FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION (See Section 10 and Rule 13)
A PROCESS FOR INACTIVATION OF VIRUSES OR VIRUS-LIKE PARTICLES
Intas Biopharmaceuticais Limited
An Indian company having its registered office at:
Plot No.: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, TaL: Sanand
Ahmedabad-382 210
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
The present invention relates to a process for inactivation of viruses or virus-like particles in the manufacturing processes of therapeutic proteins.
BACKGROUND OF THE INVENTION
The importance of recombinant proteins has increased as the methods of treating many diseases involve the use of recombinant proteins. However, large-scale production of these therapeutic proteins still remains a challenge. For example, the commercial manufacturing process must result high-yield with downstream processes producing an extremely pure product, allowing only trace amounts of impurities and preferably no contaminants.
Chinese Hamster Ovary mammalian cell lines serve as competent expression systems for the production of therapeutic proteins. However, mammalian cell systems are subjected to contamination with viruses that may be introduced through raw materials or failures in process controls. All viruses contain nucleic acid, either DNA or RNA, surrounded by a protective protein coat called a capsid. Some viruses are also enclosed by an envelope of lipid and protein molecules that is derived from the host cell membrane but includes virus proteins. Numerous types of viruses can infect mammalian cells, including RNA and DNA viruses which may be enveloped or naked. In addition, n on-infectious retroviruses like particles, produced by GHO cells, are consistently observed and quantitated by electron microscopy. Because of this, relevant viruses that are readily detected and quantitated in these cell cultures are used to characterize potential protein purification processes for their capacity to clear adventitious viral agents.
Xenotropic murine leukemia virus (XmLV) and murine minute virus (MMV) are common model viruses used to test the viral clearance efficiency of each unit operation during recombinant protein purification.
Viral inactivation ts different from viral removal because, in the former process, the surface chemistry of the virus is altered and in many cases the (now non-infective) viral particles remain in the final product. Rather than simply rendering the virus inactive, some viral inactivation processes actually denature the virus completely. Viral inactivation is used widely in the blood plasma industry.

During the manufacture of protein formulations, contamination by viruses may occur. Therefore, it is absolutely necessary to provide a step in the manufacturing process of inactivating and/or eliminating viruses (ICH Harmonized Tripartite Guideline: Viral Safety Evaluation of Biotechnology Products Derived from Cell lines of Human or Animal Origin).
In order to achieve inactivation of the viruses in the sample, it is necessary to perform "special" purification processes that will chemically alter the virus in some way. Some of the more widely used processes are as follows: Solvent/detergent inactivation, Pasteurization (heating). Acidic pH inactivation.
EP0374625 described a method of inactivating a lipid enveloped virus in a solution of biologically active therapeutic proteins; the method comprising the step of contacting the solution with caprytic acid is in the non-ionized form in an amount ranging from about 0.07 % to about 0.001 %, on a weight % basis in water.
EP0252392 described a method of preparing a biologically active protein product substantially free of active viruses comprising the steps of (a) subjecting a source for a given biologically active protein to a viral inactivation step under conditions sufficient to inactivate any virus present, and (b) subjecting the product step (a) to a protein separation step under conditions sufficient to remove biologically inactive forms of the protein.
US20080318300 describes about a method for conveniently producing a protein formulation in which viruses are inactivated, without impairing the quality of the obtained protein formulation, characterized by including the step of exposing the protein formulation contaminated with the viruses to a 0.1-2M (Molar) aqueous solution of arginine, an arginine derivative, or a mixture thereof, the aqueous solution being adjusted to pH 3.5 to 5. The present invention also provides a virus inactivation method characterized by including the step of contacting a virus-containing object with a 0.1-2M aqueous solution of arginine, an arginine derivative, or a mixture thereof. the aqueous solution being adjusted to pH 3.5 to 5.
Many proteins are sensitive to the low pH treatment, which may cause the dissociassion of the molecular subunit or the complete denaturation of protein leading to loss of biological activity of the protein. Hence low pH virus inactivation is not a suitable method for low pH sensitive protein.

