WO2021079337A1

WO2021079337A1 - Pharmaceutical formulation of anti-her2 antibody and preparation thereof

Info

Publication number: WO2021079337A1
Application number: PCT/IB2020/059985
Authority: WO
Inventors: Sanjeev Kumar Mendiratta; Sanjay Bandyopadhyay; Chintan Patel; Tushar Patel; Chandresh Bhatt
Original assignee: Cadila Healthcare Limited
Priority date: 2019-10-23
Filing date: 2020-10-23
Publication date: 2021-04-29

Abstract

The present invention provides novel stable pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab formulated in presence of arginine containing buffer(s), wherein the arginine containing buffer is selected from arginine-succinate buffer, arginine-phosphate buffer arginine-acetate buffer, arginine-aspartate buffer, arginine-citrate buffer and suitable combination thereof. The pharmaceutical formulation according to the present invention comprises pertuzumab and about 50 to about 500 mM of buffer(s) with one or more suitable excipients. Further, the present invention also provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, buffer and amino acid(s) with one or more suitable excipient(s). In one of the aspects, the present invention provides liquid or lyophilized or frozen pharmaceutical formulation of anti-HER2 antibody preferably, pertuzumab. Other aspect of the present invention includes method of making pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab.

Description

Pharmaceutical formulation of anti-HER2 antibody and preparation thereof

Field of the Invention

The invention provides stable pharmaceutical formulation comprising anti-HER2 antibody, preferably pertuzumab.

Background of the Invention

A basic principle of pharmaceutical protein formulations is that certain instabilities must be overcome. Degradation pathways of proteins can be separated into two distinct classes, involving chemical instability and physical instability. Chemical instabilities lead to the modification of the protein through bond formation or cleavage. Examples of chemical instability problems include deamidation, racemization, hydrolysis, oxidation, beta elimination and disulfide exchange. Physical instabilities, on the other hand, do not lead to covalent changes in proteins. Rather, they involve changes in the higher order structure (secondary and above) of proteins. These include denaturation, adsorption to surfaces, aggregation and precipitation (Manning et al, Pharm. Res. 6, 903 (1989)). It is generally accepted that these instabilities, which can have great effect on the commercial viability and efficacy of pharmaceutical protein formulations, can be overcome by incorporating additional components in the formulation. Protein stability can be improved by adding excipients that interact with the protein in solution to keep the protein stable. For example, salt compounds and other ionic species are very common additives to protein formulations. They prevent denaturation of proteins by binding to proteins in a non-specific manner and provides stability. Amino acids (e.g., histidine, arginine) have been shown to reduce alterations in proteins' secondary structures when used as formulation additives (Tian et al., Int'l J. Pharm. 355, 20 (2007)). Other examples of commonly used additives include polyalcohol materials such as glycerol, sugars and surfactants such as detergents, both nonionic (e.g., Tween, Pluronic) and anionic (sodium dodecyl sulfate). The near universal prevalence of additives in all liquid commercial protein formulations indicates that protein solutions without such compounds may encounter challenges with degradation due to instabilities. The primary goal of protein formulation is to maintain the stability of a given protein in its native, pharmaceutically active form over prolonged periods of time to guarantee acceptable shelf-life of the pharmaceutical protein drug. To date, biologic formulations require additional excipients to maintain protein stability. Typically, liquid pharmaceutical formulations contain multiple additives for stability. For example, a liquid formulation for patient self-administration of human growth hormone, Norditropin SimpleXx^®, contains the additives mannitol (a sugar alcohol), histidine and poloxamer 188 (a surfactant) to stabilize the hormone. Pharmaceutical additives need to be soluble, non- toxic and used at particular concentrations that provide stabilizing effects on the specific therapeutic protein. Since the stabilizing effects of additives are protein and concentration-dependent, each additive being considered for use in a pharmaceutical formulation must be carefully tested to ensure that it does not cause instability or have other negative effects on the chemical or physical make-up of the formulation. Ingredients used to stabilize the protein may cause problems with protein stability over time or with protein stability in changing environments during storage.

The present invention provides novel stable pharmaceutical formulation comprising anti- HER2 antibody, preferably pertuzumab. In the present invention, pertuzumab is formulated in the presence of buffer selected from citrate buffer, succinate buffer, phosphate buffer, arginine-citrate buffer, arginine-succinate buffer, arginine-phosphate buffer, histidine-acetate buffer and combination thereof. The formulation of the present invention provides stability to the antibody in liquid, lyophilized or frozen dosage form. This and other advantages of the invention, as well as additional inventive features, will be apparent from the descriptions of the invention provided herein. W02001/00245 describes production of exemplary humanized HER2 antibodies which bind HER2 and block ligand activation of a HER receptor. It discloses amino acid sequences of heavy and light chain of pertuzumab. W02006/044908 describes antibody formulations, such as apomab and pertuzumab, formulated in histidine-acetate buffer. It is concluded that formulation of apomab with histidine-acetate buffer is most stable formulation.

CN101199483 discloses a humanized anti-HER2 antibody formulation in histidine buffer i.e. L- histidine hydrochloride monohydrate or L- histidine monohydrate.

WO2011/012637 describes stable pharmaceutical formulation of a trastuzumab with an effective amount of at least one hyaluronidase enzyme.

WO2014/096051 describes a pharmaceutical formulation of an anti-HER2 antibody with acetate buffer. The applicants of the application have found that an acetate buffer confers significant stability to the liquid formulation, compared to a citrate buffer.

None of these documents discloses formulation of anti-HER2 antibody, preferably pertuzuamb which comprises buffer concentration in the range of about 50 mM to about 500 mM as described in the present application. None of these documents discloses formulation of anti-HER2 antibody, preferably pertuzumab, which comprises buffers selected from arginine-succinate buffer, arginine-phosphate buffer and arginine-citrate buffer. The formulation of the present invention provides stability to the antibody in liquid, lyophilized or frozen dosage form, which comprises anti-HER2 antibody, preferably pertuzumab, and buffers selected from arginine-succinate buffer, arginine- phosphate buffer and arginine-citrate buffer along with other suitable excipients such as carbohydrate(s), amino acid(s), surfactant(s), salt(s) and combination thereof.

Further, present invention also provides formulation of anti-HER2 antibody, preferably pertuzumab, buffer and amino acid(s) as a stabilizer with one or more suitable excipients. Summary of the invention

The present invention provides novel stable pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab formulated in presence of arginine containing buffer(s), wherein the arginine containing buffer is selected from arginine-succinate buffer, arginine-phosphate buffer arginine-acetate buffer, arginine-aspartate buffer, arginine- citrate buffer and suitable combination thereof. The pharmaceutical formulation according to the present invention comprises pertuzumab and about 50 to about 500 mM of buffer(s), wherein the buffer is selected from wherein the buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof. Further, the present invention also provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, buffer and amino acid(s) with one or more suitable excipient(s). In one of the aspects, the present invention provides liquid or lyophilized or frozen pharmaceutical formulation of anti-HER2 antibody preferably, pertuzumab. Other aspect of the present invention includes method of making pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab.

Brief description of drawings

Figure 1 illustrates the rate of formation of HMW species of pertuzumab protein formulated in different buffer(s) with excipients selected from amino acid, sugar, surfactant and suitable combination thereof during storage under temperature stressed condition 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Control formulation plotted herein represents rate of formation of HMW species of approved formulation of pertuzumab. Figure 2 illustrates the rate of formation of total impurities of pertuzumab protein formulated in different buffer(s) with suitable excipients selected from amino acid, sugar, surfactant and suitable combination thereof during storage under temperature stressed condition 50°C ± 2 °C, RH 75 % ± 5% as assessed by HP-SEC. Control formulation plotted herein represents rate of formation of total impurities of approved formulation of pertuzumab.

