Title of Invention	AN IMPROVED PROCESS FOR PEGYLATION OF PROTEINS
Abstract	The present invention relates to a process for improving-pegylation reaction yield of r-metHuG-CSF comprising conjugating r-metHuG-CSF to a PEG aldehyde at a free amine moiety at the N terminal end on the G-CSF in presence of a reducing agent in a pegylation buffer solution comprising a polyol having the formula C„H2o 2O„ where n is from 3 to 6, or a carbohydrate, or a derivative thereof wherein the concentration of said polyo l or carbohydrate or derivative thereof is in the range of 0.1 % to 10% w/w.

Title of Invention

AN IMPROVED PROCESS FOR PEGYLATION OF PROTEINS

Abstract

The present invention relates to a process for improving-pegylation reaction yield of r-metHuG-CSF comprising conjugating r-metHuG-CSF to a PEG aldehyde at a free amine moiety at the N terminal end on the G-CSF in presence of a reducing agent in a pegylation buffer solution comprising a polyol having the formula C„H2o 2O„ where n is from 3 to 6, or a carbohydrate, or a derivative thereof wherein the concentration of said polyo l or carbohydrate or derivative thereof is in the range of 0.1 % to 10% w/w.

Full Text	FORM 2 THE PATENTS ACT I970 (39 of 1970) & The Patents Rules, 20()3 PROVISIONAL SPECIFICATION (See Section 10 & Rule 13) 1. Title of the Invention "An Improved Process for Pegylati^n of Proteins" 2. Applicant (s) Name: USV Limited Nationality: Indian company incorporated under the Companies Act, 1956. Address: B.S.D. Marg, Station Road, Govandi, Mumbai 400 088, Maharashtra, India. 3. Preamble to the Description The following specification particularly describes the invention. TECHNICAL FIELD The present invention relates to the field of protein modification, and, more specifically, to an improved process for the attachment of water soluble polymer selectively to N terminal of proteins or analogs thereof The improved process of pegylation is characterized in that the reaction is carried out in the presence of sugar alcohol, as for example sorbitol. BACKGROUND OF INVENTION: Proteins for therapeutic use are currently available in suitable forms in adequate quantities largely as a result of the advances in recombinant DNA technologies. The availability of recombinant proteins has endangered advances in protein formulation and chemical modification. One goal of such modification is protein protection. Chemical attachment may effectively block a proteolytic enzyme from physical contact with the protein backbone itself, and thus prevent degradation. Additional advantage include, under certain circumstances, increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. One method commonly used for protein modification is by covalent attachment of water soluble polymers. Polyethylene glycol ("PEG") is one such chemical moiety which has been used in the pegylation of therapeutic protein products. The US FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations. Molecules coupled to PEG become non-toxic, nonimmunogenic, soluble in water and many organic solvents, and surfaces modified by PEG attachment become hydrophillic and protein rejecting. The FDA has approved several pegylated polypeptides as therapeutics and more are undergoing clinical investigation. In 1990, pegademase (Adagen) received approval for the treatment of severe combined immunodeficiency (SCID). Pegaspargase (Oncaspar) approved in 1994, contains the pegylated enzyme L-asparaginase, used clinically in combination with chemotherapy for the treatment of acute lymphocytic leukaemia, acute lymphpblastic leukaemia and chronic myelogenous leukaemia. In 2001, peginterferon a2b (Peglntron) became available as a once-a-week treatment for hepatitis C. Peginterferon a2a (Pegasys) approved in 2002 used a second generation, branched PEG of 40 kDa conjugated through a E-NH2 group of lysine used as spacer to interferon a2a increased the half life of IFN- a2a from 9 to 77 hours. A pegylated form of human growth hormone antagonist called pegvisomant (Somavert) was approved by FDA in 2003 for the treatment of acromegaly. Doxil, a pegylated liposomal formulation of doxorubicin was approved in 1995 for the treatment of Kaposi's sarcoma. Pegfilgrastim (Neulasta), approved in 2002, is a pegylated form of the earlier drug filgrastim (Neupogen) used for the treatment of neutropenia. Pegylation has taken 20 years to emerge as a viable pharmaceutical tool. Over the period there have been important advances in the chemistry of pegylation, in the generation of biomolecule therapeutics and in understanding PEG-biomolecule conjugates. Pegylation is now established as the method of choice for improving the pharmacokinetics and pharmacodynamics of protein pharmaceuticals. A variety of active PEGs have been prepared. mPEG succinimidyl succinate and mPEG succinimidyl carbonate were the reagents used and approved by US FDA. The regaents had the limitation of forming weak linkages between the PEG moiety and protein, potential unwanted side reactions, contamination, and restriction to low MW PEGs. The above limitations were overcome by use of mPEG-propionaldehyde which was easier to prepare. PEG aldehydes are inert