OA17700A

OA17700A - Formulation for gonadotropins.

Info

Publication number: OA17700A
Application number: OA1201600152
Authority: OA
Inventors: Chintan Patel; Mukesh Mahajan; Sanjay Bandyopadhyay; Sanjeev Kumar Mendiratta; Bhat CHANDRESH
Original assignee: Cadila Healthcare Limited
Priority date: 2013-11-12
Filing date: 2014-10-31
Publication date: 2017-09-19

Abstract

The present invention relates to a stable composition for gonadotropins. It provides a composition useful for stabilization of gonadotropins while preventing aggregation, dissociation, fragmentation and formation of oxidized species variants in solution for injection. Thus, it prevents instability of protein or polypeptide molecules caused due to aggregation or fragmentation or oxidation during or after formulation. Also, it provides a pharmaceutical composition of gonadotropins, which can be therapeutically used for the treatment of various indications either in single-dose form or in multidose form.

Description

The présent invention relates to stable pharmaceutical compositions of gonadotropins. It provides a formulation composition useful for stabilization of gonadotropins by preventing 5 aggregation, dissociation, fragmentation and formation of oxidized species variants during and after formulation. It also provides a pharmaceutical composition of gonadotropins, which can be therapeutically used for the treatment of various indications either in single-dose form or in multi-dose form.

Background ofthe invention

Therapeutic proteins or polypeptides pose a number of challenges for pharmaceutical scientists regarding their formulation and delivery. Maintaining the physical and chemical stability of protein or polypeptide molécules in solution is important to retain the biologically active conformation of the molécule, which results in providing the desired level of potency and safety of the pharmaceutical préparation for injection comprising the protein or polypeptide molécules.

Lack of physical and chemical stability may lead to significant dégradation or irréversible modifications of protein or polypeptide molécules during processing, manufacturing, transportation and storage. Protein aggregation or fragmentation in pharmaceutical préparation is associated with loss of efficacy, altered pharmacokinetics, reduced stability, limited product shelf-life, and induction of unwanted immunogenicity. Aggregation or dissociation or fragmentation or oxidation of protein or polypeptide molécules in pharmaceutical préparation severely affects the potency of the drug product. Pharmaceutical préparation comprising such functionally compromised molécule significantly alters the efficacy, bioavailability, tissue distribution pattern and pharmacokinetic profile of the drug product with higher risk of immunogenicity. In pharmaceutical préparation of protein drug product, a number 25 of excipients hâve been used with varying success to reduce such protein dégradation or modification. However, each excipient has its own limitations, and in some cases, the more effective ones are less amenable to inclusion in final formulation. Therefore, it is always challenging to establish stable formulation of sensitive protein or polypeptide molécules with a mixture of suitable inactive ingrédients or excipients of interest, for pharmaceutical use.

Here, the présent invention provides pharmaceutical composition of proteins or polypeptides, preferably gonadotropins, which provides stable formulation of the said molécules for therapeutic use either in single-dose or multi-dose form.

Follicles stimulating hormone (FSH), Luteinizing hormone (LH), Human chorionic gonadotropin (hCG) etc. are glycoproteins in nature and composed of two subunits, alpha and beta, which 35 remain held together by non-covalent forces in protein structure. Glycosylation occurs on both alpha and beta subunits at spécifie sites on the polypeptide backbone. The alpha subunit is identical among the specified gonadotropins, while beta subunit is different for each of these glycoproteins. The beta unit is responsible for the specificity of the biological activity. The subunit alone has no known biological activity. It is the formation of heterodimer that provides the biological activity of the protein molécules.

The présent invention aims to deliver novel composition for therapeutically effective amount of FSH or its variants, which provides stable formulation of FSH or its variants for pharmaceutical use either in single-dose or multi-dose form. Follitropin alpha (recombinant human folliclestimulating hormone; follitropin alfa) is a recombinant form of follicle-stimulating hormone (FSH), an endogenous gonadotrophin. Follitropin beta is a human follicle stimulating hormone (FSH) préparation of recombinant DNA origin, which consists of two non-covalently linked, nonidentical glycoproteins designated as the alpha- and beta- subunits. The alpha- and betasubunits hâve 92 and 111 amino acids. The alpha subunit is glycosylated at Asn 51 and Asn 78 while the beta subunit is glycosylated at Asn 7 and Asn 24.

