FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
A PROCESS FOR THE PURIFICATION OF GONADOTROPINS
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No:423/P/A/GH)C
Sarkhej-Bavla Highway
Moraiya, Tal: Sanand
Ahmedabad-382 210
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
This invention relates to the process for the purification of gonadotropins such as recombinant human follicle stimulating hormone and the like.
BACKGROUND OF THE INVENTION
The gonadotropins form a family of structurally related glycoprotein hormones. Typical members include chorionic gonadotropin (CG), follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). FSH, LH and TSH are present in most vertebrate species and are synthesized and secreted by the pituitary. Mostly gonadotropins are used in the treatment of infertility and reproductive disorders in both female and male patients.
For pharmaceutical use, gonadotropins may be produced recombinantly or it may be isolated from the urine of postmenopausal females.
Usually gonadotropins are used in female patients in ovulation induction (OI) and in controlled ovarian hyperstimulation (COH) for assisted reproductive technologies (ART). In a typical treatment regimen for ovulation induction, a patient is administered daily injections of gonadotropins or a variant thereof for a period of about 6 to 12 days.
Because of the importance of gonadotropins in the treatment of infertility disorders, the provision of gonadotropins of high purity and high specific activity is desirable. Gonadotropin treatment requires repeated injections. Highly purified gonadotropin preparations can be administered subcutaneously, permitting self-administration by the patient, thus increasing patient convenience and compliance.
Lynch et al. (Acta Endocrinologica, 1988, 288, 12 - 19) describe a method for purifying human goandotropins. The method involves anion and cation exchange chromatography, immunoaffinity extraction and size exclusion chromatography. The method is said to result in pituitary FSH having a specific activity of 4990 IU with 16IU/mg of LH. Protein content was determined either by dry weight or in solution by adsorption at 280nm.
WO 98/20039 describe a process for the purification of human urinary FSH starting with urinary extracts called human menopausal gonadotropins (hMG). The process uses ion-exchange chromatography on weakly basic anionic exchange resins of the DEAE type followed by affinity chromatography on resin having an anthraquinone derivative as a ligand. The process is said to yield urinary FSH free from LH and having a specific activity of 6870 IU/mg. Protein content was determined by assuming that an aqueous solution of lmg/ml of protein has an optical density of 0.62 at 277 nm, in quartz cuvettes with a 1 cm path length.

WO 00/63248 describe a process for the purification of gonadotropins, including FSH, from human urine. The process involves the following steps: ion exchange chromatography with a strong cationic resin of the type sulphopropyl, ion exchange chromatography with a strong anionic resin, and hydrophobic interaction chromatography (HIC). The high biological activity and chemically pure composition of human gonadotropins obtained by this process, are used for the treatment of infertility and are selected from the group of follitropin or menotropins, having a bioactivity greater than 2500 rU/mg protein as tested by biological assay in rats, for both FSH and LH hormones for menotropins and greater than 5000 IU/mg protein for follitropin having an FSH: LH ratio about 75:1. Pharmaceutical preparations of said gonadotropins free of contaminating materials are also comprised within the present invention.
US 5,990,288 describe a method for purifying FSH from biological samples, such as human pituitary glands or human post-menopausal urine. The process uses cation exchange chromatography on Fractogel EMD SO3-650M, followed by dye affinity chromatography on Mimetic Orange 1 resin, followed by a step of hydrophobic interaction chromatography on Bakerbond Wide Pore Hl-Propyl resin. The process is said to result in human pituitary FSH having a specific activity of 7.066 IU (immunoassay)/mg and less than 1 IU (immunoassay)/mg of LH, and a urinary FSH having a specific activity of 6,298 IU (immunoassay)/mg and less than 3 IU (immunoassay)/mg of LH. Protein content was determined by absorption at 280 nm (assuming that A<280>lcm for 1 g/1 is equal to 1).
Chiba et al. [Endocrinol. J., 1997, 44, 205-218] describe a technique for purifying canine pituitary gonadotropins, including FSH, using Concanavalin (Con) affinity chromatography, hydrophobic interaction chromatography (HIC) and immobilized metal ion chromatography with Cu++ The resulting FSH is reported to have a specific activity of 2.17 IU/g protein using a radio receptor assay for FSH for measuring biological activity and the BioRad protein assay kit (BioRad Laboratories CA USA) for determining protein content.
WO 88/10270 describe a method for the purification of human FSH from urine. The process involves immunochromatography with FSH-specific immobilized monoclonal antibodies bound to Sepharose 4B by divinyl sulphone, followed by reverse phase HPLC. The resulting FSH is free of LH and other urinary proteins and has a specific activity of 6,200 IU/mg of lyophilized powder (Steelman-Pohley method). The preparation was the first FSH preparation to be suitable for subcutaneous administration, due to its purity.
WO 01/62773 describe about a process for the purification of recombinant human Chorionic Gonadotropin (hCG) from a sample of crude recombinant hCG in the supernatant of CHO cells. The process involves the combined use of ion-exchange chromatography and reverse phase HPLC. The ion-exchange chromatography is performed twice and the final use of a size exclusion