toward water and react primarily with amines. Inertness toward water is desired, not only because of efficiency of storage, preparation, and application, but also because it permits stepwise linkage, in aqueous media, of molecules to surfaces and molecules to molecule. mPEG aldehyde has essentially all the properties of ideal PEG derivative i.e. Reactive with nucleophillic groups (typically amino) on proteins and surfaces; stable in aqueous media and on the shelf; easily prepared and characterized; and capable of coupling to proteins without reducing protein activity. US Pat No. 5,824,784 assigned to Amgen claims a substantially homogenous preparation of N-terminally monoPEGylated G-CSF or analog thereof and a method for attaching a polyethylene glycol to a G-CSF molecule wherein the PEG moeity has single aldehyde group. The pegylation process claims reacting G-CSF with polyethylene glycol under reducing alkylation conditions at a pH sufficiently aciidic to selectively activate the alpha amino group at the amino terminus of G-CSF. The process discloses the addition of a 5-fold molar excess of methoxypolyethylene glycol aldehyde of average MW, 6 kDa to a cooled (4°C) stirred solution of rhG-CSF (1 ml, 5 mg/ml) in 100 mM sodium phosphate, pH 5, containing 20 mM NaCNBH3. The stirring of the reaction mixture was continued at the same temperature The mono-MPEG-GCSF derivative was purified by ion exchange chromatography using HiLoad 16/10 S SEPHAROSE HP column and eluted with a linear 400 minute gradient from 0% to 45% 20 mM sodium acetate, pH 4, containing 1M NaCl . The % composition of N terminally mono-MPEG-GCSF obtained by reductive alkylation is not disclosed . A comparative stability analysis of N-terminally momopegylated G-CSF obtained by amide linkage (derived by using N-hydroxy succinimidyl ester of carboxymethyl methoxy polyethylene glycol as nucleophile) and the other obtained by amine linkage for 8 weeks yielded 82% purity with respect to one having amine linkage between the protein and the mPEG-aldehyde. A surprising result was observed as the amine linkage produced a material with far fewer aggregates against the one with amide linkage. efficiency.The pegylation efficiency by reductive amination of peptide sequences benefits from the use of more hydrophobic alcohols and the efficiency enhancement afforded by fluoro alcohols is most advantageous to reductive amination reactions. The % of alcohols found to be beneficial is in the range of 30% to 70% v/v. The above prior art are applicable to the synthetic peptides produced by solid phase peptide synthesis or solution phase synthesis. However the harsh conditions when alcohols are used in such a high concentration cannot be used for pegylating proteins. An essential element of the present invention is use of non-hazardous additives for increasing the pegylation efficiency specially of proteins. The handling of volatile and hazardous alcohols limits the use for large scale operations. SUMMARY OF THE INVENTION The present invention relates to the process of selective pegylation of the proteins. More specifically, the present invention relates to an improved process for pegylation of the protein by reductive alkylation in the presence of sugar additive. The present invention further relates to an improved pegylation process wherein the sugar additive is selected from a group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, xylitol. The present invention further relates to a process with an enhanced pegylation efficiency wherein the recovery of the pegylated product is atleast 80%. The present invention still further relates to a process wherein the unreacted G-CSF in the pegylation reaction is less than 1%. The present invention further relates to a process for producing monopegylated G- CSF with greater than 99% purity. The present invention further relates to a process for pegylation wherein the sugar additive is added after the buffer exchange to the pegylation buffer and the concentration maintained throughout the pegylation process. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS The manner in which the objects and advantages of the invention may be obtained will appear more fully from the detailed description and accompanying drawings, which are as follows: Figure 1: SEC-HPLC profile of USV's crude pegylated G-CSF wherein the pegylation is carried in absence of sorbitol. Figure 2: SEC-HPLC profile of USV's crude pegylated G-CSF wherein the pegylation is carried in presence of 5% sorbitol in pegylation buffer. Figure 3: Non-reducing SDS-PAGE of USV's purified pegylated G-CSF at different concentrations; Lane 1: USV's PEG-GCSF (2000 ng), Lane 2: USV's PEG-GCSF (1000 ng), Lane 3: USV's PEG-GCSF (200 ng), Lane 4: USV's PEG-GCSF (40 ng), Lane 5: USV's PEG-GCSF (20 ng), Lane 6: Neulasta (200 ng), Lane 7: Neulasta (40 ng), Lane 8: Protein MW ladder. Figure 4: SEC-HPLC profile of USV's monopegylated G-CSF with a purity of >99%. Figure 5: HPLC endoproteinase SV8 peptide mapping profile of monopegylated G-CSF; A: USV's monopegylated G-CSF, B:Neulasta. Figure 6: SEC-HPLC profile of monopegylated G-CSF with a purity of >99%; A: USV's