FORM
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
A process for the preparation and purification of filgrastim.
ONLY FOR AUTHORISED PERSONS
2. APPLICANT(S)
(a) NAME: Gennova Biopharmaceuticals Ltd.
(b) NATIONALITY: an Indian Company (b) (c)ADDRESS: P-l,IT-BT Park
MIDC Phase-2, Hinjwadi, Pune-411057, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the
manner in which it is to be performed.

4. DESCRIPTION
This invention relates to a process for the preparation and purification of human granulocyte colony stimulating factor (G-CSF), also known as filgrastim and its derivative pegylated filgrastim (pegfilgrastim), of higher purity and yield for pharmaceutical applications. Filgrastim is a cell growth factor involved in the stimulation of growth of a specific cell line (granulocytes) of the haematopoietic system. It is a 175-amino acid protein of molecular weight of 18,800 daltons. Filgrastim has been approved for use in the medicinal products for the treatment of neutropenia in cancer patients receiving myelosupressive chemotherapy. It is also indicated for the treatment of neutropenia after chemotherapy on acute myeloid leukaemia, cancer patients receiving bone marrow transfer, patients undergoing peripheral blood progenitor cell therapy and other severe chronic neutropenia.
The pharmaceutical production of G-CSF is done by recombinant DNA technology in heterogeneous organisms like Escherichia coli or mammalian cells. These methods afford large-scale production of the protein with higher purity and quantity. However, there remain lack of understanding of many aspects of recombinant DNA technology, process development and production that affect the productivity and characteristics of the final products. Therefore, there is need for new methods of preparation of recombinant DNA technology products that are improved over the existing methods and technologies. Besides, these methods being industrially suitable and cost effective for the development of

medicaments from these products.
The purification of various growth factors to homogeneity has been difficult and only few methods are known to yield high purity filgrastim and other proteins of similar nature. There has been two major barriers to the development of new methods: 1) a lack of good yield of final product if several steps are used in the purification protocols leading to uneconomic product and 2) the quality of expression system and inclusion bodies, its preparation and purification and subsequent steps of refolding and further purification, if not very clean, leading to the substandard product. Besides, purification procedures have been laborious, and the yields of pharmaceutical quality growth factor proteins low. Disclosed here is an improved method for the purification of recombinant human granulocyte colony stimulating factor (G-CSF) or filgrastim having combination of ion-exchange and gel filtration chromatography steps.
Filgrastim has been synthesized in the known art by various biotechnological methods. Several of the currently used methods have limitations in that the final product batches are not of consistent quality and quantity. This is caused due to several parameters playing parts, beginning at the complex methods of preparation of the protein from heterogeneous organisms. Also there exist many problems in scale-up and work-up of the current processes. These factors lead to the formation of impurities that can limit the use of the final products in pharmaceutical compositions. Besides, recombinant biosynthesis methods need many

controls at different levels, which create several limitations at large-scale production of these proteins. Here an economical process for the preparation of filgrastim is disclosed that uses multiple steps of chromatography with a set of buffers that gives higher purity and yield of the final product.
It is known to the skilled person in biochemical synthesis that in order to obtain highly pure final products, it is a good strategy to develop robust purification protocols and methods, besides, having selected good cell lines robustly over-expressing the target proteins. This is particularly important in the preparation of peptides and proteins synthesised using recombinant DNA technology. The principal object of this invention relates to an improved method of preparation of pharmaceutical batches of filgrastim for use in pharmaceutical compositions with improved impurity profiles for the impurities formed during the preparation. Another object of this invention relates to the use of a novel method of chromatographic separation of the protein from the its crude bacterial source using multiple steps without significant decrease in the yield at each step leading to the formation of filgrastim batches of higher purity and yield. The disclosed schemes are previously not used in this type of preparations with new chemistry of chromatography for the protein purification that significantly increases the utility of the invention in industrial applications.
Filgrastim prepared and purified by various strategies is known in the prior art. In this invention, a novel process for the purification of