We, hereinafter, provide various formulations of the desired proteins or polypeptides, preferably gonadotropins, more preferably FSH or its variants, in which the said proteins or polypeptides remain adequately stable without undergoing further aggregation or dissociation or fragmentation or oxidation or any other modifications during and after formulation. The formulations disclosed, hereinafter, can be stored for longer period of time, under suitable storage conditions and provide better stability.

Summary of the invention

The présent invention provides a liquid stable formulation containing therapeutic amount of gonadotropins, preferably FSH or its variants for the purpose of single-use or multiple-use.

In one aspect, the présent invention provides a stable liquid formulation containing a therapeutic amount of FSH or its suitable variants and suitable excipients, selected from suitable buffers, stabilizer(s), antioxidants, preservatives and other excipients optionally selected from suitable surfactants, amino acids, and tonicity agents

In another aspect, the présent invention provides a process for preparing a stable liquid formulation of FSH or its suitable variants with suitable buffer(s), stabilizer(s), antioxidants, preservatives and other excipients optionally selected from suitable surfactants, amino acids, and tonicity agents .

In further aspect, such formulations can also be, optionally lyophilized. Lyophilization 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 yet another aspect, the présent invention provides a liquid stable formulation, which comprises of about 5 pg / mL to 200 pg / mL of FSH or its variants and suitable buffers at a concentration of about 5 mM to 100 mM, suitable stabilizers in a concentration of about 0.005 % to 10%, optionally suitable surfactants at a concentration of about 0.001% to 5%, antioxidants at a concentration of about 0.001% to 1% and optionally, preservatives at a concentration of about 0.01% to 1 %, for therapeutic use either in single-dose or multi-dose form.

In another aspect, the présent invention provides a liquid stable formulation, which optionally can be in lyophilized form comprising of about 5 pg / mL to 200 pg / mL of FSH or its variants and suitable buffers at a concentration of about 5 mM to 100 mM, optionally suitable stabilizers with a concentration of about 0.005 % to 10 %, optionally suitable surfactants at a concentration of about 0.001 % to 5 %. The said lyophilized préparation is reconstituted in suitable diluent, preferably, in the presence of suitable preservatives at a concentration of about 0.01 % to 1 %, for therapeutic use either in single-dose or multi-dose form.

In one ofthe embodiments, the présent invention provides a liquid formulation buffered between pH 5 to 9.

In another embodiment, the présent invention provides a liquid formulation, which can be used for parentéral administration. Parentéral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parentéral administration and as is well known to a skilled person.

In another embodiment, the présent invention provides a liquid formulation which stabilizes the protein or polypeptide molécule in solution by preventing any further dégradation of the desired protein or polypeptide, during and after formulation. Generally, a stable formulation is the one which retains the physical stability and chemical stability and/or biological activity over a period of time, upon storage.

In a further embodiment, the présent invention provides a liquid stable formulation of FSH or its variants, which can be therapeutically used for the relevant indications.

Detailed description ofthe présent invention

The présent invention provides novel liquid stable formulation, which can optionally be lyophilized, comprising of suitable amount of therapeutic protein(s), preferably gonadotropins in suitable buffer(s), one or more suitable stabilizers, and other excipients, which are selected from antioxidants, preservatives and other excipients optionally selected from suitable surfactants, amino acids, and tonicity agents . The présent formulation stabilizes the gonadotropins during and after formulation and prevents any further dégradation or modification of protein or polypeptide, while maintaining the active biological conformation of the protein or polypeptide during and after formulation. In such embodiment the protein is gonadotropin. In such embodiment the gonadotropin is derived from either urine or can be produced by recombinant technology. In a preferred embodiment the gonadotropin is selected from FSH, LH, hCG and combination thereof. In a more preferred embodiment, gonadotropin is FSH or its variants.

In some embodiments, the FSH or its variant is generally présent in a therapeutic amount of up to 200 pg / mL. In a preferred embodiment the therapeutic amount is about 5 pg / mL to 100 pg / mL. In a more preferred embodiment the therapeutic amount is about 5 pg / mL to 50 pg / mL.