monopegylated G-CSF; B: Neulasta. Figure 7: Graph illustrating a comparison of in vitro bioactivity of USV's monopegylated G-CSF compared against Neulasta & Neupogen. DETAILED DESCRIPTION OF THE INVENTION EXAMPLES: Example -1 Concentration and diafiltration (buffer exchange) of the rh-GCSF: The liquid stock solution of rH-GCSF from USV (concentration ca 1.9 mg/ml supplied in 10mM sodium acetate, 5% sorbitol pH 4.0) which was stored at 2-8°C, was aliquoted out from the stock. The protein was then concentrated to about 6-7 mg/ ml. The sample was diluted twice the volume with l00mM sodium phosphate buffer pH 5.0 containing 20mM sodium cyanoborohydnde and 5% Sorbitol. The dilution and concentration was done for 3 times with the buffer. The final diafiltered concentration was 6-7mg/ml. The diaflltration procedure was done at 4°C in an ice bath. The concentrated solution was then stored at 4°C or taken for PEGylation reaction. The % recovery of the rh-GCSF post buffer exchange and concentration was about 95% to 97%. Example -2 Preparation of Pegylated G-CSF: The diafiltered, and the quantified protein obtained from Example 1 was taken in a 250ml glass bottle. A buffer consisting of lOOmM sodium phosphate buffer pH 5.0 with 20mM sodium cyanoborohydride and with/without 5% Sorbitol was added to the reaction mixture. The methoxy-polyethylene glycol-propionaldehyde (mPEG-aldehyde; SUNBRIGHT ME-200AL from NOF Corp., Japan) of approximately 20Kda was added to the above stirred solution of the protein. The mPEG-aldehyde was then transferred to the bottle containing protein solution. The protein concentration was maintained at 5mg/ml. The reaction mixture was then stirred at 2-8°C, overnight. The reaction mixture was quenched by addition of 40mM sodium acetate buffer pH 4.0 with and without 5% Sorbitol in the buffer (volume made to five times the reaction volume) (Figures 1 & 2). The percentage conversion of GCSF was >98% wherein the pegylation was carried out in the presence of sorbitol. Example -3 Purification using Ion exchange chromatography: The pegylated G-CSF obtained from Example-2 was loaded to a weak cation exchange column of following specification: System: AKTA UPC 100 medium pressure system The pegylated protein was eluted in a gradient mode using the following buffer system. Buffer A: 40mM Sodium acetate in 5% Sorbitol pH 4-0 Buffer B: 40mM Sodium acetate , 5% Sorbitol ,0.5M NaCl pH 4.0 The fractions containing the mono PEG-GCSF (>99%), were pooled. Tween 20 was added to these fractions prior to concentration. The sample was concentrated and diafiltered with l0mM sodium acetate, 5% Sorbitol, Tween 20. The final concentration achieved was >10mg/ml, with purity of mono PEG >99% (Figure 4). Table 1 illustrates the effect of addition of 5% sorbitol to the pegylation buffer and subsequent addition of tween-20 to the storage buffer prior to concentration. Table 1 rComparative analysis for pegylation carried in the presence and absence of sorbitol The USV's monopegylated G-CSF was characterized using 1) Non-reducing SDS-PAGE of USV's purified pegylated G-CSF (Figure 3), 2) Size exclusion chromatography HPLC (SEC-HPLC) profile of USV's monopegylated G-CSF overlayed with Neulasta (Figure 6), 3) peptide mapping analysis (Figure 5), 4) in vitro G-CSF bioassay (Figure 7), 5) in vivo testing in mice. Example 4: Biological Activity: NFS-60 cells (ATCC CRL 1838) grown in RPMI + 10% FBS were used for PEG-GCSF cell proliferation assay. Cells were plated in 96 well plate and incubated with USV's monopegylated GCSF at concentration range 10 - 100 pg/ml for 72 hours in 37°C and 5% C02 humidified incubator. 5mg/ml MTT solution made in PBS was added in each well. Plates were incubated in 37°C and 5% C02 humidified incubator for 4-5 hrs. MTT is used for quantitative determination of cellular proliferation and activation in response to PEG-GCSF. The assay is based on cleavage of the yellow tetrazolium salt MTT, to purple formazan crystals by metabolic active cells. These crystals are then dissolved by adding acidified 25% SDS solution. The solubilized formazan product was spectrophotometrically quantified using an ELISA reader at 570 run. An increase in the number of living cells resulted in an increase in the total metabolic activity in the sample. This increase directly correlates to the amount of purple formazan crystals formed, as monitored by the absorbance. Example 5: In-vivo Activity: Pharmacokinetics of pegylated GCSF was evaluated in male Sprague Dawley rats. A single subcutaneous dose (100 mcg/kg bodyweight) of pegylated GCSF (USV) was administered to the experimental animals. Blood samples were withdrawn at 0, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192 and 216 hours post dosing. The plasma was separated and concentration of GCSF was measured by ELISA using a commercially available kit. The monopegylated GCSF (USV) had an elimination half life of about 12 hours. While the present invention is described above in connection with preferred or iHustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within its spirit and scope, as defined by the appended claims. Dated this the 20th day of October 2008

