Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF RHGM-CSF

Abstract The present invention relates to a process for the preparation of rhGM-CSF by growing the yeast cells expressing rhGM-CSF in a suitable medium, which comprises of glycerol as a carbon source to yield the biomass and inducing the grown yeast cells capable of expressing the protein using alcohol in an incremental order, thereby harvesting and purifying the protein, wherein, the glycerol is fed in the range of 0.06 to 0.18 ml/Lit/min.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
"A PROCESS FOR THE PREPARATION OF A CYTOKINE"
EMCURE PHARMACEUTICAL LIMITED of Gennova Biopharmaceuticals Ltd., Plot No. 1, IT BT Park, Phase 2, MIDC, Hinjwadi, Pune - 411 057, Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:
The present invention is directed to a process for the preparation of a cytokine. In
particular the present invention relates to a process for the preparation of recombinant Human Granulocyte-Macrophage Colony Stimulating Factor (rhGM-CSF) by using methylotropic yeast, i.e., Pichia pastoris.
DESCRIPTION OF BACKGROUND AND RELATED ART:
Granulocyte-macrophage colony stimulating factor (GM-CSF) is an inflammatory cytokine secreted from epithelial cell, macrophage, T lymphocyte, endothelial cell and fibroblast having a molecular weight of about 14 kDa. GM-CSF induces development of granulocyte and macrophage precursor cells from bone marrow progenitor cells and also it is an established fact that GM-CSF plays an important role in proliferation, differentiation and functional activation of a hematopoietic stem cell, erythroblast, granulocyte, and macrophage.
Cloning and expression of recombinant human GM-CSF from various sources has been reported by Cantrell et al., Proc. Natl. Acad. Sci. USA 82:6250 (1985); Wong et al., Science 228:810 (1985); and Lee et al., Proc. Natl. Acad. Sci. USA 82:4360 (1985). Previously, human GM-CSF has been produced by recombinant means in COS (Wong et al., 1985), yeast (Ernst et al., 1987) and Namalwa cells (Okamoto et al., 1990). GM-CSF has also been expressed in tobacco, but at very low levels (James et al., 2000; Sardana et al., 2002). cDNA clones encoding rhGM-CSF were obtained by the way of selecting them from the total cDNA clones prepared from mRNA isolated from HUT-102 animal cell and T-lymphocyte cells by employing murine GM-CSF cDNA (Michael et al., P.N.A.S. 82, 6250-6254 (1984)). Another expression result of an rhGM-CSF gene in a COS-1 animal cell was published (Gordon et al., Science 228, 810-815 (1985) and Nicholas et al.,
EMBO 4,645-653 (1985)).
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With increase in the rate of occurrence of infectious diseases and also cancer, GM-CSF has been proven to be a potential therapeutic agent. GM-CSF also plays an important role in clinical management for life-threatening situations such as neutropenia, the most common toxicity of cancer chemotherapy (Dale, D. C. Colony-stimulating factors for the management of neutropenia in cancer patients. Drugs 62, (Suppl 1) 1-15 (2002))). Other oncology applications include treatment of febrile neutropenic conditions and support following bone marrow transplantation (Dale, 2002). GM-CSF has been used as a support medication, especially to accelerate the recovery of white blood cells following chemotherapy, bone marrow transplantation, before and/or after peripheral blood stem cell transplantation and also know to be used following induction chemotherapy in Acute Myelogenous Leukemia (AML). Potential applications are also under evaluation in patients with pneumonia, Crohn's fistulas, diabetic foot infections and a variety of other infectious conditions including HIV-related opportunistic infections (Dale, D. C. Colony-stimulating factors for the management of neutropenia in cancer patients. Drugs 62, (Suppl 1) 1-15 (2002)).
Though the use of GM-CSF in the treatment of cancer and other infectious diseases is highly appreciated, but the high cost of production of human GM-CSF has placed practical limits on its widespread use (Dale, D. C. Colony-stimulating factors for the management of neutropenia in cancer patients. Drugs 62, (Suppl 1) 1-15 (2002)). There is a long felt need for a high yielding manufacturing process for the production of GM-CSF. The availability of adequate quantities of GM-CSF is required for treatment of leukemia, anemias and also in bone marrow transplantation following cancer chemotherapy. Processes and methods to increase cell biomass yields, particularly maintain the production at high cell
densities is highly desirable.
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In general for expression and production of various proteins, E coli was a host of choice due to its ability to grow rapidly and ease of operation. However, its use has been limited due to its inability to make post-translational modifications. To avoid the problems regarding post translational modification imparted by E.coli, the organism chosen was S.cerevisiae, which is commonly known as Baker's yeast. But it was found that the glycol proteins expressed by S.cerevisiae can be of antigenic in nature.
