(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`I lllll llllllll II llllll lllll lllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`( 43) International Publication Date
`31 December 2003 (31.12.2003)
`
`PCT
`
`(10) International Publication Number
`WO 2004/001056 Al
`
`(51) International Patent Classification7:
`C12N 15/27
`
`C12P 21/00,
`
`Laboratories, Survey No. 47, Bachupally, Hyderabad 500
`123 (IN).
`
`(21) International Application Number:
`PCT/US2002/019945
`
`(74) Agent: CORD, Janet, I.; Ladas & Parry, 26 West 61st
`Street, New York, NY 10023 (US).
`
`(22) International Filing Date:
`
`24 June 2002 (24.06.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(71) Applicant (for all designated States except GD, US): DR.
`REDDY'S LABORATORIES LTD. [IN/lN]; 7-1-27
`Ameerpet, Hyderabad 500016 (IN).
`
`(71) Applicant (for GD only): CORD, Janet, I. [US/US]; 26
`West 61st Street, New York, NY 10023 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KOMATH, Uma,
`Devi [IN/lN]; Biotechnology Divisionn, Dr. Reddy's
`Laboratories, Survey No. 47, Bachupally, Hyderabad
`500 123 (IN). LODHA, Sanjay [IN/lN]; Biotechnology
`Divisionn, Dr. Reddy's Laboratories, Survey No. 47,
`Bachupally, Hyderabad 500 123 (IN). CHIGURUPATI,
`Jayaram [IN/lN]; Biotechnology Divisionn, Dr. Reddy's
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
`NE, SN, TD, TG).
`
`Published:
`with international search report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`iiiiiiii
`
`---iiiiiiii
`iiiiiiii -iiiiiiii --
`--
`--
`
`- !
`
`!!!!!!!
`iiiiiiii
`
`iiiiiiii ----
`
`0 (57) 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.
`
`Page 1
`
`KASHIV EXHIBIT 1005
`IPR2019-00791
`
`

`

`- 1 -
`
`PROCESS FOR PREP ARING 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
`ofneutrophils (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 0-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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Page 2
`
`

`

`-2-
`
`5
`
`10
`
`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
`15 mammalian cells. Expression ofhG-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. CuS04 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-7 5 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
`
`30
`
`20
`
`25
`
`Page 3
`
`

`

`- 3 -
`
`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 fuc., 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 chromato(cid:173)
`graphy 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 ofhG-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(cid:173)
`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 ofG-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.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Page 4
`
`

`

`-4-
`
`(EMBO J. vol 5, 871, 1986). The process describes the use of a three-step chromato(cid:173)
`
`graphy 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
`
`5
`
`(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 s~eps for the purification of G-CSF.
`
`Although the process described in patent U.S.P. 5,055,555 is simpler and economical; it
`
`10
`
`is not readily applicable to recombinant G-CSF expressed in E.coli or other cells where
`
`G-CSF is produced in the fonn 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
`
`15
`
`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.
`
`20
`
`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 ofrecombinant hG-CSF, a simple
`
`and economical process involving fewer steps and yielding high levels of active clinical
`
`25
`
`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 hun1an G-CSF from E.coli and other cells in
`
`which inclusion bodies of G-CSF are formed. The process involves culturing E.coli or
`
`30
`
`other suitable cells to high cell densities and isolating inclusion bodies containing hG(cid:173)
`
`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,
`
`Page 5
`
`

`

`- 5 -
`
`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;
`isolating the inclusion bodies (IB) containing hG-CSF
`c)
`d) solubilizing and denaturing hG-CSF in 1B 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
`
`5
`
`10
`
`15
`
`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
`20 metal ions. IB 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 maybe used during the
`refolding. PH adjustment is normally effected by use of appropriate buffers. ill 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
`
`25
`
`30
`
`Page 6
`
`

`

`- 6 -
`
`DNA are removed by an ion exchange colrum1. 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
`
`5
`
`the expression ofhG-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
`
`10 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
`
`15
`
`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
`
`20
`
`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 ofrhG-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
`
`25
`
`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 NdeI/BamHI sites
`
`30
`
`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 pr9duce a NdeI site at the 5' end
`
`Page 7
`
`

`

`- 7 -
`
`and a blunt product at the 3' end of the gene. pET3a digested with NdeI 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 lOL fermenter (BioFlo 3000, New Brunswick
`Scientific). The following process parameters were maintained throughout the run; air
`flow: 1 vessel volume per minute (vvm); 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(cid:173)
`
`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 oflow concentrations (0.05 to 0.2%) of
`nonionic detergents like Triton or Tween. The inclusion bodies along with the cell
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Page 8
`
`

`

`- 8 -
`
`debris and unbroken cells are collected as pellet after high-speed centrifugation of the
`bacterial lysate
`
`PURIFICATION
`Purification ofrhG-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 l litre oflysis buffer, i.e.
`Tris buffer, at pH8.0, lmM EDTA and lmM 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 HCl, 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.
`In the second step, the IB pellet is again finely dispersed into 1 % sodium
`deoxycholate in 50 mM Tris HCl, pH8.0, 5mM EDTA solution. The pellet to buffer
`ratio is maintained at 1 :40 to 1 :80 (w/v). The suspended IB 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 IM NaCl in
`50mm Tris HCl, pH8.0, 5mM EDTA buffer. The IB 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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Page 9
`
`

`

`- 9 -
`
`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
`
`5
`
`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 of2mg/ml. The pH is then
`
`brought down to 8.0 with acetic acid. The protein solution is diluted further with 0.1 %
`
`10
`
`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
`
`15
`
`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
`
`20
`
`density value at 280nm returns to baseline. G-CSF is eluted from this column using
`
`O. lM Tris HCl 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
`
`25
`
`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 HCl buffer
`
`30
`
`pH8.0, lOmM EDTA, lmM 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
`
`Page 10
`
`

`

`- 10 -
`
`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.
`
`EXAMPLE2
`
`5
`
`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.
`
`10
`
`In a preferred embodiment of this invention, non- ionic detergents like the Triton X(cid:173)
`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
`
`15
`
`the cell membrance. To a 25gm quantity ofIB 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 lB 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
`
`20
`
`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 IM
`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
`
`25
`
`30
`
`Page 11
`
`

`

`- 11 -
`
`third step in the wash procedure can also be carried out by using 0.5 to l .OM solution of
`
`any other ionic salt like potassium chloride, sodium sulphate, etc.
`
`EXAMPLE3
`
`This example relates to the use of a combination of sub-denaturing
`
`5
`
`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
`
`10
`
`15
`
`is added to the IB to which lN 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 of2mg/ml the pH is shifted back to 8.0 using lM
`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
`
`20
`
`done for a total of 12 to 16 hrs at room temperature.
`
`EXAMPLE4
`
`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%Tween20. 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
`
`25
`
`30
`
`sodium chloride concentrations is shown in the Table 1.
`
`Page 12
`
`

`

`- 12 -
`
`TABLE 1
`
`Molarity of NaCl
`25mM
`50mM
`lOOmM
`250mM
`500mM
`
`%recovery
`No elution
`No elution
`<1%
`<1%
`<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.
`
`5
`
`10
`
`Page 13
`
`

`

`- 13 -
`
`CLAIMS
`
`A method for expression, isolation an granulocyte colony stimulating
`1.
`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;
`
`5
`
`b) lysing said cells;
`
`isolating the inclusion bodies (IB) containing hG-CSF
`c)
`d) solubilizing and denaturing hG-CSF in lB with a combination of
`
`10
`
`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.
`
`15
`
`A method as claimed in claim 1, wherein said inclusion bodies are
`2.
`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.
`A method as claimed in claim 1, wherein the pellets are washed by a
`
`3.
`
`three step washing procedure, using non-ionic detergents.
`
`4.
`
`5.
`
`20
`
`A method as claimed in claim 3, further comprising a chelating agen