So, there exists a need for a better method or the modification of the method for mactivation of virai contaminants during recombinant protein purification without affecting its biological activity.
SUMMARY OF THE INVENTION
The present invention is directed to a method for inactivating viruses, virus like particles or fragments thereof from purified or partially purified therapeutic proteins/therapeutic solutions.
In one aspect of the invention, the method for inactivating viral contaminants in a purified or partially purified therapeutic protein/therapeutic solution comprises the following steps:
(a) Addition of stabilizers to the said solution;
(b) Maintaining the pH of the solution after adding stabilizers between 3.0 to 4.1 to produce an intermediate solution;
(c) Incubating the solution obtained in step (b) for 45 mins to 1 hour to inactivate the virus.
(d) Readjusting the pH of the solution obtained in step (c) for further chromatographic purification steps.
The methods of the invention may be applied to any therapeutic protein, including gonadotropins, cytokines (darbepoietin, erythropoietin), fusion protein including fusion antibody or an antibody and recombinant peptides sensitive to low pH comprising the forgoing as long as they exhibit the desired biological activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the comparison of dissociation form of alpha and beta subunits of recombinant human follicle stimulating hormone
Lane 1: Sample buffer
Lane 2: Control
Lane 3: Partially purified therapeutic protein solution at pH 3.0
Lane 4: Ion exchange eluate at pH 3.5
Lane 5: Ion exchange eluate at pH 3.7
Lane 6: Ion exchange eluate at pH 4.0
Lane 7: Substantially purified therapeutic protein solution at pH 7.0

Lane 8: Sample buffer
Note: Each well is loaded with 5.0 fig of the protein
Figure 2 shows the comparison of dissociation form of alpha and beta subunits of recombinant human follicle stimulating hormone with increasing inorganic salt.
Lane 1: Control at, pH 7.0
Lane 2: Substantially purified therapeutic protein solution 100 mM. pH 3.7 Lane 3: Substantially purified therapeutic protein solution 250 mM, pH 3.7 Lane 4: Substantially purified therapeutic protein solution 400 mM. pH 3.7 Lane 5: Substantially purified therapeutic protein solution S50 mM, pH 3.7 Lane 6: Substantially purified therapeutic protein solution 700 mM, pH 3.7 Lane 7: Substantially purified therapeutic protein solution 800 mM, pH 3.7 Lane 8: Substantially purified therapeutic protein solution 1000 mM, pH 3.7 Note: Each well is loaded with 5.0 μg of the protein
DESCRIPTION OF THE INVENTION
The present invention is directed to a method for inactivating viruses, virus like particles or fragments thereof from purified or partially purified therapeutic protein solutions.
The present invention provides a method for inactivation of viral contaminants during the protein purification, even wherein the proteins are susceptible to denaturation, degradation, dissociation, aggregation and other forms of damages during inactivation of viruses.
The present invention also provides a method for inactivation of viral contaminants during protein purification by lowering the pH of the solution comprising inorganic salts and one or more stabilizers which prevent the dissociation and/or aggregation of protein during the inactivation of viral contaminants.
A "therapeutic protein solution" refers to a purified aqueous solution of protein which is used for the therapeutic purpose obtained by cell culture media or microbial cell suspension. Jt can also be any intermediate step of preparing the therapeutic protein solution.
The term "inactivating" or "virus inactivation" refers to treatment of the virus containing solution with a regimen such that the contaminating viral particles are no longer infectious to cells or cannot replicate. Methods of removing and inactivating virus are discussed below.

inorganic salts such as sodium chloride, potassium chloride, calcium chloride and the like prevent the dissociation of therapeutic proteins during chromatographic purification steps.
Stabilizers, used in the present invention, are selected from the group consisting mainly of inorganic salts such as sodium chloride, optionally with amino acids such as arginine. polyols such as mannitol, sugars such as sucrose and trehalose, etc., and surfactants such as polysorbate 20 , etc.
Virus Inactivation and Removal
Inactivation of contaminating virus is a matter of great concern in the biopharmaceutical industry as production of recombinant protein and purification of proteins from plasma and other mammalian cell components has become the norm in the industry. The World Health Organization (WHO) has recently issued guidelines and has reviewed the optimal methods of inactivating and removing viruses from blood products (WHO Technical Report, Annex-4, "Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products." Series No.: 924 pi 51-224, 2004). These methods are also commonly used in the purification of recombinant therapeutic proteins.
Determination of Viral Content
Vira! inactivation measure the clearance capacity of the purification process by determining the fog reduction value (LRV) of virus, comparing the viral contaminant levels before and after the purification step, or unit operation. Determination of virus titer through viral infectivity assays is the major viral clearance evaluation method for each unit operation. All virus infectivity assays used in the process evaluation study need are validated in accordance with ICH guidelines and include proper controls for possible cytotoxic and inhibitory effects of process intermediates on the assay. The sum of the individual log reduction factors from each unit operation represents the total viral clearance capability of the purification process.
Purification of Proteins
Purification of therapeutic proteins relies on a series of steps after harvesting of cell culture media to adequately render a therapeutic pure protein solution (Current Protocols in Protein Science. "Conventional Chromatographic Separations," Ch. 8-9, John Wiley & Sons Inc., Hoboken, New Jersey). Generally, the steps of protein purification are: capture of the protein to a more

concentrated form, intermediate purification steps to remove impurities, polishing to remove additional impurities and protein variants, and virus removal and inactivation, which may be done at various points during the purification process.
An exemplary protein purification and viral inactivation process are demonstrated in purification of therapeutic heterodimeric protein solutions. The entire process consists of three or four purification steps, which includes UF/DF and two or three purification steps.. The chromatographic steps remove product-related impurities such as dissociated subuniis, oxidative impurities and other host cell impurities like Host ceil protein and DNA. It also provides potential viral clearance depending on the type of chromatography used for the purification. In addition to chromatographic viral reduction the purification process should have viral removal by filtration, and viral inactivation steps.
Although the above methodologies are useful for removal of viral contaminants, no one methodology stands out as an optimal process. Thus, there is a need to develop additional processes for the inactivation of viral contaminants from therapeutic protein solutions.
Additional aspects and details of the invention will be apparent from the following examples. which are intended to be illustrative rather than limiting,
Example 1
Initially, culture media containing a heterodimeric protein is loaded on to ion exchange chromatographic steps to purify the protein from the culture media using a standard procedure known in the art. The collected eluate is mixed with sodium chloride and pH of the solution is adjusted to 3.0 to 4.1, ideally to 3.5 to 3.9, specifically to 3.7 to produce an intermediate solution and incubated for lhr. After 1 hr incubation pH was readjusted to 7.0, and processed for next chromatographic steps.
The dissociation percentage was determined by using ATTO, SPECTRA AGRITECH SDS gel apparatus. After 1 hour incubation at low pH, sample was prepared with non reducing loading dye and loaded on the gel. Force fully dissociated protein was also loaded on to the same gel in different concentration to determine the percentage dissociation.

We Claim
J. A process for inactivating viral contaminants in purified or partially purified therapeutic protein/therapeutic solution comprises the following steps:
(a) Addition of stabilizers to the said solution;
(b) Maintaining the pH of the solution after adding stabilizers between 3.0 to 4.1 to produce an intermediate solution:
(c) Incubating the solution obtained in step (b) for 45 mins to 1 hour to inactivate the virus.
(d) Readjusting the pH of the solution obtained in step (c) for further chromatographic purification steps.

2. A process of claim 1 wherein the biologically active protein is selected from the group consisting of gonadotropins, cytokines, fusion protein including fusion antibody or an antibody and recombinant peptides.
3. A process of claim 2 wherein the biological active protein is recombinant gonadotropins.
4. A process of claim 3 wherein the gonadotropin is follicle stimulating hormone.
5. A process as claimed in claim 1 wherein the stabilizer is selected from the group consisting of inorganic salts, polyols, sugars, surfactants, amino acids, either alone or in combination thereof to the protein solution.
6. A process as claimed in claim 5 wherein the stabilizer is inorganic salt either alone or in combination thereof.
7. A process claimed in claim 6 wherein the inorganic salt is selected from the group consisting of sodium chloride, ammonium sulphate either alone or in combination thereof.
8. A process claimed in claim 1 wherein the pH of the solution obtained from step (c) is maintained between 5 and 7 for further chromatographic purification steps.

9. A process claimed in claim 1 wherein the solution obtained from step (d) is purified further by chromatographic step.
10. A process as claimed in claim 9 wherein the chromatographic step is hydrophobic interaction chromatography.