filgrastim is disclosed having several advantages over the existing processes. The method disclosed uses a multiple-step liquid chromatographic separation of the peptide prepared in recombinant £. coli cells. This strategy entirely employs ion exchange chromatography in the purification of filgrastim of higher purity and yield having several advantages like ease of scale-up of the preparation to gram scale and ease of handling the process steps as well as economy of operation. It is known in the prior art several methods of preparation of filgrastim and similar peptides. All the known methods use recombinant heterogeneous expression of the protein in bacteria; while there are reports of expression of the protein in eukaryotic cells as well. However, for the pharmaceutical application the protein of high purity is desired without compromise as to the yield and cost of the product.
The process of the disclosed invention is elaborated with Examples below. In the first step, the seed culture of required characters is prepared from a working cell bank sample. The quality of the seed culture is maintained with in-house specification to give seed of exponentially growing cells in the synchronised growth phase. This seed culture is mixed with the optimised fermentation medium in the bioreactor at an amount of 10% of the volume of the fermentation medium. The medium used for the production is optimised for ingredients and micronutrients. The bioreactor is run in the fed-batch mode with addition of glucose, and ammonia solution to control pH of the system. The culture is allowed to reach high density of 70-80 OD (optical density) units at 600 nm. The auxostat set parameters are kept at the temperature of 30 °C, pH of 7.0

and dissolved oxygen of 35% with variable agitation up to 1100 rpm and aeration up to 1-2 WM (volume of air/ volume of medium/ min). The foaming in the reactor is controlled by anti-foam agents and temperature is controlled by steam heated jacket around the vessel. At start ampicillin is added to the reactor to maintain the selection pressure on the bearing bacteria plasmid vectors. After about 20 h of the process, when cell density is sufficiently high the heterogeneous protein expression is induced by heat shock of the fermented broth at 42 °C for about 4 h. After the induction phase the fermented broth is cooled down to about 10 °C and further processed at low temperature. The cell mass is recovered by centrifugation at 14,000 g and stored at 4 °C till further processed. The cell recovered at this step contains the over-expressed filgrastim accumulated in insoluble inclusion bodies (IBs).
The cells bearing the inclusion bodies of filgrastim are disrupted physically by ultrasonication or high pressure press disruption. Briefly, the cell mass is dissolved in a lysis buffer at 10% w/v and is incubated at room temperature for about 60 min. Then is sonicated at predetermined parameters for about 40 min running about 40 cycles of sonication. The progress of cell lysis is checked by microscopy and lysate is recovered when the lysis is complete for more than 95% of the cells. Alternatively, the cell lysis is carried out in a high pressure press or homogenizer at about 1000-1500 bars of pressure. Briefly, the cell mass is resuspended in the lysis buffer at 10% w/v and passed through the press at 4 °C for about 1-3 passes till more than 95% of the cells are broken. The lysate is recovered and insoluble inclusion bodies are removed by centrifugation

for further processing.
The purification and refolding of the inclusion body protein is carried out by washing the IBs in a set of buffers serially to remove the associated biomolecules. The washing buffers removed the adsorbed biomolecules from the IBs, and then the IBs are subject to the solubilization in the buffer with high amounts of urea so that the protein is completely denatured and stretched to the primary structure. This buffer also contains reducing agent to dissociate the disulphide bonds. Then the protein is refolded to get the active recombinant protein as elaborated in the examples below.
In the next steps, the refolded protein is subjected to the chromatographic purification protocols to obtain the filgrastim of high purity suitable for pharmaceutical applications. In the first step, the crude filgrastim preparation is separated on a strong cation exchange resin to remove the impurities that do not bind to the resin. The bound protein is then released from the resin; the buffer is exchanged and again subjected to the same strong cation resin at more concentrated amount. Here elution is done in gradient run to remove the impurities eluting with the protein. In the next step, the crude protein is subjected to a weak anion exchange resin to remove the impurities that bind to the column. The flow through is collected, buffer exchanged and subjected to a weak cation exchange resin to obtain the protein of higher protein upon elution. This crude protein is once again subjected to the buffer exchange and labelled at filgrastim concentrate solution for formulation. The following examples embody the steps of the method disclosed here without any limitation on

the scope of modifications and alterations possible in steps.
The filgrastim so obtained is used as such or further converted to the pegylated filgrastim by conjugating the required type of PEG (polyethylene glycol) moieties to the NH2-moity of the N-terminus of peptide molecules and further purified with a chromatographic method to high purity. Briefly, concentrated solution having 5-6 mg/mL filgrastim was incubated with a 20-kDa monomethoxy-PEG propionaldehyde in the molar ratio of 1:5 to 1:6 in the presence of sodium cynoborohydride. The pegfilgrastim so obtained was then purified by liquid chromatography to higher purity. This invention discloses a two part process in which in the first part filgrastim of higher purity is prepared. In the second part, pegfilgrastim is prepared from the filgrastim by conjugating the required PEG moieties chemically specifically to the N-terminus of the protein.
The types of the resins used for the ion exchange and gel filtration steps used in the method described here can be replaced with other resins of similar characters. The purification method described here is depiction of an example of the purification strategy used for the preparation of filgrastim and it is not limited by the use of types of chromatographic resins shown as other similar separation media may be used equally effectively.
As depicted in Table 1, different steps of the chromatography protocol leading to fold increase in the amount of the purified filgrastim as determined by SDS-APGE densitometry/ RP-HPLC. Before subjecting the

IBs isolated and purified as disclosed, it contained more than 80% filgrastim, peptide. At the end of fourth pass ion exchange chromatography (IEC) step the purity of the filgrastim peptide was at least 98% of the total protein.
Table 1

Chromatography Step No. Amount of filgrastim Amount of total impurities
IBs >80% <20%
IEC-1 >90% <10%
IEC-2 95% 5%
IEC-3 >95% <5%
IEC-4 >98% <2%
The invention detailed above is illustrated with the following examples for the purpose showing the utility of the invention.. Embodiments below do not restrict the invention in any way from broader applications of the reaction chromatographic techniques for preparation of peptides similar to filgrastim. The teaching of this invention can also be used in the preparation of the peptides that are G-CSF analogues.
The figures depict the quality of the purified filgrastim at the various steps of the chromatographic separation method. The figures are illustrative and do not limit the broader application of the invention with regard to the quality and purity of the protein separated.

LIST OF FIGURES
Figure 1: Silver stained SDS-PAGE of filgrastim - IBs: 1) molecular weight marker (from bottom: 14, 21, 31, 45, 66, and 95 kDa); 2) IBs solubilized; 3) IBs after renaturation; 4) IBs after acidification; and 5) IBs after 0.2 μM filtration.
Figure 2: Silver stained SDS-PAGE of filgrastim - chromatography steps 1-2:1) molecular weight marker (same as Fig. 1); 2) IEC first pass input; 3) IEC first pass flow through; 4) IEC first pass wash; 5) IEC first pass elution; and 6) GFC first pass eluate.
Figure 3: Silver stained SDS-PAGE of filgrastim - chromatography steps 3: 1) molecular weight marker (same as Fig. 1); 2) IEC second pass input; 3) IEC second pass flow through; 4) IEC second pass wash; and 5) IEC second pass fraction 1.
Figure 4: Silver stained SDS-PAGE of filgrastim - chromatography steps 3: 1) molecular weight marker (same as Fig. 1); 2)-8) IEC second pass fractions 2 to 9.
Figure 5: Silver stained SDS-PAGE of filgrastim - chromatography steps 4-5: 1) molecular weight marker (same as Fig. 1); 2) IEC third pass input; 3) IEC third pass flow through; 4) IEC third pass wash; and 5) GFC second pass eluate.
Figure 6: Silver stained SDS-PAGE of filgrastim - chromatography steps'

6-7:1) molecular weight marker (same as Fig. 1); 2) IEC fourth pass eluate; and 3) GFC third pass eluate.
Figure 7: An illustrative RP-HPLC graph of filgrastim after the chromatographic final step, purity of filgrastim peptide (retention time: 11.72 min.) is more than 99%.
Figure 8: Silver stained SDS-PAGE of pegfilgrastim - after IEX and GFC chromatography: 1) molecular weight marker (same as Fig. 1); 2) non-reduced peg-filgrastim (ref. std.); 3) reduced peg-filgrastim (ref. std.); 4) non-reduced pegfilgrastim as disclosed; and 5) reduced pegfilgrastim as disclosed.
Figure 9: An illustrative size exclusion-HPLC graph of pegfilgrastim after the chromatographic final step, the purity of pegfilgrastim (retention time: 15.39 min.) is more than 96%.
EXAMPLES
Example 1: Molecular biology, biochemistry and biotechnological
methods.
The general molecular biology, biochemistry and biotechnological methods used during the procedures are known in the art. Routine techniques can be anticipated and are not described as they are available in public literature. The strain used as the bacterial host cell was derived from E. coli K-12 and the plasmids and DNA elements used for the preparation of expression vectors were retrieved from commonly

available plasmids or designed and synthesised in-house and integrated.
The amino acid sequence used for the recombinant expression of human
G-CSF in E.coli is
[N-MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPE ELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALE GISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAF ASAFQKRAGGVLVASHLQSFLEVSYRVLRHLAQP-C].
The synthetic DNA elements were based on this sequence and integrated into the plasmid expression vectors using standard methods.
Example 2: Preparation of cell line for expression of recombinant human G-CSF protein.
An artificial DNA sequence of human G-CSF gene was synthesised with optimised amino acid codes for the bacterial expression. This sequence was cloned in a bacterial expression vector containing the elements for the efficient over expression of the protein in the cell as inclusion bodies. The vector was transformed in the required bacterial cell line and clones giving robust expression in the form of inclusions bodies were isolated for further use. The promoter used for transcription promotion was bacteriophage lambda left promoter (pL) which is repressed in by the temperature sensitive constitutive repressor of transcription cl857ts. The repressor too was expressed in cis from the same plasmid vector. At the elevated temperature of about 42 °C the repressor becomes ineffective leading to the de-repression of the recombinant G-CSF gene and a high level transcription of the gene leading to over production of the recombinant product and subsequent accumulation as inclusion bodies

inside the cells. This cell line was used for further large-scale fermentation batches for the recovery of large amounts of crude filgrastim peptide. The methods used here are well known in the prior art. However, some specific elements to increase yield and quality of the final product of fermentation were incorporated as disclosed.
Example 3: Recombinant G-CSF expression in E. coli. The human G-CSF amino acid sequence was expressed under the control of a heat inducible expression system in Escherichia coli cells. As the heat induction is most effective method for the controlled production of the heterogeneous proteins in the E. coli cells. The conditions used for growth of the cell line were monitored carefully so as not to hamper the production of filgrastim and also to allow the cells to grow in a good condition for the maximum production of the foreign protein. Briefly, cells were grown at about 30 °C with plenty of oxygen supply in a bioreactor at very high cell density of about 80-100 OD units at 600 nm. The fed-batch mode was used in the reactor for the efficient production of biomass without any unnecessary degradation of cells or release of toxic by-products of fermentation. This was monitored by known methods in the art. The parameters and contents of the reactor were periodically monitored and adjusted for the effective achieving the process endpoints. The induction of the protein was carried out at elevated temperature of 42 °C for a few hours to give maximum production of the protein in the form of inclusions bodies (IB's). Then further processing of the product was done in controlled conditions at low temperatures.

Example 4: Over expression of filgrastim.
The fermentation protocols for the production of crude filgrastim in E. coli used methods as disclosed. Briefly, a two-step large-scale process was developed. In the first step, seed culture was prepared in 1000-mL culture flasks from log-phase inoculates from the working cell bank samples of the recombinant cells over a period of 24-36 h. During this time the cell density was maintained below 2-8 OD units at 600 ran. The purity and growth of the cells were monitored by microscopy and/ or cell mass measurements. The medium composition used for the fermentation was (/L): 13.3 gm of potassium dihydrogen phosphate, anhydrous; 4.0 gm of diammonium hydrogen phosphate; 1.7 gm of citric acid in water for injection. The pH was adjusted with ammonia solution to 7 ± 0.2 units. This solution also contained the appropriate amounts of micro minerals and vitamins for the optimal growth of the cells. The medium components were steam or filtered sterilised as appropriate. The glucose solution was separately added in the medium at final concentration of about 1-2% of glucose. The medium also contains ampicillin at appropriate concentration for the maintenance of the plasmid vector. For the final 150 L fed-batch large-scale fermentation of about 15 L (10% of the volume) of the above seed was used. The fermentation medium for the bioreactor was the same as seed cultures except the glucose was fed as required and pH was maintained with ammonia solution. The set parameters in the auxostat were: temperature at 30 °C; pH at 7.0; dissolved oxygen above 35%; agitation at 700-1100 rpm; aeration at 1-2 VVM. The bioreactor was run for 18-20 h to achieve the cell density of 80-

100 OD units at 600 nm (wet cell mass of 130-170 g/L). During the fermentation the ampicillin solution was fed to maintain the plasmid vector at a high copy number in the producing cells. The induction of the protein over expression was carried at 42 °C for about 4 h, keeping other parameters of the bioreactor constant. After completion of the induction phase the reactor was cooled to about 14 °C while reducing the agitation. The ferment and cell mass was subsequently maintained at about 2-8 °C during all next processing steps. The inclusion bodies containing cells were harvest by centrifugation at 14,000 g for 15 min and about 20 kg of the biomass collected from a batch of 150 L for further processing.
Example 5: Isolation of inclusion bodies.
The inclusion bodies (IBs) were isolated from the harvested cell mass by break opening the cells and separating the IBs. The harvested cell mass was resuspended in Tris-EDTA lysis buffer, pH 8.8 (10 mM, 1 mM, 1-3 mS/cm) at the concentration of 10% w/v, mixed well and incubated at room temperature for about 60 min. Then the cells were physically break opened either with ultra-sonication or high pressure press depending on the volume of the total material for the processing using standard operating protocols. The cell breaking was done in small batches of about 40 L of the material and after more than 95% cell disruption the batches were pulled for further processing. The process temperature was maintained at about 4 °C during all the steps of cell disruption. Completion of the cell disruption was checked by microscopy. The conditions used for the ultra-sonication were: amplitude: 80%; cycle time: 0.5 sec; sonication time: 40-60 min. For high pressure press: 2-3 passes at

about 1400 bars of pressure. The insoluble fractions containing IBs were separated by centrifugation at 14000 g for 50 min at 2-8 °C and collected for further processing.
Example 6: Washing of inclusion bodies.
The isolated IBs were washed with different buffers to get rid of the non specific proteins from it for further purification steps. Briefly, IBs obtained above were resuspended in a Tris-EDTA Triton buffer, pH 8.0 (2% Triton-X 100) at a 10% w/v concentration and stirred for 30 min at room temperature. Then centrifuged at 14,000 g for about 45 min at 2-8 °C and the pellet recovered. Next, the pellet was washed with Tris-EDTA-DOC buffer, pH 8.0 (2% deoxycholic acid) using the conditions as the first washing step. Then the pellet was collected and further washed with Tris-NaCl urea buffer, pH 8.0 (2 M urea). In the final step, the pellet was washed with Tris-HCl buffer, pH 8.0 (20 mM) centrifuged and collected. The pellet can be stored at -80 °C till further processed.
Example 7: Solubilization of inclusion bodies.
The washed IBs were suspended in the solubilization buffer, pH 11.5 (urea: 8 M, Tris; 20 mM, beta-mercaptoethanol 1 mM, at conductivity of 0.5 -1.5 mS/cm) at a 10% w/v concentration and stirred for 80 min at room temperature without much frothing in the solution. At this stage the IPQC (in-process quality control) parameters were: pH between 11.30-11.70, conductivity between 0.50-1.50 mS/cm and bacterial endotoxins less than 0.25 EU/mL. Then sample was centrifuged at 14,000 g for about 30 min at 2-8 °C and the supernatant recovered and filtered through a 0.45 urn fluid

filter. At this stage the IPQC parameters were: total protein between 8-12 mg/mL, purity - a major band observed around 18 kDa.
Example 8: Refolding of the crude filgrastim protein.
The solubilized IBs obtained in Example 6 were subjected to the refolding by diluting in the dilution buffer, pH 8.0 (urea: 3 M, Tris: 100 mM, at conductivity of 0.8-2.0 mS/cm). IBs in the dilution buffer were stirred for 30 min at room temperature without any frothing in the solution. Then this solution diluted with the renaturation buffer, pH 8.0 (Tris: 10 mM, polysorbate 20: 0.02%, urea 3 M, L-cysteine 2 mM, L-cystine 1 mM, at conductivity of 0.5-1.5 mS/cm) to ten times (v/v) and allowed to incubate at room temperature for 16-20 h. At this stage the IQ parameters were: total protein between 0.2-0.3 mg/mL, purity - a major band observed around 18 kDa. Then pH of the solution was adjusted to about 5.0, by 0.2 M acetic acid. This solution was subjected to clarification by serial filtration through 1 μm, 0.45 μm and 0.2 μm membrane filters. The clarified solution having protein concentration of 0.02-0.06 mg/mL may be stored at 2-8 °C till further processed. At this step the filgrastim content of the in total proteins is more than 80% SDS-PAGE electrophoresis.
Example 9: Ion exchange chromatography - first pass. The refolded protein solution as above was then subjected to the first chromatographic purification step. On a fast liquid chromatography (FLC) system a column with 1500 mL of activated strong cation exchange resin (like sulpho-propyl sepharose) was prepared with the regeneration buffer-1, pH 5.0 (RB-1, sodium acetate: 103 mM, polysorbate 80: 0.004% at

conductivity of 7-9 mS/cm, pH 5.0) and then equilibrated with equilibrium buffer-1, pH 5.0 (EB-1, sodium acetate: 10 mM, polysorbate 80: 0.004% at conductivity of 1.0-1.3 mS/cm, pH 5.0) by passing 2 column volumes (CVs) of the both buffers at a flow rate of about 250 cm/h. Then the crude refolded protein was loaded onto the column with a linear flow rate of about 200 cm/h. Followed by passing EB-1 of 2 CVs at a flow rate of about 200 cm/h. This was followed by the wash buffer (WB-1, sodium acetate: 50 mM, polysorbate 80: 0.004% at conductivity of 3-4 mS/cm, pH 5.5) at 8-10 CVs at a flow rate of 200 cm/h. Then the bound target protein was eluted by passing about 4 CVs of elution buffer-1, pH 5.5 (EUB-1, sodium acetate: 20 mM, NaCl: 500 mM, polysorbate 80: 0.004%, pH 5.5) and collected separately based on the absorbance detection at 280 ran. The eluate (E-l) was analysed for protein quantity and purity by SDS-PAGE electrophoresis. At this step the filgrastim content of the in total proteins is about 90%.
Example 10: Gel filtration chromatography - first pass.
The eluate (E-l) of the first pass IEC was then subjected to the second chromatography step. On an FLC system a column with 12 L of a GFC resin (iike Sephadex G-25 resin) was prepared with the regeneration buffer-2, pH 5.5 (RB-2,. sodium acetate: 100 mM, polysorbate 80: 0.004% at conductivity of 8-10 mS/cm, pH 5.5) and then equilibrated with equilibrium buffer-2, pH 5.5 (EB-2, sodium acetate: 20 mM, polysorbate 80: 0.004% at conductivity of 1.5-2.0 mS/cm)by passing 2 CVs of the both buffers at a flow rate of about 200 cm/h. After regeneration step the column again equilibrated with equilibrium buffer-3, pH 4.0 (EB-3, sodium acetate: 10 mM, polysorbate 80: 0.004% at conductivity of 0.15-0.28 mS/cm, pH 4.0) by passing 2 CVs at a flow rate of about 200 cm/h. Then E-l eluate was loaded onto the column in fraction of about 3 L with a linear flow rate of about 200 cm/h. Followed by a pass of EB-2 to elute of the protein. The new eluate (E-2) was collected in a sterile container based on the absorbance at 280 nm. The eluate was analysed for purity by SDS-PAGE electrophoresis.
Example 11: Ion exchange chromatography - second pass.
The eluate (E-2) was then subjected to the third chromatographic purification step. On an FLC system a regenerated column with 250 mL of a strong cation exchange resin (like sulpho-propyl sepharose) was prepared with the RB-2 and then equilibrated EB-2 by passing 2 CVs of the both buffers at a flow rate of about 120 cm/h. Before loading the eluate (E-2), if s the conductivity was adjusted to about 2.7 mS/cm with 2 M NaCl solution. Then E-2 was loaded onto the column with a linear flow rate of about 120 cm/h. Followed by passing EB-2 of 2 CVs at a flow rate of about 100 cm/h. This was followed by a gradient washing using 0-20% of elution buffer-2, pH 5.5 (EUB-2, sodium acetate: 20 mM, NaCl: 100 mM, polysorbate 80: 0.004%, pH 5.5) in EB-2 buffer at 30 CVs at a flow rate of 100 cm/h. Then the bound target protein was eluted by passing about 20 CVs 20-60% of EUB-2 in EB-2. The eluate (E-3) fractions with more than 95% of filgrastim were collected based on the absorbance at 280 nm. E-3 was diluted with EB-2 to reach the conductivity of 4.8 mS/cm for the next step. The eluate was analysed for purity by SDS-PAGE electrophoresis. At this step the filgrastim content of the in total proteins is about 95%.

Example 12: Ion exchange chromatography - third pass.
The eluate (E-3) was then subjected to the fourth chromatographic purification step. On an FLC system a regenerated column with 100 mL of a weak anion exchange resin (like DEAE-sepharose) was prepared with the RB-2 and then equilibrated with EB-2 by passing 2 CVs of the both buffers at a flow rate of about 250 cm/h. Then E-3 was loaded onto the column with a linear flow rate of about 200 cm/h. The target protein came in the flow through fractions and was detected by absorbance at 280 nm (named E-4). The E-4 was analysed for purity by SDS-PAGE electrophoresis and RP-HPLC. At this stage the filgrastim content of the in total proteins is more than 95%.
Example 13: Gel filtration chromatography - second pass. The eluate (E-4) was then subjected to the fifth chromatography step. On an FLC system a regenerated column with 12 L of a GFC resin (like Sephadex G-25 resin) was prepared with the RB-3 (sodium acetate: 100 mM, polysorbate 80: 0.004%, pH 4.0) and then equilibrated with EB-3 by passing 2 CVs of the both buffers at a linear flow rate of about 150 cm/h. Then E-4 was loaded onto the column in fraction of about 3 L with a linear flow rate of about 150 cm/h. Followed by a pass of EB-3 to elute of the protein and new eluate (E-5) was collected in a sterile container based on the absorbance at 280 nm. The eluate was analysed for purity by SDS-PAGE electrophoresis and RP-HPLC.