The liquid formulation comprises a suitable buffer along with other pharmaceutically acceptable excipients, which stabilizes the pharmaceutical préparation. Suitable buffers, which can be used are selected from those, which are known in the art and can be found in literature. In an embodiment, the suitable buffers comprise but are not limited to histidine, arginine, citrate, succinate, acetate, phosphate, tromethamine buffers and the like or their suitable mixtures such as citrate-phophate and the like.

In a preferred embodiment, the suitable buffer comprises of a phosphate buffer or a succinate buffer.

The buffers are generally used in concentrations of about 5 mM to 100 mM. In a preferred embodiment, the buffer concentration is about 10 mM to 50 mM.

In an embodiment, the liquid formulation maintains a pH value ranging from pH 5 to about pH 9 depending on the FSH or its variant being used. In a preferred embodiment, the buffer used maintains the pH of the formulation in the range of about pH 6 to pH 8. In a more preferred embodiment, the pH is maintained to about pH 7.

The liquid formulation further comprises suitable surfactants, which are pharmaceutically acceptable excipients used to protect the protein formulations against various stress conditions, like agitation, shearing, exposure to high température etc., and reduce the surface interaction e.g., liquid-air or liquid-solid interfaces, during and after formulation.. The suitable surfactants include but are not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylenepolyoxypropylene copolymer (e.g. Poloxamer, Pluronic), octanoic acid (caprylate), sodium dodecyl sulphate (SDS) and the like. In a preferred embodiment, the suitable surfactant is polyoxyethylenesorbitan-fatty acid esters (Tweens). In a more preferred embodiment, the polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). In another preferred embodiment, the suitable surfactant is polyethylene-polypropylene copolymers, which are sold under the names Pluronic (R) F68 or Poloxamer 188™. In another preferred embodiment, the suitable surfactant is alkylphenolpolyoxyethylene esters, which are sold under the trade name Triton-X.

The surfactants are generally used in concentrations of about 0.001% to 5%. In a preferred embodiment, surfactant concentration is about 0.01% to 1%.

The liquid formulation further comprises one or more pharmaceutically acceptable or suitable stabilizer(s), which protect the active pharmaceutical ingrédient from chemical and/or physical dégradation during processing, manufacturing, transportation, storage and application. In an embodiment, the stabilizers include but are not limited to suitable sugars, amino acids, polyols, polyethylene glycols (PEGs), polyethyleneimine, cyclodextrines and the like or suitable dérivative or mixtures, thereof.

In one such embodiment, the sugar is a monosaccharide or an oligosaccharide. Monosaccharide sugars include but are not limited to glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, dextran, dextrin and the like or amino sugars, like neuraminic acid or N-acetyl glucosamine and the like. An oligosaccharide includes but is not limited to sucrose, trehalose, lactose, maltose and raffinose and the like or suitable mixtures, thereof.

In another embodiment the polyols which can be used as stabilizers include but are not limited to mannitol, sorbitol, glycerol, arabitol, polyethylene glycol, propylene glycol and the like or suitable combinations thereof. In a preferred embodiment the suitable polyol is sorbitol or mannitol.

In another embodiment, polyethylenieimine can also be used as a stabilizer. In another preferred embodiment, the stabilizer is selected from sugar, polyol and suitable combination thereof. In an embodiment the stabilizer is présent in amount about 0.005 % to about 10 %.

In a more preferred embodiment, the stabilizer is Polyethylene glycol (PEG) or Polyethyleneimine. In the présent invention, polyethylene glycol having molecular weight in the range of 200 Dalton to 40,000 Dalton can be used. In a preferred embodiment, the formulation according to the présent invention contains polyethylene glycol having molecular weight in the range of 200 Dalton to 10,000 Dalton. In a preferred embodiment, polyethylene glycol is présent in amount about 0.005 % to about 10 %.