chromatography allows the purification from any residual traces of contaminants. The specific bioactivity of the highly purified hCG obtained from the process is particularly high, amounting to about 25,000 nj/mg.
WO 01/62774 describe a process for the purification of recombinant human Luteinizing Hormone (LH) from a sample of crude recombinant LH in the supernatant of CHO cells. The process comprises the combined use of ion-exchange chromatography and reverse phase HPLC. The ion-exchange chromatography and the reverse phase HPLC are performed twice and the final use of a gel permeation column allows the purification from any residual traces of contaminants. The specific bioactivity of the highly purified LH obtained from the process is particularly high, amounting to about 25.000 IU/mg.
An ongoing need remains for new methods for purifying gonadotropins such as recombinant human follicle stimulating hormone and the like with high purity and specific biological activity. In particular, there is a need for purification methods that avoid the use of the cost-intensive immunoaffinity and reverse phase chromatographic steps. Moreover these are regulatory issue of antibody or dye leaching from resins which may be present in the final product. This can easily be addressed for early product approval by avoiding the use of immunoaffinity chromatographic step in the purification process.
SUMMARY OF THE INVENTION
The objective of the present invention is to provide a new method for purifying gonadotropins such as recombinant human follicle stimulating hormone and the like.
In the first aspect, the present invention provides a method for purifying gonadotropins such as recombinant human follicle stimulating hormone and the like starting from a liquid containing the crude gonadotropins which is concentrated by ultrafiltration/ diafiltration followed by the steps:
(1) Anion exchange Chromatography
(2) Cation Exchange Chromatography
(3) Hydrophobic Interaction Chromatography
(4) Gel permeation Chromatography which may be carried out in any order.
Another objective of the present invention is to provide a pharmaceutical composition comprising the gonadotropins purified according to the said purification process with one or more pharmaceutically acceptable expients.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the present invention (i.e) a process of purification comprising the steps of
• Anion exchange chromatography
• Cation exchange chromatography
• Hydrophobic Interaction Chromatography
• Gel Permeation Chromatography
Figure 2 shows a flow chart of a specific embodiment of the present invention (i.e.) a process of purification comprising the steps of:
•• Ultrafiltration/ Diafiltration
• Anion exchange chromatography
• Ultrafiltration/ Diafiltration
• Cation exchange chromatography
• Hydrophobic Interaction Chromatography
• Ultrafiltration
• Gel Permeation Chromatography
• Nanofiltration
Fig 3 shows the SDS-PAGE profile of anion exchange chromatography
Lane 1: Blank
Lane 2: Q Sepharose Load/UFDF I Output
Lane 3: Q Sepharose eluate flow through and wash
Lane 4: Q Sepharose eluate 5-11 mS/cm Fraction
Lane 5: Reference Medicinal Product
Lane 6: Q Sepharose eluate main fraction
Lane 7: UFDFI permeate
Lane 8: Blank
Fig 4 shows the SDS-PAGE profile of Cation exchange chromatography
Lane 1: SP sepharose Eluate Fraction 2 Lane 2: SP sepharose Eluate Fraction 3 Lane 3: SP sepharose Eluate Fraction 4 Lane 4: SP sepharose Eluate Fraction 5 Lane 5: Reference Medicinal Product Lane 6: SP sepharose Eluate Fraction 6 Lane 7: SP sepharose Eluate Fraction 7 Lane 8: SP sepharose Eluate Fraction 8
Fig 5 shows the SDS-PAGE profile of hydrophobic interaction chromatography
Lane 1: PS Eluate Fraction 1 Lane 2: PS Eluate Fraction 2 Lane 3: PS Eluate Fraction 3 Lane 4: PS Eluate Fraction 4 Lane 5: PS Eluate Fraction 5 Lane 6: PS Eluate Fraction 6 Lane 7: PS Load Lane 8: PS high salt Fraction

Fig 6 shows the SDS-PAGE profile of Gel Permeation chromatography
Lane 1: GFC Eluate Fraction 1 Lane 2: GFC Eluate Fraction 2 Lane 3: GFC Eluate Fraction 3 Lane 4: GFC Eluate Fraction 4 Lane 5: GFC Eluate Fraction 5 Lane 6: GFC Eluate Fraction 6 Lane 7: Reference Medicinal Product Lane 8: 2.5 % of 5.00 Mg of Standard
Fig 7 shows the SDS-PAGE profile of Final Bulk of different batches
Lane 1: Drug Substance Batch 1 Lane 2: Drug Substance Batch 2 Lane 3: Drug Substance Batch 3 Lane 4: Drug Substance Batch 4 Lane 5: Drug Substance Batch 5 Lane 6: Reference Medicinal Product Lane 7: Marker Lane 8: Blank
Fig 8 shows the WB-IEF profile of Final Bulk of different batches
Lane 1: Drug Substance Batch 1 Lane 2: Drug Substance Batch 2 Lane 3: Drug Substance Batch 3 Lane 4: Drug Substance Batch 4 Lane 5: Reference Medicinal Product Lane 6: Drug Substance Batch 5
Fig 9 shows the comparative RP-HPLC chromatograms of in-house rHu FSH Drug substance with Reference Medicinal Product.
DESCRIPTION OF THE INVENTION
The invention provides a novel method for purifying gonadotropins such as recombinant human follicle stimulating hormone and the like from a liquid containing the crude gonadotropins such as recombinant human follicle stimulating hormone and the like, comprising the steps:
(1) Anion exchange Chromatography
(2) Cation Exchange Chromatography
(3) Hydrophobic Interaction Chromatography
(4) Gel permeation Chromatography which may be carried out in any order.
The purification method of the invention affords gonadotropins such as recombinant human follicle stimulating hormone and the like of high purity, high specific activity which may be then formulated to the final medicament. It has the advantage of affording a high degree of purity without using

reverse phase chromatography. According to the present invention the crude cell culture harvests, containing gonadotropins such as recombinant human follicle stimulating hormone and the like which forms the starting material for the purification.
The invention described herein may optionally encompass any of tangential flow filtration, concentration, diafiltration or ultrafiltration steps between the chromatographic steps.
The purification according to the present invention thus utilizes at least four chromatographic steps i.e. a) anion exchange chromatography, b) cation exchange chromatography, c) hydrophobic interaction chromatography and d) gel permeation chromatography, which may be carried out in an order.
The cultivation of gonadotropins such as recombinant human follicle stimulating hormone and the like producing host cells is done in a cell culture medium which is free of animal proteins or animal derived components.
It has been found that the gonadotropins such as recombinant human follicle stimulating hormone and the like obtained by the purification process according to the present invention has purity at least >85% and more specifically >95% wherein the purity is determined by analytical reverse phase high performance liquid chromatography.
The present invention also relates to the pharmaceutical preparation comprising the gonadotropins such as recombinant human follicle stimulating hormone and the like purified according to the present invention.
Preferably, the gonadotropin purified according to the present invention is recombinant human follicle stimulating hormone, produced in eukaryotic cells. Preferably, it is produced in mammalian cells, particularly in CHO cells. According to the conventional protocols, cell cultivation is done with commercially available culture medium.
The person skilled in the art must be familiar with the principles of chromatographic steps employed in the purification process according to the present invention; in any case, they are described in detail in the manuals or protocols of the chromatographic matrice suppliers.
Ion exchange chromatography removes highly basic isoforms, other host cell proteins and media components and cation exchange chromatography removes the host cell proteins as well as product related impurities.

Prior to the step of ion exchange chromatography, it may be desirable to carry out a step of ultrafiltration, in order to concentrate the crude FSH.
Hydrophobic Interaction chromatography is effective in removing oxidized impurities and gel permeation chromatography is used to remove the product related impurities as well as buffer exchange in formulation buffer.
The following example illustrates the present invention and the means of carrying out the invention to obtain the purified recombinant human follicle stimulating hormone.
Examples
The example described in detail below use starting material samples containing rhuFSH obtained from culture supernatant medium from a bioreactor.
Example 1
Step 1 - Clarification, concentration, dialysis and filtration of harvest
The clarified cell culture harvest is concentrated and diafiltered by Ultrafiltration. This step is carried out at about 25°C and is repeated individually for each harvest during the production cycle of the bioreactor.
Step 2 - Anion exchange chromatography on Q Sepharose FF
In this step all the proteins are captured and eluted according to their binding properties. This step is used to separate highly basic isoforms from desired isoforms. This step is carried out at about 25°C.
Step 3 - Ultrafiltration/ Diafiltration
Elute of anion exchange chromatographic step is concentrated and diafiltered. This step is carried out at about 25°C.
Step 4 - Cation Exchange Chromatography on SP Sepharose FF
In this step, host cell as well as product related impurities are removed. This step is carried out at about 25°C.
Step 5 - Hydrophobic interaction Chromatography on Phenyl Sepharose
In this step mainly oxidized impurity is removed. This step is carried out at about 25°C.

Step 6 - Concentration by Ultrafiltration
In this step protein is concentrated upto a desired concentration for the next step.
Step 7 - Gel permeation chromatography on Sephacryl S100 & Nanofiltration
In this step, the separation is based on molecular size. Elute obtained from this step, is then preferably passed through a filter to remove viral contaminants, the filtrate solution is stored in sterile bottles at about 2°C-8°C or -20°C.
Chromatographic resins
The following chromatographic resins are currently employed in the purification process. Equivalent resins can be employed as well in the purification process described in the present invention.
Step 2 - Q Sepharose FF (GE Healthcare)
Step 4 - SP Sepharose FF (GE Healthcare)
Step 5 - Phenyl Sepharose (GE Healthcare)
Step 7 - Sephacryl S100 (GE Healthcare)
The advantage of the present invention is that the purification method is devoid of a cost intensive affinity chromatography and reverse phase chromatographic step and provides highly purified gonadotropins such as recombinant human follicle stimulating hormone and the like with specific bioactivity.
The gonadotropins obtained can be formulated in the form of a liquid/lyophilized formulation with pharmaceutically acceptable excipients as known in the art.
The details of individual chromatographic steps are given below.
Step 1 - Clarification, concentration, dialysis and filtration of harvest
The clarified cell culture harvest is concentrated and diafiltered by Ultrafiltration against lOmM Tris buffer, pH 8.5 and conductivity < 1.0 mS/cm followed by loading on an anion exchange column (Q Sepharose FF) pre-equiiibrated with the same buffer.
Step 2 - Anion exchange chromatography on Q Sepharose FF
Ultrafiltration and diafiltration out put is loaded on an anion exchange column (Q Sepharose FF) pre-equilibrated with lOmM Tris buffer, pH 8.5 and conductivity < 1.0 mS/cm buffer. After washing with the equilibration buffer the bound protein is eluted with a gradient between 10 mM Tris buffer, pH 8.5 and 1M sodium chloride in the same buffer.

Step 3 - Ultrafiltration/ Diafiltration
Elute of Q Sepharose FF is concentrated and diafiltered against 2mM sodium phosphate buffer, pH 5.0 and conductivity <1.0 mS/cm and loaded to SP sepharose pre-equilibrated with 2mM sodium phosphate buffer, pH 5.0.
Step 4 - Cation Exchange Chromatography on SP Sepharose FF
Ultrafiltration and diafiltration out put is loaded on cation exchange column (SP Sepharose FF) pre-equilibrated with 2mM Phosphate buffer, pH 5.0 and conductivity < 1.0 mS/cm buffer. After washing with the equilibration buffer the bound protein is eluted with a gradient between 2mM Phosphate buffer, pH 5.0 and 1M sodium chloride in the same buffer.
Step 5 - Hydrophobic interaction Chromatography on Phenyl Sepharose
Elute of SP Sepharose FF is processed for next chromatographic step by adding 2.0 M ammonium sulphate and pH is adjusted to 7.0 with NaOH. After filtration, sample is loaded on phenyl sepharose resin pre-equilibrated with 20mM Phosphate buffer containing 2.0M ammonium sulphate, pH 7.0. After washing with the equilibration buffer the bound protein is eluted with a gradient between 20mM Phosphate buffer containing 2.0M ammonium sulphate and 20mM Phosphate buffer. pH 7.0.
Step 6 — Concentration by Ultrafiltration
The elute of Phenyl Sepharose is concentrated to get O.D280 3.00 - 4.00, after that membrane is washed with 20mM phosphate buffer, pH 7.0 and mixed with Ultrafiltration out put.
Step 7 - Gel permeation chromatography on Sephacryl S100 & Nanoflltration
The Ultrafiltration out put further loaded on gel permeation chromatography column (Sephacryl SI00) as an additional polishing step followed by nano filtration.
A novel process for the purification of gonadotropins described in the present invention has the following advantages;
1. Involves operational simplicity and robustness.
2. Avoids the usage of reverse phase and/or dye-binding affinity chromatography
3. With reduced cost.
4. Results in purity >95% with desired isoform profile and activity

We Claim
1. A process for the purification of gonadotropins from a culture supernatant produced by
culturing eukaryotic host cells in culture medium, comprising the following steps:
• Anion exchange Chromatography
• , Cation Exchange Chromatography
• Hydrophobic Interaction Chromatography
• Gel permeation Chromatography which may be carried out in any order.

2. A process of claim 1, wherein the gonadotropin is selected from the group consisting of follicle stimulating hormone, luteinizing hormone, human chorionic gonadotropin and thyroid stimulating hormone.
3. A process of claim 1, wherein Q Sepharose is used for anion exchange chromatographic step.
4. A process of claim 1, wherein SP Sepharose is used for cation exchange chromatographic step.
5. A process of claim 1, wherein Phenyl Sepharose SP is used for hydrophobic interaction chromatographic step.
6. A process of claim 1, wherein Sephacryl SI00 is used for Gel permeation chromatographic step.
7. A process of claim 1, optionally comprising at least one nanofiltration and/or viral inactivation step.
8. A process of claim 1, wherein the protein mixture is a host cell-free filtrate of the culture medium, which is subjected to diafiltration prior to an anion exchange chromatography.
9. A method for purifying gonadotropins subjecting a liquid comprising gonadotropins obtained from urine, clarified cell culture harvest or partially pure gonadotropin concentrate to;
a. ultrafiltration of said liquid and recovering a retentate;
b. subjecting said retentate to anion exchange chromatography on a resin comprising
quaternary ammonium groups and eluting a first eluate with a buffer comprising NaCl
and L-methionine at a pH between 6.5 to 9.5;
c. subjecting the eluate to from step 9b to a step of cation exchange chromatography on a
resin comprising sulpho propyl group and eluting with a buffer comprising phosphate,
NaCl and L-methionine at a pH between 4.0 to 6.0;

d. subjecting the second eluate from step 9c to hydrophobic interaction chromatography
on a resin comprising phenyl groups and eluting a third eluate with a buffer comprising
phosphate, ammonium sulfate at a pH between 5.0 to 8.0;
e. ultrafiltration of the eluate from step 9d to concentrate;
f. subjecting the ultrafiltration retentate to gel permeation chromatography on a resin
comprising sepharcryl group with a formulation buffer at a pH between 4.0 to 8.0;
g. subjecting the fourth eluate to nanofiltration to form a permeate;
A pharmaceutical composition comprising the gonadotropins with 95% purity, purified according to any one of the preceding claims with one or more pharmaceutic ally acceptable excipients.