuppressive factor in the human body, is an immunoglobulin, and is a membrane protein consisting of 268 amino acids. It was first cloned from the apoptotic mouse T cell hybridoma 2B 4.11. The immunomodulator taking Anti-PD-1 as a target for treatment comprises an Anti-PD-1monoclonal antibody (Anti-PD-1monoclonal antibody), can block the interaction between PD-1 and a ligand PD-L1 and PD-L2 thereof, and has important significance for resisting tumors, infection, autoimmune diseases, organ transplantation survival and the like. Inhibitors of programmed death protein-1 (PD-1) and its ligand (PD-L1) are the main drugs of immune checkpoint therapy, and the breadth, depth and persistence of the response are rare, and the inhibitors are the hot spots of tumor immunotherapy research in recent years. Currently marketed nivolumitumumab (nivolumab) and parbolizumab (pembrolizumab) belong to PD-1 inhibitors, and are mainly used for treating malignant tumors such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin lymphoma, urothelial cancer, microsatellite high-instability cancer and the like.

The Nwaruiuzumab injection (trade name: European Divor) is a fully human monoclonal antibody against PD-1 receptor developed by Bezish Shi Guibao Co., U.S.A.. The monoclonal antibody is approved and registered in China as the first monoclonal antibody drug taking PD-1 as a target in the approval of treatment for adult patients who are negative to Epidermal Growth Factor Receptor (EGFR) gene mutation and Anaplastic Lymphoma Kinase (ALK) and have advanced or intolerant local advanced or metastatic non-small cell lung cancer (NSCLC) after receiving platinum-containing chemotherapy in 6-15 days in 2018. The injection of the Nawuliuyuxuuzumab is approved by national drug administration for expansion indication in 2019, 10 and 8 days, and is suitable for treating patients with recurrent or metastatic Squamous Cell Carcinoma of Head and Neck (SCCHN) who have disease progression and positive tumor PD-L1 expression (the tumor cells expressing PD-L1 are more than or equal to 1%) during or after receiving treatment with platinum-containing scheme. 13 days 3 and 2020, the Nwaruliuzumab injection is approved by the Chinese drug administration for treating advanced or recurrent gastric or gastroesophageal junction adenocarcinoma patients who have received two or more systemic treatment schemes before, and is the first Chinese medicament for treating the immune tumor of advanced gastric cancer.

Currently marketed Nanwuliu monoclonal OPDIVO is a hydro-acupuncture preparation, and contains sodium citrate dihydrate, sodium chloride, mannitol, pentetic acid, polysorbate 80, hydrochloric acid, sodium hydroxide and water for injection. Like most protein molecules, nivolumetrizumab is unstable and undergoes a variety of chemical and physical degradation. Compared with the traditional synthesized small molecule drugs, the biological molecules have complex structures, such as primary, secondary, tertiary and other high-level structures. The structure of proteins, especially higher order structures, is very fragile and subject to structural changes such as denaturation, aggregation and precipitation. The maintenance of the high-order structure of antibodies is the most essential requirement for their biological activity. The products of these degradations can have a significant impact on biopharmaceutical safety. In particular, some protein aggregates can stimulate the immune response of human body, and the curative effect of biological medicines can be reduced in the mild case and even death of patients can be caused in the severe case. We found that the OPDIVO formulations that have been marketed are less stable and significantly degrade under long-term and accelerated test conditions, especially producing more impurities such as multimers and insoluble particles. The polymer and insoluble particles are regarded as quality parameters (CQA) which are very important for the safety of biopharmaceuticals, and directly influence the medication safety of the nivolumetrizumab, particularly the immunogenicity of the nivolumetrizumab, which is particularly important for long-term medication products such as the nivolumetrizumab.

Different antibody drugs need different solvent environments to maintain the stability in the processes of freezing, thawing, storage, transportation and the like, and even the antibody drugs with the same target point need different preparation formulas due to different amino acid sequences and post-translational modifications of the antibody. Accordingly, there is a need to provide alternative or improved liquid formulations of nivolumab.

Through a large number of experimental researches, the invention surprisingly discovers that in the pharmaceutical composition containing the nivaletuzumab, the thermal stability, the physical stability, the purity and the like of the preparation can be obviously improved by adopting a phosphate buffer system to replace a citrate buffer system and adopting cane sugar to replace mannitol as a sugar stabilizer, and sodium chloride is not added in the prescription, so that the storage time is effectively prolonged, and the invention has wide market application prospect.

Disclosure of Invention

The invention provides a stable anti-PD-1 antibody pharmaceutical preparation. The preparation can maintain the physical and chemical stability of the pharmaceutical composition for a long time.

Specifically, the invention provides a stable anti-PD-1 antibody pharmaceutical preparation which comprises an anti-PD-1 antibody, a buffer and a stabilizer, and the pharmaceutical preparation does not contain sodium chloride. The buffer solution is a phosphate buffer solution, and the concentration of the phosphate buffer solution is 5-50 mM, preferably 10-30 mM. The present inventors have found that the use of phosphate buffer systems results in formulations with better stability than other buffer systems.

In some embodiments of the present invention, the stabilizer is sucrose, and the concentration of sucrose is 50-150 mg/ml, preferably 60-135 mg/ml, and more preferably 80-95 mg/ml. During the research process, the inventor finds that the effect of selecting sucrose as the stabilizer is better than that of other stabilizers. And when sodium chloride is not added in the formula, the stability of the preparation is better. The sucrose can be used as a stabilizing agent and also can be used as an osmotic pressure regulator to replace the traditional isotonic agent sodium chloride. Therefore, the sucrose selected by the invention can play a role in killing two birds with one stone. In addition, when the concentration of sucrose is within the concentration range of the present invention, the stability of the formulation can be maintained while maintaining a certain osmotic pressure.

Further, the pharmaceutical formulation of the present invention further comprises a surfactant, including but not limited to tween 80, tween 20, tween 21, tween 40, tween 60, tween 61, tween 65, tween 80, tween 81, tween 85, poloxamer, triton, sodium lauryl sulfate, octyl glucoside sodium, lauryl-sulfobetaine, polyethylene glycol, polypropylene glycol, or a mixture thereof, preferably tween 80. The concentration of the surfactant is 0.01-10 mg/ml, preferably 0.1-1.0 mg/ml, and more preferably 0.1-0.5 mg/ml.

The pharmaceutical formulations of the present invention further contain a pharmaceutically acceptable amount of an antioxidant, including, but not limited to, ascorbic acid, tryptophan, methionine, glutathione, sodium thiosulfate, catalase, aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycine, citric acid, nicotinamide, deferoxamine and deoxycholate and mixtures thereof, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA) and salts thereof. The chelating agents used in the present invention may be present as the free acid or free base of the compound or as a salt, or as an anhydrate, hydrate or other solvate of the compound or corresponding salt. Calcium disodium edetate and disodium edetate are preferred, and calcium disodium edetate is more preferred. The concentration of the antioxidant is 0.001-1.0 mg/ml, preferably 0.01-0.1 mg/ml.

The anti-PD 1 antibody in the pharmaceutical preparation can keep dissolution and stability in a wider pH value range, and the suitable pH value is 5.0-6.5, preferably 5.2-6.4.

In some embodiments of the invention, the anti-PD 1 antibody is nivolumab. The concentration of the anti-PD-1 antibody is 1-50 mg/ml, preferably 5-15 mg/ml.

Preferably, the anti-PD 1 antibody pharmaceutical formulation of the present invention comprises the following components: (1) 1-50 mg/ml of nivolumitumumab; (2) 50-150 mg/ml of sucrose; (3) 5-50 mM of phosphate buffer system; (4) tween 800.01-10 mg/ml; (5) 0.001-1.0 mg/ml of calcium disodium edetate; wherein the pH value of the preparation is 5.0-6.5.

More preferably, the anti-PD 1 antibody pharmaceutical formulation of the present invention comprises the following components: (1) 5-25 mg/ml of nivolumitumumab; (2) 60-135 mg/ml of sucrose; (3) 10-30 mM of phosphate buffer system; (4) tween 800.1-1.0 mg/ml; (5) 0.01-0.1 mg/ml of calcium disodium edetate; wherein the pH value of the preparation is 5.2-6.4.

The pharmaceutical preparation of the present invention may be in the form of a water-injection preparation, a lyophilized preparation, or a preparation prepared from a lyophilized powder and water for injection by double-chamber carbo-multicell, which may be administered by subcutaneous injection (s.c), intravenous injection (i.v.), intramuscular injection (i.m.), or other parenteral (parenteral) forms.

The invention has the following beneficial effects:

according to the invention, through extensive and intensive research, the phosphate is used as a buffer system, the sucrose is used as a stabilizer, and isotonic agent sodium chloride is not added in the prescription, so that the thermal stability, the physical stability, the purity and the like of the antibody preparation can be obviously improved, and the effective period and the medication safety of the antibody preparation are prolonged.

Definition of terms

As used in the specification and claims, the following terms have the following meanings, unless otherwise specified.

As used herein, "nivolumetrizumab" includes primary drugs (e.g., commercially available) and may also include biosimilar drugs.

The term "biosimilar" (also referred to as follow-on biologicals) is well known in the art and will be readily understood by those skilled in the art when the drug is to be considered a biosimilar of nivolumab. Further, "biosimics" require official approval as marketed "biosimics" before said "biosimics" are marketed. The term "biosimilar" is commonly used to describe subsequent versions (usually from different sources) of "innovative biopharmaceutical products" (biologicals "whose drug substances are made from or derived from living organisms or are made by recombinant DNA or controlled gene expression methods) that have previously been formally granted marketing authorization. Because biologies are highly molecularly complex and are often sensitive to variations in the manufacturing process (e.g., if different cell lines are used in production), and because subsequent manufacturers typically do not obtain original molecular clones, cell banks, know the know-how in fermentation and purification processes, nor obtain the active drug substance itself (the innovator's commercial drug product only), any "biosimilar" is unlikely to be exactly the same as the innovator drug product.

As used herein, the terms "buffer," "buffer system," or "buffer solution" refer to an aqueous solution comprising a mixture of an acid (typically a weak acid) and its conjugate base, or a mixture of a base (typically a weak base) and its conjugate acid. After the addition of a small amount of strong acid or base, the pH of the "buffer" will change only slightly due to the "buffering effect" imparted by the "buffer".

"buffering system" includes one or more buffers and/or acid/base conjugates, more suitably one or more buffers and acid/base conjugates thereof, and most suitably only one buffer and acid/base conjugate thereof. Unless otherwise indicated, any concentration specified herein with respect to a "buffer system" (i.e., buffer concentration) suitably refers to the combined concentration of the buffer and/or its acid/base conjugate. In other words, the concentrations specified herein in relation to the "buffer system" suitably refer to the combined concentration of all relevant buffer substances (i.e. the substance in dynamic equilibrium with another substance, e.g. disodium hydrogen phosphate/sodium dihydrogen phosphate). Thus, a given concentration of a phosphate buffer system generally relates to a combined concentration. The overall pH of a composition including the relevant buffering system will generally reflect the equilibrium concentration of each relevant buffering substance (i.e., the buffer is in equilibrium with its acid/base conjugate).

The term "Phosphate Buffered Saline (PBS) is generally used as a solvent to solubilize the protective agent. It is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution can be Na₂HPO₄、 NaH₂PO4、KH₂PO4、K₂HPO₄One or more of NaCl and KCl due to Na₂HPO₄And KH₂PO₄They have secondary dissociation and a wide range of buffered pH values.

The term "Tm" (melting transition temperature) refers to the protein melting transition temperature. Typically the protein in solution is in an equilibrium state between its native (folded) conformation and its denatured (unfolded) state. During the heating process, the protein is unfolded with the increase of temperature. When the protein is subjected to thermal denaturation, the temperature corresponding to half unfolding is the Tm value. The higher the Tm value, the better the thermal stability of the protein molecule.

The term "multimer" (D/A) is understood to mean a non-covalent association of the same antibody, a molecule formed by the association of two or more antibodies. The antibody may be composed of homogeneous or heterogeneous multiple polypeptides to which a single chain antibody is covalently bound (e.g., disulfide bonds). The multimers of the invention are soluble in aqueous solutions. For example, a dimer is a non-specific binding of two IgG molecules. The formation of multimers is closely related to the distorting influence on the natural antibody folding and antibody structure. For example, high salt and extreme pH induce denaturation of antibodies to form multimers.

The definition "or" as used herein denotes alternatives which may be combined if appropriate, that is to say the term "or" includes each of the listed individual alternatives as well as combinations thereof.

