STABLE ANTI-PD-1 ANTIBODY PHARMACEUTICAL PREPARATION AND APPLICATION THEREOF IN MEDICINE

Abstract

A stable anti-PD-1 antibody pharmaceutical preparation and an application thereof in a medicine. The anti-PD-1 antibody pharmaceutical preparation comprises an anti-PD-1 antibody, a buffer, and can further comprise at least one type of stabilizer, and optionally can further con a surfactant. The anti-PD-1 antibody pharmaceutical preparation of the present invention can effectively suppress antibody aggregation and deamidation, thereby preventing degradation of an antibody product, obtaining a stable injected pharmaceutical preparation.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 15/761,552, filed Mar. 20, 2018, which is a Section 371 of International Application No. PCT/CN20161098982, filed Sep. 14, 2016, which was published in the Chinese language on Apr. 6, 2017, under International Publication No. WO 2017/054646 A1, which claims priority under 35 119(b) to Chinese Application No. 201510629020,X, filed Sep. 28, 2015, the disclosures of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “065825_54US2_Sequence_Listing” and a creation date of Aug. 17, 2020, and having a size of 8 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical formulation comprising an anti-PD-1 antibody or antigen-binding fragment thereof, a manufacture process of the formulation and the use of the formulation.

BACKGROUND OF THE INVENTION

There is extremely complex relationship between tumor immune escape mechanism and body's immune response to tumors. In early stage of cancer immunotherapy, tumor specific killer T cells have biological activity, but lose the capability of killing in the late stage of tumor growth. In order to enhance the patient's own immune system response to tumor to the maximum, the key of tumor immunotherapy is not only to activate the existing immune system response, but also to maintain the duration and intensity of the immune system response.

Programmed death-1 (PD1), found in 1992, is a protein receptor expressed on T cell surface, and is involved in cell apoptosis. PD-1 belongs to CD28 family, exhibits 23% amino acid homology with cytotoxic T lymphocyte antigen 4 (CTLA-4), unlike CTLA4. PD-1 is mainly expressed in activated T cells B cells and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2 respectively. New studies have detected high expression of PD-L1 protein in human tumor tissues such as breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma, and the expression level of PD-L1 is closely related to patient's clinical response and prognosis. Since PD-L1 inhibits T cell proliferation in the second signaling pathway, PD-L1 becomes a very promising new target in field of cancer immunotherapy by blocking the binding of PD-1 to PD-L1.

WO2015/085847 discloses a novel anti-PD-1 antibody, which is characterized by high affinity and long half-life, and is expected to have a better therapeutic effect on the above-mentioned diseases. However, these new anti-PD-1 antibodies are extremely unstable, and difficult to be formulated into the form of clinically available preparation. There is no PCT application describing how they are formulated. Therefore, it is necessary to carry out in-depth study of these antibodies in order to obtain a formulation for stable and convenient clinical use.

SUMMARY OF THE INVENTION

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

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

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

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

and

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

The amino acid sequences are shown in the following table:

	Anti-PD-1 antibody
Domain	Sequence	SEQ ID NO
VH	HCDR1	SYMMS	1
	HCDR2	TISGGGANTYYPDSVKG	2
	HCDR3	QLYYFDY	3
VL	LCDR1	LASQTIGTWLT	4
	LCDR2	TATSLAD	5
	LCDR3	QQVYSIPWT	6

The sequence homology may be derived from conventional methods for improving the affinity or immunogenicity or stability of the antibody or other conventional physical and chemical properties or biological activity.

A further preferred anti-PD-1 antibody comprises heavy chain amino acid sequence of SEQ ID NO: 7 and light chain amino acid sequence of SEQ ID NO: 8.

SEQ ID NO: 7
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVA
TISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
QLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSSTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV
YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
K,
SEQ ID NO: 8
DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIY
TATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTF
GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC.

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

The buffer of the present invention is one or more selected from the group consisting of acetate, citrate, succinate, and phosphate, wherein the phosphate is selected from the group consisting of sodium dihydrogen phosphate and potassium dihydrogen phosphate; the preferred buffer is acetate, and the amount thereof is not particularly limited in the embodiment of the present invention, for example, 1 to 50 mM, preferably 2 to 20 mM, more preferably 5 to 15 mM, and most preferably 10 mM.

The pH of the formulation in the present invention may range nom 4.5 to 6.0, preferably from 4.8 to 5.6, most preferably pH 5.2.

The at least one stabilizer of the present invention is selected from saccharide or amino acid. Wherein the saccharide is preferably a disaccharide selected from the group consisting of sucrose, lactose, trehalose and maltose, preferably trehalose, most preferably α,α-trehalose dihydrate. The amount of the disaccharide used is not particularly limited in the embodiment of the present invention, for example, 30 to 120 mg/ml, preferably 60 to 100 mg/ml, more preferably 85 to 95 mg/ml, most preferably 90 mg/ml.

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

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

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

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

anti-PD-1 antibody, wherein the humanized antibody comprises heavy chain amino acid sequence of SEQ ID NO: 7 and light chain amino acid sequence of SEQ ID: 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-trehalose dihydrate, 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-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.0; or

(v) 60 mg/ml α, α-trehalose dihydrate, 0.1 mg/ml polysorbate 2.0. and 20 mM acetate buffer at pH 5,2; or

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

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

(viii) 30 mg/ml α,α-trehalose dihydrate, 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 formulation may be in the form of an injection or further prepared in a lyophilized form. The lyophilized powder may be prepared by conventional methods in the art.

The present invention also provides an injection obtained by reconstitution of the lyophilized powder, and can be directly used for injection.

The pharmaceutical formulation of the present invention can effectively suppress aggregation and deamidation of antibody, thereby prevent degradation of an antibody product, obtaining a stable injectable composition which can be stored for 6 months at 25° C. and stable for 12 month at 2-8° C. Moreover, the pharmaceutical composition of the present invention has a protective effect on the oxidative degradation of the protein, and can be compatible with glass and stainless steel containers and stably present in these containers.

The pharmaceutical formulation of the present invention is used for the prophylaxis or treatment of PD-1 mediated diseases or disorders, wherein the disease or disorder is preferably cancer; more preferably a cancer expressing PD-L1; most preferably cancer selected from breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma and kidney cancer.

The use of the pharmaceutical formulation of the present invention, in the preparation of medicament for prophylaxis or treatment of PD-1 mediated disease or disorder, wherein the disease or disorder is preferably cancer; more preferably a cancer expressing PD-L1; wherein the cancer is most preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma and kidney cancer.

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

TERMS

The present invention relates to a stable pharmaceutical liquid formulation comprising a high concentration of an antibody against PD-1.

As used herein, “Antibody” refers to immunoglobulin, a four-peptide chain structure formed by two identical heavy chains and two identical light chains connected together by disulfide bond.

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

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 the antigen-binding function of an antibody can be realized by fragments of a full-length 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 domains of an antibody that mainly contribute to antigen binding. One of the most commonly used definitions for the six CDRs was provided by Kabat E. A. et al (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, Kabat definition of CDRs only applies to LCDR1, LCDR2 and LCDR3 of the light chain variable domain, as well as to HCDR1, HCDR2 and HCDR3 of the heavy chain variable domain.

In the present invention, an antibody light chain described herein comprises a light chain variable region and a light chain constant region, which can include a human or murine κ, λ chain or a variant thereof.

In the present invention, an antibody heavy chain described herein comprises a heavy chain variable region and a heavy chain constant region, which can include the heavy chain constant region of human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof

The term “chimeric antibody”, is an antibody which is formed by fusing the variable region of murine antibody with constant region of human antibody, the chimeric antibody can alleviate the murine antibody-induced immune response. To establish chimeric antibody, hybridoma secreting specific murine monoclonal antibody shall be established first, variable region gene is cloned from murine hybridoma, then constant region gene of a desired human antibody is cloned according requirement, the murine variable region gene is connected to human constant region gene to form a chimeric gene which can be inserted into an expression carrier, and finally the chimeric antibody molecule is expressed in the eukaryotic or prokaryotic system.

The term “humanized antibody”, also known as CDR-grafted antibody, refers to an antibody generated by murine CDR sequences grafted into human antibody variable region framework, comprising different types of sequences of human germline antibody framework. Humanized antibody avoids allogenic reaction induced by chimeric antibody which carrys a large number of murine components. Such framework sequences can be obtained from public DNA database covering germaline antibody gene sequences or obtained from published references.

The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the clearly effective biological activity of the active ingredients, and contain no additional components which are toxic to the subjects to which the formulation is administered.

The term “liquid” as used herein in connection with the formulation according to the invention denotes a formulation which is liquid at a temperature of at least about 2° C. to about 8° C. under atmospheric pressure.

The term “stabilizer” denotes a pharmaceutical acceptable excipient, which protects the active pharmaceutical ingredient and/or the formulation from chemical and/or physical degradation during manufacturing, storage and application. Chemical and physical degradation pathways of protein pharmaceuticals are reviewed by Cleland, J. L., M. F. Powell, et al. (1993). “The development of stable protein formulations: 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 liquid protein 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. Krishna, et al. (2003). “Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation.” Pharm Res 20(9): 1325-36. Stabilizers include but are not limited to saccharide, amino acids, polyols, surfactants, anti-oxidants, preservatives, cyclodextrines, polyethylenglycols (e.g. PEG 3000, 3350 4000, 6000), albumin (e.g. human serum albumin (HSA), bovines serum albumin (BSA)), salts (e.g. sodium chloride, magnesium chloride, calcium chloride), chelators (e.g. EDTA) as hereafter defined. The stabilizer specifically used in the present invention is selected from saccharide. More specifically, the stabilizer is selected from the group consisting of sucrose, trehalose, and sorbitol. The stabilizer may be present in the formulation in an amount of from 30 mg/ml to 100 mg/ml, preferably from 60 mg/ml to 90 mg/ml. More specifically, sucrose or trehalose was used as a stabilizer in an amount of 90 mg/ml.

A “stable” formulation is one in which the protein therein, e.g. the antibody, essentially retains its physical and chemical stability and thus its biological activity upon storage.

A “stable liquid pharmaceutical antibody formulation” is a liquid antibody formulation with no significant changes observed at a refrigerated temperature (2-8° C.) for at least 12 months, particularly 2 years, and more particularly 3 years. The criteria for stability is the following: no more than 10%, particularly 5%, of antibody monomer is degraded as measured by size exclusion chromatography (SEC-HPLC). Furthermore, the solution is colorless or clear to slightly opalescent by visual analysis. The protein concentration of the formulation has a change no more than +/−10%. No more than 10%, particularly 5% of aggregation is formed. The 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 Death 1,” “Programmed Cell Death 1,” “Protein PD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-F are used interchangeably, and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found under NCBI Reference Sequence: NM_005018.1.

The terms “anti-PD-1 antibody”, “antibody against PD-1” and “anti-PD-1 antibody” refer to an antibody that is capable of binding to PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-1, The term “binding to PD-1” as used herein means the binding of the antibody to PD-1 in either a BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in ELISA, in which purified PD-1 or PD-1 CHO transfectants are coated onto microtiter plates.

The concentration of the antibody against PD-1 comprised in the pharmaceutical formulation is in the range of 1 mg/ml to 60 mg/ml, particularly in the rang of 20 mg/ml to 50 mg/ml, most particularly of 40 mg/ml.

The term “surfactant” as used herein denotes a pharmaceutically acceptable excipient which is used to protect protein formulations against mechanical stresses like agitation and shearing. Examples of pharmaceutically acceptable surfactants include polyoxyethylensorbitan fatly acid esters (Tween), polyoxy ethylene alkyl ethers (for example those commercially obtained under the trademark Brij™) and polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic). Examples of polyoxyethylenesorbitan-fatty acid esters are polysorbate 20 (commercially obtained sander the trademark Tween 20™) and polysorbate 80 (commercially obtained under the trademark Tween 80™).

The term “buffer” as used herein denotes a pharmaceutically acceptable excipient, which 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 comprise but are not limited to histidine-buffers, citrate-buffers, succinate-buffers, acetate-buffers, arginine-buffers, phosphate-buffers or mixtures thereof. Buffers of particular interest are selected from citrate-buffer or acetate-buffer with its pH adjusted with an acid or a base known in the art. The abovementioned buffers are generally used in an amount of about 1 mM to 50 mM, preferably about 10 mM to 30 mM, and more preferably about 10 mM. Independently from the buffer used, the pH can be adjusted to a value in the range from 4.5 to 6.0 and particularly to value in the range from 4.8 to 5.6 and most particularly to pH 5.2 with an acid or a base known in the art, e.g. hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide.

In some embodiments the stable anti-PD-1 antibody pharmaceutical formulation of the present invention comprises an antioxidant as a second stabilizer. An “antioxidant” is a pharmaceutically acceptable excipient, which 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 hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, benzyl alcohol and n-acetyl cysteine.

The term “saccharide” as used herein denotes a monosaccharide or an oligosaccharide. A monosaccharide is a monomeric carbohydrate which is not hydrolysable by acids, including simple saccharide and their derivatives, e.g. aminosaccharide. Example of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid. An oligosaccharide is a carbohydrate consisting of more than one monomeric saccharide unit connected via glycosidic bond(s), either branched or in a chain. The monomeric saccharide units within an oligosaccharide can be identical or different. Depending on the number of monomeric saccharide units, the oligosaccharide is a di-, tri-, tetra- penta-saccharide, and so forth. In contrast to polysaccharides, the monosaccharides and oligosaccharides are water soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose and raffmose. In particular, saccharides are selected from sucrose and trehalose.

The term “amino acid” as used herein denotes a pharmaceutically acceptable organic molecule possessing an amino moiety located at α-position of a carboxylic group. Examples of amino acids include arginine, glycine ornithine, lysine, histidine, glutamic acid, asparagic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine, serine, proline. Amino acids are generally used in an amount of about 5 to 500 mM, particularly in an amount of about 5 to about 200 mM, and more particularly in an amount of about 100 to about 150 mM.

The term ‘stabilizer’ also includes lyoprotectants. The term “lyoprotectant” denotes a pharmaceutical acceptable excipient, which protects the labile active ingredient (e.g. a protein) against destabilizing conditions during the lyophilisation process, subsequent storage and reconstitution. Lyoprotectants comprise but are not limited to the group consisting of saccharides, polyols (such as e.g. sugar alcohols) and amino acids. In particular, lyoprotectants can be selected from the group consisting of saccharides such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, aminosaccharides such as glucosamine, galactosamine, N-methylglucosamine (“Meglumine”), 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 surprisingly found that disaccharides exhibit better effect on the stability of formulations than that of monosaccharides, polyols, and amino acids.

The pharmaceutical formulation may also contain tonicity agents. The term “tonicity agent” as used herein denotes pharmaceutically acceptable tonicity agents which are used to modulate the tonicity of the formulation. The formulation can be hypotonic, isotonic or hypertonic. Isotonicity in general relates to the relative osmostic pressure of a solution, usually relative to that of human serum. The formulation according to the invention can be hypotonic, isotonic or hypertonic, but will preferably be isotonic. An isotonic formulation, is liquid or liquid reconstituted from a solid form (e.g. from a lyophilised form) and denotes a solution having the same tonicity as that of other solution with which it is compared, such as physiologic salt solution and the serum. Suitable tonicity agents comprise but are not limited to sodium chloride, potassium chloride, glycerine and any component selected from the group of consisting of amino acids, saccharides, in particular glucose. Tonicity agents are generally used in an amount of about 5 mM to about 500 mM. As for stabilizers and tonicity agents, there is a group of compounds which can serve as both stabilizer and tonicity agent at the same time. Examples thereof can be found in the group of saccharide, amino acids, polyols, cyclodextrines, polyethyleneglycols and salts. An example of saccharide which can serve as both stabilizer and tonicity agent is trehalose.

The pharmaceutical formulation may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured by both sterilization procedures and by inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. Preservatives are generally used in an amount of about 0.001 to about 2% (w/v). Preservatives comprise but are not limited to ethanol, benzyl alcohol, phenol, in-cresol, p-chlor-m-cresol methyl or propyl parabens, benzalkonium chloride.

The stable anti-PD-1 antibody pharmaceutical formulation of the present invention can be used for the prophylaxis or treatment of a PD-1 mediated disease or disorder, wherein the disease or disorder is preferably cancer; more preferably PD-L1 expressing cancer; wherein the disease or disorder is most preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, melanoma and kidney cancer.

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

In view of their high stability, the pharmaceutical formulation according to the invention can be administered i.v. without the need of an in-line filter and is thus much more convenient to handle than conventional formulations that neat to be administered via an in-line filter. In-line filters such as Sterifix® have to be installed in the infusion line of i.v. medications to prevent any particles, air, or microorganisms that may be present in the i.v. solution or line. Particles of 5 to 20 microns and larger size have the capability of obstructing blood flow through pulmonary capillaries, which could lead to complications such as pulmonary embolism. Foreign particles can also cause phlebitis at the injection site and filters may help to reduce the incidence of phlebitis.

The 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 invention can be prepared by methods known in the art, e.g. ultrafiltration-diafiltration, dialysis, addition and mixing, lyophilisation, reconstitution, and combinations thereof. Examples of preparations of formulations according to the invention can be found herein after.

The stable anti-PD-1 antibody pharmaceutical formulation according to the invention can also be in a lyophilized form or in a liquid form reconstituted from the lyophilized form. The “lyophilized form” is manufactured by lyophilization method known in the art. The lyophilizate usually has a residual moisture content of about 0.1 to 5% (w/w) and is present as a powder or a physically stable cake. The “reconstituted form” can be obtained from the lyophilizate by a fast dissolution after addition of reconstitution medium. Suitable reconstitution media comprise but are not limited to water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g. 0.9% (w/v) NaCl), glucose solutions (e.g. 5% (w/v) glucose), surfactant-containing solutions (e.g. 0.01% (w/v) polysorbate 20) and pH-buffered solutions (e.g. phosphate-buffered solutions).

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 2 shows the effect of saccharide on the thermal stability of the anti-PD-1 antibody.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is further described in detail with reference to the following examples: however, these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

In the examples of the present invention, where specific conditions are not described, the experiments are generally conducted under conventional conditions, or under conditions proposed by the material or product manufacturers. Where the source of the reagents is not specifically given, the reagents are commercially available conventional reagents.

EXAMPLES

The preparation process of the present invention is as follows:

Step1: Samples were extracted for the detection of antibody protein concentration in the middle control, before anti PD-1 antibody stock solution was filted. After passing the middle control, the stock solution was passed through a 0.22 μm PVDF filter and the filtrate was collected. The PD-1 antibody has the heavy chain amino acid sequence shown as SEQ ID NO: 7 and the light chain amino acid sequence shown as SEQ ID NO: 8, which is prepared according to the method disclosed in WO2015/085847.

Step 2: Adjust the loading volume to 5.3 ml, the filtrate was filled in 20 ml vial with a half stopper, the loading difference was detected by sampling at the beginning, the middle and the end of the filling, respectively.

Step 3: The solution with a stopper was filled into the lyophilization chamber and lyophilized according to the &Rowing lyophilization process. After the lyophilization procedure was completed, the lyophilized powder was plugged in vacuum.

		Time	time of	Vacuum
	Temperature	setting	maintenance	degree
Parameter	(° C.)	(min)	(min)	(mbar)
Pre-frozen	−5	10	60	/
	−45	40	120	/
First drying	−5	40	2100	0.1
Second drying	25	60	400	0.01

Step 4: Open the capping machine, add alumunium cover, and roll the cover.

Step 5: Visual inspection was performed to confirm the product without collapse, accurate of loading volume and other defects. Print and past vial label; print carton label and fold the carton, perform packing and labelling.

The concentration of protein during the middle control was measured by ultraviolet spectrophotometer, and the absorption peak at 280 nm was measured (Thermo: Nanodap 2000).

0.22 μm MN filter is Millipore Millipak-100,

Filling machine is Chutian technology KGSS/2-X2 linear filling machine.

Loading volume difference was detected using electronic balance weighing (manufacturer: Sartorius, model BSA423S).

Tofflon Lyo-B (SIP.CIP) vacuum freeze diver was used for lyophilization.

Shandong Penglai DZG-130 knife type automatic capping machine was used for capping machine.

Tianjin Jingtuo YB-2A clarity detector was used for visual inspection appearance.

The HPLC (SEC and IEC) measurements in the examples were performed using an Agilent 1200DAD High Pressure Liquid Chromatograph (TSK gel SuperSW mAb HR 300×7.8 mm column and ProPac™ WCX-10 BioLC™, 250×4 mm column).

The thermal denaturation temperature (Tm) of the protein was measured using 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 nano-particle size potentiometer.

Example 1

The anti-PD-1 antibody was formulated as a preparation at pH 4.8-5.6 containing 10 mM (sodium) acetate, 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, respectively. The antibody protein concentration was 40 mg/mL. Each formulation was filtered and filled into a 20 mL neutral borosilicate glass syringe bottle at 5 mL/bottle for lyophilization, the glass bottle is sealed with a halogenated butyl rubber stopper which was used for injection and lyophilized sterile powder. The lyophilized product was stored at 25° C. and 40° C. for stability analysis. The results showed that the anti-PD-1 antibody was very stable at pH 4.8-5.6.

TABLE 1
The effect of pH on the degradation of anti-PD-1 antibody
	Storage
	conditions
Test index	and time	pH 4.8	pH 5.0	pH 5.2	pH 5.4	pH 5.6
SEC	0 point	98.0	97.9	97.9	97.9	97.9
monomer	40° C.,	97.3	97.5	97.6	97.6	97.4
(%)	35 Days
	25° C.,	97.6	97.7	97.8	97.8	97.8
	30 Days
IEC main	0 point	66.2	66.4	66.6	66.4	66.4
peak (%)	40° C.,	64.7	64.1	64.7	65.9	65.3
	35 Days
	25° C.,	64.7	65.6	65.6	65.4	66.0
	30 Days

Example 2

In the following buffer solutions, anti-PD-1 antibody formulation with a protein concentration of 1 mg/ml were prepared.

• • 1) Buffer 1: 10 mM (sodium) acetate, pH 5.0;
  • 2) Buffer 2: 10 mM disodium hydrogen phosphate (citric acid), pH 5.0;
  • 3) Buffer 3:10 mM (sodium) succinate, pH 5.0
  • 4) Buffer 4: 10 mM (sodium) citrate, pH 5.0

The thermal stability of anti PD-1 antibody in each formulation was detected by the differential scanning calorimetry (DSC). Analysis of the thermal denaturation median temperature (Tm) of the medicine showed that the stability of anti-PD-1 antibody in acetate buffer salt system is obviously better than that in succinate, citrate, disodium hydrogen phosphate buffer system. The result was shown in FIG. 1.

Example 3

The PD-1 antibody formulations comprising a protein concentration of 1 mg/mL and different saccharides of different concentrations were screened using DSC technology:

• • 1) Buffer 1: 10 mM (sodium) acetate, 90 mg/mL sucrose, pH 5.2;
  • 2) Buffer 2: 10 mM (odium) 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, 90mg/mL α,α-trehalose dihydrate, pH 5.7.

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

Example 4

Anti-PD-1 antibody formulations comprising a PD-1 antibody protein concentration of 40 mg/mL, 10 mM (sodium) acetate, 90 mg/mLα,α-trehalose dihydrate and pH of 5.2 were prepared in the following buffer solutions which contain different concentrations of surfactant:

• • 1) Without 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 in 20 mL vials with 5 mL/vial, and the vials were sealed with plastic film stopper. The drug was placed on a 25° C. thermostat shaker and shaken at 500 rpm. The results of stabilization showed that 0.1-0.4 mg/mL polysorbate 20 effectively prevented the anti-PD-1 antibody from aggregation and the taxation large particles.

TABLE 2
The effect of surfactant on the aggregation of anti-
PD-1 antibodies at 25° C. and 500 rpm shaking
	Shaking
	Time
Test Index	(hour)	1	2	3	4	5	6
Appearance	0	Clear	Clear	Clear	Clear	Clear	Clear
	0.2	Turbid	Clear	Clear	Clear	Clear	Clear
	0	N/A	Clear	Clear	Clear	Clear	Clear
	6	N/A	Clear	Clear	Clear	Clear	Clear
	24	N/A	Clear	Clear	Clear	Clear	Clear
DLS Average	0	6.222	6.740	6.224	6.728	6.174	6.444
Diameter (nm)	0.2	8.102	7.725	6.384	6.642	6.598	6.574
	2	N/A	7.794	6.475	6.764	6.570	6.610
	6	N/A	7.435	6.286	6.710	6.280	6.750
	24	N/A	6.812	6.295	6.801	6.287	7.418
SEC Monomer	0	97.9	98.3	97.9	98.1	97.9	98.0
(%)	0.2	97.9	98.3	98.0	98.1	98.0	98.0
	2	N/A	98.2	98.0	98.1	98.0	98.0
	6	N/A	97.9	97.6	98.0	97.7	97.0
	24	N/A	97.3	97.6	97.9	97.7	96.3

Example 5

Anti-PD-1 antibody was prepared at 40 mg/mL in a solution comprising 10 mM (sodium) acetate, 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20 at pH 5.2. The formulation was filled in 20 mL vial with 5 mL/vial and lyophilized and the vial was sealed with lyophilization stopper. The lyophilized samples were placed for 10 days under 4500±500 Lx light irradiation or placed for 10 days at 40±2° C., or subjected to a cycle of low temperature 4° C.˜40° C. for 3 times, or a cycle of freeze-thaw at −20° C.˜40° C. for 3 times. Drug stability was assessed by protein content, purity, and activity assay, and the results showed that the anti PD-1 antibody was stable in this prescription and could still meet the requirement even after the treatment of strong light, high temperature or low temperature and freeze-thaw cycles.

TABLE 3
Stability of anti PD-1 antibody formulations whem exposed to
strong light, high or low temperature and freeze-thaw cycles
					Low	Freezing
				High	Temper-	and
	Acceptable	Null	Strong	Temper-	ature	thawing
Test Index	Limit	Point	Light	ature	Cycle	Cycle
Protein Content	200.0 ± 20.0	195.4	197.5	201.0	197.5	190.5
(mg/vial)	mg/vial
SEC Monomer (%)	≥93.0%	97.6	97.4	97.2	97.6	97.6
IEC main peak (%)	≥55.0%	66.5	64.2	64.1	65.2	65.5
Non-reducing CE-	≥90.0%	95.6	95.8	95.7	94.9	95.1
SDS main peak (%)
Reducing CE-SDS	≥93.0%	98.2	98.1	98.2	97.9	98.2
main peak (%)
Activity (%)	70%~130%	92	104	108	91	85

Example 6

Anti-PD-1 antibody was prepared at 40 mg/mL in a solution comprising 10 mM (sodium) acetate, 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20 at pH 5.2. The formulations were filled in glass bottles and 316L stainless steel tanks respectively, and placed for 24 hours at room temperature. Protein content and purity analysis showed that anti-PD-1 antibody was stable within 24 hours. The preparation is compatible with the 316L stainless steel tank.

TABLE 4
The stability of anti PD-1 antibody in stainless steel tank.
		storage
	Quality	Time		316 L Stainless
Test Index	Standard	(hour)	Glass	Steel Tank
Protein	40.0 ± 4.0	0	39.4	39.4
Concentration	mg/mL	3	39.2	39.0
(mg/mL)		6	38.9	39.0
		24	39.3	38.7
SEC Monomer	≥93.0%	0	96.2	96.2
(%)		3	96.1	96.1
		6	96.1	96.1
		24	96.8	96.5
IEC Main peak	≥55.0%	0	68.2	68.2
(%)		3	68.1	68.0
		6	68.3	68.5
		24	68.0	67.5

Example 7

Anti-PD-1 antibody was prepared at 40 mg/mL in a solution comprising 10 mM (sodium) acetate, 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20 at pH 5.2. The formulation was filled into a 20 ml vial with 5 mL vial and was lyophilized at primary drying temperature of −15° C., −10° C. and −5° C. respectively, and the vial was. sealed with a lyophilization stopper. The lyophilized product was stored at 25° C. and its stability was analyzed. The results show that the temperature at −10° C.˜−5° C. is the best primary drying temperature for lyophilization process.

TABLE 5
The stability of anti PD-1 antibody formulation
prepared with different primary drying process.
Primary	Storage	SEC	DLS	Water
drying	Time	Monomer	Average	Content
temperature	(Month)	Purity (%)	Diameter (nm)	(%)
−15°	C.	0	97.8	7.177	1.5
		1	97.9	7.172	N/A
		2	97.8	10.097	N/A
−10°	C.	0	97.9	7.991	1.2
		1	97.9	7.318	N/A
		2	97.5	7.349	N/A
−5°	C.	0	98.0	8.120	1.0
		1	97.6	7.781	N/A
		2	97.5	9.018	N/A

Example 8 Other Formulations

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

• • (i) 90 mg/mL α,α-trehalose dihydrate, and 10 mM acetate buffer at pH 5.2;
  • (ii) 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2;
  • (iii) 90 gm/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 20 mM acetate buffer at pH 5.4;
  • (iv) 60 mg/mL α,α-trehalose dihydrate, 0.4 mg/mL polysorbate 20, and 20 mM acetate buffer at pH 5.0;
  • (v) 60 mg/mL α,α-trehalose dihydrate, 0.1 mg/mL polysorbate 20 and 20 mM acetate buffer at pH 5.2;
  • (vi) 60mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2;
  • (vii) 30 mg/mL α,α-trehalose dihydrate, 0.4 mg/mL polysorbate 20, and 10 mM acetate buffer a pH 4.8;
  • (viii) 30 mg/mL α,α-trehalose dihydrate 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 was within 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 embodiment that can be performed may be selected from, but not limit to the following combinations:

• • (1) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 2.0, and 10 mM acetate buffer at pH 5.2;
  • (2) Anti PD-1 antibody 1 mg/mL, 30 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 4.5;
  • (3) Anti PD-1 antibody 20 mg/mL, 60 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 1 mM acetate buffer at pH 4.8;
  • (4) Anti PD-1 antibody 35 mg/mL, 85 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 2 mM acetate buffer at pH 5.6;
  • (5) Anti PD-1 antibody 45 mg/mL, 95 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 5 mM acetate buffer at pH 6.0;
  • (6) Anti PD-1 antibody 50 mg/mL, 100 mg/mL α,α-trehalose dihydrate, 0.2 mg/mL polysorbate 20, and 15 mM acetate buffer at pH 5.2;
  • (7) Anti PD-1 antibody 60 mg/mL, 90 mg/mL sucrose, 0.2 mg/mL polysorbate 400, and 30 mM acetate buffer at pH 5.2;
  • (8) Anti PD-1 antibody 40 mg/mL, 90 mg/mL lactose, 0.2 mg/mL polysorbate 60, and 50 mM acetate buffer at pH 4.5;
  • (9) Anti PD-1 antibody 40 mg/mL, 90 mg/mL trehalose, 0.2 mg/mL polysorbate 80 and 10 mM acetate buffer at pH 5.2;
  • (10) Anti PD-1 antibody 40 mg/mL, 90 mg/mL, maltose, 0.2 mg/mL polyoxyethylene hydrogenated castor oil, and 10 mM acetate buffer at pH 5.2;
  • (11) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 0.01 mg/mL glycerin fatty acid ester, and 10 mM acetate buffer at pH 5.2;
  • (12) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 0.05 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2;
  • (13) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 0.4 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2;
  • (14) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 0.5 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2;
  • (15) Anti PD-1 antibody 40 mg/mL, 90 mg/mL α,α-trehalose dihydrate, 1 mg/mL polysorbate 20, and 10 mM acetate buffer at pH 5.2.

Claims

1. An anti-PD-1 antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 7 and alight chain having the amino acid sequence of SEQ ID NO: 8.

2. A pharmaceutical composition comprising the anti-PD-1 antibody of claim 1.

3. The pharmaceutical composition of claim 2, being an injectable pharmaceutical formulation further comprising water for injection.

4. A lyophilized powder prepared from the pharmaceutical formulation of claim 2.

5. A method of blocking the binding of PD-1 to PD-L1 in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 2.

6. The method of claim 5, wherein the subject is in need of a treatment of a cancer selected from the group consisting of breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, and melanoma.

7. The method of claim 5, wherein the pharmaceutical composition is an injectable pharmaceutical formulation further comprising water for injection.

8. A method of blocking the binding of PD-1 to PD-L1 in a subject in need thereof, comprising administering to the subject a stable pharmaceutical formulation comprising:

(i) an anti-PD-1 antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 7 and a light chain having the amino acid sequence of SEQ ID NO: 8;

(ii) a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a succinate buffer, and a phosphate buffer;

(iii) a stabilizer selected from the group consisting of a saccharide and an amino acid, and

(iv) a surfactant selected from the group consisting of polyoxyethylene hydrogenated castor oil, a glycerol fatty acid ester, and a polyoxyethylene sorbitan fatty acid ester.

9. The method of claim 8, wherein the concentration of the anti-PD-1 antibody in the pharmaceutical formulation is in the range of 1 mg/ml to 60 mg/ml.

10. The method of claim 8, wherein the buffer is the acetate buffer.

11. The method of claim 10, wherein the concentration of the buffer is 1 mM to 50 mM.

12. The method of claim 8, wherein the pH of the formulation ranges from 4.5 to 6.0.

13. The method of claim 8, wherein the at least one stabilizer comprises a disaccharide selected from the group consisting of sucrose, lactose, trehalose, and maltose.

14. The method of claim 13, wherein the disaccharide is at concentration from 30 mg/ml to 120 mg/ml.

15. The method of claim 8, wherein the surfactant is the polyoxyethylene sorbitan fatty acid ester selected from the group consisting of polysorbate 20, 40, 60 and 80.

16. The method of claim 15, wherein the surfactant is at a concentration from 0.01 mg/ml to 1 mg/ml.

17. The method of claim 8, wherein the pharmaceutical formulation is an injectable pharmaceutical formulation winch further comprises water for injection.

18. The method of claim 8, wherein the subject is in need of a treatment of a cancer selected from the group consisting of breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, and melanoma.

19. A method of treating a cancer selected from the group consisting of breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, and melanoma in a subject in need thereof, comprising administering to the subject a pharmaceutical formulation comprising:

(a) an anti-PD-1 antibody, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 7, and a light chain having the amino acid sequence of SEQ ID: 8; and

(b) at least one of: (i) 90 mg/ml α,α-trehalose dihydrate, 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 10 mM acetate buffer at pH 5.2; or (iii) 90 mg/ml α,αtrehalose dihydrate, 0.2 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.4; or (iv) 60 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.0; or (v) 60 mg/ml α,α-trehalose dihydrate, 0.1 mg/ml polysorbate 20, and 20 mM acetate buffer at pH 5.2; or (vi) 60 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 5.2; or (vii) 30 mg/ml α,α-trehalose dihydrate, 0.4 mg/ml polysorbate 20, and 10 mM acetate buffer at pH 4.8; or (viii) 30 mg/ml α,α-trehalose dihydrate, 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; or (x) 90 mg/ml α,α-trehalose dihydrate, 0.2 mg/ml polysorbate 20, 10 mM acetate buffer, and the pH of the formulation is 5.2, wherein the concentration of the anti-PD-1 antibody in (x) is 40 mg/ml.

20. The method of claim 19, wherein the pharmaceutical formulation is an injectable pharmaceutical formulation which further comprises water for injection.