In another embodiment, cyclodextrines or dérivatives thereof, which can be used as stabilizers, include but are not limited to a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or their hydroxypropylated, hydroxyethylated, ethylated or methylated dérivatives thereof or Sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) or branched cyclodextrins or cyclodextrin polymers or suitable mixture thereof. In a preferred embodiment the suitable cyclodextrin variant is hydroxypropylated cyclo beta-dextrin (ΗΡ-β-CD).

In a preferred embodiment, the cyclodextrin or dérivative is présent in amount about 0.2 % to about 10 %. In another such embodiment, the amino acids which can be used as stabilizers or antioxidants include but are not limited to arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, méthionine, serine, proline, cysteine / cystine and the like or suitable combination of any of the above. In a preferred embodiment, the suitable amino acid is méthionine or cysteine or glycine or tryptophan or combination, thereof.

In an embodiment, the amino acid is présent in amount about 0.01 % to 10 %. Here, a skilled person can also use ascorbic acid or EDTA or combination, thereof, as an antioxidant(s) separately or in combination with other antioxidant(s) in the said formulation. The antioxidant according to the current invention is présent in the concentration range of 0.001% to 1%, preferably, 0.01% to 0.5%.

In one of the embodiments, the stable liquid formulation comprises preservatives selected from hydroxybenzens (phénol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol and the like), paraben (methyl, ethyl, propyl, butyl and the like), sodium benzoate, benzyl benzoate, benzalkonium chloride, benzéthonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof. In a preferred embodiment, the preservative is selected from phénol, paraben, sodium benzoate, benzyl benzoate and mixture thereof.

In a preferred embodiment, the preservative is présent in amount about 0.01% to 1%. In a more preferred embodiment, the preservative is présent in amount of 0.01% to 0.5%.

In another embodiment, the liquid formulation optionally comprises tonicity agents such as sodium chloride or potassium chloride. In a preferred embodiment, the tonicity agent is sodium chloride, which is présent in amount about 10 mM to about 150 mM.

Phosphoric acid or sodium hydroxide can be used in a suitable amount to adjust the desired pH of the formulation.

The formulation may additionally further comprises one or more suitable other excipients, which are well known to a person skilled in the art.

In some embodiments, the liquid formulation maintains the storage stability in terms of not allowing any further protein dégradation or modifications as compared to the initial.

In some embodiments, the liquid formulation maintains the stability during the process of formulation.

In one ofthe embodiments, the stable liquid formulation with said excipients can be prepared for combination of FSH and LH or FSH and hCG or LH and hCG.

To estimate the level of high molecular weight species or aggregates and low molecular weight or dissociated species, analytical HP-size exclusion chromatography was performed. To analyze oxidized species variants or purity of desired protein a person skilled in the art can use reversed-phase HPLC. In-vivo or in-vitro biological assay can be performed to check the biological activity of the desired protein. A person skilled in the art can use other analytical tools/techniques known in the art to check the physico-chemical as well as biological properties ofthe desired protein.

The said analytical methods used in the présent invention are well known to a skilled person and a brief description ofthe same is provided below merelyforthe sake of reference only.

ΗΡ-Size exclusion chromatography (HP-SEC):

Samples were analyzed to estimate the high molecular weight species or aggregates and low molecular weight or dissociated species by HP-size exclusion chromatography (HP-SEC) using TSK gel G3000 SWXL column (7.8 mm I.D χ 30 cm L). Samples were loaded and eluted isocratically using sodium phosphate buffer at a flow rate of 0.5 mL / min. Elution was monitored at UV215 nm.

The following non-limiting examples describe the different formulations, which can be prepared as per the présent invention. It will be appreciated that other excipients may be added to these formulations and these are within the scope of a person skilled in the art.

The présent invention is illustrated further through the following examples which are provided for illustration purpose and should not be construed as being a limitation to the scope of the invention.