Therefore the selection of host organism is critical for production of proteins with desired post-translational modifications and devoid of any antigenic effect. Pichia pastoris is the recent option for the production of the proteins. (High yield production from Pichia pastoris yeast; a protocol for bench top fermentation, New Brunswick Scientific, web site www.nbsc.com).
Prior attempts do disclose various host cells such as bacteria, plants, human cell lines and yeast been capable of use in production of GM-CSF such as for example E-coli, tobacco, cereal, CHO cells, sacciromyces cervesiea, Pichia pastoris.
However none of them demonstrate the large scale production of GM-CSF. The supply demand of GM-CSF is increasing due to the lack of efficient large scale production processes giving high yielding product.
Wu J.M et al. has disclosed combined use of Glyceraldehyde-3-phosphate dehydrogenase (GAP) and alcohol oxidase (AOX 1) promoters, wherein Pichia pastoris strain was generated capable of inducing both the promoters constitutively to express rhGM-CSF (Wu J.M et al., Enzyme and Microbial
Technology (2003), 33(4), 453-459)), and has reported to be superior to GAP alone in expressing recombinant rhGM-CSF. However it does not disclose large scale production using the generated strain.
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Mukerjee et al. (Appl. Microbiol Biotechnol, 69; 650-657 (2006)) discloses a fed batch culture for the production of rhGM-CSF in Pichia pastoris strain having GAP as a promoter which on induction is capable of expressing rhGM-CSF.
As per Mukherjee et al, the dry cell weight yield of GM-CSF is 98gm/litre. The process makes use of GAP promoter instead of the AOX 1 promoter to avoid use of methanol and avoid any cell suppression caused by methanol during production of GM-CSF. Additionally, the process optimization as reported suggests use of a complex media comprising casamino acids and yeast extract in order to obtain GM-CSF. The process makes use of a complex feed, which comprises amino acids and yeast extract which result in higher load of impurities in the final product GM-CSF and require intensive purification procedures. The low yield coupled with such intensive purification requirements makes the entire process commercially unviable.Thus, there is a need in the art to provide a process whereby the overall yield of rhGM-CSf is increased and the process is commercially viable.
Accordingly, the present invention provides a process for production of rhGM-CSF on a large scale. The process of the invention not only results in high yield but is also user-friendly and cost effective.
OBJECTS OF THE INVENTION:
The main object of the present invention is to provide a process for large scale production of rhGM-CSF.
Yet another object of the invention is to provide a process yielding high per volumetric productivity of rhGM-CSF in substantially constant environment.
Further, the object of the invention is to provide with a less cumbersome process for purification of rhGM-CSF.
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SUMMARY OF THE INVENTION:
Accoringly, the present invention provides an improved process for the preparation of rhGM-CSF, comprising the steps of:
a) growing methyloptropic yeast cells expressing rhGM-CSF in a medium
comprising a nitrogen source and glycerol as carbon source,
b) inducing the yeast cells to express rhGM-CSF by adding alcohol and
c) harvesting the rhGM-CSF and optionally purifying the same;
wherein, glycerol is fed at the rate of 0.05 to 0.2 ml/Lit/min.and alcohol is fed in increments.
In one aspect of the invention, the methylotropic yeast cells used for the production of the protein is Pichia pastoris.
In another aspect of the invention, the yeast cells are grown in a medium comprising as carbon source, nitrogen source, organic and inorganic salts suitable for the growth of methylotropic yeasts such as Pichia pastoris.
In yet another aspect of the invention, GM-CSF is isolated and purified by the combination of reverse phase chromatography, ion exchange chromatography and gel filtration chromatography.
In yet another aspect alcohol is fed at an incremental rate of 0.04 to 0.08 ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/min/Lit for 5-10 hours, which is followed by a range of 0.18 to 0.24 ml/min/Lit till the end.
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DETAILED DESCRIPTION OF THE INVENTION:
Some of the terms used in the present invention are discussed and explained herebelow:
