FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See section 10 and rale 13)
LIQUID FORMULATION OF GONADOTROPINS
Intas Bio pharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 213
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
The present invention relates to an aqueous formulation of human Gonadotropins which are stabilized to maintain its activity for a prolonged period of time for therapeutic use to guarantee a reasonable shelf life. More specifically, the present invention relates to an aqueous formulation of gonadotropin (e.g. FSH, TSH, LH, hCG. hMG) comprising a therapeutically effective amount of gonadotropin stabilized in a buffer containing one or more stabilizer, a non-ionic surfactant, an antioxidant, optionally with one or more preservative' and optionally an inorganic salt
BACKGROUND OF THE INVENTION
Gonadotropins form a family of structurally related glycoprotein hormones. Typical members include chorionic gonadotropin (hCG), Follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). Gonadotropins are synthesized and secreted by the pituitary gland in most vertebrate species. Structurally, the gonadotropins are heterodimers composed of two dissimilar subunits, namely alpha and beta, which are associated by non-covalent bonds. The alpha subunit is essentially identical for each member of the gonadotropin family. The beta subunits are different for each protein, i.e. hCG, FSH, TSH and LH, but show considerable homology in structure. In human, the alpha subunit consists of 92 amino acid residues, where the beta subunit varies in size for each protein: 111 residues in FSH, 121 residues in LH, 118 residues in TSH and 145 residues in hCG. ((Combamous, Y. (1992), Endocrine Reviews, 13, 670-691; Lustbader, J. W. et al. (1993), Endocrine Reviews, 14, 291-311).
Purified FSH, alone or in combination with semi-purified human menopausal gonadotropins containing a mixture of FSH and LH, has been used to stimulate the development of ovarian follicles which is required for assisted reproduction techniques such as the IVF (in vitro fertilization) method.
Until the 1980's, a primary source of hFSH was urine of pregnant women. A further purified form of high-purity; urine-derived FSH was introduced in the 199CFs. Later, in 1998, a recombinant FSH molecule was developed and since then it is widely used for the treatment of infertility. With the advent of recombinant DNA technology, it became possible to produce

human FSH in cell cultures transfected with the nucleic acid sequences coding for the alpha and beta chain. DNA sequences coding for the alpha and beta chains and methods for producing recombinant FSH have been disclosed in WO88/10270, WO86/04589 and EP0735139. FSH stimulates the growth and maturation of the ovarian follicles in females. Therefore, FSH is a critical hormone responsible for ovulation of females and is used iri the infertility treatment of anovulatory infertile women.
Generally, proteins have a very short half-life, and undergo denaturation upon exposure to various external factors such as high pressure, physical or mechanical stress, organic solvents, contact with water-air interface and microbial contamination etc. The denaturation of proteins may lead to aggregation of monomers, dissociation of subunits, oxidation and adsorption on the surfaces of vessels. Denaturation of proteins usually causes irreversible loss of native properties including bioactivity.
In the bio pharmaceutical industry, the long term storage of gonadotropins, prepared using recombinant DNA technology in aqueous formulations, is generally a difficult task. This is particularly true fo'r pure gonadotropins in relatively dilute solutions. To overcome the stability problem of proteins in aqueous formulations, gonadotropin products are stabilized via lyophilization (freeze-drying). For example, U.S. Patent No. 5270057 discloses a lyophilized formulation comprising gonadotropin stabilized with polycarboxylic acid or a salt thereof, preferably citric acid and a non-reducing disaccharide, sucrose. U.S. Patent No. 5650390 discloses a lyophilized formulation comprising FSH, LH or hCG stabilized by means of a combination of sucrose and glycine. Commercially, human menopausal gonadotropin (hMG) (mixture of FSH and LH in an approximate ratio of 1:1 and human chorionic gonadotropin (hCG) are available in lyophilized form, under the trade names Humegon® and Pregnyl® respectively, from Schering Plough. However, lyophilized products are inconvenient because they need to be reconstituted in Water for Injection (WFI) prior to use. Further, the production process of the lyophilized products involves a freeze-drying step which could result in freeze-injury to the protein lyophilized.
U.S. Patent No. 5929028 discloses a liquid formulation of gonadotropin, which is prepared by dissolving gonadotropin using a diluent composed of a stabilizing amount of polycarboxylic

