WO2004001056A1 - Process for preparing g-csf - Google Patents

Abstract

A simple, economic and scaleable process for the purification of recombinant human G-CSF expressed in E. coli cells is provided. The steps include lysing the microorganism, separating the inclusion bodies containing G-CSF, a multi step washing procedure for inclusion bodies to remove protein, LPS, and other host cell impurities, refolding at basic pH and chromatography.

Description

PROCESS FOR PREPARING G-CSF FIELD OF THE INVENTION The present invention relates to recovery of recombinant human granulocyte-colony stimulating factor (G-CSF) from cells in which it has been expressed. More specifically, the invention is directed to a simplified process for the preparation of an essentially endotoxin free G-CSF solution for therapeutic applications. "Essentially endotoxin free" means that the levels of endotoxins are very low and within the specification limits for therapeutic proteins. G-CSF used in the present invention is typically derived from a genetically engineered prokaryotic organism containing a recombinant plasmic carrying the human G-CSF gene.

BACKGROUND OF THE INVENTION G-CSF, is used in reconstituting normal blood cell populations following chemotherapy or radiation. G-CSF may also be used in potentiating immune responsiveness to infectious pathogens or in the treatment of certain leukemias (Bronchud, M.H. et. al. Br. J. Cancer 1987; 56: 809-813 and Morstyn, G. et. al, Lancet 1988; 1: 667-672). Human G-CSF is one of the hemopoietic growth factors which plays an important role in stimulating proliferation, differentiation and functional activation of neutrophils (Metcalf, D., The Hemopoietic Colony stimulating Factors, Elsevier,

Amsterdam, 1984) both in vitro and in vivo (Zsebo, K.M., et. al., Immunobiology 1986; 172:175-184 and Cohen, A.M., et. al, Proc. Natl. Acad. Sci. U.S.A. 1987; 84:2484- 2488).

G-CSF protein has only one single O-glycosylation site at threonine 133; absence of glycosylation at this residue was not found to affect the stability of the protein. For many protein therapeutics where glycosylation of the protein is known to affect stability, it is necessary to undertake cloning and expression in yeast or mammalian cells, using appropriate expression vectors. In the case of G-CSF, the recombinant protein expressed in E.coli was found to have the same specific activity as the native protein (Oh-eda et. al. 1990 J. Biol. Chem. 256, 11432-11435, Hill et. al. 1993 Proc. Nat. Acad. Sci. USA 90. 5167-5171, and Arakawa et. al. 1993 J. Protein Chem. 12, 525-531).

The form in which G-CSF is produced depends at least in part on the type of cell in which it is produced. In some cells, typically eucaryotic cells, it is normally produced in a soluble form and secreted. In others, particularly procaryotic cells such as those of E. coli, it forms inclusion bodies, inclusion bodies are aggregated extremely dense protein structures. They typically have a partial secondary structure and are commonly found in the cytoplasm of E. coli. It is understood that a combination of factors relating to the physiological state of the host cell and the growth conditions affect the formation of such inclusion bodies. The formation of inclusion bodies of G-CSF in E. coli and other cells presents problems in using such commonly utilized host cells for the production of G-CSF since it complicates the recovery of a useful product from the cell in which it is produced. Effective ways of doing this are therefore desirable. Recombinant human G-CSF has been produced by expressing a rhG-

CSF gene in E.coli and purifying it to homogeneity. The following U.S. Patents 4,810,643; 4,999,291; 5,055,555; 5,849,883; 5,582,823; 5,580,755; and 5,830,705, describe various aspects of recombinant expression and purification of the h-GCSF protein from various expression systems ranging from bacterial cells to yeast and mammalian cells. Expression of hG-CSF as inclusion bodies in a bacterial system is described in U.S. Patent 4,810,643 (Souza, assigned to Kirin-Amgen Inc., May 7, 1989). In the preferred recovery technique, a suspension of broken E. coli cells in which hpG-CSF had been expressed was centrifuged and the pellet was resuspended in deoxycholate (DOC), EDTA,, and 50 Tris at pH 9. This suspension was centrifuged, resuspended and centrifuged again. The pellet was solubilized in Sarkosyl (sodium lauryl sarcosine, an ionic detergent) and Tris (tris(hydroxymethyl) aminomethane) at pH 8. CuSO₄ was added and the mixture was stirred, and then centrifuged. Acetone was added to the supernatant. This mixture was put on ice and then centrifuged. The pellet was dissolved in guanidine and sodium acetate at pH 4, and put over a G-25 column equilibrated and run in 20 mM sodium acetate at pH 5.4. The hpG-CSF peak was pooled and put on a CM-cellulose column equilibrated in 20 mM sodium acetate at pH 5.4. After loading, the column was washed with sodium acetate at pH 5.4 and with sodium chloride, and then the column was eluted with 20 mM sodium acetate at pH 5.4 and with 37 mM sodium chloride. Part of this eluent was concentrated to and applied to a G-75 column equilibrated and run in 20 mM sodium acetate and 100 mM sodium chloride at pH 5.4. The peak fractions were pooled and filter sterilized. In this protocol, a high amount of detergent and chaotropic agent is used in the solubilization step for inclusion bodies, and hence additional steps are included for the removal of these agents in the process. With multiple handling steps, the overall yields are also expected to be lower.

U.S. Patent 5,849,883 (Boone et al., assigned to Amgen Inc., December 15, 1998) describes recovering G-CSF from a microorganism in which it is produced by lysing the microorganism, separating insoluble material containing G-CSF from soluble proteinaceous material, optionally extracting the material with deoxycholate, solubilizing and oxidizing the G-CSF in the presence of a denaturant solubilizing agent, removing the denaturant, subjecting the residual G-CSF to ion exchange chromatography and recovering purified G-CSF. The solubilization and oxidation step is effected using Sarkosyl and Tris followed by treatment with copper sulphate. The product is stated to have the same primary structure and one or more of the biological properties of naturally-occurring bovine G-CSF. It is indicated that a wide range of host cells may be used to produce the G-CSF and it is noted that G-CSF is produced in insoluble form in E. coli. The U.S. Patent 5,055,555, describes a simplified process for purification of recombinant hG-CSF. Although it is stated that rhG-CSF expressed in bacterial or fungal cells may be used, the technique described relies on secretion of the protein and so as a practical matter is applicable only in yeast and mammalian expression systems where G-CSF is secreted into the medium. For bacterially expressed G-CSF in inclusion bodies, sodium chloride precipitation of the protein is not a feasible step.

Other related patents describing production of hG-CSF include U.S. Patent 5,714,581, which discusses the polypeptide derivatives of hG-CSF, and U.S. Patent 5,681,720 which discloses information on the DNA encoding the various hG-CSF containing plasmids and expression of these in host cells. European Patents EP 0335423, EP 0272703, EP 0459630 and EP 0256843 disclose amino acid modifications of G-CSF, their expression and biological activities. British 2213821 discusses the construction of a synthetic human G-CSF gene. Australian Patent Publication No. AU- A-76380/91 reports the construction of various muteins of G-CSF and their comparative activities. Various other methods have been reported in scientific literature for the purification of G-CSF expressed in E.coli, yeast or CHO cells. A method of purification of G-CSF from CHU-2 conditioned medium (human oral carcinoma cell line), which is known to produce G-CSF constitutively was developed by Nomura et. al. (EMBO J. vol 5, 871,1986). The process describes the use of a three-step chromatography procedure after concentration and ultrafiltration of the conditioned medium. Patented publications for the process of purifying recombinant G-CSF from E.coli were mainly assigned to Kirin/Amgen (WO-A-8703689), Biogen (WO-A-8702060), Amgen (U.S.P. 5,849,883 and U.S.P. 4,810,643), Chugai Seiyaku Kabushiki Kaisha (WO-A- 8604605 and WO-A-8604506) and Sassenfeld (U.S.P. 5,055,555).

The various purification protocols discussed in the above patents mention multiple chromatography and other steps for the purification of G-CSF. Although the process described in patent U.S.P. 5,055,555 is simpler and economical; it is not readily applicable to recombinant G-CSF expressed in E.coli or other cells where G-CSF is produced in the form of an inclusion body. . The above method was developed for soluble G-CSF expressed and secreted by recombinant yeast into the fermentation medium. For E.coli derived G-CSF, solubilization of inclusion bodies and refolding of G-CSF are additional steps to be taken into account. Besides, obtaining a therapeutic grade preparation of G-CSF free of lipopolysaccharide endotoxins, which are likely contaminants when E.coli is used as a host, in a simple, scaleable procedure has not been reported so far. On a commercial scale, yield losses from a multi-step process becomes highly significant. Hence a simplified procedure with fewer steps will give higher yields in a shorter time, besides being economical. None of the above-related patents disclose a simpler and economical method for the production of G-CSF at a commercially viable scale. The processes described are complex, lengthy and unit costs are high.

In an effort to purify large quantities of recombinant hG-CSF, a simple and economical process involving fewer steps and yielding high levels of active clinical grade protein has been developed.

SUMMARY OF THE INVENTION The present invention provides a simple and cost effective process for purifying large quantities of recombinant human G-CSF from E.coli and other cells in which inclusion bodies of G-CSF are formed. The process involves culturing E.coli or other suitable cells to high cell densities and isolating inclusion bodies containing hG- CSF by lysing the cells. G-CSF is isolated from IB by initially denaturing the protein and then refolding back to produce active hG-CSF.

Accordingly, the present invention provides a A method for expression, isolation and purification of human granulocyte colony stimulating factor (hG-CSF) wherein said hG-CSF has a methionine residue at N-terminus from a recombinant microorganism, which method comprises: a) culturing hG-CSF producing recombinant cells in which over- expressed hG-CSF accumulates as inclusion bodies (IB) to high cell density; b) lysing said cells; c) isolating the inclusion bodies (IB) containing hG-CSF d) solubilizing and denaturing hG-CSF in IB with a combination of solubilizing agent and high alkaline pH e) refolding hG-CSF by a two step method f) subjecting the hG-CSF to ion exchange chromatography g) recovering purified hG-CSF.

Preferably, the inclusion bodies containing hG-CSF are recovered from the cells by lysing them by high pressure homogenization or sonication. IB pellets can be obtained from the lysate, for example by centifugation. The pellets are then preferably washed. We have found that repeated washing, for example by a three step washing procedure, is particularly useful. Such washing is effected using non-ionic detergents. These are useful to solubilize cell wall components that may at first adhere to the inclusion bodies. Chelating agents such as EDTA may also be used to help remove metal ions. LB pellets are then preferably reformed, for example by centrifugation of the wash liquid into which the IB component was dispersed during removal of extraneous material. Following the washing step, the G-CSF present in the IBs is solubilized. This is most conveniently effected at high pH using urea but other materials such as guanidimium hydrochloride, may be used if desired. Suitable pHs are in the range 10 to 12.5, for example in the range 11 to 12, preferably about 12. As noted above, IB's have some secondary structure. However, to optimize the usefulness of the product obtained, the solubilized G-CSF should be refolded. This is conveniently effected by holding the solubilized G-CSF at a pH of from 8.0 to 8.5 for a period of from 6 to 12 hrs and then lowering the pH into the range 4.0 to 5.0 for a further period of from 4 to 10 hours. If desired, surface active agents may be used during the refolding. PH adjustment is normally effected by use of appropriate buffers. In a preferred embodiment of this invention, hG-CSF is purified to homogeneity using a single ion-exchange chromatography. All the contaminants like endotoxins and host DNA are removed by an ion exchange column. Cation exchange chromatography or anion exchange chromatography may be used.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a restriction map of pET G-CSF expression vector, which directs the expression of hG-CSF

Figure 2 is the nucleotide and amino acid sequence of rhG-CSF

DETAILED DESCRIPTION OF THE INVENTION A simple and innovative method for the purification of rhG-CSF has been developed. Preferably, hG-CSF is produced by recombinant methods using microbial (fungal or bacterial) expression systems. The hG-CSF gene is isolated from a known source and ligated to a suitable expression vector, which is then used to transform an appropriate host strain. The recombinant microbial strain is then grown by fermentation under suitable conditions promoting the maximum expression of the desired protein. The process described in the present invention can be applied to purify recombinant G-CSF as well as analysis of G-CSF that have similar physico-chemical characteristics as the native protein.

SOURCE OF RHG-CSF GENE 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 expression of rhG-CSF are disclosed in European Patent Application No.220, 520 and PCT Patent Application Nos. W088/01297 and W087/01132.

In one embodiment briefly, a cDNA clone was constructed from the human bladder carcinoma cell line No. 5637. U.S. Patent 5,055,555 reports that this cell line has been deposited with ATCC as ATCC HBT-9, the current availability of this material is not known. It was originally deposited under restrictive conditions. Oligonucleotide primers specific for the G-CSF gene were synthesized and used to amplify the gene product by RT-PCR. This was then cloned into the Ndel BamHI sites of the expression vector pET3a, which carries the ampicillin antibiotic resistance marker. This plasmid was then used to transform strain BL21(DE3) of E.coli. pET3a (Novagen Inc.) carries the phage T7 promoter which is used to drive expression of the G-CSF gene. The primers for G-CSF were designed to produce a Ndel site at the 5' end and a blunt product at the 3' end of the gene. pET3a digested with Ndel and BamHI (made blunt using klenow polymerase) was then used for this cloning. Strain BL21(DE3) (Novagen Inc.), which carries a chromosomal copy of the phage T7 RNA polymerase, was transformed with the plasmic constructed for G-CSF production. Hosts for production of recombinant hG-CSF by the method of the present invention are those in which G-CSF is produced in inclusion bodies. The preferred host is E. coli, but inclusion bodies are also produced in other types of cells including those of the genera Saccharomyces and Bacillus.

FERMENTATION Fermentation of the recombinant E. coli strains containing the G-CSF gene is done under conditions optimized for maximum expression. Briefly, fermentation was carried out in a 10L fermentor (BioFlo 3000, New Brunswick Scientific). The following process parameters were maintained throughout the run; air flow: 1 vessel volume per minute (wm); agitation: 300 — 800 rpm; Dissolved oxygen (D.O.): > 40%; pH: 6.80; temperature: 37°C.

Production medium used was a synthetic medium, containing glucose as carbon source and yeast extract as additional nitrogen source. The medium was supplemented with trace metals and ampicillin was used as a selection marker.

This medium was inoculated with seed culture and the culture allowed to grow. When the carbon source got exhausted, controlled feeding was initiated with a medium containing glucose and yeast extract. Once the desired cell density was obtained, the cells were induced for expression of G-CSF by adding isopropyl- thiogalactoside (IPTG).

At the end of induction period of about 8 to 10 hours, the batch was harvested. The cell density obtained was 40 - 60 g/L (wet weight). G-CSF yield is 3 to 3.5 g/L of fermentation broth and represents 30 to 40 percent of total protein. G-CSF is produced in E.coli cells as inclusion bodies (IBs), inclusion bodies are recoverd from the harvested cells. Lysis can be carried out by passing a cooled (temperature below 5°C) cell suspension through a high pressure homogenizer or through a sonicater for a few cycles till nearly complete lysis of bacterial cells is obtained or by grinding the cells in the presence of glass beads. Lysis can also be carried out using Tris buffer or phosphate buffer in the presence or absence of low concentrations (0.05 to 0.2%) of nonionic detergents like Triton or Tween. The inclusion bodies along with the cell debris and unbroken cells are collected as pellet after high-speed centrifugation of the bacterial lysate

PURIFICATION Purification of rhG-CSF from the harvested E.coli cells is done by a simple three step procedure involving lysis of the cells, washing of inclusion bodies and ion exchange chromatography. The highlight of the simplified purification process is a three step washing procedure for IBs. The frozen cell paste is suspended in lysis buffer, for example, 50gms of cell paste is suspended in 500ml to 1 litre of lysis buffer, i.e. Tris buffer, at pH8.0, lmM EDTA and ImM phenyl methyl sulfonyl fluoride (PMSF). Repeated washings of the above pellet is done by fine dispersion homo- genization and centrifugation in a combination of buffers; chosen so as to minimize solubilization and loss of IB, with a complete loss of all other protein and non-protein components that make up the non IB part of the pellet. The final washed IB pellet so obtained is essentially free of endotoxins, host cells proteins and host DNA. The purified IB pellet of G-CSF, which is essentially pure G-CSF, is then ready to be solubilized, refolded to native form and concentrated by ion exchange chromatography.

The first step in the three step washing procedure of IBs involves suspension of the IB pellet in 2% Triton X-100 in 50mM Tris HC1, pH8.0 and 5mM EDTA by fine dispersion methods at a pellet to buffer ratio of 1 :40 to 1:80 (w/v). The pellet is suspended in the above buffer at RT, stirred and re-pelleted over a period of 30 to 60 minutes. The IB solution is re-pelleted using a centrifuge. the second step, the IB pellet is again finely dispersed into 1% sodium deoxycholate in 50 mM Tris HC1, pH8.0, 5mM EDTA solution. The pellet to buffer ratio is maintained at 1 :40 to 1 : 80 (w/v). The suspended LB solution is stirred at RT and repelleted over a period of 30 to 60 minutes.

In the third wash step, the wash buffer has a composition of 1M NaCl in 50mm Tris HC1, pH8.0, 5mM EDTA buffer. The LB pellet after the second wash step, is again finely dispersed in this buffer, stirred at RT and re-pelleted under conditions similar to the previous two wash steps. The G-CSF protein in the final IB pellet is found to be >95% to 98% pure, when analyzed by SDS-PAGE.

The endotoxin levels, host cell protein and DNA contamination levels in the IB pellet itself (when checked upon solubilization and refolding) was found to be very low and within the specification limits for therapeutic proteins. The DNA contamination referred to above is DNA from the host.

The IB pellet is solubilized using a combination of a denaturant and high alkaline pH. The uniqueness of this method is that a sub-denaturing concentration of urea is chosen (2M) and the IB is solubilized by shifting the pH of the IB suspension in 2M urea to in the pH range of pH 11 to 12.5. Complete solubilization of the IB pellet was obtained under these conditions. The solubilized pellet is stirred for 30 min. at RT at pH12.0, after diluting the protein to a concentration of 2mg/ml. The pH is then brought down to 8.0 with acetic acid. The protein solution is diluted further with 0.1% polysorbate 20 for refolding. Refolding of the protein is carried out at RT for 12 - 16 hrs. The pH of the refolded protein solution is shifted to 4.5 with sodium acetate buffer for loading on an ion exchange column.

6M Guanidine hydrochloride can also be used as a denaturant, although additional steps to reduce the conductivity of GdnHCl need to be included before refolding the denatured protein.

ION EXCHANGE CHROMATOGRAPHY A radial flow column is packed with SP - Sepharose (Pharmacia) matrix, which is equilibrated with 25mM sodium acetate buffer, pH4.5. The refolded protein solution is loaded on this column and washed with equilibration buffer till the optical density value at 280nm returns to baseline. G-CSF is eluted from this column using 0.1M Tris HC1 buffer at pH8.0. The recovery of G-CSF under these elution conditions was found to be maximal, 3 to 5 times more than with NaCl at pH4.5.

G-CSF eluted in Tris buffer is a clear solution at a concentration of around 0.3 to 0.5 mg/ml. Since G-CSF is not very stable at neutral pH, for long term storage, it is advisable to diafilter the protein solution using a low pH buffer, either phosphate, citrate or acetate, at pH values preferably below 4.5.

EXAMPLE 1 This example shows the preparation of inclusion bodies from the cell pellet. Bacterial cell pellet after harvesting is suspended in 50mM Tris HC1 buffer pH8.0, lOmM EDTA, ImM PMSF at a pellet to buffer ratio of 1 : 10. Lysis can be done either by high-pressure homogenization or sonication, by multiple passes, keeping the temperature below 4°C and monitoring the OD at 600nm for complete lysis. The lysate obtained is centrifuged at high speed 26000xg in Beckman JA-20 rotor for 30 minutes at 4°C to pellet the inclusion bodies. A 1 :2 dilution of the lysate before centrifugation helps to reduce viscosity and to get a better yield of inclusion bodies. The recovered IB pellet weight is generally about 25% of the wet weight of the bacterial cell pellet.

EXAMPLE 2 This example relates to the purification of inclusion bodies by a repeated wash procedure. The inclusion bodies containing G-CSF get pelleted along with the cell debris during the centrifugation step after homogenization of the cells. Purification of G-CSF inclusion bodies from the contaminating E.coli proteins and other cellular components is achieved by a simple multi-step wash procedure, which is faster, economical and easier to handle than chromatography steps for purifying the impurities, fn a preferred embodiment of this invention, non- ionic detergents like the Triton X- series or Polysorbate 20 are used at high concentrations to solubilise the bacterial cell wall components that contaminate the IB preparation. Addition of 5mM EDTA also helps to chelate divalent metal ions, which help to maintain the structural integrity of the cell membrance. To a 25gm quantity of IB pellet, the wash buffer containing 2% detergent and 5mM EDTA at pH8.0 in 25mM Tris buffer are added at a pellet to buffer ratio of 1 :40 (w/v). Fine dispersion of the pellet combined with vortexing helps solubilise the impurities from the IB differentially. Under the wash conditions specified, G-CSF does not get solubilized from the inclusion bodies. Selective enrichment of the IB pellet for G-CSF is obtained. Separation of the soluble fraction from the particulate fraction can be accomplished by centrifugation, filtration or other similar methods. In the preferred embodiment of the present invention, the IB suspensions in wash buffer are centrifuged at 26000xg for 20 min. in SLA 1500 rotor at 4°C to collect the pellet. This step can be repeated twice under identical conditions to remove impurities from the IB pellet that did not get solubilized completely in the first instance. A second wash procedure incorporates 1% sodium deoxycholate solution in WB. The IB pellet to wash buffer ratio is maintained between 1 :20 to 1:80 (w/v) followed by fine dispersion homogenization, stirring and centrifugation. This strips the IB pellet of any residual cell debris particles, especially lipopolysaccharides units that contribute to the unacceptable levels of endotoxins in protein preparations from E.coli. The third step in the wash procedure using 1M sodium chloride in WB helps to elute nucleic acids or any other contaminants that are non-specifically bound to the G-CSF protein in the IB pellet by ionic interactions. This third step in the wash procedure can also be carried out by using 0.5 to 1.0M solution of any other ionic salt like potassium chloride, sodium sulphate, etc.

EXAMPLE 3 This example relates to the use of a combination of sub-denaturing concentrations of urea with alkaline pH for the solubilization of G-CSF from the inclusion bodies. The washed IB pellet is solubilized with urea at concentrations ranging from 2M to 6M. In a preferred embodiment of this invention, 2M urea in water is added to the IB to which IN NaOH is added drop wise to shift the pH briefly to between 11 and 12.5, preferably pH 12.0. After stirring the solubilized IB at pH 12.0 for 30 min, at a protein concentration of 2mg/ml the pH is shifted back to 8.0 using IM acetic acid and a concomitant dilution of protein to 0.2mg/ml with 0.1 % Polysorbate 20 in water at pH8.0 for refolding.

Refolding of the solubilized protein can be done in two steps in the concentration ranges from 0.05mg/ml to 0.2mg/ml in the presence or absence of the detergent. In a preferred embodiment of this invention, refolding of G-CSF is carried out in the first step between pH 8.0 and 8.5 for 6 hrs and then at low pH between pH values 4.0 to 5.0 at protein concentrations ranging from 0.05mg/ml to 0.2mg/ml for another 6 to 8 hrs. The pH shift to between 4.0 and 5.0 can be achieved using sodium acetate or sodium phosphate buffers of low conductivity. Refolding of the protein is done for a total of 12 to 16 hrs at room temperature.

EXAMPLE 4 This example relates to a single step chromatography procedure as a final polishing step for the protein. The refolded G-CSF binds to the cation exchange column in pH range 4.0 to 5.0, preferably at 4.5. In the present invention the chromatography procedure has been optimized for maximum recovery. The column run in the radial flow format at higher flow rates for elution was found to increase the recovery of the protein from the column. Loading of the sample was done in 25mM sodium acetate buffer, pH 4.5 with 0.1% Tween 20. Washing of the column is done with the same buffer without the detergent till the optical density at 280nm comes to baseline. Elution of the protein from the column was done using various concentrations of sodium chloride in the equilibration buffer. The percentage recovery of the protein with various sodium chloride concentrations is shown in the Table 1. TABLE 1

The cation exchanger can be selected from a group of various polymer based matrices like cellulose, agarose, dextran or a synthetic polymer based. The functional groups can be sulfonate, sulfopropyl or carboxymethyl.

Claims

C L A I M S

1. A method for expression, isolation an granulocyte colony stimulating factor (hG-CSF) wherein said hG-CSF has a methionine residue at N-terminus from a recombinant microorganism, which method comprises: a) culturing hG-CSF producing recombinant cells in which over- expressed hG-CSF accumulates as inclusion bodies (IB) to high cell density; b) lysing said cells; c) isolating the inclusion bodies (IB) containing hG-CSF d) solubilizing and denaturing hG-CSF in IB with a combination of solubilizing agent and high alkaline pH e) refolding hG-CSF by a two step method f) subjecting the hG-CSF to ion exchange chromatography g) recovering purified hG-CSF.

2. A method as claimed in claim 1, wherein said inclusion bodies are isolated by lysing the cells in which they were produced by high pressure homogenization or sonication and IB pellets formed from the lysate by centifugation.

3. A method as claimed in claim 1, wherein the pellets are washed by a three step washing procedure, using non-ionic detergents.

4. A method as claimed in claim 3, further comprising a chelating agent. 5. A method as claimed in claim 1, wherein solubilization of G-CSF is effected by using urea at a pH in the range 10 to 12.

5

6. A method as claimed in claim 5, wherein solubilization is effected using sub denaturing concentrations of urea, in the absence of detergents, chaotropes or reducing agents by a) solubilizing G-CSF in 2M urea to a final concentration of 2mg/ml and b) adjusting the pH to between 10 to 12 and stir at 20 - 25°C for 30 - 60 min.

7. A method as claimed in claim 5, wherein the pH used is about 12.

8. A method as claimed in claim 1, wherein refolding of said solubilized G-

CSF is effected by holding the solubilized G-CSF at a pH of from 8.0 to 8.5 for a period of from 6 to 12 hours and then lowering the pH into the range 4.0 to 5.0 for a further period of from 4 to 10 hours.

9. A method of claim 8, where refolding of G-CSF is accomplished by a two step procedure comprising a) diluting the sample for refolding with water of pH8.0 to 8.5 containing 0.05% to 0.2% polysorbate to a final concentration of 0.2mg/ml and allowing the diluted sample to stand for 6 hrs at 20 - 25°C in the absence of oxidizing agents and b) adjusting the pH to 4.5 and conductivity to lmS/cm and then allowing the sample to stand for further 6 hrs at 20 - 25°C.

10. A method as claimed in claim 1, wheein step (f) is effected by a single ion- exchange chromatography.

11. A method of claim 10, where a single step column chromatography step is used to purify the protein to homogeneity by a) binding the refolded G-CSF to a cation exchange matrix in column chromatography at acidic pH and b) eluting the pure G-CSF using Tris buffer under alkaline condition.

12. A method as claimed in claim 11, wherein said cation exchange matrix is

SP sepharose.