Figure 3 illustrates the rate of formation of HMW species of pertuzumab protein formulated in 50 mM of histidine-acetate buffer with excipients selected from sugar, amino acid, surfactant and suitable combination thereof during storage under temperature stressed condition 50°C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Control formulation plotted herein represents rate of formation of HMW species of approved formulation of pertuzumab.

Figure 4 illustrates the rate of formation of total impurities of pertuzumab protein formulated in 50 mM of histidine-acetate buffer with excipients selected from sugar, amino acid, surfactant and suitable combination thereof during storage under temperature stressed condition 50°C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Control formulation plotted herein represents rate of formation of total impurities of approved formulation of pertuzumab.

Embodiments of the invention

In one embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and buffer(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine- acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine- citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof.

In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and arginine containing buffer(s), wherein arginine containing buffer is selected from arginine buffer, arginine- citrate buffer, arginine- succinate buffer, arginine-phosphate buffer, arginine-acetate buffer, arginine-aspartate buffer and suitable combination thereof.

In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and about 50 mM or more of buffer(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine- citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof. In one of the embodiments, about 50 mM or more of buffer(s) according to the present invention is the buffer concentration in the range of about 50 mM to about 500 mM. In another embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, buffer and amino acid(s) with one or more suitable excipient(s).

In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody without buffer(s).

In another embodiment, the pharmaceutical formulation according to the present invention has pH in the range of about 4.0 to about 8.0, preferably about 4.5 to about 7.5, more preferably about pH 6.0.

In second embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, buffer(s) and one or more suitable excipient(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine- citrate buffer, succinate buffer (e.g. succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof.

In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, arginine containing buffer(s) and one or more suitable excipient(s), wherein arginine containing buffer is selected from arginine buffer, arginine-citrate buffer, arginine-succinate buffer, arginine-phosphate buffer, arginine- acetate buffer, arginine-aspartate buffer and suitable combination thereof. In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, about 50 mM or more of buffer(s) and one or more suitable excipient(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof. In one of the embodiments, about 50 mM or more of buffer(s) according to the present invention is the buffer concentration in the range of about 50 mM to about 500 mM.

In third embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, buffer(s) selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine- acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof, wherein the formulation further comprises one or more suitable excipient(s) selected from suitable carbohydrate(s), suitable surfactant(s), suitable salt(s), suitable amino acid(s), suitable chelating agent(s), suitable anti oxidants), suitable tonicity modifier(s), glycerin, water for injection (WFI) and suitable combination thereof and the like.

In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, arginine containing buffer(s) selected from arginine buffer, arginine-citrate buffer, arginine-succinate buffer, arginine-phosphate buffer, arginine- acetate buffer, arginine-aspartate buffer and suitable combination thereof, wherein the formulation further comprises one or more suitable excipient(s) selected from suitable carbohydrate(s), suitable surfactant(s), suitable salt(s), suitable amino acid(s), suitable chelating agent(s), suitable anti-oxidant(s), suitable tonicity modifier(s), glycerin, water for injection (WFI) and suitable combination thereof and the like. In one of the embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, about 50 mM or more of buffer(s) and one or more suitable excipient(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof, wherein the formulation further comprises one or more suitable excipient(s) selected from suitable carbohydrate(s), suitable surfactant(s), suitable salt(s), suitable amino acid(s), suitable chelating agent(s), suitable antimicrobial agent(s), suitable anti-oxidant(s), suitable tonicity modifier(s), glycerin, water for injection (WFI) and suitable combination thereof and the like. In one of the embodiments, about 50 mM or more of buffer(s) according to the present invention is the buffer concentration in the range of about 50 mM to about 500 mM.

In further embodiment, the carbohydrate of present invention is sugar(s) and / or polyol(s) selected from dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D-mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof, preferably sucrose or trehalose or sorbitol. In one more embodiment, the surfactant of present invention is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene- polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine, sodium dodecyl sulphate (SDS) and suitable combination thereof and the like, more preferably polysorbate 20 or polysorbate 80.

In one more embodiment, the amino acid of present invention is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof and the like, preferably L-arginine, L-proline or its suitable salt form.

In one more embodiment, chelating agent of present invention is selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene glycol-bis( b- aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

In one of the embodiments, the formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention may comprise stabilizer(s). The stabibzer(s) of present invention is selected from surfactant(s), sugar(s), amino acid(s), polyol(s) and the like or suitable derivative or mixtures thereof, preferably surfactant(s), sugar(s) and / or amino acid(s). In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises amino acid as a stabilizer, wherein amino acid is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and combination thereof.

In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises sugar(s) and / or polyol(s) as a stabilizer, wherein sugar(s) and / or polyol(s) is selected from dextran, trehalose, raffinose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, mannitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, fructose, maltose, galactose, glucose, D-mannose, sorbose, cellobiose, melezitose, maltodextrins, starches, lactitol, xylitol, pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof. In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises surfactant(s) as a stabilizer, wherein surfactant is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton- X), poly oxy ethyl ene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine, sodium dodecyl sulphate (SDS) and suitable combination thereof and the like.

In one more embodiment, the salts of present invention are preferably selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate. Skilled artisan can use sugar(s) or polyol(s) or amino acid(s) as the tonicity modifiers instead of salts.

In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, arginine containing buffer(s) and sugar(s). In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and arginine containing buffer(s), sugar(s), surfactant(s) and suitable combination thereof.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- citrate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- succinate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20. In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- phosphate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20.

In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, arginine containing buffer(s) and amino acid(s).

In one more preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and arginine containing buffer(s), amino acid(s), surfactant(s) and suitable combination thereof. In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- citrate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- succinate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- phosphate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20. In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, arginine containing buffer(s), sugar(s) and amino acid(s).

In further preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and arginine containing buffer(s), sugar(s), amino acid(s), surfactant(s) and suitable combination thereof.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- citrate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- succinate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 29 mM of arginine- phosphate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20. In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM or more of buffer(s) and sugar(s).

In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM to about 500 mM of buffer(s) and sugar(s).

In one of the preferred alternate embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM or more of buffer(s), sugar(s), surfactant(s) and suitable combination thereof. In one of the preferred alternate embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM to about 500 mM of buffer(s), sugar(s), surfactant(s) and suitable combination thereof. In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 50 mM of histidine- acetate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20.

In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM or more of buffer(s) and amino acid(s).

In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM to about 500 mM of buffer(s) and amino acid(s).

In one more preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM or more of buffer(s), amino acid(s), surfactant(s) and suitable combination thereof.

In one more preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM to about 500 mM of buffer(s), amino acid(s), surfactant(s) and suitable combination thereof.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 50 mM of histidine- acetate buffer, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20. In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM or more of buffer(s), sugar(s) and amino acid(s). In one of the preferred embodiments, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody, about 50 mM to about 500 mM of buffer(s), sugar(s) and amino acid(s).

In further preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM or more of buffer(s), sugar(s), amino acid(s), surfactant(s) and suitable combination thereof.

In further preferred embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of pertuzumab antibody and about 50 mM to about 500 mM of buffer(s), sugar(s), amino acid(s), surfactant(s) and suitable combination thereof.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 50 mM of histidine- acetate buffer, 117 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20.