Example 1

Active Ingrédient
FSH	44 pg / mL
Inactive Ingrédients
Sodium phosphate	10 mM
Polyethylene Glycol	0.1 %
EDTA	0.1 %
Sodium benzoate	0.3 %

Follitropin alfa was purified as per the technique known in the art. In this example, the purified

Follitropin alfa was formulated in sodium phosphate buffer, further comprising polyethylene glycol, EDTA and sodium benzoate at desired concentrations, as described above. pH of the formulation medium was maintained around pH 7.0. If required, pH of the formulation can be adjusted using O-phosphoric acid or sodium hydroxide. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level. The formulated bulk was distributed in suitable container-closure Systems (like vials, cartridges, syringes etc.) for storage. Similarly, a person skilled in the art can also formulate the composition for Follitropin beta. Upon formulation, samples were analyzed for the presence of high molecular weight species or aggregates and dissociated or fragmented species variants by ΗΡ-Size exclusion chromatography (HP-SEC). A person skilled in the art can analyze said parameters at various température conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed condition (higher température).

Example 2

Active Ingrédient
FSH	44 pg / mL
Inactive Ingrédients
Sodium phosphate	10 mM
Arginine	0.5 %
Polyethylene Glycol	0.1 %
EDTA	0.1 %
Sodium benzoate	0.3 %

Follitropin alfa was purified as per the technique known in the art. In this example, the purified Follitropin alfa was formulated in sodium phosphate buffer, further comprising arginine, polyethylene glycol, EDTA and sodium benzoate at desired concentrations, as described above. pH of the formulation medium was maintained around pH 7.0. If required, pH of the formulation can be adjusted using O-phosphoric acid or sodium hydroxide. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level. The formulated bulk was distributed in suitable container-closure Systems (like vials, cartridges, syringes etc.) for storage. Similarly, a person skilled in the art can also formulate the composition for Follitropin beta. Upon formulation, samples were analyzed for the presence of high molecular weight species or aggregates and dissociated or fragmented species variants by ΗΡ-Size exclusion chromatography. A person skilled in the art can analyze said parameters at various température conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed condition (higher température).

Example 3

Active Ingrédient
FSH	44 pg / mL
Inactive Ingrédients
Sodium phosphate	10 mM
Méthionine	0.01 %
Polyethylene Glycol	0.1 %
EDTA	0.1 %
Sodium benzoate	0.3 %

Follitropin alfa was purified as per the technique known in the art. In this example, the purified Follitropin alfa was formulated in sodium phosphate buffer, further comprising méthionine, polyethylene glycol, EDTA and sodium benzoate at desired concentrations, as described above.

pH of the formulation medium was maintained around pH 7.0. If required, pH of the formulation can be adjusted using O-phosphoric acid or sodium hydroxide. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level. The formulated bulk was distributed in suitable container-closure Systems (like vials, cartridges, syringes etc.) for storage. Similarly, a person skilled in the art can 10 also formulate the composition for Follitropin beta. Upon formulation, samples were analyzed for the presence of high molecular weight species or aggregates and dissociated or fragmented species variants by ΗΡ-Size exclusion chromatography. A person skilled in the art can analyze said parameters at various température conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed condition (higher 15 température).

Example 4

Active Ingrédient

FSH	44 pg / mL
Inactive Ingrédients
Sodium phosphate	10 mM
Sucrose	6 %
Méthionine	0.01 %
Polyethylene Glycol	0.1 %
EDTA	0.1 %
Phénol	0.3 %

Follitropin alfa was purified as per the technique known in the art. In this example, the purified Follitropin alfa was formulated in sodium phosphate buffer, further comprising sucrose, méthionine, polyethylene glycol, EDTA and phénol at desired concentrations, as described above. pH of the formulation medium was maintained around pH 7.0. If required, pH of the formulation can be adjusted using O-phosphoric acid or sodium hydroxide. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level. The formulated bulk was distributed in suitable 5 container-closure Systems (like vials, cartridges, syringes etc.) for storage. Similarly, a person skilled in the art can also formulate the composition for Follitropin beta. Upon formulation, samples were analyzed for the presence of high molecular weight species or aggregates and dissociated or fragmented species variants by ΗΡ-Size exclusion chromatography. A person skilled in the art can analyze said parameters at various température conditions like Real-Time 10 storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed condition (higher température).