Drawings

FIG. 1 is a schematic diagram of the folding state of a protein as a function of temperature.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Example 1 buffer System examination

Biopharmaceuticals can be subjected to various damaging conditions during production, transportation, storage and use, such as high temperature, light, and freeze-thawing. To ensure safety, they must be able to maintain structural integrity, particularly integrity of advanced structures. Multimers (aggregates) and insoluble microparticles (particles) of biopharmaceuticals are very critical factors in eliciting the immune response to biopharmaceuticals. The appearance of biopharmaceuticals is also a very important indicator. The pharmacopoeia of various countries stipulates that biopharmaceuticals must not contain visible foreign substances, because the presence or absence and the amount of microparticles in biopharmaceuticals are directly related to the safety of biopharmaceuticals, and they are considered to be one of the most important factors causing immune response of biopharmaceuticals.

SEC-HPLC is an analytical method for separation using the pore size of the gel pores versus the size of the sample analyzed. The larger molecules in the sample are completely excluded by not entering the pores of the gel, and pass through the column only along the spaces between the porous gel particles, and are therefore first eluted from the column by the mobile phase. Molecules with smaller molecular weight can enter the gel pores, are more strongly retained in the column and are not easy to elute, so that the separation of solute molecules with different sizes is achieved. The analysis of biopharmaceuticals is mainly used to measure the content of multimers (aggregates), mainly reflected in the loss of monomer content and the increase in multimer content.

We designed several formulations 1-5 (see table 1). Wherein the preparation 1 is a buffer system (citric acid buffer system) of a product preparation on the market; the preparation 2 is a phosphate buffer system; formulation 3 is a histidine buffer system; formulation 4 is an acetate buffer system; formulation 5 is a succinate buffer system. The resulting formulations 1-5 were prepared according to the concentrations of the formulation components in table 1 for high temperature stability studies. Appearance and SEC-HPLC purity test results at 0 are shown in Table 2. The appearance and SEC-HPLC purity test results at 37 ℃ for 14 days are shown in Table 3.

TABLE 1 investigation of different buffer systems (unit: mg/ml)

TABLE 2 recipe 0d results

TABLE 3 high temperature 37. + -. 2 ℃ 14d test results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. From the results, it can be seen that after 2 weeks of high temperature testing, visible foreign matter appeared in each formulation sample, but the SEC purity of formulation 2 could be maintained at a higher level. The experimental data show that compared with a buffer system adopting citrate (citrate), histidine, succinic acid and hydrochloride, when phosphate is adopted as the buffer system, the SEC has higher purity and better stability, and the polymer can be kept at a lower level.

Example 2 influence of sugar species on the stability of formulations

We designed several formulations 6-8 (see Table 4). Wherein formulation 1 is mannitol in a marketed product formulation; formulation 2 was formulated using sucrose at an osmolality instead of mannitol as formulation 1; formulation 3 was prepared using trehalose at an osmolality in place of mannitol in formulation 1. The formulations 6-8, prepared according to the concentrations of the formulation components in table 4, were subjected to high temperature stability studies. The appearance and SEC-HPLC purity measurements at 37 ℃ for 14 days are shown in Table 5.

TABLE 4 formulation of different sugar types

TABLE 5 high temperature 37. + -. 2 ℃ 14d test results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. As can be seen from the results, after 2 weeks of the high temperature test, the sample of formulation 8 showed visible foreign matter, no change in formulations 6 and 7, and higher SEC purity of formulation 7. The experimental data show that the SEC purity of the preparation is higher when sucrose is used as the structure protective agent, and the physical stability of the preparation can be maintained.

EXAMPLE 3 stability study (I)

We designed several formulations 9-12 (see Table 6). Wherein the preparation 9 is the main component of the preparation of the products on the market; formulation 10 is a formulation containing high concentrations of mannitol without sodium chloride; formulation 11 was formulated using sucrose at an osmolality instead of mannitol of formulation 10; formulation 12 was formulated using sucrose at an osmolality instead of mannitol as in formulation 9. The appearance and SEC-HPLC purity measurements at 37 ℃ for 14 days are shown in Table 7.

TABLE 6 amounts of the components contained in the respective formulations (unit: mg/ml)

TABLE 7 high temperature 37. + -. 2 ℃ 14d test results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. As can be seen from the results, after 2 weeks of the high temperature test, visible foreign matter appeared in the samples of formulation 9 and formulation 12, no change was observed in formulation 10 and formulation 11, and the SEC purity of formulation 11 was high. The above experimental data show that the stability of the nivolumab preparation can be significantly improved by using the structure protective agent instead of sodium chloride, and when sucrose is used as the structure protective agent (preparation 11), the stability of the preparation is significantly higher than that of the preparation using mannitol as the structure protective agent (preparation 10).

Example 4 stability study (two) (concentration of Nawuliuyumab 10mg/ml)

We designed several formulations, recipes 13-18 (see Table 8). Wherein the preparation 13 is the main component of the product preparation on the market; the preparation 14 is prepared by using sucrose with equal molar osmotic pressure to replace mannitol and sodium chloride of the preparation 13; formulation 15 was formulated using sucrose at equimolar osmotic pressure instead of mannitol of formulation 13; formulation 16 was formulated using a phosphate buffer system instead of the citrate buffer system of formulation 13; formulation 17 was formulated using a phosphate buffer system instead of the citrate buffer system of formulation 14; formulation 18 is a formulation formulated using a phosphate buffer system instead of the citrate buffer system of formulation 15. The Tm value at 0 day is shown in Table 9; the results of the appearance and SEC-HPLC purity at 37 ℃ for 28 days and the change value detection at 0 day are shown in Table 10.

TABLE 8 stabilizers and buffers of different compositions (unit: mg/ml)

TABLE 9 observation results of 0-day appearance, visible foreign matter, SEC-HPLC purity and Tm value of each preparation

TABLE 10 high temperature 37. + -. 2 ℃ 28d appearance, visible impurities and SEC-HPLC purity results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. From the results, it was found that, after 4 weeks of the high temperature test, visible foreign substances appeared in the samples of formulation 13, formulation 15, formulation 16, and formulation 18, no change was observed in formulation 14 and formulation 17, and the SEC purity of formulation 17 was less changed and the Tm value was higher. The above experimental data show that the formulation using sucrose as a structure protectant and phosphate as a buffer system (formulation 17) has significantly higher stability than formulations in other conditions.

EXAMPLE 5 investigation of different classes of antioxidants (Nawuliuyumab concentration 10mg/ml)

We designed several formulations 19-22 (see Table 11). Wherein the preparation 19 is a composition of a product preparation on the market; the preparation 20 is prepared by substituting pentaric acid of the preparation 19 with EDTA.2Na serving as an antioxidant; formulation 21 is an optimized formulation component; the preparation 22 is prepared by substituting EDTA.2Na of the preparation 21 with EDTA.CaNa serving as an antioxidant. The appearance and SEC-HPLC purity measurements at 37 ℃ for 14 days are shown in Table 12.

TABLE 11 investigation of the different classes of antioxidants (units: mg/ml)

TABLE 12 high temperature 37. + -. 2 ℃ 14d test results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. As can be seen from the results, after 2 weeks of the high temperature test, visible foreign matter appeared in the samples of the preparation 19 and the preparation 20, and there was no significant difference in SEC purity; there was no change in visible foreign matter for formulation 21 and formulation 22, and the SEC purity of formulation 22 was higher. The experimental data show that when the preparation is prepared by using the original prescription, the stability of the pentetic acid and the EDTA.2Na on the preparation are the same, and the two have no obvious difference; in the optimized formula, the preparation (preparation 21) prepared by EDTA.CaNa as the antioxidant has higher SEC purity than the preparation (preparation 21) prepared by EDTA.2Na as the antioxidant, and the EDTA.CaNa has no side effect of reducing blood calcium reported by EDTA.2Na, so the EDTA.CaNa as the antioxidant has better safety and stability than the EDTA.2Na.

Example 6 comparison of stability of marketed formulation with self-developing agent

We designed several formulations 23-25 (see Table 13). Wherein preparation 23 is a marketed product preparation; the preparation 24 is a preparation prepared by adopting a marketed preparation auxiliary material from a self-grinding API; the preparation 25 is a preparation prepared by optimizing the composition of the preparation. The 0 day appearance and SEC-HPLC purity results are shown in Table 14; the results of appearance and SEC-HPLC purity change at 37 ℃ for 14 days are shown in Table 15.

TABLE 13 formulation on the market and self-developed formulation (unit: mg/ml)

TABLE 140 days appearance and SEC-HPLC purity results

TABLE 15 high temperature 37. + -. 2 ℃ 14d appearance and SEC-HPLC purity results

The appearance and visible foreign matter of each formulation sample after the high temperature test were visually observed. As can be seen from the results, after 2 weeks of the high temperature test, a visible foreign substance was present in formulation 24, and no change was observed in formulations 23 and 25; formulation 23 had a lower initial SEC-HPLC purity. The results of the above data indicate that the commercial formulation SEC-HPLC purity is low, while the optimized formulation (formulation 25) can keep the SEC-HPLC purity at a high level at 0 days of formulation and the high temperature growth rate is comparable to that of the commercial formulation; if the self-developed API was formulated using the marketed formulation recipe (formulation 24), both physical stability and SEC-HPLC purity were poor. Therefore, the optimized prescription can obviously improve the stability of the preparation.

Example 7 examination of the range of phosphate levels

TABLE 16 formulation recipe for different phosphate dosages

TABLE 17 high temperature 37. + -. 2 ℃ 28d appearance and SEC-HPLC purity results

According to the experimental results, the stability of the preparation is better when the concentration of the phosphate is 10-30 mM.

Example 8 sucrose dosage Range examination

Table 18 formulation of different sucrose dosages

TABLE 19 high temperature 37. + -. 2 ℃ 28d appearance and SEC-HPLC purity results

According to experimental results, the stability of the preparation is excellent when the concentration of sucrose is 75-93 mg/ml. EXAMPLE 9 examination of the amount of Tween 80

TABLE 20 formulation recipe for different amounts of Tween 80

TABLE 21 high temperature 37. + -. 2 ℃ 28d appearance and SEC-HPLC purity results

According to experimental results, the stability of the preparation is better when the concentration of the Tween 80 is 0.14-0.26 mg/ml.

Example 10 range of EDTA. CaNa

Table 22 formulation with different edta

TABLE 23 high temperature 37. + -. 2 ℃ 28d appearance and SEC-HPLC purity results

The experimental result shows that the stability of the preparation is better when the concentration of EDTA.CaNa is 0.024-0.056 mg/ml.

Example 11pH Range examination

TABLE 24 formulation at different pH ranges

TABLE 25 high temperature 40. + -. 2 ℃ 28d appearance and SEC-HPLC purity results

The experimental results show that the stability of the preparation has little difference when the pH value is 5.2-6.4.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A stable pharmaceutical preparation of an anti-PD-1 antibody, comprising an anti-PD-1 antibody, a buffer and a stabilizer, wherein the pharmaceutical preparation does not contain sodium chloride.

2. The pharmaceutical preparation according to claim 1, wherein the buffer is a phosphate buffer; optionally, the concentration of the phosphate buffer is 5-50 mM.

3. The pharmaceutical preparation according to claim 1, wherein the stabilizer is sucrose; optionally, the concentration of sucrose ranges from 50 to 150 mg/mL.

4. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation further comprises a surfactant.

5. pharmaceutical preparation according to claim 4, is characterized in that, described surfactant is selected from Tween 80, Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 65, at least one of Tween 80, Tween 81, Tween 85, poloxamer, triton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octyl glycoside, lauryl-sulfobetaine;

Preferably, described surfactant is selected from Tween 80, Tween 20, poloxamer;

More preferably, the surfactant is selected from Tween 80.

6. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation further comprises an antioxidant.

7. The pharmaceutical preparation according to claim 6, wherein the antioxidant is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, nitrilotriacetic acid, ascorbic acid, tryptophan, methionine, Glutathione, sodium thiosulfate, catalase, aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, citric acid, nicotinamide, deferoxamine, and their salts;

Preferably, the antioxidant is selected from EDTA.2Na, EDTA.2Na, and EDTA.CaNa;

More preferably, the antioxidant is EDTA.CaNa.

8 . The pharmaceutical preparation according to claim 1 , wherein the pH value of the pharmaceutical preparation is 5.0-6.5. 9 .

9. The pharmaceutical preparation of claim 1, wherein the anti-PD-1 antibody is nivolumab.

10. Use of the pharmaceutical preparation of any one of claims 1-9 in the treatment of tumors.