Full Text

FORM 2
THE PATENTS ACT I970
(39 of 1970)
&
The Patents Rules, 20()3
PROVISIONAL SPECIFICATION
(See Section 10 & Rule 13)
1. Title of the Invention
"An Improved Process for Pegylati^n of Proteins"

2. Applicant (s)
Name: USV Limited Nationality: Indian company incorporated under the Companies Act, 1956.
Address: B.S.D. Marg, Station Road, Govandi, Mumbai 400 088, Maharashtra, India.
3. Preamble to the Description
The following specification particularly describes the invention.

TECHNICAL FIELD
The present invention relates to the field of protein modification, and, more specifically, to an improved process for the attachment of water soluble polymer selectively to N terminal of proteins or analogs thereof The improved process of pegylation is characterized in that the reaction is carried out in the presence of sugar alcohol, as for example sorbitol.
BACKGROUND OF INVENTION:
Proteins for therapeutic use are currently available in suitable forms in adequate quantities largely as a result of the advances in recombinant DNA technologies. The availability of recombinant proteins has endangered advances in protein formulation and chemical modification. One goal of such modification is protein protection. Chemical attachment may effectively block a proteolytic enzyme from physical contact with the protein backbone itself, and thus prevent degradation. Additional advantage include, under certain circumstances, increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. One method commonly used for protein modification is by covalent attachment of water soluble polymers.
Polyethylene glycol ("PEG") is one such chemical moiety which has been used in the pegylation of therapeutic protein products. The US FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations. Molecules coupled to PEG become non-toxic, nonimmunogenic, soluble in water and many organic solvents, and surfaces modified by PEG attachment become hydrophillic and protein rejecting. The FDA has approved several pegylated polypeptides as therapeutics and more are undergoing clinical investigation. In 1990, pegademase (Adagen) received approval

for the treatment of severe combined immunodeficiency (SCID). Pegaspargase (Oncaspar) approved in 1994, contains the pegylated enzyme L-asparaginase, used clinically in combination with chemotherapy for the treatment of acute lymphocytic leukaemia, acute lymphpblastic leukaemia and chronic myelogenous leukaemia. In 2001, peginterferon a2b (Peglntron) became available as a once-a-week treatment for hepatitis C. Peginterferon a2a (Pegasys) approved in 2002 used a second generation, branched PEG of 40 kDa conjugated through a E-NH2 group of lysine used as spacer to interferon a2a increased the half life of IFN- a2a from 9 to 77 hours. A pegylated form of human growth hormone antagonist called pegvisomant (Somavert) was approved by FDA in 2003 for the treatment of acromegaly. Doxil, a pegylated liposomal formulation of doxorubicin was approved in 1995 for the treatment of Kaposi's sarcoma. Pegfilgrastim (Neulasta), approved in 2002, is a pegylated form of the earlier drug filgrastim (Neupogen) used for the treatment of neutropenia. Pegylation has taken 20 years to emerge as a viable pharmaceutical tool. Over the period there have been important advances in the chemistry of pegylation, in the generation of biomolecule therapeutics and in understanding PEG-biomolecule conjugates. Pegylation is now established as the method of choice for improving the pharmacokinetics and pharmacodynamics of protein pharmaceuticals. A variety of active PEGs have been prepared. mPEG succinimidyl succinate and mPEG succinimidyl carbonate were the reagents used and approved by US FDA. The regaents had the limitation of forming weak linkages between the PEG moiety and protein, potential unwanted side reactions, contamination, and restriction to low MW PEGs. The above limitations were overcome by use of mPEG-propionaldehyde which was easier to prepare. PEG aldehydes are inert toward water and react primarily with amines. Inertness toward water is desired, not only because of efficiency of storage, preparation, and application, but also because it permits stepwise linkage, in aqueous

media, of molecules to surfaces and molecules to molecule. mPEG aldehyde has essentially all the properties of ideal PEG derivative i.e. Reactive with nucleophillic groups (typically amino) on proteins and surfaces; stable in aqueous media and on the shelf; easily prepared and characterized; and capable of coupling to proteins without reducing protein activity.
US Pat No. 5,824,784 assigned to Amgen claims a substantially homogenous preparation of N-terminally monoPEGylated G-CSF or analog thereof and a method for attaching a polyethylene glycol to a G-CSF molecule wherein the PEG moeity has single aldehyde group. The pegylation process claims reacting G-CSF with polyethylene glycol under reducing alkylation conditions at a pH sufficiently aciidic to selectively activate the alpha amino group at the amino terminus of G-CSF. The process discloses the addition of a 5-fold molar excess of methoxypolyethylene glycol aldehyde of average MW, 6 kDa to a cooled (4°C) stirred solution of rhG-CSF (1 ml, 5 mg/ml) in 100 mM sodium phosphate, pH 5, containing 20 mM NaCNBH3. The stirring of the reaction mixture was continued at the same temperature The mono-MPEG-GCSF derivative was purified by ion exchange chromatography using HiLoad 16/10 S SEPHAROSE HP column and eluted with a linear 400 minute gradient from 0% to 45% 20 mM sodium acetate, pH 4, containing 1M NaCl . The % composition of N terminally mono-MPEG-GCSF obtained by reductive alkylation is not disclosed . A comparative stability analysis of N-terminally momopegylated G-CSF obtained by amide linkage (derived by using N-hydroxy succinimidyl ester of carboxymethyl methoxy polyethylene glycol as nucleophile) and the other obtained by amine linkage for 8 weeks yielded 82% purity with respect to one having amine linkage between the protein and the mPEG-aldehyde. A surprising result was observed as the amine linkage produced a material with far fewer aggregates against the one with amide linkage.

efficiency.The pegylation efficiency by reductive amination of peptide sequences benefits from the use of more hydrophobic alcohols and the efficiency enhancement afforded by fluoro alcohols is most advantageous to reductive amination reactions. The % of alcohols found to be beneficial is in the range of 30% to 70% v/v. The above prior art are applicable to the synthetic peptides produced by solid phase peptide synthesis or solution phase synthesis. However the harsh conditions when alcohols are used in such a high concentration cannot be used for pegylating proteins. An essential element of the present invention is use of non-hazardous additives for increasing the pegylation efficiency specially of proteins. The handling of volatile and hazardous alcohols limits the use for large scale operations.
SUMMARY OF THE INVENTION
The present invention relates to the process of selective pegylation of the proteins.
More specifically, the present invention relates to an improved process for pegylation
of the protein by reductive alkylation in the presence of sugar additive.
The present invention further relates to an improved pegylation process wherein the
sugar additive is selected from a group consisting of sorbitol, mannitol, sucrose,
trehalose, glycerol, xylitol.
The present invention further relates to a process with an enhanced pegylation
efficiency wherein the recovery of the pegylated product is atleast 80%.
The present invention still further relates to a process wherein the unreacted G-CSF in
the pegylation reaction is less than 1%.
The present invention further relates to a process for producing monopegylated G-
CSF with greater than 99% purity.

The present invention further relates to a process for pegylation wherein the sugar additive is added after the buffer exchange to the pegylation buffer and the concentration maintained throughout the pegylation process.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
The manner in which the objects and advantages of the invention may be obtained will appear more fully from the detailed description and accompanying drawings, which are as follows:
Figure 1: SEC-HPLC profile of USV's crude pegylated G-CSF wherein the pegylation is carried in absence of sorbitol.
Figure 2: SEC-HPLC profile of USV's crude pegylated G-CSF wherein the pegylation is carried in presence of 5% sorbitol in pegylation buffer.
Figure 3: Non-reducing SDS-PAGE of USV's purified pegylated G-CSF at different concentrations; Lane 1: USV's PEG-GCSF (2000 ng), Lane 2: USV's PEG-GCSF (1000 ng), Lane 3: USV's PEG-GCSF (200 ng), Lane 4: USV's PEG-GCSF (40 ng), Lane 5: USV's PEG-GCSF (20 ng), Lane 6: Neulasta (200 ng), Lane 7: Neulasta (40 ng), Lane 8: Protein MW ladder.
Figure 4: SEC-HPLC profile of USV's monopegylated G-CSF with a purity of >99%.
Figure 5: HPLC endoproteinase SV8 peptide mapping profile of monopegylated G-CSF; A: USV's monopegylated G-CSF, B:Neulasta.

Figure 6: SEC-HPLC profile of monopegylated G-CSF with a purity of >99%; A: USV's monopegylated G-CSF; B: Neulasta.
Figure 7: Graph illustrating a comparison of in vitro bioactivity of USV's monopegylated G-CSF compared against Neulasta & Neupogen.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES:
Example -1
Concentration and diafiltration (buffer exchange) of the rh-GCSF:
The liquid stock solution of rH-GCSF from USV (concentration ca 1.9 mg/ml supplied in 10mM sodium acetate, 5% sorbitol pH 4.0) which was stored at 2-8°C, was aliquoted out from the stock. The protein was then concentrated to about 6-7 mg/ ml. The sample was diluted twice the volume with l00mM sodium phosphate buffer pH 5.0 containing 20mM sodium cyanoborohydnde and 5% Sorbitol. The dilution and concentration was done for 3 times with the buffer. The final diafiltered concentration was 6-7mg/ml. The diaflltration procedure was done at 4°C in an ice bath. The concentrated solution was then stored at 4°C or taken for PEGylation reaction. The % recovery of the rh-GCSF post buffer exchange and concentration was about 95% to 97%.
Example -2
Preparation of Pegylated G-CSF:
The diafiltered, and the quantified protein obtained from Example 1 was taken in a 250ml glass bottle. A buffer consisting of lOOmM sodium phosphate buffer pH 5.0

with 20mM sodium cyanoborohydride and with/without 5% Sorbitol was added to the reaction mixture. The methoxy-polyethylene glycol-propionaldehyde (mPEG-aldehyde; SUNBRIGHT ME-200AL from NOF Corp., Japan) of approximately 20Kda was added to the above stirred solution of the protein. The mPEG-aldehyde was then transferred to the bottle containing protein solution. The protein concentration was maintained at 5mg/ml. The reaction mixture was then stirred at 2-8°C, overnight. The reaction mixture was quenched by addition of 40mM sodium acetate buffer pH 4.0 with and without 5% Sorbitol in the buffer (volume made to five times the reaction volume) (Figures 1 & 2).
The percentage conversion of GCSF was >98% wherein the pegylation was carried out in the presence of sorbitol.
Example -3
Purification using Ion exchange chromatography:
The pegylated G-CSF obtained from Example-2 was loaded to a weak cation
exchange column of following specification:
System: AKTA UPC 100 medium pressure system
The pegylated protein was eluted in a gradient mode using the following buffer
system.
Buffer A: 40mM Sodium acetate in 5% Sorbitol pH 4-0
Buffer B: 40mM Sodium acetate , 5% Sorbitol ,0.5M NaCl pH 4.0
The fractions containing the mono PEG-GCSF (>99%), were pooled. Tween 20 was
added to these fractions prior to concentration. The sample was concentrated and
diafiltered with l0mM sodium acetate, 5% Sorbitol, Tween 20. The final
concentration achieved was >10mg/ml, with purity of mono PEG >99% (Figure 4).

Table 1 illustrates the effect of addition of 5% sorbitol to the pegylation buffer and subsequent addition of tween-20 to the storage buffer prior to concentration. Table 1 rComparative analysis for pegylation carried in the presence and absence of sorbitol

The USV's monopegylated G-CSF was characterized using 1) Non-reducing SDS-PAGE of USV's purified pegylated G-CSF (Figure 3), 2) Size exclusion chromatography HPLC (SEC-HPLC) profile of USV's monopegylated G-CSF overlayed with Neulasta (Figure 6), 3) peptide mapping analysis (Figure 5), 4) in vitro G-CSF bioassay (Figure 7), 5) in vivo testing in mice.

Example 4: Biological Activity:
NFS-60 cells (ATCC CRL 1838) grown in RPMI + 10% FBS were used for PEG-GCSF cell proliferation assay. Cells were plated in 96 well plate and incubated with USV's monopegylated GCSF at concentration range 10 - 100 pg/ml for 72 hours in 37°C and 5% C02 humidified incubator. 5mg/ml MTT solution made in PBS was added in each well. Plates were incubated in 37°C and 5% C02 humidified incubator for 4-5 hrs.
MTT is used for quantitative determination of cellular proliferation and activation in response to PEG-GCSF. The assay is based on cleavage of the yellow tetrazolium salt MTT, to purple formazan crystals by metabolic active cells. These crystals are then dissolved by adding acidified 25% SDS solution.
The solubilized formazan product was spectrophotometrically quantified using an ELISA reader at 570 run. An increase in the number of living cells resulted in an increase in the total metabolic activity in the sample. This increase directly correlates to the amount of purple formazan crystals formed, as monitored by the absorbance. Example 5: In-vivo Activity:
Pharmacokinetics of pegylated GCSF was evaluated in male Sprague Dawley rats. A single subcutaneous dose (100 mcg/kg bodyweight) of pegylated GCSF (USV) was administered to the experimental animals. Blood samples were withdrawn at 0, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192 and 216 hours post dosing. The plasma was separated and concentration of GCSF was measured by ELISA using a commercially available kit. The monopegylated GCSF (USV) had an elimination half life of about 12 hours.

While the present invention is described above in connection with preferred or iHustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within its spirit and scope, as defined by the appended claims.
Dated this the 20th day of October 2008

Documents:

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Patent Number

271091

Indian Patent Application Number

2261/MUM/2008

PG Journal Number

06/2016

Publication Date

05-Feb-2016

Grant Date

02-Feb-2016

Date of Filing

20-Oct-2008

Name of Patentee

USV PRIVATE LIMITED

Applicant Address

B.S.D. MARG, STATION ROAD,GOVANDI,MUMBAI

Inventors:

#	Inventor's Name	Inventor's Address
1	MOHE NIKHIL UMESH	USV LIMITED, B.S.D MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
2	TARUR RADHAKRISHNAN VENKATASUBRAMANIAN	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
3	PALIWAL DINESH KUMAR	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
4	SAKSENA DIVYA LAL	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400 088, MAHARASHTRA, INDIA.
5	PATIL NILESH DAGDU	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
6	CHANDRAKESAN MURALIDHARAN	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
7	PAWAR DIGAMBER SHRIPATI	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
8	SHEKHAWAT RAKESH	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.
9	KHARE ARUNA	USV LIMITED B.S.D. MARG, STATION ROAD, GOVANDI, MUMBAI-400088, MAHARASHTRA, INDIA.

PCT International Classification Number

A61K47/48

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA