Title of Invention

METHOD FOR PRODUCTION OF A THERAPEUTICALLY PURE GRANULOCYTE MACROPHAGE - COLONY STIMULATING FACTOR

Abstract

A simple and scaleable process is provided for obtaining recombinant human GM-CSF expressed in microbial cells, secreting mature GM-CSF, culturing these cells and purifying the protein in good yield and of a therapeutic grade quality. The steps include cloning, fermentation and purification of the protein. The purified protein is got by a process that involves lysing the microorganism, extraction of the processed and unprocessed forms of GM-CSF from the membrane pellet and selectively purifying the processed form of the protein from the unprocessed form by a two-step chromatography procedure. The purity of the processed form of GM-CSF (without signal peptide) obtained has endotoxin units, host cell proteins and host cell DNA within the specified compendial limits for therapeutic proteins.

Full Text

Field of the Invention The present invention relates to a method for production of a therapeutically pure granulocyte macrophage-colony stimulating factor (GM-CSF). More specifically, the instant invention relates to the production of a recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF with a heterologous PelB signal peptide in Escherichia coli (E.coli), processing of the pre-GM-CSF form by the bacterial host by cleaving the signal sequence and extracting the processed form from the membrane pellet fraction of the cell lysate for final purification. More specifically, the invention is directed to a recombinant E.coli strain harboring a vector construct comprising the mature GM-CSF coding sequence fused to the PelB signal peptide and a simplified process for the purification of an essentially endotoxin free, processed GM-CSF protein for therapeutic applications from a periplasmic but insoluble cellular localization. Background of the Invention Human GM-CSF belongs to the family of endogenous colony stimulating factors (CSFs) that stimulate the growth of hematopoietic progenitor cells of the myeloid and erythroid lineages. It is a naturally occurring hematopoietic growth factor (cytokine) that is produced by T cells, macrophages, fibroblasts and endothelial cells. As the name indicates, it was originally defined by its ability to stimulate the proliferation and differentiation of granulocyte/macrophage progenitor cells, but later found to have the same activity on progenitor cells of other hematopoietic lineages (Metcalf D. et al. Blood 55: 138-147 (1980)) and also cells of non-hematopoietic origin like human bone marrow fibroblasts (Dedhar, S et al, Proc Natl Acad Sci (USA) 85: 9253-9257,1988). It is a 22kDa glycoprotein in its native form and a 14.4 kDa protein in its non-glycosylated recombinant form with clinical uses in fighting infection for example in patients with chemotherapy- induced neutropenia. Endogenous colony-stimulating factors (CSFs) are a class of glycoproteins that act on hematopoietic cells by binding to specific cell surface receptors to stimulate proliferation, differentiation, commitment and cellular function activity. GM-CSF "turns on" the immune system by stimulating production of the myeloid progenitor stem cells of neutrophils, monocytes, macrophages, eosinophils, and dendritic cells. It also has a role in fiinctional cell activities, such as T cell activation. Increased monocyte activity generates macrophages and allows for increased phagocytosis that may prevent or diminish the occurrence of life-threatening bacterial and fungal infections. Endogenous myeloid colony-stimulating factors (CSFs) have demonstrated the ability to enhance the clinical management of immuno-suppressed patients with cancer. These agents are associated with significant decreases in chemotherapy-associated infections, antibiotic use, length of hospital stay and mortality. Clinical trials suggest that GM-CSF has clinical benefits beyond enhancing neutrophil recovery, including shortening the duration of mucositis and diarrhea, stimulating dendritic cells, preventing infection, acting as an adjuvant vaccine agent, and facilitating antitumor activity. In addition, this cytokine also plays a vital role in the body's ability to mount an immune response. Thus, this growth factor is also being explored as a modulator of immune response in treatments for cancer, AIDS and infectious diseases. Various experimental studies and also clinical trials are underway wherein GM-CSF is being used as a vaccine adjuvant (US patent 5,679,356). Naturally occurring mature GM-CSF is a glycoprotein containing 127 