FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
METHOD OF CHROMATOGRAPHIC ANALYSIS Op A PROTEIN SOLUTION
Intas Biopharmaceuticals 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
This invention relates to the methods for the analysis of proteins. More specifically, it relates to the quantitative determination of protein's impurities by chromatographic methods.
BACKGROUND OF THE INVENTION
Proteins are very much sensitive to physicochemical factors such as temperature, pressure and undergo deformation such as denaturation, aggregation of monomers, dissociation of dimmers and adsorption on the surfaces of vessels. Consequently, these forms lose the properties of native proteins and biological activity gets affected. Deformation of proteins is irreversible and can hardly recover their native properties to the initial state.
Generally dosage of recombinant proteins is very less, such as in micrograms; the precise quantitation of protein and its associated impurities becomes very important as the injected dose has direct relationship with its activity in human body. HPLC (High Performance Liquid Chromatography) is the most reliable method for the purity analysis as well as quantitation of the protein. Generally protein analysis on HPLC is done either at room temperature or at higher temperature (up to 60°C). Analysis at these temperatures may generate impurities in the sample and which are considered as a part of impurities for the said protein that may not give the exact purity profile of the protein. The analysis of the protein at 4°C to 15°C (Storage temperature) can minimize the probability of impurity generation due to temperature and allow us to analyze the protein in its real state. So, the obtained impurity profile in a given condition will be a true reflection of the impurities present in the sample solution.
In particular, proteins which have a heterodimeric structure consisting of two different subunits associated with covalent or non-covalent bonds are more difficult to quantify at lower dose amounts compared to monomeric proteins. When injected in the HPLC column, dissociation of subunits takes place and the total amount of protein gets divided into two halves. In order to have equivalent sensitivity of the HPLC method for dimeric or multimeric proteins when compared to monomeric proteins, the sensitivity of method must be multiple fold of the number of subunits present in the molecule. For example, if a particular method has the sensitivity of 5 ng (nanogram) for monomeric proteins detection, then the method for dimeric and trimeric protein should have sensitivity of l0ng and 15ng respectively, provided the subunits are getting separated by HPLC Method.

Method of analysis for the quantification of proteins and impurities must be sensitive enough to detect the minimal level with accuracy and precision. Dose accuracy of protein is very much dependent on the quantification method which ultimately reflects its bioactivity.
An example of an analytical assay that may be used with proteins is size exclusive chromatography (SEC), in which a protein in aqueous solution is passed over a solid or gel phase that separates mixtures of protein by differences in their molecular weight. The resulting chromatogram shows one or more peaks associated with a given protein(s) in a sample, which may be identified by molecular weight. The area under a peak associated with a given protein can be used to quantify the amount of that protein in the sample.
Another example of an analytical assay that may be used with proteins is reverse phase high performance liquid chromatography (RP-HPLC). A sample containing the pharmaceutical protein is passed through a column which separates the protein from impurities like oxidation, deamidation and degradation. The protein and any impurities present in the sample elute as separate peaks on a chromatogram.
European Patent No. EP 1625147 Bl discloses about an improved method of chromatographic analysis of protein samples in which the SEC (Size Exclusion Chromatography) method is used for the quantitative determination of Follicle stimulating hormone. In this method the column was maintained at approximately at 4°C throughout the analysis.
In Olijve et al. [Mol. Human Reprod. 2(5), 1996, 371-382 Fig. 8] explained that the purity of rhFSH was determined by several complementary methods including sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, high performance size exclusion chromatography (HP-SEC), Elisa and DNA hybridization. In this publication, C4 RP-HPLC is used for separation of rHu-FSH (recombinant human Follicle Stimulating Hormone) into it's α- and β- subunits. The two subunit structure of rHu-FSH was further demonstrated using hydrophobic interaction chromatography on a C4 reverse phase HPLC column. The above method has been used for the purity analysis of rHu-FSH bulk only where the resolution of oxidized alpha subunit has been shown but it does not say anything about the oxidation of beta subunit. Due to the low sensitivity, the method cannot be used for purity analysis of the drug product.
It would be desirable to have a better chromatographic method of protein analysis for quantifying protein and/or assessing purity, in which assay precision, accuracy and reproducibility are

improved by avoiding variations due to the analysis at room temperature or other than storage temperature of the protein.
SUMMARY OF THE INVENTION
Accordingly the invention provides a chromatographic method for the analysis of a protein in a sample for quantifying the protein, wherein the method comprises:
• A step of performing a chromatographic separation by maintaining the column temperature between 4°C to 15°C.
• A step of data manipulation to determine the quantity of the protein, wherein the quantity of the protein is determined using the data from calibration with a standard.
There is also provided a chromatographic method which can be done either in one chromatographic column or in dual chromatographic columns connected in tandem.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 - RP-HPLC profile of rHu-FSH bulk
Peak - 1 - Beta subunit of rHu-FSH
Peak - 2 - Oxidized alpha subunit of rHu-FSH
Peak - 3 - Alpha subunit of rHu-FSH
Fig. 2 - RP-HPLC profile of oxidized rHu-FSH
Peak- 1 - Oxidized beta subunit of rHu-FSH
Peak - 2 - Beta subunit of rHu-FSH
Peak - 3 - Oxidized alpha subunit of rHu-FSH
Peak - 4 - Alpha subunit of rHu-FSH
Fig. 3A - RP-HPLC profile of oxidized rHu-FSH"
Peak - 1 - Oxidized beta subunit of rHu-FSH
Peak - 2 - Oxidized alpha subunit of rHu-FSH
Peak - 3 - Alpha subunit of rHu-FSH

Fig. 3B RP-HPLC profile of partially oxidized rHu-FSH Peak - J & 2 - Oxidized beta submit of rHu-FSH Peak - 3 - Beta subunit of rHu-FSH Peak - 4 to 10 - Oxidized alpha subunit of rHu-FSH Peak - 11 - Alpha subunit of rHu-FSH
DESCRIPTION OF THE INVENTION
The present invention relates to a chromatographic method of analysis of protein for quantification and/or purity determination, where precision, accuracy and reproducibility of the method were established.
The present invention discloses a method of analysis for quantification and/or purity determination of protein in a sample, wherein, the method comprises a step of chromatography at a temperature ranging from 4°C - 15°C. The method preferably has a step of RP-HPLC using single or dual columns in combination having different column chemistries (depending on target protein). The chromatography step is followed by data analysis to determine the quantity and purity of the protein. For example, the lowest dose of recombinant human follicle stimulating hormone (rHu-FSH) available in market is 44 microgram/ml Considering 100 microlitres as maximum injection volume in RP-HPLC, only 4.4 microgram can be injected on the column. In order to determine the 1% impurity in the sample, one should have a suitable method to quantitate at 44 nanogram (ng) (1% of 4.4 microgram (μg) which is present in 100 microlitres injection volume).
The method according to the present invention can be used essentially with any protein sample e.g. cytokines, glycoprotein, heterodimeric proteins, gonadotropins.
The following example will further illustrate about the invention in greater detail and are not intended to limit the scope of the invention.
Example 1 -RP-HPLC method of quantity and purity determination of rHu-FSH
Method detail:
HPLC system: Agilent 1200 series
Column: Dual column (C4 column combined with C18 column)

Temperature: 4°C Flow rate: 0.3 ml/minute Detection: UV at 210 nm Injection Volume: 6 microlitres Method time table:

S.No. Time (Minutes) %B %A
1. 0 18 82
2. 12 18 82
3. 34 40 60
4. 38 18 82
5. 57 18 82
Outlet of C4 column (250 mm length, 4.6 mm diameter and 5 micron particle size with 300 angstrom porosity) was attached to inlet of C18 column (100 mm length, 2.1 mm diameter and 3 micron particle size with 300 angstrom porosity) and the outlet of C18 column was attached to UV (Ultraviolet) detector. Column was equilibrated for almost half an hour to get the baseline with initial gradient. The sample was injected in desired volume and the area of sample was used to determine the amount of protein using calibration curve prepared with reference medicinal standard (Gonal F RFF®marketed by Sereno).
Forceful oxidation was performed using hydrogen peroxide to find the resolution of oxidized impurities. Oxidation of both subunits is clearly distinguishable and quantifiable.
Methionine is the most prone amino acid for oxidation. Total 8 oxidized species are possible in FSH if oxidation occurs at Methionine only. Possible oxidized species are as follows
1. Oxidation at Met29 in alfa subunit
2. Oxidation at Met47 in alfa subunit
3. Oxidation at Met71 in alfa subunit
4. Oxidation at Met29 and Met 47 in alfa subunit
5. Oxidation at Met29 and Met 71 in alfa subunit
6. Oxidation at Met47 and Met 71 in alfa subunit
7. Oxidation at Met29, Met 47 and Met71 in alfa subunit

8. Oxidation at Met 109 in beta subunit
In conclusion, the method described in the present invention has a better precision than the current existing analytical assays, and generates accurate results statistically equivalent to those obtained with the current assay.
The method described here is having the following advantages:
• No sample preparation is involved.
• Protein samples can be analyzed at storage temperature which usually ranges from 2°C to 8°C.
• This method ensures that no impurity gets generated due to chromatographic conditions (i.e.) at ambient or higher temperature usually ranging from 25°C to 60°C.
This method is very sensitive in which protein can be quantified in nanogram with more than 90% accuracy.

We claim
1. A chromatographic method for the analysis of a protein in a sample for quantifying the
protein wherein the method comprises
• A step of performing a chromatographic separation by maintaining the column temperature between 4°C to 15°C.
• A step of data manipulation to determine the quantity of the protein, wherein the quantity of the protein is determined using the data from calibration with a standard.

2. A method of claim 1. further comprising the step of preparing a diluted sample for bringing the protein concentration to a level acceptable for the chromatographic system used.
3. A chromatographic method according to any one of the preceding claims wherein the chromatography is reverse phase high performance liquid chromatography.
4. A chromatographic method according to any one of the preceding claims wherein the protein on which analysis is carried out is selected from the group consisting of dimeric protein or a glycoprotein.
5. A chromatographic method according to claim 4 wherein the protein on which analysis is carried out is follicle stimulating hormone.
6. A chromatographic method according to any one of the preceding claims wherein the analysis is done by using either one chromatographic column' or dual chromatographic columns connected in tandem.