Example 5

Active Ingrédient
FSH	44 pg / mL
Inactive Ingrédients
Sodium phosphate	10 mM
Polyethylene Glycol	0.1 %
Sodium chloride	100 mM
EDTA	0.1 %
Sodium benzoate	0.3 %

Follitropin alfa was purified as per the technique known in the art. In this example, the purified Follitropin alfa was formulated in sodium phosphate buffer, further comprising polyethylene 15 glycol, sodium chloride, EDTA and sodium benzoate at desired concentrations, as described above. pH of the formulation medium was maintained around pH 7.0. If required, pH of the formulation can be adjusted using O-phosphoric acid or sodium hydroxide. Excipients were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level. The formulated bulk was distributed in suitable 20 container-closure Systems (like vials, cartridges, syringes etc.) for storage. Similarly, a person skilled in the art can also formulate the composition for Follitropin beta. Upon formulation, samples were analyzed for the presence of high molecular weight species or aggregates and dissociated or fragmented species variants by ΗΡ-Size exclusion chromatography. A person skilled in the art can analyze said parameters at various température conditions like Real-Time 25 storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed condition (higher température).

Follitropin alfa is formulated in different compositions as described above and exposed to higher température to check dégradation over the period of time. Results of HP-SEC analysis, obtained for the different samples are shown below in Table 1. No significant increase in either high molecular weight species (HMWs) or aggregates and low molecular weight (LMWs) or dissociated species observed over the period of time when Follitropin alfa is exposed to higher température with formulation compositions as described with different examples above.

Table 1: Results of HP-SEC analysis obtained with samples of different formulation 5 compositions

Formulations	Exposed to higher température (40 °C)
	Initial	3^rd Day	7^th Day	15^th Day
Example 1	HMWs /Aggregates	0.03 %	0.02 %	0.02 %	-
Principal peak (/β intact)	99.97 %	99.98 %	99.98 %	-
Dissociated species / LMWs	ND	ND	ND	-
Example 2	HMWs/Aggregates	0.01 %	0.03 %	0.02 %	0.02 %
Principal peak (/β intact)	99.99 %	99.97 %	99.98 %	99.94 %
Dissociated species / LMWs	ND	ND	ND	0.04 %
Example 3	HMWs/Aggregates	0.03 %	0.02 %	0.03 %	0.02 %
Principal peak (/β intact)	99.97 %	99.98 %	99.97 %	99.91 %
Dissociated species / LMWs	ND	ND	ND	0.07 %
Example 4	HMWs/Aggregates	0.05 %	0.06 %	0.05 %	0.02 %
Principal peak (/β intact)	99.95 %	99.94 %	99.83 %	98.81 %
Dissociated species / LMWs	ND	ND	0.12%	1.18%
Example 5	HMWs/Aggregates	0.05 %	0.02 %	0.02 %	-
Principal peak (/β intact)	99.95 %	99.98 %	99.98 %	-
Dissociated species / LMWs	ND	ND	ND

ND - Not détectable; HMWs - High molecular weight species; LMWs - Low molecular weight species

Various compositions as mentioned in the preceding description and examples can also be prepared for FSH or its variant protein using similar process. FSH protein formulated with 10 different compositions described in the présent invention can also be stored between +2 °C and +8 °C for long term storage in suitable container-closure Systems (like vials, cartridges, syringes etc.).

Table 2: Formulation compositions of Follitropin

Example 6	Example 7
Active Ingrédient	Active Ingrédient
Follitropin	44 pg / mL	Follitropin	44 pg / mL

Inactive Ingrédients	Inactive Ingrédients
Sodium phosphate	10 mM	Sodium phosphate	10 mM
Sucrose	6 %	Méthionine	0.01 %
Polyethylene Glycol	0.1 %	Sucrose	6 %
EDTA	0.1 %	Polyethylene Glycol	0.1 %
Phénol	0.3 %	EDTA	0.1 %
		Phénol	0.3 %

Results of HP-SEC analysis, obtained for different samples stored between +2 °C and +8 °C are shown below in Table 3. No significant increase in either high molecular weight species (HMWs) or aggregates and low molecular weight (LMWs) or dissociated species observed over the period of time when Follitropin is stored at +2°C to +8 °C with different formulation 5 composition exemplified in Table 2.

Table 3

Results of HP-SEC analysis obtained with samples of different formulation compositions

Formulations	Stored between +2 °C and +8 °C
	Initial	After 12 Months
Example 6	HMWs /Aggregates	0.2 %	0.2 %
Principal peak (/β intact)	99.8 %	99.7 %
Dissociated species / LMWs	ND	0.01 %
Example 7	H MWs/Aggregates	0.4 %	0.3 %
Principal peak (/β intact)	99.5 %	99.7 %
Dissociated species / LMWs	ND	0.01 %

Similarly, a person skilled in the art can also formulate the other gonadotropins such as LH or HCG according to the présent invention.

Various compositions can be prepared using the excipients disclosed in the spécification and following similar processes as mentioned in the preceding Examples. The other suitable compositions that can be used for stabilization of the FSH or its variant protein are mentioned in 15 the below Table 3.

Table 3
Example 8 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Méthionine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1%-0.5% Phénol	Example 9 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% -0.5 % Phénol	Example 10 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1%-0.5% Phénol
Example 11 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1%-0.5% Phénol	Example 12 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Méthionine 0.1 -10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% -0.5 % Phénol	Example 13 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% -0.5 % Phénol
Example 14 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 5-100 mM Méthionine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% -0.5 % Phénol	Example 15 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 -10 % Sucrose 5-100 mM Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% -0.5 % Phénol	Example 16 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 5-100 mM Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% -0.5 % Phénol

Example 17 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate butter of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 -10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% -0.5 % Phénol	Example 18 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Méthionine 0.1 - 10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1%-0.5% Phénol	Example 19 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 00.001 - 1% Polysorbate 20 0.01 - 5 % EDTA 0.1%-0.5% Phénol
Example 20 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 -10% Sucrose 0.1 - 10 % Méthionine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 21 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 22 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben
Example 23 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 -10 % Sucrose 0.1 -10 % Méthionine 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 24 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1-10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 25 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben
Example 26 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0	Example 27 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0	Example 28 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0

0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben
Example 29 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 30 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben	Example 31 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 00.001 - 1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Paraben
Example 32 5-200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 33 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 34 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate
Example 35 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium	Example 36 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 -10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium	Example 37 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA

Benzoate	Benzoate	0.1% - 0.5 % Sodium Benzoate
Example 38 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 -10 % Sucrose 0.1 -10 % Méthionine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 39 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 40 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1-10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate
Example 41 5-200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Méthionine 0.1 - 10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 42 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate	Example 43 5-200 pg/mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 00.001 - 1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Sodium Benzoate
Example 44 5-200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 45 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA Ifa, - 0.5 % Benzyl Benzoate	Example 46 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate

Example 47 5-200 pg/mLFSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 48 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 49 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1-10 % Tryptophan 0.01 - 5 % Polyethylene Glycol 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate
Example 50 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Méthionine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 51 5-200 pg/mLFSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 -10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 52 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate
Example 53 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 54 5-200 pg/mLFSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1-10 % Tryptophan 0.001 -1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate	Example 55 5 - 200 pg / mL FSH 5 - 100 mM Sodium phosphate buffer of pH 7.0 0.1 - 10 % Sucrose 0.1 - 10 % Méthionine 0.1 - 10 % Cysteine 0.1 - 10 % Tryptophan 00.001 - 1% Polysorbate 20 0.01 - 5 % EDTA 0.1% - 0.5 % Benzyl Benzoate

Similar formulations can be prepared using 5-100 mM of acetate buffer (sodium acetate acetic acid) or of succinate buffer or of citrate buffer (sodium citrate-citric acid) or of Phosphate buffered saline or of Arginine buffer or of citrate-phosphate buffer or of histidine buffer and the like with pH range of about pH 5.0 to pH 9.0.

Similar formulation can be prepared using 0.01 % to 10 % of raffinose or of trehalose or of sorbitol or of dextran or of cyclodextrin or of mannitol.

Similar formulation can be prepared using 0.001% to 5% of pluronics (poloxamers) alone or in combination with Polyethylene Glycol or polysorbates.

Similar formulation can be prepared using ascorbic acid in a suitable concentration.

Similar formulation can be prepared using tonicity agents with suitable concentrations.

Similar formulation can also be prepared for other gonadotropins, like LH or its variants and hCG or its variants or combination thereof. A skilled person can préparé similar formulation for combination of gonadotropins selected from LH or its variant and FSH or its variant, hCG or its variant and FSH or its variant.

The formulations of the présent invention can be used for the treatment in which activity of 10 gonadotropin is detrimental.

Claims

1. A liquid pharmaceutical formulation comprising an effective amount of gonadotropin in a buffer system, polyethylene glycol as a stabilizer, suitable preservative and optionally other suitable excipients.

2. The formulation as claimed in claim 1, wherein the buffer system is selected from histidine-buffer, arginine-buffer, citrate-buffer, succinate-buffer, acetate-buffer, phosphate-buffer, tromethamine buffers, citrate-phosphate buffer and the like or suitable mixture thereof or wherein the buffer is in the concentration of 5 mM to about 100 mM,

3. The formulation as claimed in claim 1, wherein the polyethylene glycol is présent in the concentration of 0.005% to 10%.

4. The formulation as claimed in claim 1, wherein the preservative is selected from hydroxybenzenes (phénol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol and the like), paraben (methyl, ethyl, propyl, butyl and the like), sodium benzoate, benzyl benzoate, benzalkonium chloride, benzéthonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof, preferably, sodium benzoate or phénol or wherein the preservative is présent in a concentration of 0.01% to 1%.

5. The formulation as claimed in claim 1, wherein other suitable excipients can be selected from antioxidants, other stabilizers, surfactants, tonicity agents and suitable combination thereof.

6. The formulation as claimed in claim 5, wherein the antioxidant is selected from EDTA, ascorbic acid or their suitable combination or wherein the antioxidant is présent in the concentration of 0.001% to 1% or wherein the surfactant is selected from polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers, alkylphenylpolyoxyethylene ethers, polyoxyethylene-polyoxypropylene copolymer and sodium dodecyl sulphate (SDS), preferably polysorbate 80 or polyoxyethylenepolyoxypropylene copolymer or wherein the surfactant is présent in the concentration of 0.001 % to about 1 % or wherein the tonicity agent is sodium chloride or potassium chloride or wherein the tonicity agent is présent in amount about 10 mM to about 150 mM or wherein the stabilizer is selected from suitable sugars, polyols, amino acids and suitable combination thereof.

7. The formulation as claimed in claim 6, wherein the amino acid is selected from arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, méthionine, serine, proline, cysteine / cystine and suitable combination thereof or wherein the amino acid is présent in the concentration of 0.01% to 1% or wherein the sugar is selected from glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, dextran, dextrin, sucrose, trehalose, lactose, maltose, raffinose and suitable mixtures thereof or wherein the sugar is présent in the concentration of 0.005% to 10%.

8. The formulation as claimed in claims 1, wherein the concentration of gonadotropin is between 5 pg/ml to 200 pg/ml or wherein the pH of the formulation is between pH 6 and pH 8.

9. A formulation as claimed in claims 1 comprising:

a) 5-200 pg/ml of gonadotropin,

b) 0.005-10% polyethylene glycol

c) a buffer system for maintaining the pH between 6 and 8

d) 0.01-1% of preservative;

e) optionally with other suitable excipients selected from antioxidants selected from EDTA, ascorbic acid and combination thereof.

10. The formulation as claimed in claim 9, wherein the buffer system is a phosphate or succinate or acetate or citrate-phosphate buffer system, the preservative is sodium benzoate or phénol or m-cresol, the surfactant and/or tonicity agent is selected from polysorbate 80 and/or sodium chloride respectively and gonadotropin is selected from follicle stimulating hormone or variant thereof, luteinizing hormone or variant thereof, human chorionic gonadotropin hormone or variant thereof and wherein the follicle stimulating hormone is selected from the follitropin alfa, follitropin beta..

11. A formulation as claimed in claim 1 comprising:

a) 44 pg/ml of follitropin alfa,

b) 0.1% polyethylene glycol,

c) 10 mM phosphate buffer with a pH about 7.

d) 0.1 % of EDTA

e) 0.3% of sodium benzoate

f) 100 mM of sodium chloride