amino acids and two disulfide bonds. The availability of GM-CSF from its natural source is extremely limited because of its presence in trace quantities. This hindered the biochemical characterization of the cytokine until the time when it was expressed using a recombinant expression system. The cloning and expression of human GM-CSF has been reported from various sources 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). The mammalian cell-expressed GM-CSF is present in different glycosylated forms ranging in size from 14 to 35 kilo Daltons. Some forms of GM-CSF may contain two N-linked carbohydrate groups and/or three 0-linked carbohydrate groups explaining the apparent size heterogeneity. The GM-CSF expressed in CHO cells is biologically active. However, the biological activity is enhanced by 20-fold upon enzymatic removal of the carbohydrate residues. Thus, indicating that the unglycosylated form may be superior for clinical use (Moonen P. J., et al, 1987 (Proc natl Acad Sci USA). Ernst J. F. et al., 1987 (Bio/Technol. 5: 831-834) described a process for secreting GM-CSF using the alpha mating factor signal peptide in the yeast system. The yeast Saccharomyces cerevisiae-expressed GM-CSF is secreted as a heterogenous mixture of glycoproteins ranging in size from 35 to 100 kilo Daltons. This heterogeneity due to varying degree of glycosylation in the yeast cells poses several problems for therapeutic applications of this cytokine. The variable and excess amounts of carbohydrate in the recombinant GM-CSF results in more complex purification procedures resulting in low yields besides having the possibility of developing allergic reactions to the foreign carbohydrate chains in the recipients. Moreover presence of dibasic residues in native GM-CSF sequence provides a cleavage site for the yeast protease like KEX 2. This necessitates site directed mutations of such sites to obliterate KEX 2 cleavage site while maintaining the same bioactivity (the drug LEUKINE.^^^ from Immunex Corporation, is a yeast-derived recombinant human GM-CSF that differs from the endogenous human GM-CSF by having a leucine instead of an arginine at position 23, besides having different carbohydrate moiety. Human GM-CSF in unglycosylated form has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli (Burgess, A.W., et al., 1987 (Blood 58: 43-51)). The £". co//-expressed GM-CSF formed inclusion bodies in the cytoplasm and also had an extra methionine residue at the N-terminus. The conversion of the biologically inactive GM-CSF to the bioactive form required in vitro oxidative refolding. The refolded Exoli-expressed human GM-CSF is still not equivalent to an unglycosylated form of native GM-CSF due to the presence of an amino terminal methionine in the £. coli produced protein. US patents 5,942,221; 5,908763; 5,891429; 5,391,485 and 5,393,870 describe isolation and cloning of the coding sequence of human GM-CSF and its recombinant expression in heterologous hosts like mammalian cells, yeast and E. coli. Human granulocyte macrophage - colony stimulating factor (GM-CSF) produced by recombinant DNA technology in E.coli has been shown to be similar to the glycosylated native protein in its therapeutic action. A commercially available product Leucomax® (Novartis/Schering), produced in E,coli, with 127 amino acids, molecular weight of 14.4 kDa has been successfully used clinically to stimulate the progenitor cells in the bone marrow to multiply, differentiate and enhance neutrophil recovery. U.S. Patent 5,047,504 (Boone; Thomas C, assigned to Amgen Inc., February 17, 1989) describes recovering GM-CSF from a microorganism in which it is produced by lysing the microorganism, separating insoluble material containing GM-CSF from soluble proteinaceous material, extracting the material with a chaotropic agent, refolding and oxidizing the GM-CSF in the presence of a glutathione, precipitating the incorrectly folded GM-CSF, removing the denaturant and subjecting the residual GM-CSF to ion exchange and reverse phase chromatography to recover purified GM-CSF. The patent however does not mention whether the protein is free of N-terminal methionine and since no additional attempt has been made to exclude this possibility it can be assumed that this protein is not in the final processed form, without the N-terminal methionine, required for therapeutic use. Besides, the process described is lengthy due to the refolding and oxidation steps involved since the expression of the protein is in inclusion bodies in the cytoplasm. The U.S. Patent 5,136,024 describes a method for extracting GM-CSF from bacterial cells by treating it with an acid to lower the pH to a value between 1.5 and 3.0 and then neutralizing the suspension to a pH between 6 and 9. Although GM-CSF in this case was cloned with the signal peptide and hence the processed form of GM-CSF without the N-terminal methionine is likely to be obtained after purification but the procedure mentioned is only for extraction by acidification and neutralization and does not mention purification of the protein to homogeneity for therapeutic use. Hence, this only describes a single step involving extraction of GM-CSF from the bacterial cells, without any mention of the quality of the GM-CSF protein obtained after this procedure. Another related patent that describes production of GM-CSF is U.S. Patent 5,391,706. It discusses the purification of GM-CSF from bacteria to 95% homogeneity by a four-step chromatography process that includes QAE, dye-ligand affinity, gel filtration and reverse phase steps. A related patent to the same assignee (Sobering Corporation), US 5,451,662 describes an ion-exchange separation procedure near the isoelectric point of the protein called delta isoelectric point chromatography. In this patent also no mention is made of the form (processed or unprocessed) of GM-CSF that is purified and its therapeutic quality. Wong et al. Science Vol.228,pp, 810-815 (1985) and Kaushansky et al, Proc. Natl. Acad. Sci. USA, Vol. 83,pp. 3101-3105 (1986) have described the production of GM-CSF in mammalian cells. Burgess et al.. Vol. 69,pp 43-51 (1987) describes the purification of GM-CSF produced in £. coli. Various methods have been disclosed for cloning and expressing GM-CSF in microbial cells and extracting the processed GM-CSF (without the N-terminal methionine and the Exoli signal peptide) from the periplasm of the host cells. The isolation of periplasm is a carefully controlled process that is not easily scaleable for production. Besides, the expression level of the soluble recombinant protein that can be achieved in the periplasm is not very high making the process scale economically inefficient. The processes described do have room for improvement because of various limitations such as over-glycosylation when expressed in yeast systems, the presence of an extra N-terminus methionine when expressed in the cytoplasm as inclusion bodies in E,coU. Expression in inclusion bodies also necessitates subsequent in-vitro refolding procedures that result in low yields of biologically active species and complex and expensive purification procedures. In view of this situation, there is a continuing need to develop improved recombinant DNA systems and economical high yielding processes for production of therapeutic grade GM-CSF. An effort has been made to develop a more efficient method that can ensure the yield of processed recombinant GM-CSF in good quantities with the retention of biological activity and essentially free of endotoxin for therapeutic use. This metiiod can be used to purify large quantities of recombinant human GM-CSF expressed in the periplasm of £.co//, by a simple and economical process involving fewer steps and higher final yields. Summary of the Invention The present invention provides a method of production of human GM-CSF in a bacterial host by transforming it with a genetically engineered plasmid that contains tiie gene for human GM-CSF with the heterologus signal peptide, culturing these recombinant microbial cells under favorable conditions for enhanced expression of the protein and purifying it to therapeutic endotoxin-free grade by a simple, scaleable two-step chromatography process that does not include periplasmic extraction or solubilization and refolding from inclusion bodies. Accordingly, the present invention provides a method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor (GM-CSF), wherein said GM-CSF is secreted in its mature form fix>m a recombinant bacteria, has no methionine residue at N-terminus, and the method comprises: a) cloning the human GM-CSF gene in-frame with Pel B signal peptide in an E.coli strain b) producing elevated expression levels of the processed protein by altering the induction conditions c) culturing GM-CSF producing recombinant cells in which over-expressed GM-CSF gets processed during secretion into the periplasm and gets associated with the cell membranes. d) lysing said cells and isolating the membrane fraction containing GM-CSF e) extracting the processed and unprocessed forms of the membrane-bound GM-CSF for purification f) subjecting the mixed GM-CSF forms to ion exchange chromatography g) recovering the purified processed form of GM-CSF after a reverse phase or HIC chromatography of therapeutic grade. Preferably, the membrane pellet fraction containing GM-CSF is recovered from the cells by lysing them by high-pressure homogenization or sonication. The membrane pellets can be obtained from the lysate, for example by centrifixgation. The pellets are then subjected to a repeated washing procedure to remove endotoxins. Such washing is affected by using either or a combination of a non-ionic detergent and a chaotropic agent. The processed GM-CSF that is bound to the cell membrane after secretion is then extracted by a combination of a low concentration of a chaotropic agent and any ionic salt. The extracted soluble GM-CSF is buffer exchanged and directly loaded onto an anion exchange column for partial purification and enrichment of the collected fraction with GM-CSF. The eluate from this column is then purified to homogeneity on a hydrophobic column which when eluted with the buffer gives a homogeneous protein of GM-CSF with a recovery of almost 80% of the processed form that was initially bound to the membrane fraction. Brief Description of the Acconwanvins Drawines The invention will now be described with reference to the accompanying drawings. Figure 1 is a map of £. coli expression vector, which directs the secretion of GM-CSF Figure 2 is the nucleotide and amino acid sequence of mature GM-CSF Figure 3 is the elution profile of GM-CSF after chromatography-1 Figure 4 is the elution profile after chromatography-II showing separation of processed and unprocessed GM-CSF Figure 5 is the SDS-PAGE analysis of final purified GM-CSF under reducing and non-reducing conditions showing a single processed GM-CSF band and the absence of the unprocessed GM-CSF and other host cell impurities Detaiied Description of the Invention with accampanvins drawinss: The present invention provides a method for the production of a fully processed form of human GM-CSF by recombinant methods using a microbial expression system with improved culturing conditions and a simple and scaleable purification process for an endotoxin-free preparation of the purified protein. The process described in the present invention can be applied to the production of recombinant GM-CSF for therapeutic purposes. Source of GM-CSF gene, cloning and expression Appropriate cloning strategies and expression vectors for use with bacterial or fungal hosts are described by Sambrook et. al. (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. 1989) and Pouwels et. al (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Suitable methods for cloning and expression of GM-CSF in various hosts are disclosed in US patents 5,908,763; 5,891,429; and 5,391,485. In the present embodiment, the mature coding portion of the GM-CSF gene is isolated from a human lymphocyte cell line. Total RNA is extracted from the cells and converted into cDNA. An aliquot of the synthesized cDNA is used as a template for amplifying the desired DNA fragment of GM-CSF coding sequence using appropriately designed specific oligonucleotide primers. The GM-CSF amplicon is cloned at the Bam HI and blunt fragment into the expression vector in frame with the Pel B signal sequence at its 5' end to form the plasmid construct pTMF02 as shown in Fig:l. A promoter inducible either by IPTG/ lactose / arabinose vsdll drive the transcription of the Pel B-mature hGM-CSF coding sequence. The complete nucleotide sequence of the GM-CSF gene is given in Fig: 2. E, coli host strains harboring the afore described plasmid construct will secrete the mature GM-CSF into the periplasmic space when grown using optimized media conditions. The optimized culture conditions comprises of induction with an inducer of the promoter like IPTG, arabinose or lactose or related sugar molecules, the presence of a reducing agent like glutathione, DTT, L-cysteine or related compounds having thiol groups to facilitate reshuffling of incorrect disulfide bonds and growth at low temperatures of 20 to 28'C for slower growth kinetics. The combinatorial effect of the above media and culture conditions leads to efficient processing and secretion of GM-CSF suitable for production of therapeutic grade mature GM-CSF. Fermentation ' ^^. ^L ^ tJ^'^^ Cuhivation of recombinant E. co// strain containing human GM-CSF gene is done under conditions optimized for maximum heterologous protein expression. Fermentation is carried out in a 10-litre fermentor vessel. The following parameters are maintained during the run V7Z., air flow: I vessel volume per minute (ww), agitation: 300-800 rpm, dissolved oxygen (DO): >40% and temperature around 37^C during cell growth, which is shifted to 20 -28'C prior to the addition of the inducer and maintained as such till harvest. Production medium used is a complex medium like Terrific Broth, containing glycerol as carbon source, yeast extract and tryptone as nitrogen sources. Ampicillin and chloramphenicol are used for selection. The medium is inoculated with a seed culture and allowed to grow till the desired cell density is obtained and the temperature is gradually shifted to between 20 and 28'^C, for acclimatization, and maintained thereon /.e., during induction and post-induction growth. Expression of GM-CSF is achieved by feeding of the inducer selected from the family of sugars that can induce the promoter at concentrations ranging from 0.1 mM to lOmM, while a reducing agent is added in a concentration range of 0. ImM to 20mM to improve the yield of processed GM-CSF. At the end of induction period (--7 h), the batch is harvested. The cellular biomass obtained is atleast 40±10 g/1 (wet wt). The secreted GM-CSF is directed to the £". coli periplasm by a signal sequence that gets processed during the process. However, a fraction of the GM-CSF molecules remain unprocessed and retain the signal sequence which are separated from the processed form during the protein purification process. Purification Purification of GM -CSF from the harvested E. coli cells is done by a simple procedure involving lysis of the cells, solubilization of membrane bound secreted GM-CSF followed by a two-step chromatography procedure. The highlight of the simplified purification process is a selective purification step from the membrane fraction for the recovery- of the processed GM-CSF protein from a mixture of the processed and unprocessed GM-CSF protein. The unprocessed form containing the signal peptide shows a differential solubility and binding behaviour on the chromatography column. The harvested cell pellet is suspended in lysis buffer, for example, 50gms of cell paste is suspended in 500ml to I litre of lysis buffer, i.e. 25mM to 50mM Tris buffer, at pH8.0, ImM to lOmM EDTA and 0.5mM to ImM phenyl methyl sulfonyl fluoride (PMSF) and lysed by high pressure homogenization or by repeated short pulses on a sonicator. The process is conducted in an ice bath and continued till complete cell lysis is obtained as monitored by the stabilization of ODeoo value of the lysate. The cell lysate is centrifuged in a refrigerated highspeed centrifuge to pellet the cell debris along with the membrane fraction. This cell pellet is rid of contaminating cell wall lipids and endotoxins by washing the fraction with either a non-ionic or an ionic detergent or a combination of both. The non-ionic detergent can be chosen from a class belonging to Triton or Tween series and the ionic from a class belonging to the any of the anionic detergents. The pellet thus obtained has the processed and unprocessed GM-CSF bound to the membrane, which can be easily solubilized in native form by treating it with a high molarity salt solution or a mild chaotropic solution or a combination of both. The solubilized protein fraction is recovered by centriftigation and buffer exchanged with the equilibration buffer of the first chromatography column step to match the conductivity and pH values with the column loading conditions. A chromatography column is packed with an anionic matrix selected from a group of Q, DEAE or QAE ligands. This is equilibrated with a suitable buffer in the pH range of 7.0 to 8.5 and the loaded at low conductivities. The solubilized sample is loaded on to the column and washed with equilibration buffer till the optical density value at 280nm returns to baseline. GM-CSF is eluted from this column using a linear gradient of salt preferably sodium chloride from 0 M to 1.0 M concentration in the equilibration buffer. The processed and unprocessed GM-CSF elutes from this column as a distinct single peak as shown in 1 ig 3. Most of the GM-CSF loaded onto the column can be recovered almost quantitatively with this elution step without any significant drop in yield. The processed mature form of GM-CSF (without the signal peptide) can be selectively purified to homogeneity from a mixture of processed and unprocessed forms by a second chromatography step on either a reverse phase HPLC column or a hydrophobic interaction chromatography column. The protein eluate after the first chromatography step is treated with trifluoroacetic acid in the range of 0.1 to 0.2 % to lower the pH to a value below 3.0 and is loaded onto a butyl, octyl or octadecyl reverse phase HPLC semi-prep column preferably butyl or oct>l column equilibrated with a suitable concentration of TFA in water. The bound GM-CSF protein is eluted from the column by a linear gradient from 20% to 100% B. The processed GM-CSt protein elutes from the column as a single distinct peak. Alternately, the sample can be loaded onto a hydrophobic interaction column having the octyl or butyl functional groups ai medium to high conductivity range. Both forms of GM-CSF bind to the column under these conditions and the processed form can be selectively eluted by lowering the conductivity of the elution buffer. The unprocessed form being more hydrophobic needs a greater reduction in conductivity to elute form the column. This brings about a clear separation of the processed form from the unprocessed form of GM-CSF as shown in Fig 4. The GM-CSl protein obtained after elution from the reverse phase or HlC column is free of ilic unprocessed form of GM-CSF and also other E, coli host cell related impurities when analyzed under reducing and non-reducing conditions on SDS-poly acrvlamide uel electrophoresis ( SDS-PAGE) as shown in Fig 5. EXAMPLE 1 This shows that high yields of processed GM-CSF are obtainable under conditions where an inducer in combination with a reducing agent at appropriate temperature drives the transcription of the GM-CSF coding sequence with the signal peptide. Inducers can be chosen from a family of sugar molecules like IPTG, arabinose or lactose or their analogs at concentration ranges from ImM to lOmM.The microbial host strain harboring the plasm id construct secretes mature GM-CSF into the periplasmic space when grown initially at around 37 degrees Centigrade using the optimal media conditions. The presence of a reducing agent containing one or more thiol groups in the concentration range of ImM to 20mM will facilitate reshuffling of incorrect disulfide bonds and hence better secretion of the processed form. Alternately, lower growth temperatures in the range of 20 to 28 degrees Centigrade can also give slower growth kinetics and more efficient refolding. A combination of the above parameters may also be chosen to give effective secretion. The above parameters can be tried either in isolation or in combination to obtain efficient processing and secretion of GM-CSI suitable as a starting material for production of therapeutic grade of processed GM-CSF. EXAMPLE 2 This example shows the preparation of membrane pellet after lysis of bacterial cells. Bacterial cell pellet after h^^esting is suspended in 50mM Tris HCl buffer pH8.0, lOmM EDTA, ImM PMSF at a pellet to buffer ratio in the range of 1:5 to 1:20, more preferably in the range of 1: 10 and 1: 15. Lysis of cells is accomplished using a high-pressure homogenizer or sonicator, with multiple passes, keeping the temperature below 4 degrees ( and monitoring the OD at 600nm for complete lysis. The lysate obtained is centrifuged at high speed 12000 to 16000xg for 15 to 30 minutes at 2 to 8 degrees C to pellet the cell debns and membrane fraction. EXAMPLE 3 This example relates to the purification of solubilized GM-CSF extracted from the membrane pellet. Solubilization is achieved by the addition of high molarity salt solution to the pelieu preferably in the range of 0.5 - 2M NaCl . Alternately non-denaturing concentrations ol chaotropic agents or detergents can be used either as such or in combination with a sali solution. The solubilized fraction containing GM-CSF (having some amount of the unprocessed form of GM-CSF along with the processed GM-CSF) is loaded onto a chromatography column, packed with an anion exchanger selected from a group ot various polymer based matrices like cellulose, agarose, dextran or a synthetic polymer based. I he functional groups can be Q, DEAE or QAE. GM-CSF binds to the column in the pH range of 7.0 to 8.5 and can be eluted with good recovery by using a gradient of 0 to IM NaCl in the equilibration buffer. The processed protein elutes along with a small contamination of the unprocessed protein which can be eliminated completely in the next chromatography step. EXAMPLE 4 This example relates to the use of reverse phase HPLC or hydrophobic interacluMi chromatography for the final purification of processed GM-CSF protein. To the eluaie froni the anion exchange column, trifluoro acetic acid is added to a final concentration in the ransje of 0.1 to 0.2 % to lower the pH and is loaded onto a C-4 reverse phase HPLC semi-prep column equilibrated with 0.05% to 0.2% TFA in water. The bound GM-CSF protein is eluted from the column by a linear gradient from 20% to 100% B, where B is preferably 0.05^0 to 0.2%TFAin 70-95% acetonitrile or isopropanol in water. Alternately, the sample can be loaded onto a hydrophobic interaction column having the oct\ 1 or butyl functional groups at medium to high conductivity range in the range of 60 to 150mS in Tris or phosphate buffer in the pH range of 7.0 to 9.0. The high conductivity values can be obtained by the addition of sodium chloride, sodium sulphate or ammonium sulphate salts GM-CSF binds to the column under these conditions and can be selectively eluled b\ lowering the conductivity of the elution buffer. The GM-CSF protein obtained after eluiioi) from the reverse phase or HIC column is free of the unprocessed form of GM-CSF and most of the host protein related contaminants. The purity level conforms to the standards required for therapeutic proteins with endotoxin units in the range of We claim: 1. A method for production of a therapeutically pure granulocyte macrophage - colony stimulating factor (GM-CSF), wherein said GM-CSF is secreted in its mature form fix>m a recombinant bacteria, has no methionine residue at N-terminus, and ihc method comprises: a. cloning the human GM-CSF gene in-frame with Pel B signal peptide in an E.coli stram b. producing elevated expression levels of the processed protein by altering the induction conditions c. culturing GM-CSF producing recombinant cells in which over-expressed GM-CSF gets processed during secretion into the periplasm and gels associated with the cell membranes, d. lysing said cells and isolating the membrane fraction containing GM-CSF e. extracting the processed and unprocessed forms of the membrane-bound GM- CSF for purification f subjecting the mixed GM-CSF foitns to ion exchange chromatography g, recovering the purified processed form of GM-CSF after a reverse phase or HIC chromatography of therapeutic grade. 2. A method as in claim 1 wherein the conditions in step (b) involve the addition of a weak inducer, a reducing agent and a low temperature in any combination 3. A method as in claim 1, wherein the said membrane fraction in step (e) is extracted by a salt, a chaotrope or a combination ofboth in solution. 4. A method as claimed in claim 3, wherein the membrane fraction is washed using a non-ionic or ionic detergent or a combination ofboth before extraction. 5. A method as claimed in claim 1, wherein the solubilized GM-CSF in step (f) is bulTei exchanged to get the sample to tiie conditions required for loading on an anion exchange column. 6. A method as claimed in claim 1, wherein the solubilized GM-CSF in step (!) containing the unprocessed and processed forms are loaded onto an anion exchange column for separation of host cell related impurities 7. A method as claimed in claim 5, wherein the pH used is between 7.0 and 8.5. 8. A method as claimed in claim 6, wherein the step is used to enrich the sample fraction with the processed form of GM-CSF. 9. A method of claim 1, where in step (g) a reverse phase or hydrophobic interaction chromatography step is used to purify the processed GM-CSF protein, without the signal peptide, to homogeneity by, a) binding the processed and unprocessed forms of GM-CSF to a reverse phase butyl or octyl matrix at pH 2.0 to 3.0 and eluting the processed form selectively by increasing the hydrophobicity of the eluting solvent by the addition of acetonitrile or isopropanol at acidic pH and / or b) binding the processed and unprocessed forms to a hydrophobic interaction matrix in the pH range of 7.0 to 9.0 and a conductivity of 50 to 200mS and eluting the processed form selectively by decreasing the conductivity oftlie elution buffer. 10. A method as claimed in claim 9, wherein the said reverse phase matrix in step (a) is C-4 bound to a suitable HPLC matrix. 11. A method as claimed in claim 9, wherein the hydrophobic interaction matrix in slc[) (b) is a butyl group bound to a suitable chromatography matrix. 12. A purified form of GM-CSF without methionic residue whenever prepared by the method as claimed in claims 1 to 11.

Documents:

1343-che-2004 abstract duplicate.pdf

1343-che-2004 drawings duplicate.pdf

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1343-che-2004 description (complete) duplicate.pdf

1343-che-2004-abstract.pdf

1343-che-2004-claims.pdf

1343-che-2004-correspondnece-others.pdf

1343-che-2004-correspondnece-po.pdf

1343-che-2004-description(complete).pdf

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