"Methylotrophic yeast" includes yeast cells that make use of methanol as a sole carbon source when glycerol or glucose is not found as a food source in the medium. There are four known genera of methylotrophic yeast (Hansenula, Pichia, Candida, and Torulopsis), which share a common metabolic pathway that enables these to use alcohol methanol as a sole carbon source these are recombinant organisms, which can be used to produce large quantities of specialized proteins. These methylotropic organisms capable of producing a particular protein make use of alcohol to express the protein. The expression system of these organisms uses promoters such as AOX and MOX etc. capable of controlling the gene that codes the enzyme necessary for metabolization of alcohol. A number of proteins have been produced using this system, including tetanus toxin fragment, Bordatella pertussus petactin, human serum albumin and lysozyme. In a transcriptionally regulated response to alcohol induction, several of the enzymes are rapidly synthesized at high levels.
In accordance with the present invention, the media may comprise a carbon source, nitrogen sourceand organic and inorganic salts to sustain growth of the micro-organisms. Also, depending upon the growth requirement of a particular organism, media may be supplemented with trace elements and any vitamins.
The nitrogen source may be an organic or inorganic source. Nitrogen source includes but is not limited to yeast extract, peptone, ammonium salts, nitrates, and the preferred nitrogen source is ammonium dihydrogen phosphate and ammonium ferrous sulphate. Carbon source may include but is not limited to glucose, xylose, glycerol, and the preferred carbon source is glycerol.
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rhGM-CSF is secreted as a 127-amino acid (14.65 kDa) glycoprotein with a final molecular mass in the range of 18-25 kDa, depending on its glycosylation. Removal of oligosaccharides does not reduce the biological activity of rhGM-CSF, confirming their implication only in the half-life prolongation of the protein and not in receptor binding. The polypeptide structure of rhGM-CSF is represented from mature N-terminal residue (Ala-18) to the C terminus of the protein. Two regions of rhGM-CSF between Thr-95 and Thr-111 were found to be critical for its hematopoietic activity. After analysis, GM-CSF displays characteristics of an amphiphilic helix between Asn-34 and Asp-55, one of the two regions predicted to be involved in binding to the GM-CSF receptor. (Kenneth Kaushansky et al, Proc. Natl. Acad. Sci. USA, vol.86, pp. 1213-1217, feb. 1989, Biochemistry).
Thus, the invention provides an improved process for the preparation of rhGM-CSF, comprising the steps of:
a) growing methyloptropic yeast cells expressing rhGM-CSF in a medium
comprising a nitrogen source and glycerol as carbon source,
b) inducing the yeast cells to express rhGM-CSF by adding alcohol and
c) harvesting the rhGM-CSF and optionally purifying the same;
wherein, glycerol is fed at the rate of 0.05 to 0.2 ml/Lit/min.and alcohol is fed in increments
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In an aspect, alcohol is fed in an incremental order at a rate range of 0.04 to 0.08 ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/min/Lit for 5-10 hours, which is followed by a rate range of 0.18 to 0.24 ml/min/Lit till the end.
Preferably, the instant invention makes use of fed-batch type of fermentation. A "fed-batch" method of fermentation is similar to typical batch method, except that the substrate is added in increments as the fermentation progresses. Fed-batch fermentation is useful when catabolite repression may inhibit yeast cell metabolism, and when it is desirable to have limited amounts of substrate in the medium. Typically, the measurement of the substrate concentration in a fed-batch system is estimated on the basis of the changes of measurable factors reflecting metabolism, such as pH, dissolved oxygen, the partial pressure of waste gases (e.g., CO2), and the like. General methods for performing batch, fed-batch, and continuous methods of fermentation are well known to those of skill in the art. (Brock, T. D., Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (Sinauer Associates, Inc. 1989), Demain, A. L. and Davies, J. E. (1999) and Manual of Industrial Microbiology and Biotechnology, 2nd Edition (ASM Press 1999), and Hewitt et al., J. Biotechnol. 75:251(1999).
For protein production, Pichia cells are cultured in medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 °C to 35°C. A suitable medium of the present invention is a soluble, medium comprising glycerol as a carbon source, inorganic salts such as ammonia (nitrogen source), potassium, phosphate, iron and biotin. .Preferably, the medium avoids use of polypeptides or peptides, such as yeast extracts. Preferably the medium consists of certain recited components for e.g., water, glycerol, inorganic ammonia, potassium, phosphate, iron and the like.
