HK1238156B - Stable anti-pd-1 antibody pharmaceutical preparation and application thereof in medicine - Google Patents

Description

An anti-PD-1 antibody preparation and its application in medicine

Technical Field

The present invention relates to a pharmaceutical preparation comprising an anti-PD-1 antibody and an antigen-binding fragment thereof, a preparation method thereof, and uses of the preparation.

Background Art

There is a complex relationship between tumor immune escape mechanisms and the body's immune response to tumors. Tumor-specific killer T cells are biologically active in the early stages of tumor immunotherapy, but lose their ability to kill tumors as the tumor grows. Therefore, tumor immunotherapy aims to maximize the patient's immune system response to the tumor. This not only requires activating the body's existing immune system response, but also maintaining its duration and intensity, which are the key to successful tumor immunotherapy.

Programmed death-1 (PD-1) is a protein receptor discovered in 1992 that is expressed on the surface of T cells and is involved in the process of apoptosis. PD-1 belongs to the CD28 family and shares 23% amino acid identity with cytotoxic T lymphocyte antigen 4 (CTLA-4). However, its expression differs from CTLA and is primarily expressed on activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. Recent studies have found high expression of PD-L1 in human tumor tissues, including breast, lung, gastric, intestinal, renal, and melanoma cancers, and PD-L1 expression levels are closely correlated with clinical and prognostic factors. Because PD-L1 acts as a secondary signaling pathway to inhibit T cell proliferation, blocking the PD-L1/PD-1 binding pathway has become a promising emerging target in tumor immunotherapy.

WO2015/085847 discloses a new class of anti-PD-1 antibodies characterized by high affinity and long half-life, which are expected to have improved efficacy against the aforementioned diseases. However, these new anti-PD-1 antibodies are extremely unstable, making them difficult to formulate into clinically viable formulations. The PCT does not provide any specific description of how to formulate them. Therefore, further research is necessary to develop stable, clinically feasible formulations of these antibodies.

Summary of the Invention

The object of the present invention is to provide a stable anti-PD-1 antibody preparation.

The stable pharmaceutical formulation of the present invention comprises an anti-PD-1 antibody and an antigen-binding fragment thereof, a buffer, and may further comprise at least one stabilizer and, optionally, a surfactant.

In the pharmaceutical preparation of the present invention, the anti-PD-1 antibody and antigen-binding fragment thereof comprises any one or more CDR region sequences selected from the following or an amino acid sequence having at least 85% sequence homology thereto:

Antibody heavy chain variable region HCDR region sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; and

Antibody light chain variable region LCDR region sequence: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.

The amino acid sequence is shown in the following table:

The sequence homology can be screened using conventional methods in order to improve the affinity, immunogenicity, stability, or other conventional physicochemical properties and biological activities of the antibody.

A further preferred anti-PD-1 antibody has a heavy chain of SEQ ID NO: 7 and a light chain of SEQ ID NO: 8:

SEQ ID NO: 7

SEQ ID NO: 8

The concentration of the anti-PD-1 antibody can be 1-60 mg/ml, preferably 20-50 mg/ml, more preferably 35-45 mg/ml, and most preferably 40 mg/ml.

The buffering agent of the present invention is selected from one or more of acetate, citrate, succinate, and phosphate, and the phosphate can be selected from sodium dihydrogen phosphate and potassium dihydrogen phosphate; the preferred buffering agent is acetate, and its dosage is not particularly limited. In the embodiment of the present invention, for example, it is 1 to 50 mM, preferably 2 to 20 mM, more preferably 5-15 mM, and most preferably 10 mM.

The pH value of the pharmaceutical preparation of the present invention may be in the range of 4.5-6.0, preferably 4.8-5.6, and the most preferred pH value is 5.2.

The at least one stabilizer of the present invention is selected from a sugar or an amino acid. The sugar is preferably a disaccharide selected from sucrose, lactic acid, trehalose, and maltose, preferably trehalose, and most preferably α,α-trehalose dihydrate. The amount of the disaccharide is not particularly limited. In embodiments of the present invention, it is, for example, 30 to 120 mg/ml, preferably 60 to 100 mg/ml, more preferably 85 to 95 mg/ml, and most preferably 90 mg/ml.

The surfactant of the present invention can be selected from polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, and the polyoxyethylene sorbitan fatty acid ester can be selected from polysorbate 20, 40, 60 or 80. The amount of the surfactant used does not need to be particularly limited. In the embodiment of the present invention, it is, for example, 0.01 to 1 mg/ml, preferably 0.05 to 0.5 mg/ml, more preferably 0.1 to 0.4 mg/ml, and most preferably 0.2 mg/ml.

The stable pharmaceutical preparation of the present invention is an injectable pharmaceutical preparation.

In one embodiment of the present invention, the stable pharmaceutical formulation consists of an anti-PD-1 antibody, a buffer, a disaccharide, a surfactant, and optionally includes water.

In one embodiment of the present invention, the stable pharmaceutical formulation comprises:

An anti-PD-1 antibody, the human antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8; and

(i) 90 mg/ml α,α-trehalose dihydrate and 10 mM acetate buffer at pH 5.2; or

(ii) 90 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2; or

(iii) 90 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.4; or

(iv) 60 mg/ml α,α-dihydrate trehalose, 0.4 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.0; or

(v) 60 mg/ml α,α-dihydrate trehalose, 0.1 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.2; or

(vi) 60 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2; or

(vii) 30 mg/ml α,α-dihydrate trehalose, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 4.8; or

(viii) 30 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 30 mM acetate buffer at pH 5.2; or

(ix) 30 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.6.

The injectable pharmaceutical preparation can be an injection solution or further prepared into a freeze-dried powder. The freeze-dried powder can be prepared by conventional methods in the art.

The present invention also provides an injection solution formed by reconstructing the freeze-dried powder after reconstitution, which can be directly used for injection.

The pharmaceutical preparation of the present invention can effectively inhibit antibody aggregation and deamidation, thereby preventing degradation of the antibody product, resulting in a stable injectable composition that can be stored for 6 months at 25°C and for 12 months at 2-8°C. Furthermore, the pharmaceutical composition of the present invention protects against protein oxidative degradation and is compatible with glass and stainless steel containers, maintaining stability in these containers.

The pharmaceutical preparation of the present invention is used to prevent or treat PD-1-mediated diseases or conditions, wherein the disease is preferably cancer; more preferably, it is a cancer expressing PD-L1; the cancer is most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma; and most preferably, non-small cell lung cancer, melanoma, and kidney cancer.

The pharmaceutical preparation of the present invention is used for preparing a drug for preventing or treating a PD-1-mediated disease or condition, wherein the disease is preferably cancer; more preferably, a cancer expressing PD-L1; most preferably, the cancer is breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, or melanoma; and most preferably, non-small cell lung cancer, melanoma, and kidney cancer.

A method for preventing or treating a PD-1-mediated disease or condition, wherein the disease is preferably cancer; more preferably a cancer expressing PD-L1; most preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma, and kidney cancer, comprising administering the pharmaceutical formulation of the present invention.

the term

The present invention relates to a stable liquid pharmaceutical preparation comprising a high concentration of antibodies against PD-1.

The “antibody” mentioned in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to LCDR1, LCDR2, and LCDR3 of the light chain variable domain, and HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting murine CDR sequences into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of murine protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.

The term "pharmaceutical formulation" means a preparation that is in a form that permits the biological activity of the active ingredient to be clearly effective and that contains no additional components that are toxic to a subject to which the formulation is to be administered.

The term "liquid" as used herein in connection with a formulation according to the present invention refers to a formulation that is liquid at a temperature of at least about 2°C to about 8°C at atmospheric pressure.

The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacturing, storage and use. By Cleland, J.L., M.F. Powell, et al. (1993). "The development of stable protein fomulations: a close look at protein aggregation, deamidation, and oxidation." Crit Rev Ther Drug Carrier Syst 10(4): 307-77, Wang, W. (1999). "Instability, stabilization, and formulation of liquidprotein pharmaceuticals." Int J Pharm 185(2): 129-88., Wang, W. (2000). "Lyophilization and development of solid protein pharmaceuticals." Int J Pharm 203 (1-2): 1-60. and Chi, E.Y., S. Krishnan, et al. (2003). "Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation." Pharm Resgregation. 20(9):1325-36 reviews the chemical and physical degradation pathways of protein drugs. Stabilizers include, but are not limited to, sugars as defined below, amino acids, polyols, surfactants, antioxidants, preservatives, cyclodextrins, polyethylene glycols, such as PEG 3000, 3350, 4000, 6000, albumins, such as human serum albumin (HSA), bovine serum albumin (BSA), salts, such as sodium chloride, magnesium chloride, calcium chloride, chelating agents, such as EDTA, as defined below. The stabilizers specifically used in the present invention are selected from sugars. More specifically, the stabilizers are selected from sucrose, trehalose, and sorbitol. The stabilizer can be present in the formulation in an amount of 30 mg/ml to 100 mg/ml, preferably in an amount of 60 mg/ml to 90 mg/ml. More specifically, sucrose or trehalose is used as a stabilizer in an amount of 90 mg/ml.

A "stable" formulation is one in which the protein (eg, antibody) contained therein substantially retains its physical and chemical stability, and thus its biological activity, upon storage.

"Stable liquid pharmaceutical antibody formulations" are liquid antibody formulations that are not significantly altered for at least 12 months, particularly 2 years, and more particularly 3 years at refrigerated temperatures (2-8°C). The criteria for stability are as follows: when measured by size exclusion chromatography (SEC-HPLC), no more than 10%, particularly 5%, of the antibody monomers are degraded. In addition, by visual analysis, the solution is colorless or clear to a slight milky white. The protein concentration of the formulation has a change of no more than +/- 10%. No more than 10%, particularly 5%, of aggregation is formed. Stability is measured by methods known in the art such as UV spectroscopy, size exclusion chromatography (SEC-HPLC), ion exchange chromatography (IE-HPLC), turbidimetry, and visual inspection.

The terms "programmed cell death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-I" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one common epitope with PD-1. The complete PD-1 sequence can be found at NCBI Reference Sequence: NM_005018.1.

The terms "anti-PD-1 antibody," "antibodies against PD-1," and "antibodies against PD-1 protein" refer to antibodies that bind to PD-1 protein with sufficient affinity so that the antibodies can be used as diagnostic and/or therapeutic agents in targeting PD-1 protein. As used herein, the term "binding to PD-1 protein" refers to the binding of an antibody to PD-1 protein in a BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in an ELISA, wherein purified PD-1 protein or PD-1 protein CHO transfectants are coated on microtiter plates.

The concentration of the antibody against PD-1 protein contained in the pharmaceutical preparation is in the range of 1 mg/ml to 60 mg/ml, preferably in the range of 20 mg/ml to 50 mg/ml, and most preferably 40 mg/ml.

The term "surfactant" as used herein refers to a pharmaceutically acceptable excipient for protecting protein formulations from physical stress (such as stirring and shearing). Examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tweens), polyoxyethylene alkyl ethers (such as those sold under the trademark Brij ^™ ), and polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic). Examples of polyoxyethylene sorbitan fatty acid esters include polysorbate 20 (sold under the trademark Tween 20 ^™ ) and polysorbate 80 (sold under the trademark Tween 80 ^™ ).

The term "buffer" as used herein refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer, or a mixture thereof. Buffers of particular interest include citrate buffer or acetate buffer, which are pH-regulated with an acid or base known in the art. The above-mentioned buffer is typically used in an amount of about 1 mM to 50 mM, preferably about 10 mM to 30 mM, most preferably 10 mM. Independent of the buffer used, the pH can be adjusted to a value within the range of 4.5-6.0, particularly to a value within the range of 4.8-5.6, most particularly to pH 5.2, with an acid or base known in the art (e.g., hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide).

In certain embodiments, the stabilized anti-PD-1 antibody pharmaceutical formulations of the present invention may further comprise an antioxidant as a second stabilizer. An "antioxidant" is a pharmaceutically acceptable excipient that prevents oxidation of the active pharmaceutical ingredient. Antioxidants include, but are not limited to, chelating agents such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, benzyl alcohol, and n-acetylcysteine.

The term "sugar" as used herein refers to a monosaccharide or oligosaccharide. Monosaccharides are monomeric carbohydrates that cannot be hydrolyzed by acid, including monosaccharides and their derivatives, such as amino sugars. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid. Oligosaccharides are branched or straight-chain carbohydrates consisting of more than one monomeric sugar unit connected via glycosidic bonds. The monomeric sugar units in an oligosaccharide can be the same or different. Depending on the number of monomeric sugar units, oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, and the like. Unlike polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose, and raffinose. Specifically, the sugar is selected from sucrose and trehalose.

As used herein, the term "amino acid" refers to a pharmaceutically acceptable organic molecule having an amino group located at the alpha position relative to the carboxyl group. Examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. Amino acids are typically used in amounts of about 5-500 mM, particularly in amounts of about 5 to about 200 mM, and more particularly in amounts of about 100 to about 150 mM.

The term "stabilizer" also includes cryoprotectants. The term "cryoprotectant" refers to a pharmaceutically acceptable excipient that protects unstable active ingredients (e.g., proteins) from destabilizing conditions during freeze-drying, subsequent storage, and reconstitution. Cryoprotectants include, but are not limited to, sugars, polyols (e.g., sugar alcohols), and amino acids. Specifically, cryoprotectants can be selected from: sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine, galactosamine, N-methylglucosamine ("methylglucamine"), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine, or mixtures thereof. The cryoprotectant is preferably a disaccharide. The present invention has surprisingly found that disaccharides are superior to monosaccharides, polyols, and amino acids in terms of the stability of the formulation.

The pharmaceutical formulation may also contain a tonicity agent. As used herein, the term "tonicity agent" refers to a pharmaceutically acceptable tonicity agent used to adjust the tonicity of the formulation. The formulation may be hypotonic, isotonic, or hypertonic. Isotonicity generally refers to the relative osmotic pressure of a solution, often relative to the osmotic pressure of human serum. The formulation according to the present invention may be hypotonic, isotonic, or hypertonic, but is preferably isotonic. An isotonic formulation is a liquid or a liquid reconstituted from a solid form (e.g., from a lyophilized form) and refers to a solution that has the same tonicity as some other solution to which it is compared (such as physiological saline solution and serum). Suitable tonicity agents include, but are not limited to, sodium chloride, potassium chloride, glycerol, and any component selected from the following: amino acids, sugars, especially glucose. Tonicity agents are typically used in amounts of about 5 mM to about 500 mM. Within stabilizers and tonicity agents, there is a group of compounds that can act in two ways, i.e., they can be both stabilizers and tonicity agents. Examples of these can be found in the following group: sugars, amino acids, polyols, cyclodextrins, polyglycols and salts. An example of a sugar that can be both a stabilizer and a tonicity agent is trehalose.

The pharmaceutical preparation may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Sterilization procedures and the inclusion of various antibacterial and antifungal agents (e.g., methylparaben, chlorobutanol, phenol, sorbic acid, etc.) ensure that microorganisms are prevented from occurring. Preservatives are typically used in amounts ranging from about 0.001 to about 2% (w/v). Preservatives include, but are not limited to, ethanol, benzyl alcohol, phenol, m-cresol, parachloro-m-cresol, methylparaben or propylparaben, and benzalkonium chloride.

The stable pharmaceutical formulation of the antibody against PD-1 protein according to the present invention can be used to prevent or treat PD-1-mediated diseases or conditions, wherein the disease is preferably cancer; more preferably, cancer expressing PD-L1; most preferably, the cancer is breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma; most preferably, non-small cell lung cancer, melanoma, and renal cancer.

The stable anti-PD-1 antibody pharmaceutical formulation according to the present invention can be administered by intravenous (i.v.), subcutaneous (s.c.) or any other parenteral administration route, such as those known in the pharmaceutical art.

In view of their high stability, the pharmaceutical preparation according to the present invention can be intravenously administered without the need for an in-line filter, and thus is much more convenient to operate than conventional preparations that could be administered with an in-line filter. The in-line filter must be installed in the infusion line of an intravenous drug to prevent the administration of any particles, air or microorganisms that may be present in the intravenous solution or line. 5-20 micron-sized and larger particles have the ability to block the blood flow through the pulmonary capillaries, which can cause complications such as pulmonary embolism. Foreign particles can also cause phlebitis at the injection site, and the filter can help reduce the incidence of phlebitis.

Stable formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

The stable anti-PD-1 antibody pharmaceutical formulation according to the present invention can be prepared by methods known in the art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, freeze drying, reconstitution and combinations thereof. Examples of preparations according to the present invention can be found below.

The stable anti-PD-1 antibody pharmaceutical formulation according to the present invention can also be in a lyophilized form or in a liquid form reconstituted from a lyophilized form. The "lyophilized form" is prepared by freeze-drying methods known in the art. The lyophilized powder often has a residual water content of about 0.1-5% (w/w) and exists as a powder or a physically stable cake. A "reconstituted form" can be obtained from the lyophilized powder by rapid dissolution after adding a reconstitution medium. Suitable reconstitution media include, but are not limited to: water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% (w/v) glucose), a solution containing a surfactant (e.g., 0.01% (w/v) polysorbate 20, and a pH buffer solution (e.g., acetate buffer solution).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of buffer system on the thermal stability of anti-PD-1 antibody

FIG2 shows the effect of sugars on the thermal stability of anti-PD-1 antibodies.

DETAILED DESCRIPTION

The present invention is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Experimental methods in the examples of the present invention where specific conditions are not specified are generally performed under conventional conditions or the conditions recommended by the raw material or product manufacturers. Reagents where the specific sources are not specified are conventional reagents purchased from the market.

Example

The preparation process of the present invention is as follows:

Step 1: Before filtering the anti-PD-1 antibody stock solution, sample the solution for protein concentration. After passing the control, filter the solution through a 0.22μm PVDF filter, and collect the filtrate. The PD-1 antibody has the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8, and was prepared according to the method disclosed in WO2015/085847.

Step 2: Adjust the filling volume to 5.3 ml, fill the filtrate into a 20 ml vial, half-stopper it, and take samples at the beginning, middle and end of filling to detect the difference in filling volume.

Step 3: Place the filled and stoppered liquid into the freeze-drying chamber and freeze-dry it according to the following freeze-drying process. After the freeze-drying process is completed, vacuum stopper is added.

Step 4: Turn on the capping machine, add aluminum caps, and start capping.

Step 5: Visual inspection to confirm that the product has no defects such as collapse, inaccurate filling, etc. Print and affix vial labels; print carton labels, fold cartons, pack boxes, and apply box labels.

The concentration of the control protein was determined by detecting the absorption peak at 280 nm using an ultraviolet spectrophotometer (manufacturer: Thermo, model Nanodrop 2000).

The 0.22μm PVDF filter element uses Millipak-100.

The filling machine is Chutian Technology KGS8/2-X2 linear filling and stoppering machine.

The central control loading difference detection uses an electronic balance (manufacturer: Sartorius, model BSA423S) for weighing.

Freeze drying was performed using a Tofflon Lyo-B (SIP, CIP) vacuum freeze dryer.

The capping machine uses Shandong Penglai DZG-130 knife-type automatic capping machine.

The visual inspection of the appearance was performed using Tianjin Jingtuo YB-2A clarity tester.

In the examples, HPLC (SEC and IEC) detection was performed using an Agilent 1200DAD high pressure liquid chromatograph (TSK gelSuperSW mAb HR 300×7.8 mm chromatographic column and ProPac ^™ WCX-10 BioLC ^™ , 250×4 mm chromatographic column).

The protein thermal denaturation temperature (Tm) was measured using a GE MicroCal VP-Capillary DSC differential scanning calorimeter.

The average particle size of DLS (Dynamic Light Scattering) was measured using a Malvern Zetasizer Nano ZS nanoparticle size analyzer.

Example 1

Anti-PD-1 antibodies were formulated into formulations containing 10 mM sodium acetate, 90 mg/mL α,α-dihydrate trehalose, and 0.2 mg/mL polysorbate 20 at a pH of 4.8-5.6, with an antibody protein concentration of 40 mg/mL. Each formulation was filtered and filled into 20 mL neutral borosilicate glass injection vials at 5 mL/vial for lyophilization. The vials were sealed with halogenated butyl rubber stoppers using freeze-dried sterile powder for injection. The lyophilized products were stored at 25°C and 40°C for stability analysis. The results showed that the anti-PD-1 antibodies were very stable at pH 4.8-5.6.

Table 1. Effect of pH on the degradation of anti-PD-1 antibodies

Example 2

Prepare the anti-PD-1 antibody preparation at a protein concentration of 1 mg/mL in the following buffer:

Buffer 1: 10 mM sodium acetate, pH 5.0.

2) Buffer 2: 10 mM sodium hydrogen phosphate (citric acid), pH 5.0;

3) Buffer 3: 10 mM sodium succinate, pH 5.0;

4) Buffer 4: 10 mM sodium citric acid, pH 5.0.

Differential scanning calorimetry (DSC) was used to measure the thermal stability of the anti-PD-1 antibody in each formulation. Analysis of the thermal denaturation midpoint temperature (Tm) of the drug showed, as shown in Figure 1, that the stability of the anti-PD-1 antibody in acetate buffer was significantly better than in succinate, citrate, and disodium hydrogen phosphate/citrate buffer systems.

Example 3

DSC technology was used to screen the PD-1 antibody formulation with a protein concentration of 1 mg/mL and formulations containing different sugars and concentrations:

Buffer 1: 10 mM sodium acetate, 90 mg/mL sucrose, pH 5.2.

Buffer 2: 10 mM sodium acetate, 30 mg/mL α,α-trehalose dihydrate, pH 5.2.

3) Buffer 3: 10 mM sodium acetate, 60 mg/mL α,α-trehalose dihydrate, pH 5.2;

4) Buffer 4: 10 mM sodium acetate, 90 mg/mL α,α-trehalose dihydrate, pH 5.2;

2 , the Tm value shows that the thermal stability of the anti-PD-1 antibody is best when the concentration of α,α-dihydrate trehalose reaches 90 mg/mL.

Example 4

Anti-PD-1 antibody formulations were prepared at a PD-1 antibody protein concentration of 40 mg/mL in a buffer containing 10 mM sodium acetate, 90 mg/mL α,α-dihydrate trehalose, and pH 5.2 in the following surfactant buffers:

1) Does not contain surfactant;

2) 0.1 mg/mL polysorbate 20;

3) 0.2 mg/mL polysorbate 20;

4) 0.3 mg/mL polysorbate 20;

5) 0.4 mg/mL polysorbate 20;

6) 0.2 mg/mL polysorbate 80;

Each formulation was filled into 20mL vials at a rate of 5mL/vial and sealed with a film-coated rubber stopper. The vials were shaken at 500 rpm on a 25°C thermostat. Stability results demonstrated that 0.1-0.4mg/mL of polysorbate 20 effectively prevented the aggregation of anti-PD-1 antibodies and the formation of large, agglomerated particles.

Table 2. Effect of surfactants on the aggregation of anti-PD-1 antibodies at 25°C and 500 rpm shaking

Example 5

The anti-PD-1 antibody was formulated at 40 mg/mL in 10 mM sodium acetate, 90 mg/mL α,α-trehalose dihydrate, and 0.2 mg/mL polysorbate 20, pH 5.2. The antibody was filled into 20 mL vials at 5 mL/vial and lyophilized, then sealed with lyophilization stoppers. The lyophilized samples were exposed to strong light at 4500 ± 500 L/min, or to a high temperature at 40 ± 2°C for 10 days, or subjected to three cycles of low temperature between 4°C and 40°C, or three freeze-thaw cycles between -20°C and 40°C. Drug stability was assessed using protein content, purity, and activity assays. The results demonstrated that the anti-PD-1 antibody was relatively stable in this formulation and remained stable after exposure to strong light, high or low temperatures, and freeze-thaw cycles.

Table 3. Stability of anti-PD-1 antibody formulations under intense light, high or low temperatures, and freeze-thaw cycles

Example 6

The anti-PD-1 antibody was formulated at 40 mg/mL in 10 mM sodium acetate, 90 mg/mL α,α-trehalose dihydrate, and 0.2 mg/mL polysorbate 20, pH 5.2. The formulations were filled into glass bottles and 316L stainless steel tanks, respectively, and left at room temperature for 24 hours. Protein content and purity analysis showed that the anti-PD-1 antibody was stable over a 24-hour period. The formulation was compatible with the 316L stainless steel tank.

Table 4. Stability of anti-PD-1 antibodies in stainless steel tanks

Example 7

Anti-PD-1 antibody was prepared at 40 mg/mL in 10 mM sodium acetate, 90 mg/mL α,α-trehalose dihydrate, and 0.2 mg/mL polysorbate 20, pH 5.2. 20 mL vials were filled at 5 mL/vial and lyophilized at primary drying temperatures of -15°C, -10°C, and -5°C, respectively, and sealed with lyophilized rubber stoppers. The lyophilized product was stored at 25°C for stability analysis. Results showed that the optimal primary drying temperature for the lyophilization process was between -10°C and -5°C.

Table 5 Stability of anti-PD-1 antibody preparations prepared using different primary drying processes

Example 8 Other formulations

The stable pharmaceutical formulation provided by the present invention comprises: an anti-PD-1 antibody (heavy chain amino acid sequence of SEQ ID NO: 7 and light chain amino acid sequence of SEQ ID NO: 8); and a combination of a stabilizing buffer selected from the following:

(i) 90 mg/ml α,α-trehalose dihydrate, and 10 mM acetate buffer, pH 5.2;

(ii) 90 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(iii) 90 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.4;

(iv) 60 mg/ml α,α-dihydrate trehalose, 0.4 mg/ml polysorbate 20, and 20 mM acetate buffer, pH 5.0;

(v) 60 mg/ml α,α-dihydrate trehalose, 0.1 mg/ml polysorbate 20, and 20 mM acetate buffer, pH 5.2;

(vi) 60 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(vii) 30 mg/ml α,α-dihydrate trehalose, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 4.8;

(viii) 30 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 30 mM acetate buffer at pH 5.2; or

(ix) 30 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.6.

In the above examples, the concentration of anti-PD-1 antibody is in the range of 1 mg/ml to 60 mg/ml, preferably 20-50 mg/ml, more preferably 35-45 mg/ml, and most preferably 40 mg/ml. The feasible schemes may be selected from, but not limited to, the following combinations:

(1) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(2) 1 mg/ml anti-PD-1 antibody, 30 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 4.5;

(3) 20 mg/ml anti-PD-1 antibody, 60 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 1 mM acetate buffer at pH 4.8;

(4) 35 mg/ml anti-PD-1 antibody, 85 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 2 mM acetate buffer at pH 5.6;

(5) 45 mg/ml anti-PD-1 antibody, 95 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 5 mM acetate buffer at pH 6.0;

(6) 50 mg/ml anti-PD-1 antibody, 100 mg/ml α,α-dihydrate trehalose, 0.2 mg/ml polysorbate 20, and 15 mM acetate buffer at pH 5.2;

(7) 60 mg/ml anti-PD-1 antibody, 90 mg/ml sucrose, 0.2 mg/ml polysorbate 400, and 30 mM acetate buffer at pH 5.2;

(8) 40 mg/ml anti-PD-1 antibody, 90 mg/ml lactose, 0.2 mg/ml polysorbate 60, and 50 mM acetate buffer at pH 4.5;

(9) 40 mg/ml anti-PD-1 antibody, 90 mg/ml trehalose, 0.2 mg/ml polysorbate 80, and 10 mM acetate buffer at pH 5.2;

(10) 40 mg/ml anti-PD-1 antibody, 90 mg/ml maltose, 0.2 mg/ml polyoxyethylene hydrogenated castor oil, and 10 mM acetate buffer at pH 5.2;

(11) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 0.01 mg/ml glycerol fatty acid ester, and 10 mM acetate buffer at pH 5.2;

(12) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 0.05 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(13) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(14) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 0.5 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2;

(15) 40 mg/ml anti-PD-1 antibody, 90 mg/ml α,α-dihydrate trehalose, 1 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2.

Claims (30)

1. A stable anti-PD-1 antibody drug formulation, comprising the following components: 1 mg/ml - 60 mg/ml anti-PD-1 antibody; 30mg/ml-120mg/ml α,α-trehalose dihydrate; 0.01 mg/ml - 1 mg/ml polysorbate 20; and 2mM-30mM acetate buffer; The anti-PD-1 antibody comprises the heavy chain shown in SEQ ID NO:7 and the light chain shown in SEQ ID NO:8; and the pH value of the anti-PD-1 antibody drug formulation is 4.5-6.0. 2. The pharmaceutical formulation according to claim 1, wherein the concentration of the anti-PD-1 antibody is 20 mg/ml to 50 mg/ml. 3. The pharmaceutical formulation according to claim 2, wherein the concentration of the anti-PD-1 antibody is 35 mg/ml to 45 mg/ml. 4. The pharmaceutical formulation according to claim 3, wherein the concentration of the anti-PD-1 antibody is 40 mg/ml. 5. The pharmaceutical formulation according to claim 1, wherein the concentration of the acetate buffer is 5 mM-15 mM. 6. The pharmaceutical formulation according to claim 5, wherein the concentration of the acetate buffer is 10 mM. 7. The pharmaceutical preparation according to claim 1, wherein the pH value of the preparation is 4.8-5.6. 8. The pharmaceutical preparation according to claim 1, wherein the pH value of the preparation is 5.2. 9. The pharmaceutical preparation according to claim 1, wherein the concentration of α,α-trehalose dihydrate is from 60 mg/ml to 100 mg/ml. 10. The pharmaceutical preparation according to claim 8, wherein the concentration of α,α-trehalose dihydrate is from 85 mg/ml to 95 mg/ml. 11. The pharmaceutical preparation according to claim 10, wherein the concentration of α,α-trehalose dihydrate is 90 mg/ml. 12. The pharmaceutical formulation according to claim 1, wherein the concentration of polysorbate 20 is from 0.05 mg/ml to 0.5 mg/ml. 13. The pharmaceutical formulation according to claim 12, wherein the concentration of polysorbate 20 is from 0.1 mg/ml to 0.4 mg/ml. 14. The pharmaceutical formulation according to claim 13, wherein the concentration of polysorbate 20 is 0.2 mg/ml. 15. The pharmaceutical formulation of claim 1, comprising: an anti-PD-1 antibody, said antibody comprising the heavy chain amino acid sequence of SEQ ID NO:7 and the light chain amino acid sequence of SEQ ID NO:8; and (i) 90 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2; or (ii) 90 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.4; or (iii) 60 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.0; or (vi) 60 mg/ml α,α-trehalose dihydrate, 0.1 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.2; or (v) 60 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2; or (vi) 30 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 4.8; or (vii) 30 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 30 mM acetate buffer at pH 5.2; or (viii) 30 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.6. 16. The stable anti-PD-1 antibody pharmaceutical formulation according to any one of claims 1-15, wherein the pharmaceutical formulation is an injectable pharmaceutical formulation and further comprises water for injection. 17. A lyophilized powder obtained from the pharmaceutical preparation of claim 16. 18. The freeze-dried powder according to claim 17, wherein the optimal drying temperature for the freeze-drying process is -10℃ to -5℃. 19. The lyophilized powder of claim 17, and the injection solution obtained by reconstitution therefrom. 20. A stable anti-PD-1 antibody pharmaceutical preparation according to any one of claims 1-15, for the prevention or treatment of PD-1 mediated diseases or conditions. 21. The stable anti-PD-1 antibody drug formulation according to claim 20, wherein the disease is cancer. 22. The stable anti-PD-1 antibody drug formulation according to claim 21, wherein the cancer is a cancer expressing PD-L1. 23. The stable anti-PD-1 antibody drug formulation according to claim 21, wherein the cancer is selected from breast cancer, lung cancer, gastric cancer, colorectal cancer, kidney cancer, and melanoma. 24. The stable anti-PD-1 antibody drug formulation according to claim 21, wherein the cancer is selected from non-small cell lung cancer, melanoma, and renal cell carcinoma. 25. Use of a stable anti-PD-1 antibody pharmaceutical formulation according to any one of claims 1-16, or of a lyophilized powder according to any one of claims 17-19, for the preparation of a pharmaceutical product. 26. The use according to claim 25, wherein the medicament is used for the prevention or treatment of PD-1 mediated diseases or conditions. 27. The use according to claim 26, wherein the disease is cancer. 28. The use according to claim 27, wherein the cancer is a cancer expressing PD-L1. 29. The use according to claim 27, wherein the cancer is selected from breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma. 30. The use according to claim 29, wherein the cancer is selected from non-small cell lung cancer, melanoma, and kidney cancer.