acid or a salt thereof, a stabilizing amount of a thioether compound, a non-reducing disaccharide like sucrose and a non-ionic surfactant. According to this art, sodium citrate is described as the most preferred form of polycarboxylic acid or a salt thereof and the formulation contains 1.47% sodium citrate. This composition should be stored refrigerated at 2-8 °C until dispensed. Upon dispensing, the product may be stored at 2-8°C until the expiration date, or at or below 25°C (77°F) for three months, whichever occurs first.
European Patent Application No; EP 1610822 A2 describes the pharmaceutical formulations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and mixtures of FSH and LH, and methods of producing such formulations. The invention describes a liquid or freeze-dried formulation of FSH or LH or FSH and LH comprising a surfactant selected from Pluronic®F77, Pluronic®F87, Pluronic®F88, and Pluronic®F68. The composition as described in this patent application should be stored refrigerated at 2-8°C until dispensed. Upon reconstitution with the diluent, the product may be stored by the patient at 2-8°C until the expiration date, or at room temperature (20-25°C) for upto three months or until the expiration date, whichever occurs first
PCT patent application no. WO 1996/029095 A3 discloses a gonadotropin containing liquid pharmaceutical compositions. More precisely, it concerns liquid formulations of hCG stabilized with a polyalcohol or a non-reducing sugar. The composition is stabilized with mahnitol in phosphate buffer at pH 7. Such compositions are ready to be injected and, therefore, the step of reconstitution of the lyophilised powder is avoided, thus simplifying the way of use.
PCT patent application No. WO 2000/004913 discloses a formulation comprising FSH or.an FSH variant, containing an alpha subunit and a beta subunit, and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluents. The formulation further comprises isotonic agent and a physiologically acceptable buffer such as phosphate buffer saline.
PCT patent application no. WO 2007/037607 Al discloses an aqueous formulation of human Follicle stimulating hormone (FSH) which is stabilized to maintain the activity of FSH for a
b

prolonged period of time. The patent application claims an aqueous formulation comprising a therapeutically effective amount of FSH stabilized in a phosphate buffer containing glycine, methionine and a non-ionic surfactant, preferably polysorbate 20. which is capable of maintaining the activity of FSH for an extended period of time.
PCT patent application no. WO 2009/098318 Al discloses a liquid pharmaceutical composition comprising a follicle stimulating hormone polypeptide and benzalkonium chloride and benzyl alcohol as preservatives. The composition further comprises optionally one or more additional pharmaceutically acceptable excipients. In one embodiment, the composition contains methionine as an antioxidant. The composition shows good storage stability when stored at 2-8°C. This FSH aqueous formulation comprising of Polysorbate 20 as a surfactant, mannitol as a tonicity modifier, phosphate as a buffer, methionine as a stabilizing agent, and benzyl alcohol and benzalkonium chloride as preservatives prevents protein loss also and dissociation of the protein into constituent monomers, thereby, stabilizing FSH for a prolonged period of time.
Currently, in the market, there are two commercial recombinant human FSH liquid products; Gonal-F RTF® from Merck Serono and Follistim AQ® from Schering Plough, in phosphate and citrate buffer respectively at pH 7.0. Ovidrel® (250 meg hCG /0.5 ml) single dose Pre-filled Syringe is also available from Sereno. Pergoveris® from Sereno containing follitropin alfa (r-hFSH) and lutropin alfa (r-hLH) is also formulated in phosphate buffer.
These gonadotropin products are highly prone to oxidative damage. The product insert for Gonal-f® RTF Pen states that under current storage conditions at 2-8 °C, it may contain up to 10% of oxidized follitropin alfa. Such a large amount of oxidative impurities may decrease the potency of the product and hence the therapeutic efficacy of the product.
The nature of additives and stabilizers in commercial protein formulations can vary. However, the common feature of the commercial formulations of gonadotropin products, both in powder and in aqueous form, is the presence of methionine as an anti-oxidant. An antioxidant is usually required to prevent the product from oxidation. To the best of our knowledge, as of now, all the commercial products and prior art available for gonadotropin formulations contemplate methionine as an anti-oxidant which is ineffective in preventing the oxidation as

in the case of Gonal-F-RFF pen (based on manufacturer product information sheet). Consequently, there remains a need in the art to minimize oxidation related impurities by using more effective anti-oxidant(s) or their combinations when stored at 2-8 °C. These impurities could limit the effective shelf-life of the product.
SUMMARY OF THE INVENTION
The present invention provides a stable liquid formulation comprises of therapeutically effective amount of a human gonadotropin, glycine as a stabilizer and cysteine as an antioxidant in citrate buffer to maintain a pH of the formulation in the range of 6.5 to 7.2.
In one aspect, a stable liquid formulation comprises of therapeutically effective amount of a human gonadotropin, glycine as a stabilizer and cysteine as an anti-oxidant in citrate buffer to maintain a pH of the formulation in the range of 6.5 to 7.2, non-ionic surfactant and a preservative.
In another aspect, wherein the suitable concentration of citrate buffer is 1 to 100 mM, a suitable concentration of glycine is 1 mM to 500 mM and a suitable concentration of cysteine is 0.01 to 5 mg/ml.
A stable liquid formulation of the present invention has a pH between 6.5 and 9.
In a further aspect, the non-ionic surfactant of the gonadotropin formulation is selected from the group consisting of a polysorbate-based non-ionic surfactant and a poloxamer-based non-ionic surfactant or a combination thereof.
In a further aspect, the formulation optionally consists of preservative selected from group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, alkylparaben, benzethonium chloride, sodium dehydroacetate and thimerosal or a combination thereof.

The gonadotropin of the present invention is naturally occurring gonadotropins or produced by recombinant gonadotropins selected from the group consisting of follicle stimulating hormone, luteinizing hormone and chorionic gonadotropin or in combination thereof.
Where appropriate, the formulation may further include an isotonic agent. Specific examples of the isotonic agent may include water-soluble inorganic salts such as sodium chloride and . calcium chloride may be used alone or in any combination thereof.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows the comparison of dissociation form of alpha and beta subunits of recombinant human follicle stimulating hormone composition after 1 hour at 55°C
Lane 1: Gel-filtration chromatography output
Lane 2: F-l
Lane 3: F-2
Lane 4: F-3
Lane 5: F-4
Lane 6: F-5
Lane 7: F-6
Lane 8: 10% dissociated (by boiling) output
' Figure 1 shows the comparison of dissociation form of alpha and beta subunits of recombinant human follicle stimulating hormone composition after 2 hours at 55°C Lane 1: Gel-filtration chromatography output Lane 2: F-l Lane 3: F-2 Lane 4: F-3 Lane 5: F-4 Lane 6: F-5 Lane 7: F-6 Lane 8: 10% dissociated (by boiling) output

Figure 3 shows RP-HPLC Chromatogram of FSH formulations containing different concentrations of cysteine
Figure 4 shows EF profie of FSH after 15 days at 40°C on Gradient gel (pH 3-9) Lane 1: FSH in Citrate buffer and Cysteine Lane 2: FSH only in Citrate buffer Lane 3: Composition given in Table 4 Lane 4: FSH in Citrate buffer and Poloxamer Lane 5: FSH in Citrate buffer and phenol Lane 6: Standard
Figure 5 shows the SDS-PAGE profile of FSH formulation after 15 days at 40°C
Lane 1: Same composition as given in table 4 with polysorbate 20 instead of
Poloxamer 407
Lane 2: Same composition as given in table 4 with polysorbate 80 instead of
Poloxamer 407
Lane 3: Composition as given in Table 4
Lane 4: Standard
Lane 5: 5% dissociated Standard
Figure 6 shows the SDS-PAGE profile of HCG formulation after 2 hours at 55°C
Lane 1: RMP
Lane 2: Formulation as given in table 6 (Initial, 0 time)
Lane 3: formulation as given in table 6
DESCRIPTION OF THE INVENTION
The present invention is to provide an aqueous formulation of human gonadotropins (hFSH, hCG, hLH, hMG, TSH) stabilized to maintain its activity for a prolonged period of time to guarantee a reasonable shelf-life.

Human Follicle stimulating hormone (hFSH) is produced by recombinant DNA technology. FSH being a complex heterodimeric protein, a eukaryotic cell line has been selected for expression work (Chinese hamster ovary cells). The pharmaceutical preparation of recombinant human FSH (rFSH) differs from that of human menopausal gonadotropin (hMG) and the first generation of urinary FSH (uFSH) in terms of source of bulk materials, purity, specific activity, batch to batch consistency and complete absence of luteinzing hormone activity.
Buffers are suitable for maintaining pH of the formulation. The buffer system which may be used comprises phosphate buffer, succinate buffer, citrate buffer, histidine and the like, either alone or in suitable combination giving desired pH range from 6 to 8.
Anti-oxidant as used herein refers to a chemical that reduces the quantity of oxidized human gonadotropin impurity/ species within a solution. An anti-oxidant used in the present invention is selected from the group consisting of cysteine, methionine, sodium bisulfite, salts of ethylenediamine tetraacetic acid (EDTA), butylated hydroxytoluene (BHT). and butylated hydroxyl anisole (BAH) and combinations thereof. The most preferred anti-oxidant among these is cysteine.
Non-ionic surfactant is used in the present invention, in order to prevent adsorption of gonadotropins or its variant on the surface of the vial, ampoule, carpoule, cartridge or syringe. Non-ionic surfactants lower surface tension of a protein solution, thereby preventing its adsorption or aggregation on to a hydrophobic surface. Preferred examples of non-ionic surfactant that can be used in the present invention may include a polysorbate-based non-ionic surfactant and a poloxamer-based non-ionic surfactant, either alone or in combination.
Stabilizers used in the present invention, are selected from the group consisting of amino acids such as glycine and alanine, and the like either alone or in combination thereof, monosaccharide such as glucose and mannose, and the like either alone or in combination thereof, disaccharides such as sucrose, trehalose, and maltose, and the like either alone or in combination thereof, sugar alcohols such as mannitol and xylitol, and the like either alone or in combination thereof, and polysaccharides such as dextran, and the like either alone or in

combination thereof. The presence of sugars and sugar alcohols protect the molecules during storage at relatively high temperature.
Inorganic salt such as sodium chloride, potassium chloride, calcium chloride, etc. may also be used to adjust the osmolality in the range of 250-350 mOsm. However, the use of salts in the present invention is optional.
Preservative refers to a composition or substance added to a' formulation to act as a bacteriostatic agent A preserved gonadotropin containing formulation of the present invention preferably meets statutory or regulatory guidelines for preservative effectiveness to be a commercially viable multi-use product, preferably in humans. Preservatives used in the present invention are selected from the group consisting of phenol, m-cresol. p-cresol, o-cresol, chlorocresol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzethonium chloride, sodium dehydroacetate or thimerosal, either alone or in combination thereof. However, in the present invention, the use of preservative is optional and is preferred while designing multi-dose formulations.
Oxidation is caused by reactive oxidative species and leads to irreversible changes in its chemical or physical structure during storage for prolonged period of time. The use of cysteine in the present invention imparts the enhanced oxidative stability, i.e. oxidation is less than 5 % with cysteine whereas with methionine, the product shows greater oxidative damage considering the same storage period and temperature.
Following examples illustrate the pharmaceutical compositions described in the present invention and the means of carrying out the invention to obtain a thermally stable pharmaceutical form of human Follicle Stimulating Hormone.
Example 1: Selection of stabilizer
Six different liquid formulations of rHu-FSH were prepared as per following Table 1 with six different stabilizers viz, lactose, trehalose, sucrose, mannitol, sorbitol and glycine. Cysteine was used as an anti-oxidant and phenol was used as preservative. Citrate buffer was used at concentration of 25mM.

Formulated solution was sterilized by using 0.2 μm filter under laminar air flow and incubated at 55 °C for 2 Hr. The FSH formulations were analyzed after I hour and 2 hours incubation by SDS-PAGE to select the stabilizer that can provide best protection against dissociation under stress conditions.
Figure 3 and Figure 2 depicts the effect of different stabilizers on stability of FSH after 1 Hour and 2 Hours at 55°C respectively. Glycine had shown better stability among all the sugars.
Table 1: Compositions of recombinant FSH

Formulation Code
Ingredients F-1 F-2 F-3 F-4 | F-5 F-6
Buffer Name and pH Citrate Buffer (25mM), pH 7.0
Drug (mg/ml) FSH 0.3
Stabilizers Lactose 175mM - - - - -

Trehalose - 175mM - - - -

Sucrose - - 175mM - - -

Mannitol - - - 175mM - -

Sorbitol - - - - 175mM -

Glycine - - - - - 175mM
Surfactant (mg/ml) Poloxamer
407 0.1
Anti-Oxidant Cysteine 0.1
Preservative (mg/ml) Phenol 3.0
E xample 2: Effect of Cysteine concentration
Liquid formulations of rHu-FSH were prepared as per following Table 2 with three different concentrations of cysteine with and without the addition of hydrogen peroxide (H2O2). Citrate buffer was used at a concentration of 25mM.
Formulated solutions were sterilized by using 0.2 μm filter under laminar air flow and incubated at 55 °C for 4 hours. The FSH formulations were analyzed after 4 hour incubation by RP-HPLC to determine the effect of different concentration on oxidation of FSH under stress conditions. In Table 3, percentage oxidation of a and (J subunits of FSH has been presented. Figure 4 shows RP-HPLC chromatograms to indicate the effect of different concentration of cysteine on oxidation of FSH molecule.

Table 2: Compositions of recombinant FSH

Formulation Code
Ingredients F-7 F-8 F-9 F-10 F-11 F-12
Buffer Name and pH Citrate Buffer (25mM), pH 7.0
Drug (mg/ml) FSH 0.3
Surfactant (mg/ml) Polysorbate 20 0.1
Stabilizer Glycine 175mM
Anti-Oxidant (mg/ml) Cysteine 0.1 0.25 0.5 0.1 0.25 0.5
H202 Treatment (0.005 % w/v) √ √ √ X X X
Preservative (mg/ml) Phenol 3.0
Table 3

Formulation Code % Oxid ation

a~ Subunit P- Subunit
F-7 15.19 No oxidation
F-8 7.87

F-9 4.89

F-10 4.57

F-11 3.58

F-12 3.15

Example 3
a) Preparation of hFSH aqueous formulation with Cysteine & Glycine
Liquid formulation comprising recombinant human FSH having the composition given in Table 4 was prepared. Formulated solution was sterilized by using 0.2 μm filter under laminar air flow and stored at 40 °C for 15 days. The FSH formulation was analyzed by RP-HPLC and SDS-PAGE (Oxidation).
Table 4

Sr. No. Ingredient Concentration
1. Follitropin (hFSH) 0,05 mg/ml
2. Glycine 175mM
3. Sodium Citrate Dihydrate 7.35 mg/ml
4. Cvsteine 0.5 mg/ml
5. Poloxamer 407 0.1 mg/ml
6. Phenol 3.0 mg/ml .

b) Stability test of hFSH aqueous formulations
The biological tests have been performed in compliance with the regulations of the European
Pharmacopeia.
i. pH
pH of the formulation given in Table 4 was determined and it was found to be 6.9 and pH of
RMP was 6.95.
ii. Osmolality
Osmolality of the formulation given in Table 4 was determined by using Osmometer (Advance instrument Inc.. No: 3250). Osmolality of the formulation (Table 4) was 308 mOsm and RMP was 290mOsm. iii. Isoelectric focusing
Isoelectric focusing of formulation given in Table 4 was done on a gel with pH gradient 3-9
after 15 days at 40°C along with RMP. As shown in the Figure 4 (Lane 4 and Lane 6),
numbers and position of all the bands of formulation (Table 1) were matching with that of
RMP.
iv. Reverse Phase HPLC analysis for oxidation
The aqueous formulation given in Table 4 was evaluated for the oxidation of FSH using a
reverse phase HPLC method. The result of RP-HPLC in terms of % oxidation has been
reported in table 5. It was found that FSH formulation given in table 4.had shown 18.9%
oxidation whereas RMP had shown 23.6% oxidation after incubation of 14 days at 40°C.
v. Dissociation
FSH formulation given in Table 4, the percentage of free subunit was evaluated by SDS-PAGE. Measurements were made after 14 days storage at 40°C. The results are reported in Table 5 and shown in Figure 5.
Table 5 - Analytical parameters for a liquid formulation of FSH

Assay Composition given in Table 4 RMP

Zero time at 40°C 14 days at 40°C Zero time at 40°C 14 days at 40°C
Reverse Phase HPLC analysis for Oxidation 4.0% 18.9% 3.36% 23.6%
Dissociation -1% -6% -1% -10%

Example 4
a) Preparation of hCG aqueous formulation with Cysteine & Glycine
Liquid formulation comprising recombinant human chorionic gonadotropin having the composition given in Table 6 was prepared. Formulated solution was sterilized by using 0.2 μm filter under laminar air flow and stored at 55 °C for 2 hours. The hCG formulation was analyzed by RP-HPLC and SDS-PAGE (Oxidation). .
Table 6

Sr. No. Ingredient Concentration (mg/ml)
1. Human Chorionic gonadotropin 0.05
2. Glvcine 22.5
3. Sodium Citrate
Dihydrate 7.35
4. Cysteine 0.5
5. Poloxamer 407 0.1
6. Phenol . 3.0
i. pH
pH of the formulation given in Table 6 was determined and it was found to be 6.97 and pH of
RMP was 6.98.
ii. Osmolality
Osmolality of the formulation given in Table 6 was determined by using Osmometer (Advance instrument Inc., No: 3250).. Osmolality of the formulation (Table 6) was 366 mOsm and RMP was 340mOsm. iii. Reverse Phase HPLC analysis for oxidation
The aqueous formulation given in Table 6 was evaluated for the oxidation of hCG using a reverse phase HPLC method. The result of RP-HPLC in terms of % oxidation has been reported in table 7. It was found that HCG formulation given in table 6 had shown 4.1% oxidation whereas RMP had shown 9.1% oxidation after incubation of 2 hours at 55°C.

iv. Dissociation
For a formulation given in Table 6 the percentage of free subunit was evaluated by SDS-PAGE. Measurements were made after 2 hours at 55 0C. , The results are reported in Table 7 and shown in Figure 6. From Figure 6, it is visible that the dissociation of the formulation given in Table 6 is lesser than the RMP.
Table 7 -Analytical parameters for a liquid formulation of hCG

Assay . Composition eiven in Table 6 RMP

Zero time at 40°C 2 hours at
55°C Zero time at 40°C 2 hours at
55°C
Reverse Phase HPLC analysis for Oxidation 2.82 4.1 2.56 9.1
As apparent from the above description, an agueous formultion of the present invention comprising citrate as buffer, non-reducing sugar as a stabilizer, a polysorbate-based non-ionic surfactant and a poloxamer-based non-ionic surfactant as a surfactant, cysteine as an anti-oxidizing agent, and phenol as a preservative according to the invention prevents dissociation, oxidation, denaturation and can stably maintain the activity of gonadotropin(s) for a prolonged period of time even at a higher temperature.
The formulation prepared by the said invention comprises an effective amount of biologically active gonadotropin(s) which can be used in treating infertility in humans. They are preferably used as injectable aqueous solutions.
A novel formulation of gonadotropins described in the present invention offers the following advantages:
1. Use of cysteine imparts stability against oxidative damage, i.e. oxidation is less than 5 % with cysteine whereas with methionine, the product shows greater oxidative damage considering the same storage period and temperature.
2. Provide better stability to the aqueous formulation for a prolonged period of time guaranteeing longer shelf-life than the compositions reported as prior art.

Claims
1. A stable liquid formulation comprising of therapeutically effective amount of a human gonadotropin, glycine as a stabilizer and cysteine as an anti-oxidant in citrate buffer to maintain a pH of the formulation in the range of 6.5 to 7.2.
2. A stable liquid formulation comprising of therapeutically effective amount of a human gonadotropin, glycine as a stabilizer and cysteine as an anti-oxidant in citrate buffer to maintain a pH of the formulation in the range of 6.5 to 7.2, non-ionic surfactant and a preservative.
3. A stable liquid formulation of claim 1 & 2 wherein the gonadotropin is, naturally occurring gonadotropins or produced by recombinant gonadotropins selected from the . group consisting of follicle stimulating hormone, luteinizing hormone and chorionic gonadotropin or in combination thereof.
4. A stable liquid formulation of claim 2 wherein the non-ionic surfactant is selected from the group consisting of a polysorbate-based non-ionic surfactant, a poloxamer-based non-ionic surfactant or a combination thereof.
5. A stable liquid formulation of claim 2 wherein the preservative is selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, alkylparaben, benzethonium chloride, sodium dehydroacetate and thimerosal or a combination thereof.
6. A stable liquid formulation according to claim 1 & 2, wherein the citrate buffer has salt concentration of ImM to 100 mM and a pH of 6.5 - 9.
7. A stable liquid formulation according to claim 1 & 2, wherein the glycine concentration is 1 mM to 500 mM.

8. A stable liquid formulation according to claim 1 & 2, wherein the cysteine concentration is 0.01 mg/ml to 5 mg/ml.
9. A stable1 liquid formulation comprising of therapeutically effective amount of a human gonadotropin in a range of 2 - 2000μg/ml, glycine as a stabilizer in the range of 10 -300 mM, cysteine as an anti-oxidant in the range of 0.05-5 mg/ml, poloxamer in the range of 0.1 to 5 mg/ml, phenol in the range of 1-5 mg/ml in citrate buffer in the range of 1 mM to 50mM to maintain a pH of the formulation in the range of 6.5 to 7.2