In one of the specific embodiments of this invention, the present invention provides the pharmaceutical formulation comprising 30 mg / mL of pertuzumab, 50 mM of histidine- acetate buffer, 119 mM of trehalose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20. In fourth embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably, pertuzumab, according to the present invention is in liquid dosage form, lyophilized dosage form or frozen dosage form, preferably in liquid form.

In further embodiment, lyophilized formulation of anti-HER2 antibody preferably, pertuzumab of the present invention is prepared by lyophibzation process, which can be performed by a skilled person using the techniques available in the art, which includes various steps like freezing, annealing, primary drying and secondary drying.

In one more embodiment, the pharmaceutical formulation of present invention maintains structural integrity of anti-HER2 antibody upon multiple freezing cycles. In a preferred embodiment, the pharmaceutical formulation of present invention maintains structural integrity of anti-HER2 antibody at least after two freeze-thaw cycles, preferably, maintains structural integrity of anti-HER2 antibody at least after five freeze-thaw cycles. In one of the embodiments, the pharmaceutical formulation of the present invention maintains structural integrity of anti-HER2 antibody at least after ten freeze- thaw cycles.

In preferred embodiment, the pharmaceutical formulation of present invention maintains structural integrity of pertuzumab antibody upon multiple freezing cycles. Preferably, the pharmaceutical formulation of present invention maintains structural integrity of pertuzumab antibody at least after two freeze-thaw cycles, preferably, maintains structural integrity of pertuzumab antibody at least after five freeze-thaw cycles. In one of the embodiments, the pharmaceutical formulation of the present invention maintains structural integrity of pertuzumab antibody at least after ten freeze-thaw cycles.

In furthermore embodiment, the formulation according to the present invention provides anti-HER2 antibody, preferably, pertuzumab formulation which is stable under 2 - 8 °C for at least one month. In one of the embodiments, the formulation according to the present invention provides anti-HER2 antibody, preferably, pertuzumab formulation which is stable under 2 - 8 °C for at least 12 months.

The pharmaceutical formulation of the present invention may be suitable for any use, including both in vitro and in vivo uses. In one embodiment, the formulation of the present invention is suitable for administration to a subject via a mode of administration, including, but not limited to, subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, and intramuscular administration. The formulation of the present invention may be used in the treatment of a disease or disorder in a subject. Definitions

In order that the present invention may be more readily understood, certain terms are first defined.

The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered. The term “pharmaceutical formulation”, “formulation”, “pharmaceutical composition”, or “composition” can be used here interchangeably. In a pharmacological sense, in the context of the present invention, a “therapeutically effective amount” or “effective amount” of an anti-HER2 antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the anti- HER2 antibody is effective. A “disorder” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions, which predisposes the subject to the disorder in question.

The terms “patient” and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes animals. The term “Animal” refers to a human or non-human animal, including, but not limited to, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and non-human primates, including, but not limited to, monkeys, chimpanzees and other apes and monkey species. The term does not denote a particular age. Thus, adult, juvenile and newborn individuals are of interest.

The “lyophilized formulation” is a dosage form, which is prepared by lyophilization or freeze drying process. The lyophilization was performed with conventional lyophilization technique known in the literatures involving steps such as freezing, primary drying, secondary drying and optionally annealing.

The “frozen composition of anti-HER2 antibody or pertuzumab” is a dosage form in which freezing of aqueous solution of anti-HER2 antibody or pertuzumab is conducted at below -15 °C. As used herein, the term “bulking agent” is intended to mean a compound used to add bulk to the reconstitutable solid and/or assist in the control of the properties of the formulation during preparation. Such compounds include, by way of example and without limitation, dextran, trehalose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol, dimethyl sulfoxide, glycerol, albumin, calcium lactobionate, and others known to those of ordinary skilled in the art.

The term “buffer” or “buffer solution” or “buffer system” refers to generally aqueous solution comprising a mixture of an acid (usually a weak acid, e.g. succinic acid) and conjugate base (e.g. arginine). A buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the “buffering agent(s)”. The pH of a “buffer solution” will change very only slightly upon addition of a small quantity of strong acid or base due to the “buffering effect” imparted by the “buffering agent”.

The term “buffer system” comprises one or more buffering agent(s) and/or an acid/base conjugate(s) thereof, and more suitably comprises one buffering agent only and an acid/base conjugate thereof. The overall pH of the composition comprising the relevant buffer system is generally a reflection of the equilibrium concentration of each of the relevant buffering species (i.e. the balance of buffering agent(s) to acid/base conjugate(s) thereof).

The term “buffering agent” refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution. A buffering agent maintain the pH of a given solution at or near to a pre- determined value, and the buffering agents are generally chosen to complement the pre- determined value. A buffering agent is suitably a single compound which gives rise to a desired buffering effect, especially when said buffering agent is mixed with (and suitably capable of proton exchange with) an appropriate amount (depending on the pre-determined pH desired) of “acid/base conjugate”, or if the required amount of “acid/base conjugate” is formed in situ - this may be achieved by adding strong acid or base until the required pH is reached. The term “buffering agent” and “buffers” can be used here interchangeably. The term “arginine containing buffer” refers to the “buffer” or “buffer solution” or “buffer system” wherein one of the buffering agent is arginine either L-arginine or L- arginine HC1.

The term “acid / base conjugate” refers to the conjugate acid or conjugate base (whichever is relevant at a particular pH) of a particular “buffering agent”.

The term “stabilizers” as used herein generally includes agents, which provide stability to the protein from freezing-induced stresses. Examples of stabilizers include polyol(s) such as mannitol or sorbitol, saccharide(s) such as sucrose, trehalose, surfactant(s) such as polysorbate, poloxamer or polyethylene glycol, amino acid(s) such as arginine, proline, glycine, and the like.

A “stable” formulation is one in which the protein therein essentially retains its physical stability and /or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. Preferably, the formulation is stable under 2 - 8 °C for at least one month. Furthermore, the formulation is preferably stable following freezing (to, e.g., -70 °C) and thawing of the formulation, for example following 1, 2 or 3 cycles of freezing and thawing. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino- terminal or carboxy- terminal sequence analysis; mass spectrometric analysis; SDS- PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

The term “excipient” refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain osmolality and pH of the pharmaceutical preparations. Examples of used excipients include, but are not limited to, suitable carbohydrate(s), suitable surfactant(s), suitable salt(s), suitable amino acid(s), suitable chelating agent(s) and combination thereof.

The term “tonicity modifier” is intended to mean a compound or compounds that can be used to adjust the tonicity of a pharmaceutical formulation. Suitable tonicity modifiers include glycerin, lactose, mannitol, dextrose, sodium chloride, magnesium sulfate, magnesium chloride, sodium sulfate, sorbitol, trehalose, sucrose, raffinose, maltose and others known to those or ordinary skilled in the art. Cryoprotectant also contribute to the tonicity of the formulations.

Anti-HER2 antibody as mentioned herein refers to a humanized antibody that inhibits the dimerization of HER2 with other HER receptors, thereby inhibiting ligand- driven phosphorylation and activation, and downstream activation of the RAS and AKT pathways. Preferably, an anti-HER2 antibody according to the current invention is pertuzumab.

The term “high-molecular-weight-species” or “HMWs” of pertuzumab as used herein refers to aggregates of pertuzumab that has a molecular weight greater than that of intact pertuzumab (e.g. where the intact pertuzumab has a molecular weight of about 145,197 Da measuring its peptide chains only). The HMWs can be detected by size exclusion high performance liquid chromatography (SE-HPLC) assay.