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Accordingly, the present invention utilizes a medium used for the growth of the yeast cells, which comprises of salts such as ammonium dihydrogen phosphate, calcium chloride.2H20, potassium sulphate, magnesium sulphate, glycerol and water for injection (WFI).
The media is supplemented with micro elements such as ammonium ferrous sulphate, copper chloride, manganese sulphate, EDTA and zinc sulphate, preferably dissolved in water; trace elements such as sodium molybdate, cobalt chloride, boric acid, nickel sulphate, potassium iodide dissolved in water for injection (WFI) may also be added. Additionally calcium chloride may also be added and also vitamin solution comprised of D-Biotin dissolved in isopropanol can be added.
Preferably, the growth phase of the cells is carried out in the fermenter containing the medium comprising ingredients adequately present for the growth of the cells, wherein the glycerol is fed in the range of 0.06 to 0.18 ml/Lit/min. In the fermenter the dissolved oxygen (DO) is maintained adequately in order to obtain the cell biomass.
Preferably, the invention employs the alcohol for induction of AOX gene, which in turn induces expression of protein in the biomass, preferably methanol is used having a flow rate in an incremental order at a rate range of 0.04 to 0.08 ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/min/Lit for 5-10 hours, which is followed by a rate range of 0.18 to 0.24 ml/min/Lit till the end.
Optionally, if glycerol is fed as a percentage solution in a suitable solvent such as water then the feed range would vary according to the concentration.
In the fermenter the dissolved oxygen is maintained in the range of 25-50 %, preferably in the range of 30-40 %.
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According to the present invention, the parameters used in the present invention are so devised and controlled so as to achieve high biomass.
The preferred parameters for obtaining the improved yield are selected during the fermentation process in the fermenter are as enlisted below:
a. Controlled feeding of glycerol in the range of 0.06 to 0.18 ml/Lit/min.
b. Methanol feeding in an incremental order at a rate range of 0.04 to 0.08
ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/min/Lit for 5-10 hours, which is
followed by a rate range of 0.18 to 0.24 ml/min/Lit till the end.
c. The dissolved oxygen (DO) is maintained in the range of 25-50 %, preferably in
the range of 30-40%.
d. Temperature is maintained in the range of 25-35° C, preferably in the range of
28-32°
The present invention provides a process for the large scale production of rhGM-CSF, which has the advantages of:
a. High per unit volumetric productivity due to high cell density and
metabolism.
b. Provides essentially constant environment
c. Less cumbersome, since the medium used is a medium having no organic
source to hinder the downstream process.
d. Purification load is less on the final product.
e. Facilitates the isolation of desired protein because there are not many
endogenous proteins formed.
f. The product is not antigenic in nature, free of toxic material for therapeutic use.
g. Even the size of the bioreactor can be small; more quantity of production and
final product is achieved due to more biomass formation.
h. The product formed is with desirable glycosylation and low variability of molecular weight and having the product with longer half life.
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i.The process so devised does not allow synthesis of proteolytic enzymes thereby A providing high yield of protein.
General Process:
Accordingly, herein described is a general processfor the preparation of hGM CSF using Pichia pastoris, utilizing glycerol feeding and induction of cells by methanol for production of the protein, which comprises of a) growing yeast cells; b) production phase c) harvesting, isolation and purification of rhGM-CSF..
1) Inoculum build up:
GM-CSF can be produced by making use of Pichia pastoris cells capable of producing GM-CSF, . The initial growth of the Pichia cells is achieved by incubating the cells for required period of time in the suitable medium and then the grown cells can be used for further growth using suitable medium to be inoculated in the fermenter. During the inoculum build up, the cells are grown under conditions and for a period of time that is maximum for the growth of the inoculum. This inoculum build up is further transferred to the fermenter for production phase.
2) Production phase:
The fermenter was sterilized containing a suitable medium such as a defined medium used for the growth of the yeast cells, which comprises of ammonium dihydrogen phosphate, calcium chloride.2H20, potassium sulphate, magnesium sulphate, glycerol and water for injection (WFI). The media may be supplemented with micro elements such as of ammonium ferrous sulphate, copper chloride, manganese sulphate, EDTA and zinc sulphate, preferably dissolved in water, trace elements such as sodium molybdate, cobalt chloride, boric acid, nickel sulphate, potassium iodide dissolved in water for injection (WFI) may also be added. Additionally calcium
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chloride may also be added and also vitamin solution comprised of D-Biotin dissolved in isopropanol can be added. The pH of the medium may be maintained at 5.5 and with temperature at 30° C. The fermenter is inoculated
with the grown inoculum and the fermentation process is continued with a feed of a carbon source such as glycerol at a rate, which is in the range of 0.06 to 0.18 ml/Lit/min. Following glycerol feed, there can be induction of the cells to express the protein. Optionally, the induction may be done by the use of an alcohol such as methanol, which is fed in an incremental order of 0.04 to 0.08 ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/nun/Lit for 5-10 hours, which is followed by a rate range of 0.18 to 0.24 ml/min/Lit till the end.
The dissolved oxygen (DO) is maintained in the range of 25-50 %, preferably in the range of 3040%.
Following the production of the protein during the fermentation process, the cytokine of interest can be recovered from the fermented cell broth using techniques, which are well established in the art.
3) Harvesting, isolation and purification:
The protein of interest preferably is recovered from the fermented cell broth as a secreted polypeptide, although it may also be recovered from production medium containing the cell broth and secreted protein.
Further, the harvest can be centrifuged to remove particulate cell debris. The cytokine thereafter is isolated and purified from contaminant soluble proteins and polypeptides, with either or the combination of following procedures such as by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration using, for example, SEPHADEX G-75.TM.
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In a preferred embodiment, the following chromatographic procedures being exemplary of suitable purification procedures: by reverse phase chromatography, ion exchange chromatography and gel filtration chromatography. The sequence of the same may be interchangeable.
Initially the harvest may be subjected to centrifugation and the supernatant can be acidified with suitable acid, such as but not limited to tribromo acetic acid, trichloro acetic acid, triflouro acetic acid along with hydrochloric acid, sulphuric acid, where the preferably used acids are triflouro acetic acid (TFA) and hydrochloric acid.
The acidified supernatant was further centrifuged under similar conditions and the collected supernatant was filtered through 0.2 um sterile filter. The filtrate obtained may be subjected to column chromatography using reverse phase chromatography, ion exchange chromatography and gel filtration chromatography or the combination thereof. The column can be eluted using different elution buffers having desirable pH range based on the column used and appropriate affinity of the desired protein and the impurities.
The protein of the present invention may be produced by grown yeast cells, which express the desired protein by inducing with methanol and providing with favorable conditions in the fermenter. For instance, the process operation for the large or small scale production of proteins is potentially useful within the context of the present invention.
The present invention is further illustrated by the following, non-limiting examples, which should not be construed to limit the scope of the present invention in any manner as they are only intended to illustrate the concept of the invention.
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Example 1 Inoculum build up:
Materials and procedure: The host used was recombinant Pichia pastoris. The medium (1.5 L) used comprised of ammonium dihydrogen phosphate (30 g), calcium chloride.2H20 (1.39 g), potassium sulphate (27.3 g), magnesium sulphate 7H2O (22.32 g), glycerol (30 g) dissolved in water for injection (WFI), having a pH range 5 to 6.
The media is supplemented with micro elements solution (12 ml) comprising of ammonium ferrous sulphate (72 mg), copper chloride (6 mg), manganese sulphate (30 mg), EDTA (72 mg), zinc sulphate (22.5 mg) dissolved in water for injection (WFI), trace elements solution (3 ml) comprising of sodium molybdate (0.72 mg), cobalt chloride (0.72 mg), boric acid (0.72 mg), nickel sulphate (0.72 mg), potassium iodide (0.72 mg) dissolved in water for injection (WFI), calcium chloride solution (3 ml) comprising of calcium chloride (1050 mg) dissolved in water for injection (WFI) and vitamin solution (3 ml) comprised of D-Biotin (0.42 mg) dissolved in isopropanol. Flasks were used for carrying out the growth.
Process: Inoculum build up was done using Pichia pastoris cells from manufacture's working cell bank (MWCB). A flask was taken with 100 ml medium supplemented with micro elements solution, trace elements solution, calcium chloride solution and vitamin solution. The content of the vial containing cell culture was added in the 100 ml medium containing the supplements and was allowed to incubate at 30 °C for 36 to 44 hrs. The cell culture achieved after the required period of incubation was inoculated in other 14 flasks containing 100 ml medium with supplements and allowed to incubate at 28-30 °C for 20-28 hours. After incubation, the contents of all the flasks were
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pooled together aseptically and used as seed for production of the protein in the fermenter.
Example 2 Production phase:
Materials and procedure: The fermenter used in the process was a batch fermenter (Biostat- C, Braun Biotech International) with capacity of 30 L. The medium used in the fermenter was a medium (15 L), which comprised of ammonium dihydrogen phosphate (300 g), calcium chloride.2H20 (13.9 g), potassium sulphate (273 g), magnesium sulphate 7H2O (75 g), glycerol (600 g) dissolved in water for injection (WFI), having a pH range 5 to 6. The media is supplemented with micro elements solution (160 ml) comprising of ammonium ferrous sulphate (960 mg), copper chloride (80 mg), manganese sulphate (400 mg), EDTA (960 mg), zinc sulphate (300 mg) dissolved in water for injection (WFI), trace elements solution (40 ml) comprising of sodium molybdate (9.6 mg), cobalt chloride (9.6 mg), boric acid (9.6 mg), nickel sulphate (9.6 mg), potassium iodide (9.6 mg) dissolved in water for injection (WFI), calcium chloride solution (40 ml) comprising of calcium chloride (14 g) dissolved in water for injection (WFI) and vitamin solution (40 ml) comprised of D-Biotin (5.6 mg)
dissolved in isopropanol
Glycerol feed solution (1500 ml) comprised of 750 ml of glycerol dissolved in
water for injection (WFI).
Process: The fermenter was sterilized containing the medium (15 L) without the supplements by reaching the temperature up to 121 °C with holding time for 20 mins and pressure 1.2 kg/cm2. Following sterilization, the parameters in the fermenter were set at, temperature 30 °C and pH 5.5. The fermenter was allowed to cool and the supplements were added and the seed was also
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inoculated aseptically after cooling. After DO calibration; glycerol feeding was started at a rate of 0.13 ml/min/Lit for 4 hours.
Following glycerol feed, after an interval of 2-4 hrs, the induction with methanol solution was started to allow the cells to produce the desired protein. The induction was done with certain flow rate having duration of 0.04 to 0.08 ml/min/Lit for 0-4 hours and 0.10 to 0.16 ml/min/Lit for 5-10 hours, which is followed by a rate range of 0.18 to 0.24 ml/min/Lit till the end. After the completion of induction, i.e. 70 hours the batch was stopped and the harvest containing the protein was removed aseptically from the fermenter and the dry cell weight was calculated to be 140-180 g/Lit.
Further, isolation and purification was done by the standard procedures known in the art, while using reverse phase chromatography, ion exchange chromatography and gel filtration chromatography to give 70-80 mg/lit. The purity of rhGM-CSF was found to be more than 95 %, which was then confirmed by SDS-PAGE and HPLC.
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WE CLAIM:
1. An improved process for the preparation of rhGM-CSF, comprising the steps
of:
a) growing methyloptropic yeast cells expressing rhGM-CSF in a medium
comprising a nitrogen source and glycerol as carbon source,
b) inducing the yeast cells to express rhGM-CSF by adding alcohol and
c) harvesting the rhGM-CSF and optionally purifying the same;
wherein, glycerol is fed at the rate of 0.05 to 0.2 ml/Lit/min.and alcohol is fed in increments
2. A process as claimed in claim 1, wherein the glycerol is fed in the range of 0.09 to 0.15 ml/min/Lit.
3. A process as claimed in claim 1, wherein the alcohol is fed in an incremental order at a rate of 0.04 to 0.08 ml/min/Lit for initial 0-4 hours, 0.10 to 0.16 ml/min/Lit for 5-10 hours and 0.18 to 0.24 ml/min/Lit till the end.
4. A process as claimed in claim 1, wherein the alcohol is methanol.
5. A process as claimed in claim 1 wherein the methyloptropic yeast is Pichia pastoris.
6. A process as claimed in claim 1, wherein the rhGM-CSF is isolated and
purified by chromatographic techniques selected from reverse phase
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chromatography, ion exchange chromatography and gel filtration chromatography or a combination thereof.
7. A process for the preparation of rhGM-CSF, as described herein with respect to