Example 14: Ion exchange chromatography - fourth pass.
The eluate (E-5) was then subjected to the sixth chromatographic purification step. On an FLC system a regenerated column with 50 mL of a weak cation exchange resin (like CM-sepharose) was prepared with RB-3 and then equilibrated with EB-3 by passing 2 CVs of the both buffers at a flow rate of about 50 cm/h. Then E-5 was loaded onto the column with a linear flow rate of about 200 cm/h. Followed by passing 2 CVs of EB-3 at the flow rate of 50 cm/h. this was followed washing with WB-1 at the flow rate of 250 cm/h. Then the bound target protein was eluted with EUB-1. The target protein came in the flow through fractions (E-6) and was detected by absorbance at 280 nm. The eluate (E-6) was analysed for purity by SDS-PAGE and RP-HPLC.
Example 15: Gel filtration chromatography - third pass. The eluate (E-6) was then subjected to the seventh chromatography step. On an FLC system a regenerated column with 1 L of a GFC resin (like Sephadex G-25) was prepared with the RB-3 and then equilibrated with EB-4, pH 4.0 (sodium acetate: 10 mM, polysorbate 80: 0.01%) by passing 2 CVs of the both buffers at a linear flow rate of about 150 cm/h. Then E-6 was loaded onto the column in fraction of about 200 mL with a linear flow rate of about 150 cm/h. Followed by a pass of EB-4 to elute the protein and new eluate (E-7) was collected in a sterile container based on the absorbance at 280 nm. E-7 was further filter sterilised through 0.2 urn membrane filter and designated as filgrastim concentrated solution having the protein concentration between 2-5 mg/mL. The eluate was analysed for purity by SDS-PAGE electrophoresis and RP-HPLC. After this step the filgrastim content of the in total proteins is at least 98%.

Example 16: HPLC analysis of filgrastim.
The purity and level of impurities in filgrastim during the purification process and at the end was tested by a reverse phase HPLC method. The method parameters were: column: octadecylsilyl (C-18) silica gel (5 μm beads with 20 nm pores) of ID = 2 mm and L = 10 cm; buffer system: mobile phase A - 0.5 mL of trifluoroacetic acid in 950 mL of water and 50 mL of acetonitrile and mobile phase B - 0.5 mL of trifluoroacetic acid in 50 mL of water and 950 mL of acetonitrile; column temperature: 60 °C; gradient: 3-90% of A in B and back to 3% of A in 65 min; and detector: UV at 215 nm. The purity of the final product after the serial chromatographic separation was at least 98% by the RP-HPLC method.
Example 17: Preparation of pegfilgrastim.
The filgrastim obtained as in Example 15 was subjected to the PEGylation for the preparation of pegfilgrastim. About 18 g of filgrastim in solution (5-6 mg/mL) was incubated with 99 g of a 20 kDa monomethoxy-PEG propionaldehyde (m-PEG-ALD) in the presence of sodium cynoborohydride (20 mM final concentration) for 4-5 h at 25 °C On completion the reaction was stopped with glycine solution (100 mM final concentration). The conjugated protein was stored at 2-8 °C till further processed. This conjugated protein was purified by liquid chromatography to obtain monopegylated filgrastim called pegfilgrastim. On an FLC system a regenerated column with 500 mL of a strong cation exchange resin (like Capto S-sepharose) was prepared with regeneration buffer {200 mM sodium acetate, pH 4.0) and then equilibrated with

equilibrium buffer-PFl (20 mM sodium acetate, pH 4.0) by passing 2 CVs of the both buffers at a flow rate of 200-400 cm/h. Then about 3 L of the conjugate solution was loaded onto the colurnn with a linear flow rate of about 200-400 cm/h. Then the column was washed with 2 CVs of EB-PF1. Then the conjugated protein was eluted with elution buffer (20 mM sodium acetate, 0.5 M NaCl, 0.01% polysorbate 20, pH 5.0) and detected by absorbance at 280 nm. This step is used to concentrate the pegylated molecules. This was followed by a GFC step using an acrylic resin (like Sepharyl-200) for the fractionation of the pegylated molecules to get highly homogeneous fraction of monopegylated G-CSF Fraction with more than 96% pegfilgrastim were collected, pulled and analysed for purity by SDS-PAGE and RP-HFLC and found to be more than 96% pure.
Example 19: HPLC analysis of pegfilgrastim.
The purity and the level of impurities in pegfilgrastim at the end of purification was tested by a size exclusion-FiPLC method. The method parameters were - column: hydrophilic silica based (5 μm beads with 25 rim pores) of ID = 7.8 mm and L = 30 cm; isocratic mobile phase -12.8 g of sodium hydrogen phosphate, 19.1 g of disodium hydrogen phosphate, and 50 mL of ethanol in water for injection qS to 1 L, pH 6.8; column temperature: 30 °C; flow rate: 0.5 mL/ min run time: 30 min; and detector: UV at 215 nm. The purity of the final product was at least 96% by the method.

5. CLAIMS
We claim:
1. A process of the preparation of pharmaceutical batches of filgrastim/ comprising the steps of:
a) expressing the human granulocyte colony stimulating factor protein in a prokaryotic cell line at a high amount in the form of inclusion bodies;
b) isolating the said inclusion bodies from cells by mechanical cell disruption followed by separation;
c) cleaning and dissolving the said inclusion bodies to obtain the crude preparation of the over-expressed protein;
d) subjecting the said crude protein preparation to a chromatographic separation protocol comprising steps of:

1) capturing and separating the said protein by a strong cation exchange chromatography;
2) changing the buffered solvent of the said protein by gel filtration chromatography to another buffered solvent;
3) capturing and separating the said protein by a strong cation exchange chromatography using a gradient washing and elution;
4) purifying the said protein by a weak anion exchange chromatography to remove the impurities by capturing on the resin;

5) changing the buffered solvent of the said protein by gel filtration chromatography to another buffered solvent;
6) purifying the said protein by capturing the impurities by a weak cation exchange chromatography; and
7) changing the buffered solvent of the said protein by gel filtration chromatography to another buffered solvent to obtain filgrastim of higher concentration and purity;
e) formulating filgrastim so obtained into the batches with suitable stabilizing and buffering agents.
2. A process of Claim 1, wherein prokaryote used is Escherichia coli.
3. A process of Claim 1, wherein a strong cation exchanger used in
steps d) 1, and d) 3 is a sulpho-propyl resin.
4. A process of Claim 1, wherein a weak anion exchanger is use in
s*ep d) 4 is a diethylaminoethyl resin.
5. A process of Claim 1, wherein a weak cation exchanger is use in
step d) 6 is a carboxymethyl resin.
6. A process of Claim 1, wherein resin used in the gel filtration
chromatography in steps d) 2, d) 5 and d) 7 is a cross-linked dextran.
7. A process of the purification of pharmaceutical batches of
filgrastim, comprising at least three cation exchange chromatographic steps and at least one anion exchange chromatographic step, wherein the said anion exchange

chromatographic step is immediately before the last cation
exchange chromatographic step. 8. A process of claim 7, wherein at feast one cation exchange chromatographic step employs gradient elution of the said Protein.
9. A process of claim 7, wherein gel filtration chromatographic steps
are used in optional order between ion exchange chromatographic steps.
10. A process of claim 7, wherein strong and weak cation exchange
resins and a weak anion exchange resin are used.
11. A process of claim 7, wherein the said weak cation exchange
chromatographic step the last step in the order.
12. A process of preparation of pharmaceutical batches pegfilgrastim,
wherein filgrastim prepared by processes of claim 1 and 7 is conjugated to a polyethylene glycol moiety by N-terminus.
13. A process of the purification of pegfilgrastim according to claim 12,
comprising at least one cation exchange chromatographic step and at least one gel filtration chromatographic step.
14. A process for preparing a pharmaceutical formulation, comprising
combining pharmaceutical batches of filgrastim or Pegfilgrastim made by the process of Claims 1, 7 and 13, with at least one pharmaceutically acceptable excipient.
15. A process for the preparation of filgrastim or pegfilgrastim
subbstantially as described with reference to the examples.

6. DATE AND SIGNATURE
Dated this 4th day of September 2010.

(Dr. Parag Kinge) For the applicant, Gennova Biopharmaceuticais Ltd.
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2 8 SEP 2010