The term “low-molecular-weight-species” or “LMWs” of pertuzumab as used herein refers to a fragment of pertuzumab that has a molecular weight less than that of intact pertuzumab (e.g. where the intact pertuzumab has a molecular weight of about 145,197 Da measuring its peptide chains only). The LMWs can be detected by size exclusion high performance liquid chromatography (SE-HPLC) assay.

The term “Total impurities” or “T_impumiCs” of pertuzumab as used herein refers to total of aggregates of pertuzumab (HMWs) and fragment of pertuzumab (LMWs). T_impumiCs can be measured by size exclusion high performance liquid chromatography (SE-HPLC) assay.

The term “rate of formation of HMW species” or “rate of aggregates” or “A_t/A” as used herein refers to a ratio of HMWs of pertuzumab at given time point (A_t) to HMWs of pertuzumab at an initial time point (Ai). A_t/Ai for the illustrative formulations prepared according to the current invention is presented in figures 1 and 3.

The term “rate of formation of total impurities” or “Ti_mpurities t / T_impumiCs i” as used herein refers to a ratio of total impurities of pertuzumab at given time point (Timpurities t) to Timpurities of pertuzumab at an initial time point (Ti_mp_Urities 0· Ti purities t / Timpurities i for the illustrative formulations prepared according to the current invention is presented in figures 2 and 4.

Detailed description of the present invention

The present invention provides novel pharmaceutical formulation of therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab and buffer(s). Buffers of the present invention are selected from phosphate buffer (e.g., sodium phosphate etc), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate etc.), arginine-citrate buffer, succinate buffer (e.g. succinic acid, sodium succinate etc.), arginine- succinate buffer, acetate buffer, arginine- acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and combination thereof. Such combination(s) of buffer according to the current invention can be histidine-acetate buffer, citrate-phosphate buffer, arginine-succinate buffer and the like. The present invention also provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and arginine containing buffer(s), wherein arginine containing buffer is selected from arginine buffer, arginine-citrate buffer, arginine-succinate buffer, arginine-phosphate buffer, arginine-acetate buffer, arginine-aspartate buffer and suitable combination thereof. Further, The present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and about 50 mM or more of buffer(s), wherein buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine- citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine- succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof. In one of the embodiments, about 50 mM or more of buffer(s) according to the present invention is the buffer concentration in the range of about 50 mM to about 500 mM, preferably in the range of about 50 mM to about 100 mM. The buffer concentration in the range of about 50 mM to about 500 mM according to present disclosure include each integer and non-integer number between a particular range. For Example, the buffer concentration in the range of about 50 mM to about 500 mM includes about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 250 mM. Preferred antibody according to the present invention is pertuzumab.

In another embodiment, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, buffer and amino acid(s) with one or more suitable excipient(s).

In one of the preferred embodiments, the present invention provides pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody, buffer and amino acid(s) with one or more suitable excipient(s) wherein the buffer is selected from phosphate buffer (e.g., sodium phosphate, etc.), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine-histidine buffer, glycine buffer, arginine buffer, citrate buffer (e.g., sodium citrate, etc.), arginine-citrate buffer, succinate buffer (e.g., succinic acid, sodium succinate, etc.), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof and amino acid is proline. In one of the preferred embodiments, the buffer according to the present invention is phosphate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine-phosphate buffer. In one of the preferred embodiments, the buffer according to the present invention is histidine buffer. In one of the preferred embodiments, the buffer according to the present invention is histidine-acetate buffer. In one of the preferred embodiments, the buffer according to the present invention is histidine-succinate buffer. In one of the preferred embodiments, the buffer according to the present invention is histidine-citrate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine-histidine buffer.

In one of the preferred embodiments, the buffer according to the present invention is glycine buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine buffer. In one of the preferred embodiments, the buffer according to the present invention is citrate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine- citrate buffer. In one of the preferred embodiments, the buffer according to the present invention is succinate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine- succinate buffer. In one of the preferred embodiments, the buffer according to the present invention is acetate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine-acetate buffer. In one of the preferred embodiments, the buffer according to the present invention is arginine-aspartate buffer. In one of the preferred embodiments, the buffer according to the present invention is tromethamine buffer.

The pharmaceutical formulation of present invention further comprises other suitable excipient(s) such as carbohydrate(s), surfactant(s), amino acid(s), salt(s), chelating agent(s) and combination thereof. The current invention further provides methods of making the composition of anti-HER2 antibody, preferably of pertuzumab. The composition according to the present invention finds therapeutic use in single-dose form or in multi-dose form. In one of the embodiments, the pharmaceutical formulation according to the present invention may comprises suitable preservatives. The present invention includes stable frozen, liquid or lyophilized anti-HER2 antibody composition, preferably pertuzumab composition. The frozen, liquid or lyophilized composition of anti-HER2 antibody or pertuzumab according to the present invention comprises buffer(s), sugar(s), an amino acid(s) and / or other suitable excipients. The lyophilization process to prepare formulation of the present invention can be performed by a skilled person using the techniques available in the art, which includes various steps such as freezing, primary drying, secondary drying and optionally annealing.

In one of the aspects, the formulation of the present invention has improved stability either in frozen or lyophilized or liquid dosage form.

In a further aspect, the formulation of the present invention maintains stability after at least one freeze-thaw cycle. In another aspect, the pharmaceutical formulation of present invention maintains structural integrity of pertuzumab antibody upon multiple freezing cycles. Preferably, the pharmaceutical formulation of present invention maintains structural integrity of pertuzumab antibody at least after two freeze-thaw cycles, preferably, maintains structural integrity of pertuzumab antibody at least after five freeze-thaw cycles. In one of the aspects, the pharmaceutical formulation of present invention maintains structural integrity of anti-HER2 antibody at least after ten freeze- thaw cycles.

In further describing the embodiments of the present disclosure, composition (e.g. frozen or lyophilized or liquid) and methods of making composition of present invention are described in-details.

The present invention provides novel pharmaceutical formulation of therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, buffer and other suitable excipients. The present invention also provides novel pharmaceutical formulation of therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, arginine containing buffer and other suitable excipients. Further, the present invention provides the pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab antibody and about 50 mM or more of buffer(s) and other suitable excipients. In one of the embodiments, about 50 mM or more of buffer(s) according to the present invention is the buffer concentration in the range of about 50 mM to about 500 mM, preferably in the range of about 50 mM to about 100 mM. The buffer concentration range of about 50 mM to about 500 mM according to present disclosure include each integer and non-integer number between a particular range. For Example, the buffer concentration range of about 50 mM to about 500 mM includes about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 250 mM.

The therapeutically effective amount of antibody according to the present invention is in the range of 1 mg / mL to 400 mg / mL, preferably 10 mg / mL to 350 mg / mL, more preferably 30 mg / mL. In one of the embodiments, the present invention provides pharmaceutical formulation may comprise combination of anti-HER2 antibodies or combination of anti-HER2 antibody with non-antibody molecule or antibody drug conjugate of anti-HER2 antibody.

Buffers suitable for use in the compositions of the present disclosure include buffers that are compatible with the anti-HER2 antibody, preferably pertuzumab antibody and suitable for administration to a subject, e.g. by injection, preferably subcutaneous administration or intravenous administration. Examples of suitable buffers include, but are not limited to phosphate buffer (e.g., sodium phosphate etc), arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine- citrate buffer, glycine buffer, arginine buffer, arginine-histidine buffer, citrate buffer (e.g., sodium citrate etc), arginine-citrate buffer, succinate buffer (e.g. succinic acid, sodium succinate etc), arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and combination thereof and the like. Such combination(s) of buffer according to the current invention can be histidine-acetate buffer, citrate-phosphate buffer, arginine-succinate buffer and the like. In one of the aspects, arginine containing buffers according to the present invention is selected from arginine buffer, arginine- citrate buffer, arginine- succinate buffer, arginine-phosphate buffer, arginine-acetate buffer, arginine-aspartate buffer and combination thereof arginine-phosphate buffer, arginine-succinate buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, arginine- citrate buffer and other such buffers according to the present invention can be prepared by a skilled person by method known in the art. For example, arginine-phosphate buffer is prepared with the use of L-arginine and o- phosphoric acid, arginine-succinate buffer is prepared with the use of L-arginine and succinic acid. Similarly, arginine- citrate is prepared with the use of L-arginine base and citric acid. Histidine-acetate buffer is prepared with the use of L-histidine and acetic acid, histidine-succinate buffer is prepared with the use of L-histidine and succinic acid, similarly, histidine-citrate is prepared with the use of L-histidine and citric acid. In further aspect of the present invention, buffer(s) can be used in combination with amino acid(s) to prepare stable pharmaceutical formulation of pertuzumab. In one embodiment, the present invention provides pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, arginine containing buffer(s) and other suitable excipients wherein buffer is selected from arginine buffer, arginine-citrate buffer, arginine-succinate buffer, arginine-phosphate buffer, arginine-acetate buffer, arginine-aspartate buffer and suitable combination thereof. In one of the embodiments, the present invention provides pharmaceutical formulation comprising therapeutically effective amount of anti-HER2 antibody, preferably pertuzumab, buffer(s) and other suitable excipients wherein buffer is selected from sodium phosphate, sodium succinate, sodium citrate and combination thereof.

The buffers included in the pharmaceutical formulation according to the present invention may be a buffer that maintains the pH of the composition at a pH, ranging from about pH 4.0 to about pH 8.0. The pH range of about 4 to about 8 according to the present invention includes about pH 4.0, about pH 5.0, about pH 6.0, about pH 7.0 or about pH 8.0 or any non-integer number in between them (e.g., about pH 4.5 or about pH 5.3 or about pH 6.8 and the like). In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention having a pH in the range of about pH 5.0 to about pH 7.5. In a preferred embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention having a pH in the range of about pH 5.5 to about pH 7.5. About pH 5.5 to about pH 7.5 according to the present invention includes about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.3, about pH 7.5 or any non-integer or integer number in between them. In a more preferred embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention having a pH of about 6.0. pH of about 6.0 according to the present invention is pH 5.8 to pH 6.3.

In one embodiment, the concentration of arginine containing buffer according to the present invention is in the range of about 1 mM to about 500 mM. Preferably, the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 1 mM, about 5 mM, about 10 mM, about 11 mM, about 17 mM, about 20 mM, about 24 mM, about 29 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM. In one of the embodiments, the concentration of buffer when present in combination with amino acid according to the present invention is in the range of about 1 mM to about 500 mM. Preferably, the buffer concentration according to the present invention is in the range of about 1 mM to about 100 mM. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 1 mM, about 5 mM, about 10 mM, about 11 mM, about 17 mM, about 20 mM, about 24 mM, about 29 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM. In one of the embodiments, formulation according to the present invention does not contain buffer.

In one embodiment, the buffering agent(s) or buffering species or buffering component(s) according to the present invention is present in amount about 0.01 mg / mL to about 100 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular range. For Example, the concentration range of about 0.01 mg / mL, about 0.1 mg / mL, about 1.0 mg / mL, about 1.08 mg / mL, about 1.28 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL, about 100 mg / mL. The compositions of the present disclosure, in addition to the pertuzumab antibody and buffer(s) may include amino acid. As described above, other excipient that may be included in the compositions of the present disclosure is an amino acid. Amino acids suitable for use in the compositions of the present disclosure include amino acids that are compatible with the anti-HER2 antibody, preferably pertuzumab and suitable for administration to a subject, e.g., by injection or intravenous administration. Examples of suitable amino acids include, but are not limited to, arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form or combination of any of the above. In one embodiment, the suitable amino acid is selected from arginine, proline or its suitable salt form.

In one of the preferred embodiments, the amino acid according to the present invention is arginine. In one of the preferred embodiments, the amino acid according to the present invention is glycine. In one of the preferred embodiments, the amino acid according to the present invention is asparagine. In one of the preferred embodiments, the amino acid according to the present invention is glutamine. In one of the preferred embodiments, the amino acid according to the present invention is lysine. In one of the preferred embodiments, the amino acid according to the present invention is threonine. In one of the preferred embodiments, the amino acid according to the present invention is histidine. In one of the preferred embodiments, the amino acid according to the present invention is glutamic acid. In one of the preferred embodiments, the amino acid according to the present invention is aspartic acid. In one of the preferred embodiments, the amino acid according to the present invention is isoleucine. In one of the preferred embodiments, the amino acid according to the present invention is valine. In one of the preferred embodiments, the amino acid according to the present invention is leucine. In one of the preferred embodiments, the amino acid according to the present invention is alanine. In one of the preferred embodiments, the amino acid according to the present invention is phenylalanine. In one of the preferred embodiments, the amino acid according to the present invention is tyrosine. In one of the preferred embodiments, the amino acid according to the present invention is tryptophan. In one of the preferred embodiments, the amino acid according to the present invention is methionine. In one of the preferred embodiments, the amino acid according to the present invention is serine. In one of the preferred embodiments, the amino acid according to the present invention is proline. In one of the preferred embodiments, the amino acid according to the present invention is cysteine.

Amino acid according to the present invention is in L-form (e.g., L- arginine, L- proline). Amino acid according to the present invention can be a part of buffer as a potential counter ion. For example, arginine buffer, arginine-phosphate buffer, arginine- succinate buffer, arginine-citrate buffer, arginine-acetate, arginine-aspartate, histidine buffer, histidine-acetate buffer. In a preferred embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises L- arginine or its suitable salt form. In one of the preferred embodiments, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises L- proline or its suitable salt form in addition to buffer(s).

In another embodiment, the amino acid according to the present invention is present in amount about 0.01 mg / mL to about 100 mg / mL, preferably, the amino acid is present in amount about 0.01 mg / mL to about 50 mg / mL, more preferably the amino acid is present in amount about 0.1 mg / mL to about 10 mg / mL. In one more preferred embodiment, the amino acid according to the present invention is present in amount about 15 mg / mL to about 50 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 0.1 mg / mL to about 100 mg / mL includes about 0.1 mg / mL, about 0.2 mg / mL, about 0.5 mg / mL, about 1.0 mg / mL, about 2.0 mg / mL, about 5.0 mg / mL, about 10.5 mg / mL, about 20.0 mg / mL, about 30.0 mg / mL about 50.0 mg / mL, about 75.0 mg / mL, about 100.0 mg / mL or any integer or non-integer number in between them. In one of the embodiments, the present invention provides a composition of anti-HER2 antibody, preferably pertuzumab comprising amino acid in the range of about 0.1 mg / mL to about 100 mg / mL, preferably about 0.5 mg / mL to about 10.0 mg / mL. In a preferred embodiment, the present invention provides a composition of anti-HER2 antibody, preferably, pertuzumab comprising L- arginine at a concentration of about 5 mg / mL. In one of the preferred embodiments, the present invention provides a composition of anti-HER2 antibody, preferably pertuzumab comprising L- proline in the range of about 15 mg / mL to about 50 mg / mL. In preferred embodiment, the present invention provides a composition of anti-HER2 antibody, preferably pertuzumab comprising proline at a concentration about 20 mg / mL or about 30 mg / mL.

The compositions of the present disclosure, in addition to the pertuzumab antibody, buffer(s) and amino acid, may include carbohydrate. In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises carbohydrate. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may also be employed. Some carbohydrate excipients of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. For instance, the composition may include a sugar. Preferred sugars suitable for use in the compositions of the present invention include sugars that are compatible with anti- HER2 antibody, preferably pertuzumab antibody and suitable for administration to a subject, e.g., by injection or intravenous administration. Examples of suitable sugars include, but not limited to, sucrose, mannitol, sorbitol, maltose, trehalose, hydroxypropyl-beta-cyclodextrin (b-HPCD), lactose, glucose, fructose, galactose, glucosamine, and the like, and combination thereof. In certain instances, the sugar is a disaccharide. For example, the disaccharide may be sucrose or trehalose. In a one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises trehalose or sucrose or sorbitol.

In one of the preferred embodiments, the carbohydrate according to the present invention is trehalose. In one of the preferred embodiments, the carbohydrate according to the present invention is raffinose. In one of the preferred embodiments, the carbohydrate according to the present invention is sucrose. In one of the preferred embodiments, the carbohydrate according to the present invention is sorbitol. In one of the preferred embodiments, the carbohydrate according to the present invention is mannitol. In one of the preferred embodiments, the carbohydrate according to the present invention is hydroxypropyl-beta-cyclodextrin (b-HPCD). In one of the embodiments, the sugar concentration according to the present invention is in the range of about 1 mM to about 1000 mM, preferably, about 10 mM to about 800 mM. The concentration range according to present disclosure include each integer and non-integer number between particular range. For Example, the concentration range of about 1 mM to about 1000 mM includes about 10 mM, about 25 mM, about 50 mM, about 58 mM, about 80 mM, about 100 mM, about 108 mM, about 117 mM, about 119 mM, about 120 mM, about 150 mM, about 175 mM, about 205 mM, about 350 mM, about 450 mM, about 550 mM, about 800 mM or any integer or non-integer number in between them. In another embodiment, the sugar concentration according to the present invention is in the range of about 0.01 mg / mL to about 250 mg / mL. Preferably, the sugar concentration according to the present invention is in the range of about 10 mg / mL to about 60 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular range. For Example, the concentration range of about 0.1 mg / mL to about 250 mg / mL includes about 0.1 mg / mL, about 0.5 mg / mL, about 1.0 mg / mL, about 2.0 mg / mL, about 5.0 mg / mL, about 10.5 mg / mL, about 20.0 mg / mL , about 40.0 mg / mL, about 41.1 mg / mL, about 45.0 mg / mL, about 50.0 mg / mL, about 75.0 mg / mL, about 100.0 mg / mL, about 250 mg / mL or any integer or non-integer number in between them.

The compositions of the present disclosure, in addition to the pertuzumab antibody and buffer, may include surfactant. Examples of surfactants according to the present invention include, but not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alky lphenylpolyoxy ethylene ethers (e.g. Triton- X), poly oxy ethyl ene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine (PEI), sodium dodecyl sulphate (SDS) and the like. In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises surfactant, wherein the surfactant is polyoxyethylensorbitan-fatty acid esters (Tweens). In a more preferred embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprises polysorbate 20 or polysorbate 80.

In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylensorbitan fatty acid esters (Tween). In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylene alkyl ethers (e.g. Brij). In one of the preferred embodiments, the surfactant according to the present invention is alkylphenylpolyoxyethylene ethers (e.g. Triton-X). In one of the preferred embodiments, the surfactant according to the present invention is polyoxyethylene- polyoxypropylene copolymer (e.g. Poloxamer, Pluronic). In one of the preferred embodiments, the surfactant according to the present invention is polyethylene glycol (PEG). In one of the preferred embodiments, the surfactant according to the present invention is polyethyleneimine. In one of the preferred embodiments, the surfactant according to the present invention is sodium dodecyl sulphate (SDS). In one of the preferred embodiments, the surfactant according to the present invention is polysorbate 20. In one of the preferred embodiments, the surfactant according to the present invention is polysorbate 80.

The polysorbate 20 is sold under the trademark Tween 20™. In another embodiment, the surfactant according to the present invention is in the range of about 0.001 mg / mL to about 5 mg / mL. Preferably, the surfactant concentration according to the present invention is in the range of about 0.01 mg / mL to about 2 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular range. Lor Example, the concentration range of about 0.001 mg / mL to about 5 mg / mL include about 0.001 mg / mL, about 0.005 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.5 mg / mL, about 1.0 mg / mL, about 2.0 mg / mL, about 5.0 mg / mL, or any integer or non-integer number in between them.

The pertuzumab formulation of the present invention comprising pertuzumab, buffer(s) and sugar(s). The pertuzumab formulation of the present invention comprising pertuzumab, buffer(s), sugar(s) and surfactant(s). In one of the aspects, the pertuzumab formulation of the present invention comprising pertuzumab, buffer(s) and amino acid(s). In one of the aspects, the pertuzumab formulation of the present invention comprising pertuzumab, buffer(s), amino acid(s) and surfactant(s). In one more aspect, pertuzumab formulation of the present invention comprising pertuzumab, buffer(s), sugar(s) and amino acid(s). In one more aspect, pertuzumab formulation of the present invention comprising pertuzumab, buffer(s), sugar(s), amino acid(s) and surfactant(s). Buffer(s), sugar(s), amino acid(s) and surfactant(s) for the pertuzumab formulation of the present invention are as described herein above.

Compositions of the present disclosure may also include other components, such as additional pharmaceutically acceptable excipients or a dosage delivery vehicle as part of the composition. Additional excipients according to the present invention may include, but are not limited to, antimicrobial agents, anti-oxidants, water (e.g., water for injection (WFI)), alcohols, glycerin, vegetable oils, phospholipids, acids, bases, tonicity modifiers, and any combination thereof. Examples of tonicity modifiers according to the present invention may include, but are not limited to, sodium chloride, potassium chloride, potassium sulfate and sodium sulfate. Sugar or sugar alcohol can be used as tonicity modifiers instead of salt. In one embodiment, the pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to the present invention comprising tonicity modifiers selected from sodium chloride, potassium chloride, mannitol, sorbitol, sucrose, raffinose, trehalose, potassium sulfate and sodium sulfate. Acids or bases may also be present in composition of the present disclosure. Acids or bases according to the present invention can be a part of buffering agent or acid / base conjugate. Examples of acid according to the present invention may include, but are not limited to hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and any combination thereof. Examples of bases according to the present invention include, but are not limited to sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium succinate, sodium format, sodium sulfate, potassium sulfate, potassium fumarate, and any combination thereof.

In one more embodiment, chelating agent of present invention is selected from EDTA, pentetic acid and EGTA.

The pharmaceutical formulation of anti-HER2 antibody, preferably pertuzumab according to present invention may be administered to the subject in particular dosage. The “dosage” according to the present invention refers to amount of active ingredient administered to the subject in single dose or multiple dose. The term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations. The term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture.

In certain instances, the composition (e.g., frozen or lyophilized or liquid) is a sterile composition. By “sterile” is meant that there are substantially no microbial contaminants (e.g., fungi, bacteria, viruses, spore forms, etc.). In one instance, the composition (e.g., frozen or lyophilized or liquid) contains low level of bacterial endotoxins. The present invention provides pharmaceutical composition of anti-HER2 antibody, preferably pertuzumab that is substantially free of particles and / or aggregates and prevents the formation of particles and / or aggregates during storage and / or transport. The present invention provides pharmaceutical composition of pertuzumab that is stable for at least one month under +2 °C to +8 °C storage condition. In one of the aspects, the pharmaceutical composition of pertuzumab according to the present invention provides anti-HER2 antibody, preferably, pertuzumab formulation which is stable for at least 12 months under +2 °C to +8 °C storage condition. Samples stored under +2 °C to +8 °C storage condition were assessed for purity of pertuzumab by HP-SEC. In one of the embodiments, the present invention provides pharmaceutical composition of anti-HER2 antibody, preferably pertuzumab that has been observed to show reduced rate of formation of HMW species compared to the control formulation. The pertuzumab formulations of the present invention were observed to show reduced rate and extent of the formation of HMW species when compared to the control formulation in temperature-dependent stress study. Rate of formation of HMW species formed was observed under temperature stressed condition at 50 °C ± 2 °C, RH 75 % ± 5 % for one month.

In one of the embodiments, the present invention provides pharmaceutical composition of anti-HER2 antibody, preferably pertuzumab that has been observed to show reduced rate of formation of total impurities (Ti_mpurities) compared to the control formulation. The pertuzumab formulations of the present invention were observed to show reduced rate and extent of the formation of the total impurities when compared to the control formulation. Rate of T_impumiCs formed was observed under temperature stressed condition at 50 °C ± 2 °C, RH 75 % ± 5 % for one month. To estimate the level of aggregates formation and level of total impurities formation, analytical HP-size exclusion chromatography (HP-SEC) was performed. The said analytical method used in the present invention is well known to a skilled person and a brief description of the same is provided below for the sake of reference.

HP-Size exclusion chromatography (HP-SEC): Samples were analyzed to estimate the aggregates by HP-size exclusion chromatography (HP-SEC) using TSK gel G3000 SWXL column (7.8 mm I.D x 30 cm L). Samples were loaded and eluted isocratically in the presence of sodium phosphate buffer of pH 6.8, at a flow rate of 0.5 mL / min. Elution was monitored with UV214 _nm detection.

A method of making lyophilized formulation of the present invention comprising steps of freezing, primary drying, secondary drying and optionally with the step of annealing. In a preferred embodiment, the present invention provides method of making lyophilized formulation of anti-HER2 antibody, preferably pertuzumab, comprises steps of freezing, primary drying and secondary drying.

The formulations of the invention may be suitable for any use, including both in vitro and in vivo uses. In one embodiment, the formulation of the present invention is suitable for administration to a subject via a mode of administration, including, but not limited to, subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, and intramuscular administration. The formulations of the invention may be used in the treatment of a disorder in a subject. Also included in the invention are devices that may be used to deliver the formulation of the invention. Examples of such devices include, but are not limited to, a syringe, a pen, an implant, a needle-free injection device and a patch.

Examples The following non-limiting examples describe different formulations of pertuzumab, which can be prepared as per the present invention. It will be appreciated that other excipients can also be added as necessary to these formulations and such addition of excipients are considered to be within the scope of a person skilled in the art and are to be included within the scope of the present invention. The following examples describe experiments relating to present invention.

Example 1: Pertuzumab formulations with different buffers at different concentration in combination with sugar and surfactant

Pertuzumab was formulated in arginine-citrate buffer of pH 6.0 ± 0.5 comprising sucrose at desired concentration(s), as described above. Other excipient(s), like polysorbate can be added as shown in Table 1. In the same manner, formulation 2, formulation 3 and formulation 4 were prepared in arginine-succinate buffer, arginine- phosphate buffer and histidine-acetate buffer, respectively. A person skilled in the art can prepare pertuzumab formulation and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.

Stability of pertuzumab in the said formulations was evaluated through repeated freezing and thawing study. Further, some of the formulations (formulation 1 and formulation 3) were evaluated at real-time storage condition between +2 °C and +8 °C for at least 12 months. Formulation 4 was evaluated under temperature stress conditions at 50 °C ± 2 °C; RH 75 % ± 5 % for one month to assess the extent of degradation of pertuzumab, mainly in terms of formation of HMW species as compared to the control pertuzumab formulation. Control formulation represents approved formulation of pertuzumab that is known in the art. Samples exposed to different experimental conditions as specified above were assessed for purity of pertuzumab by HP- SEC. Results are summarized in Table 4, Table 5, Table 6 and illustrated in Figure 1 and Figure 2. Figures 1 and 2 show that the rate of formation of HMW species and total impurities (HMW plus LMW species) of pertuzumab in pharmaceutical formulation comprising 50 mM of histidine- acetate buffer with sugar. The said formulations were observed to show reduced rate and extent of the formation of HMW species and total impurities when compared to the control formulation. Example 2: Pertuzumab formulations with arginine containing buffers in combination with sugar, amino acid and / or surfactant

Pertuzumab was formulated in arginine-citrate buffer comprising proline at desired concentration(s), as indicated in Table 2. Other excipient(s), like polysorbate and / or sugar can be added as shown in Table 2. Formulations with arginine-succinate buffer and arginine-phosphate buffer were prepared in the same manner as described with arginine-citrate buffer. A person skilled in the art can prepare pertuzumab formulation and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.

Stability of pertuzumab in the said formulations was evaluated through repeated freezing and thawing study. Degradation of pertuzumab in said formulations were also evaluated under temperature stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % for one month to assess the extent of degradation of pertuzumab, mainly in terms of formation of HMW species and compared to the control pertuzumab formulation. Control formulation represents approved formulation of pertuzumab. Samples exposed to different experimental conditions as specified above were assessed for purity of pertuzumab by HP-SEC. Results are summarized in Table 6, and illustrated in Figures 1 and 2. Figures 1 and 2 show that the rate of formation of HMW species and total impurities (HMW plus LMW species) of pertuzumab in pharmaceutical formulation comprising arginine containing buffer or 50 mM of histidine-acetate buffer with proline and / or sugar. The said formulations were observed to show reduced rate and extent of the formation of HMW species and total impurities when compared to the control formulation.

Example 3: Pertuzumab formulations with 50 mM buffer in combination with sugar, amino acid and / or surfactant

Pertuzumab was formulated in 50 mM of histidine-acetate buffer comprising proline at desired concentration(s), as described above. Other excipient(s), like polysorbate and / or sugar can be added as shown in Table 3. A person skilled in the art can prepare pertuzumab formulation and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.

Stability of pertuzumab in the said formulations was evaluated through repeated freezing and thawing study. Degradation of pertuzumab in said formulations were also evaluated under temperature stress conditions at 50 °C ± 2 °C, RH 75 % ± 5 % for one month to assess the extent of degradation of pertuzumab, mainly in terms of formation of HMW species and compared to the control pertuzumab formulation. Control formulation represents approved formulation of pertuzumab. Samples exposed to different experimental conditions as specified above were assessed for purity of pertuzumab by HP-SEC. Results are summarized in Table 6, and illustrated in Figures 3 and 4. Figures 3 and 4 show that the rate of formation of HMW species and total impurities (HMW plus FMW species) of pertuzumab in pharmaceutical formulation comprising arginine containing buffer or 50 mM of histidine-acetate buffer with proline and / or sugar. The said formulations were observed to show reduced rate and extent of the formation of HMW species and total impurities when compared to the control formulation.

A person skilled in the art can further evaluate formulated pertuzumab as described in Examples 1 to 3 at various temperature conditions like, between +2 °C and +8 °C, between +25 °C and +30 °C, under stress conditions at about +40 °C or above. Results and discussion:

Table 4: Purity of pertuzumab in various formulations upon repeated freeze-thaw stress

Results illustrated in Table 4 demonstrate that pertuzumab in formulations comprising arginine containing buffer can withstand multiple freeze-thaw cycles without any significant degradation in terms of formation of HMW or LMW size species variants. Table 5: Purity of pertuzumab in various arginine containing buffered formulations under real-time storage condition

Data shown in Table 5 demonstrate the purity of pertuzumab in arginine containing buffered formulations under real-time storage condition analyzed up to 12 months. No significant change in purity of pertuzumab was observed with F-l and F-3 formulations as compared to that of the control formulation indicating the stable compositions of pertuzumab in the presence of arginine containing buffered formulations.

Table 6: Purity of pertuzumab in various formulations upon repeated freeze-thaw stress

Results obtained from the freeze-thaw study, summarized in Table 6 demonstrate that purity of pertuzumab in F-4, F-5, F-6, F-7, F-8, F-9, F-10, F-ll, F-12 and F-13 formulations remains unaltered even after extensive freeze-thaw stress (at least 10 cycles) without any further degradation in terms of formation of HMW or LMW size species variants. Incorporation by reference

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

We claim

1. A stable pharmaceutical formulation of pertuzumab comprising a therapeutically effective amount of pertuzumab and arginine containing buffer, optionally with one or more suitable excipients.

2. The pharmaceutical formulation as claimed in claim 1 wherein the buffer is selected from arginine-phosphate buffer, arginine-histidine buffer, arginine buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-citrate buffer, arginine-aspartate buffer and suitable combination thereof.

3. A stable pharmaceutical formulation of pertuzumab comprising a therapeutically effective amount of pertuzumab and buffer in the range of about 50 mM to about 500 mM with one or more suitable excipients.

4. A stable pharmaceutical formulation of pertuzumab comprising a therapeutically effective amount of pertuzumab, buffer, amino acid with one or more suitable excipients.

5. The pharmaceutical formulation as claimed in claim 3 or 4 wherein the buffer is selected from phosphate buffer, arginine-phosphate buffer, histidine buffer, histidine-acetate buffer, histidine-succinate buffer, histidine-citrate buffer, glycine buffer, arginine buffer, arginine-histidine buffer, citrate buffer, arginine- citrate buffer, succinate buffer, arginine-succinate buffer, acetate buffer, arginine-acetate buffer, arginine-glutamate buffer, arginine-aspartate buffer, tromethamine buffer and suitable combination thereof.

6. The pharmaceutical formulation as claimed in claim 1 or 4, wherein the concentration of buffer is in the range of about 1 mM to about 500 mM, preferably in the range of about 1 mM to about 100 mM.

7. The pharmaceutical formulation as claimed in claim 1, 3 or claim 4, wherein suitable excipient is selected from carbohydrate(s), amino acid(s), surfactant(s), salt(s), chelating agent(s), anti-oxidant(s), glycerin, tonicity modifier(s), sterile water for injection and suitable combination thereof.

8. The pharmaceutical formulation as claimed in claim 7, wherein the carbohydrate(s) is sugar(s) or derivatized sugar(s) or polyol(s) selected from fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrins, dextrans, starches, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, hydroxypropyl-beta-cyclodextrin (b-HPCD) and suitable combination thereof, preferably selected from trehalose and sucrose.

9. The pharmaceutical formulation as claimed in claim 8, wherein the concentration of sugar or polyol is in the range of about 1 mM to about 1000 mM, preferably in the range of about 10 mM to about 800 mM.

10. The pharmaceutical formulation as claimed in claim 4 or 7, wherein the amino acid(s) is selected from arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine, their suitable salt form and suitable combination thereof, preferably selected from arginine, proline, their suitable salt form and suitable combination thereof.

11. The pharmaceutical formulation as claimed in claim 10, wherein the amino acid is in the range of about 0.01 mg / mL to about 100 mg / mL, preferably in the range of about 0.01 mg / mL to about 50 mg / mL.

12. The pharmaceutical formulation as claimed in claim 7, wherein the surfactant(s) is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), poly oxy ethyl ene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), polyethylene glycol (PEG), polyethyleneimine (PEI), sodium dodecyl sulphate (SDS)and suitable combination thereof, preferably polyoxyethylensorbitan-fatty acid esters (Tweens), more preferably selected from polysorbate 20 or polysorbate 80.

13. The pharmaceutical formulation as claimed in claim 12, wherein the surfactant is in the range of about 0.001 mg / mL to about 5 mg / mL, preferably in the range of about 0.01 mg / mL to about 2 mg / mL.

14. The pharmaceutical formulation as claimed in claim 7, wherein salt(s) is selected from sodium chloride, potassium chloride, potassium sulfate and sodium sulfate.

15. The pharmaceutical formulation as claimed in claim 7, wherein chelating agent(s) is selected from ethylenediaminetetraacetic acid (EDTA), pentetic acid and ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

16. The pharmaceutical formulation as claimed in any preceding claim has a pH in the range of about pH 4.0 to about pH 8.0, preferably about pH 4.5 to about pH

7.5, more preferably about pH 6.0.

17. The pharmaceutical formulation as claimed in claim 1, 3 or claim 4, wherein the therapeutic amount of pertuzumab is ranging from about 1 mg / mL to about 400 mg / mL, preferably in the range of about 10 mg / mL to about 350 mg / mL, more preferably about 30 mg / mL.

18. The pharmaceutical formulation as claimed in any preceding claims is in liquid dosage form, lyophilized dosage form or frozen dosage form, preferably in liquid form.

19. The lyophilized formulation as claimed in claim 18 is prepared by a method comprising following steps: (a) Lreezing, (b) Primary drying, (c) Secondary drying, and optionally (d) Annealing.

20. The pharmaceutical formulation as claimed in any of preceding claims maintains structural integrity of pertuzumab upon multiple freeze-thaw cycles, preferably at least after two freeze-thaw cycles, more preferably, at least after five freeze-thaw cycles.

21. The pharmaceutical formulation as claimed in any of preceding claims maintains stability under + 2 °C to + 8 °C storage condition for at least one month.

22. The pharmaceutical formulation as claimed in any of preceding claims is administered to a subject by subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, or intramuscular administration.

23. The pharmaceutical formulation as claimed in any of preceding claims selected from a) 30 mg / mL of pertuzumab, 29 mM of arginine-citrate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20; b) 30 mg / mL of pertuzumab, 29 mM of arginine-succinate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20; c) 30 mg / mL of pertuzumab, 29 mM of arginine-phosphate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20; d) 30 mg / mL of pertuzumab, 29 mM of arginine-citrate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20; e) 30 mg / mL of pertuzumab, 29 mM of arginine-succinate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20; f) 30 mg / mL of pertuzumab, 29 mM of arginine-phosphate buffer, 30 mg / mL of proline and 0.2 mg / mL of polysorbate 20; g) 30 mg / mL of pertuzumab, 29 mM of arginine- citrate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20; h) 30 mg / mL of pertuzumab, 29 mM of arginine-succinate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20; i) 30 mg / mL of pertuzumab, 29 mM of arginine-phosphate buffer, 58 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20; j) 30 mg / mL of pertuzumab, 50 mM of histidine-acetate buffer, 120 mM of sucrose and 0.2 mg / mL of polysorbate 20; k) 30 mg / mL of pertuzumab, 50 mM of histidine-acetate buffer, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20; l) 30 mg / mL of pertuzumab, 50 mM of histidine-acetate buffer, 117 mM of sucrose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20 and m) 30 mg / mL of pertuzumab, 50 mM of histidine-acetate buffer, 119 mM of trehalose, 20 mg / mL of proline and 0.2 mg / mL of polysorbate 20.