foregoing examples.
Dated this 3rd day of January, 2007

19

ABSTRACT
"A PROCESS FOR THE PREPARATION OF A CYTOKINE"
The present invention relates to a process for the preparation of rhGM-CSF by growing the yeast cells expressing rhGM-CSF in a suitable medium, which comprises of glycerol as a carbon source to yield the biomass and inducing the grown yeast cells capable of expressing the protein using alcohol in an incremental order, thereby harvesting and purifying the protein, wherein, the glycerol is fed in the range of 0.06 to 0.18 ml/Lit/min.
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Documents:

27-MUM-2007-ABSTRACT(19-10-2010).pdf

27-MUM-2007-ABSTRACT(5-1-2007).pdf

27-MUM-2007-ABSTRACT(GRANTED)-(26-5-2011).pdf

27-mum-2007-abstract.doc

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27-MUM-2007-CLAIMS(5-1-2007).pdf

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27-MUM-2007-CLAIMS(AMENDED)-(23-3-2011).pdf

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27-MUM-2007-CLAIMS(GRANTED)-(26-5-2011).pdf

27-MUM-2007-CLAIMS(MARKED COPY)-(11-5-2011).pdf

27-MUM-2007-CLAIMS(MARKED COPY)-(23-3-2011).pdf

27-MUM-2007-CLAIMS(MARKED COPY)-(24-3-2011).pdf

27-mum-2007-claims.doc

27-mum-2007-claims.pdf

27-MUM-2007-CORRESPONDENCE(11-5-2011).pdf

27-MUM-2007-CORRESPONDENCE(2-9-2011).pdf

27-MUM-2007-CORRESPONDENCE(24-3-2011).pdf

27-MUM-2007-CORRESPONDENCE(25-10-2010).pdf

27-mum-2007-correspondence(31-1-2007).pdf

27-MUM-2007-CORRESPONDENCE(IPO)-(26-5-2011).pdf

27-mum-2007-correspondence-received.pdf

27-mum-2007-description (complete).pdf

27-MUM-2007-DESCRIPTION(GRANTED)-(26-5-2011).pdf

27-MUM-2007-FORM 1(19-10-2010).pdf

27-mum-2007-form 1(25-1-2007).pdf

27-MUM-2007-FORM 1(5-1-2007).pdf

27-mum-2007-form 13(1)-(19-10-2010).pdf

27-mum-2007-form 13(1-8-2011).pdf

27-mum-2007-form 13(19-1-2007).pdf

27-mum-2007-form 13(19-10-2010).pdf

27-mum-2007-form 18(31-1-2007).pdf

27-MUM-2007-FORM 2(GRANTED)-(26-5-2011).pdf

27-MUM-2007-FORM 2(TITLE PAGE)-(19-10-2010).pdf

27-MUM-2007-FORM 2(TITLE PAGE)-(5-1-2007).pdf

27-MUM-2007-FORM 2(TITLE PAGE)-(GRANTED)-(26-5-2011).pdf

27-mum-2007-form 26(25-1-2007).pdf

27-MUM-2007-FORM 26(25-10-2010).pdf

27-MUM-2007-FORM 3(19-10-2010).pdf

27-MUM-2007-FORM 3(5-1-2007).pdf

27-MUM-2007-FORM 5(19-10-2010).pdf

27-MUM-2007-FORM 5(5-1-2007).pdf

27-mum-2007-form-1.pdf

27-mum-2007-form-2.doc

27-mum-2007-form-2.pdf

27-mum-2007-form-3.pdf

27-mum-2007-form-5.pdf

27-MUM-2007-REPLY TO EXAMINATION REPORT(19-10-2010).pdf

27-MUM-2007-REPLY TO EXAMINATION REPORT(23-3-2011).pdf

27-MUM-2007-WO INTERNATIONAL PUBLICATION REPORT(5-1-2007).pdf


Patent Number 247851
Indian Patent Application Number 27/MUM/2007
PG Journal Number 22/2011
Publication Date 03-Jun-2011
Grant Date 26-May-2011
Date of Filing 05-Jan-2007
Name of Patentee GENNOVA BIOPHARMACEUTICALS LTD.
Applicant Address T 184, MIDC, BHOSARI, PUNE- 411 026, MAHARASHTRA
Inventors:
# Inventor's Name Inventor's Address
1 MAHESHWARI KUMAR MISHRA Gennova Biopharmaceuticals Ltd,PLOT NO.1, IT BT PARK, PHASE 2, MIDC, HINJWADI, PUNE- 411057.
2 SANJAY SINGH Gennova Biopharmaceuticals Ltd., Plot No.1, IT BT Park, Phase 2, MIDC, Hinjwadi, Pune- 411057.
3 SATISH RAMANLAL MEHTA of Gennova Biopharmaceuticals Ltd., Plot No.1, IT BT Park, Phase 2, MIDC, Hinjwadi, Pune- 411057.
PCT International Classification Number C07K14/52
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA