(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`( 43) International Publication Date
`21 September 2006 (21.09.2006)
`
`(51) International Patent Classification:
`C07K 14153 (2006.01)
`
`PCT
`
`(21) International Application Number:
`PCT /IN2006/000090
`
`(22) International Filing Date: 13 M,m:h 2006 (13.03.2006)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`274/CHE/2005
`
`17 March 2005 (17.03.2005)
`
`IN
`
`(71) Applicant
`(for all designated States except US):
`ZENOTECH LABORATORIES LIMITED [IN/IN];
`8-3-677 I 15, Skd Nagar, Srinagar Colony, Hyderabad,
`Andhra Pradesh 500073 (IN).
`
`1111111111111111 IIIIII IIIII 11111111111111111111111111111111111 IIIII IIIII IIII IIIIIII IIII IIII IIII
`
`(10) International Publication Number
`WO 2006/097944 A2
`500073 (IN). NUVVULA, Ashok, Kumar [IN/IN]; Clo
`Zenotech Laboratories Limited, 8-3-677 / 15, Skd Nagar,
`Srinagar Colony, Hyderabad, Andhra Pradesh 500073
`(IN). MOVVA, Srilalitha [IN/IN]; Clo Zenotech Labora(cid:173)
`tories Limited, 8-3-677 / 15, Skd Nagar, Srinagar Colony,
`Hyderabad, Andhra Pradesh 500 073 (IN). KARRA,
`Sreenivasu [IN/IN]; Clo Zenotech Laboratories Limited,
`8-3-677 I 15, Skd Nagar, Srinagar Colony, Hyderabad,
`Andhra Pradesh 500073 (IN). SAMADDAR, Mitali
`[IN/IN]; Clo Zenotech Laboratories Limited, 8-3-677 / 15,
`Skd Nagar, Srinagar Colony, Hyderabad, Andhra Pradesh
`500073 (IN). CHIGURUPATI, Jayaram [IN/IN]; Clo
`Zenotech Laboratories Limited, 8-3-677 / 15, Skd Nagar,
`Srinagar Colony, Hyderabad, Andhra Pradesh 500073
`(IN).
`(74) Agents: ANAND, Pravin et al.; Anand And Anand Advo(cid:173)
`cates, B-41 Nizamuddin East, New Delhi 110013 (IN).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KOMATH, Uma,
`Devi [IN/IN]; c/o Zenotech Laboratories Limited, 8-3-677
`/ 15. Skd Nagar, Srinagar Colony, Hyderabad, Andhra
`Pradesh 500073 (IN). NANDAMURI, Anupama [IN/IN];
`Clo Zenotech Laboratories Limited, 8-3-677 / 15. Skd
`Nagar, Srinagar Colony, Hyderabad, Andhra Pradesh
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, Fl,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KN, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`LY, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI,
`
`[Continued on next page}
`
`-iiiiiiii
`iiiiiiii ---
`= ---------------------------------------------
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`!!!!!!!!
`
`(54) Title: PROCESS FOR THE PURIFICATION OF RECOMBINANT GRANULOCYTE-COLONY STIMULATING FACTOR
`
`is the SDS-PAGE profile of purified G-CSF after chromatography steps
`
`Lanes
`
`2
`
`3
`
`Lane I: G-CSF Reducing conditions
`
`Lane 2: G-CSF Non Reducing
`
`Lane 3: Molecular weight markers
`
`iiiiiiii
`
`!!!!!!!! -
`iiiiiiii ----
`
`!!!!!!!!
`iiiiiiii
`
`"'1'
`
`"'1' °" r--...
`°" Q ---\0
`
`Q
`(57) Abstract: The present invention describes a novel process for large scale purification of therapeutic grade quality of recombi-
`0
`nant human G-CSF from microbial cells, wherein the protein is expressed as inclusion bodies. The process involves the novel use
`M of Hydrophobic Interaction Chromatography (HIC) step to purify G-CSF eluted from a cation exchange column. A combination of
`0 these two chromatography steps provides good purity and yields which are essential for a production scale process. The host cell
`
`: , related contaminants like proteins, DNA and endotoxins are estimated to be within the specifications outlined by the drug regulatory
`;;, authorities.
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`1111111111111111 IIIIII IIIII 11111111111111111111111111111111111 IIIII IIIII IIII IIIIIII IIII IIII IIII
`
`NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG,
`SK, SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US,
`UZ, VC, VN, YU, ZA, ZM, 'Evv'.
`
`(84) Designated States ( unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`as to the identity of the inventor (Rule 4.17(i))
`as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.17(ii))
`of inventorship (Rule 4.17(iv))
`
`Published:
`without international search report and to be republished
`upon receipt of that 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.
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`Process For The Purification Of Recombinant Granulocyte-Colony
`Stimulating Factor.
`FIELD OF THE INVENTION
`
`The present invention relates to a novel process for the isolation of
`therapeutically pure and biologically active recombinant human granulocyte - colony
`stimulating factor (G-CSF) from inclusion bodies expressed in microbial cells. The
`process leads to the purification of biologically active G-CSF in high yields, free from
`its oligomeric fom1s and other host cell proteins. More specifically, the invention is
`directed to a process for the large-scale production of a therapeutically pure and
`biologically actin: G-CSF protein in solution by the use of hydrophobic intera..:tJt)ll
`chromatography.
`
`BACKGROUND OF THE INVENTION
`
`Human granulocyte - colony stimulating !'actor belongs to c1 group or C\)h_)ny
`stimulating factors lhal play an important role in stimulating the di/Terentiatil)n ,111d
`proliferation or hematopoietic precursor cells and activation of mature neutrnph1 Is. G(cid:173)
`CSF is capable of supporting neutrophil proliferation in vitro and in l'il'o. Large
`quantities of recombinant G-CSF have been produced in genetically engineered
`Escherichia coli and have been successfi.illy used in the clinic to treat cancer patients
`suffering from chemotherapy-induced neutropenia. £. coli produced G-CSF is a 175
`amino acid polypeptide chain containing an extra methionine at its N-terminus. This
`protein lws been produced by expressing a G- CSF gene in E. coli and purirying it to
`homogeneity.
`
`Many earlier patents have described various aspects or recombinant expression
`and purification cif the G-CSF protein from different expression systems rangi 11g from
`bacterial cells to yeast and mammalian cells. Some of the processes ckscribed arc
`multi-step processes where losses in yield at the end of the puri lication process can be
`significant. /\. rew other purirication processes described in pc1tcnt literature appear to
`be simpler but 1w mention is made of the therapeutic grade quality or the puri lied
`material. Hence. there is a need for a simplified prncedure that is easily scakablc for
`production. gives higher yields and produces purified G-CSF protein ol'thcrnpeutic
`grade qtmlity. In c1n earlier patent ror a process (\\"O 04001056A l) ror G-CSF. we
`have addressed most or the limitations or lengthy processes described in scientific
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`In this patent we have tried to develop a novel process by the inclusion of
`literature.
`a unique hydrophobic interaction chromatography step that has not been hitherto
`described for the purification of G-CSF. The G-CSF protein purified by the two-step
`chromatography process that includes a hydrophobic interaction clu-omatography step
`as described in this patent, is free from higher multimcric aggregates of G-CSF. other
`host cell-related proteins, DNA and endotoxins.
`Purification method described in US 5,532,341 (Karl Welte, Sloan Kettering
`Institute) describes the purification of pluripotent granulocyte - colony stimulating
`factor from conditioned media using a three step chromatography process involving
`DEAE, gel iiltration and reverse phase columns. The protein puriliecl by this method
`was biologically active and hnmogenous. The procedure however was applicable to
`the G-CSF protein that \\·as secreted in cell culture supernatants and concentrated by
`ammonium sulphate precipitation. The suitability l1f'this process fr1r G-CSF
`solubilizecl and refolded from inclusion bodies has not been demonstrated.
`In an international patent WO 03/051922 A 1 assigned to Gaberc Porekar and
`Mena rt, the purification of G-CSF is described by the use of an immobilized metal
`affinity chromatography (IMAC).matrix. This step is coupled to cation exchange and
`gel filtration chromatography steps to get a biologically active and pure G-CS F protein
`in solution. Although simple, but it still .incorporates a miniml1m of three
`chromatography steps for final purification and the final yields or the protein are not
`clearly evident.
`A simplified process for purification of recombinant G-CSF is mentioned in
`U.S. 5,055,555 patent. The purification method described applies to G-CSF protein
`secreted into the medium when expressed in yeast or mammalian expression systems.
`The protein is partially purified on a cation exchanger and precipitated from pooled
`column fractions by using high concentrations or sodium chloride in the range of 1.5 to
`2.5M. For G-CSF recovered f'rom inclusion bodies in bacteria, sodium chloride
`precipitation or the protein increases the aggregation status of the protein and hence
`getting it back intl) solution arter that, is likely to be a cumbersome process. Besides,
`this process docs not assure the therapeutic grade purity of the protein.
`In the U.S. Patent 5,849,883 assigned to Amgen Inc., the process describes
`recovering human and bovine G-CSF expressed as inclusion bodies from a microbial
`system. The inclusion bodies are stated to he solubilised with Sarkosyl and purified
`2
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`using CM-Sepharose, a cation exchange chromatography step. In US 5,830,705
`
`assigned to Amgen, a process for the putification of G-CSF from COS cells is
`
`described. Since G-CSF is not expressed as inclusion bodies the method for purifying
`
`the recombinant protein is in principle different from an E.coli expressed protein. The
`
`US 5,714,581 and 5,681,720 patents on G-CSF describe the various deletion and
`
`substitution derivatives of the protein and methods for producing these deri\·atives
`
`using microorganisms. In earlier patents assigned to Amgen Inc., U.S. 4,810.643 and
`
`US 4,999,291 methods for G-CSF extraction from inclusion bodies produced in£. coli
`
`cells are described. The protein in inclusion bodies is extraded with deoxycholate
`
`(DOC), solubilized with an ionic detergent (Sarkosyl) and refolded. The refolded
`
`protein is purified on a CM-column followed by a G- 75 gel filtration chromatography
`
`step. The European Patent EP 0243153 (Immunex Co1voration) describes molecular
`
`level modifications to the human G-CSF and related mutant cDNAs for increasing
`
`expression in microbial systems and processes for making the proteins using these
`
`systems. Purification of cru9e G-CSF produced in supernatants of,HBT 563- cells is
`
`achieved by ammonium sulphate precipitation followed by chromatography on gel
`
`filtration and preparative reverse phase -HPLC Cl)\umns. In EP 0215126 patent
`
`assigned to Chugai Sciyaku Kabushiki Kaisha, the G-CSF protein is purified on an
`
`Ultrogel AcA54 column followed by precipitation of non-GCSF proteins \\·ith 30% n(cid:173)
`propanol. The supe111atant or 30% n-propanol precipitation step is loaded onto 8 C- l 8
`reverse plrnse column and eluted with 40% n- propm10l to get a purified prNein
`
`preparntion.
`
`The patents EP O 169566, WO 8604506 and WO 8604605 assigned tL' Chugai
`
`Seiyaku Kabushiki Kaisha describes a novel CSF having the ability to pronwtc
`
`differentiation and proliferation of bone marrow cells, the human gene encoding a
`
`polypeptide with G-CSF activity and a method for l~btaining recombinant expression
`
`of the same. WO 8703689 and EP 0237545 are patents by Kirin -Amgen for G-CSF.
`The former one describes immunological procedures associated with the production or
`murinc monoclonal antibodies for the detection l,f G-CSF in biological fluids .incl the
`
`latter presents 1wlynuclcnticlc sequences coding !1.,r the human G-CSF and their
`
`analogs. The European patents EP 0272703, EP 0-1-59630 and EP 0256843 disclose
`amino acid mndific<1tions or G-CSF, their expressiL,n and biological activities. British
`patent 2213821 discusses the construction or a synthetic human G-CSF gene.
`
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`Australian Patent AU-A-76380/91 reports the construction of various muteins of
`G-CSF and their cmnparative activities. The US 5,580,755 and US 5,582,823 patents
`illustrate DNA sequences that encode part or all of the polypeptide sequence of G-CSF
`and their characterization.
`Scientific literature describes various methods to purify G-CSF expressed in
`bacterial, yeast or mammalian cells but the multi-step processes are meant for small(cid:173)
`scale isolation of the protein for characterization purposes only. The processes may not
`be easily scaleable for production and is not likely to yield a protein of therapeutic
`grade purity.
`
`'
`
`The different purification protocols discussed in the above patent literature
`involves multiple chromatography steps chiefly ion-exchange followed by reverse
`phase or gel filtration chromatograp}1y step. Purification of G-CSF protein on 8
`hydrophobic internction chromatography column lrns not been described so far.
`Hydrophobic interaction chromatography (HIC) involves the use of high mol8rities or
`.
`salt in the protein solution but at _concentrations that are below _their precipitation
`points. At these salt concentrations, certain ligands, which under nornrnl salt
`conditions would not adsorb these proteins, become excellent adsorbents. The
`principle for protein adso1vtion to HIC is complementary to ion exchange and gel
`filtration chromatography methods. The use of high molarities of salt restricts its use to
`those proteins that can withstand high conductivities. Highly hydrophobic proteins like
`G-CSF are practically unstable at such conductivities and hence the use of
`hydrophobic interaction chromatography is generally ruled out as a purification option.
`Here, we describe the use of HIC step to further purify G-CSF eluted from a C8tion
`exchange chromatography step. This novel combination of cation exchange and
`hydrophobic interaction chromatography steps has not been reported so far in patent
`literature. Besides being novel, the process also shows good recoveries and is easily
`scaleable for industrial production of therapeutic grade G-CSF.
`SUMMARY OF THE INVENTION
`The present invention provides a method for large scale purification of
`therapeutic grade recombinant human G-CSF, said method comprising the steps of:
`isolating inclusion bodies containing G-CSF from microbial cells
`solubilizing said G-CSF protein from isolated inclusion bodies
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`refolding ~he said solubilized G-CSF protein to obtain active folded
`protein
`
`subjecting the said refolded G-CSF protein to two step chromatography
`wherein the said refolded G-CSF protein is first subjected to cation
`exchange clu·omatography followed by hydrophobic interaction
`chromatography
`
`to obtain purified therapeutic grade G-CSF protein
`The said G-CSF isolated from inclusion bodies is solubilized in a concentration
`of urea or guanidinium hydrochloride at alkaline pH and refolded at an acidic pH for 6
`lo 16 hrs al room temperature.
`The said refolded protein is bound to a sulphonate, carboxymethyl or
`sulphopropyl runctional group containing chromatography m<1trices.
`The ion exchange column is run in the pH range or3.5 to 5.5 using buffers or
`citrate, phosph<1le or acetate salts in the molarity range or 5mM to 50mM.
`The said protein bound to the cation exchange group is eluted by increasing
`the ionic strength of the buffer by the addition or chloride, citrate or sulphate salts in
`the pH range of 4.0 to 6.0.
`The G-CSF containing protein solution eluted l'rom the cation exchange
`column is purified using hydrophobic 'interaction chromatography on resins having
`butyl, octyl or phenyl fimctional groups.
`The said column is equilibrated with buffers in the pH range of 4.0 to 7.0
`containing ammonium sulphate salts in the molarity range of 0.25 M to 1.0 M.
`The said column is eluted by decreasing the concentration of ammonium
`sulphate salt in the buffer and by optionally increasing the concentration of ethanol
`from 2 tl) 2(J<¼i for enhanced recoveries.
`BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
`The im-cntion will now be described with reference to the accompanying
`drawings.
`Figure l is a restriction map or f.cofi expression vector, which directs the
`expression or G-CSF.
`Figure 2 is the complete nucleotide sequence of G-CSF and the cleri\·ed amino
`acid sequence.
`
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`Figure 3 is the chromatography profile of G-CSF after ion exchange
`chromatography
`Figure 4 is the chromatography profile of G-CSF after hydrophobic interaction
`chromatography
`Figure 5 is the SDS-PAGE profile of G-CSF after purification
`DETAILED DESCRIPTION OF THE INVENTION
`A simple and novel method involving a combination of ion exchange and
`hydrophobic interaction chromatography steps has been developed for large-scale
`purification of G-CSF solubilized from inclusion bodies expressed in microbial cells.
`The G-CSF protein in this case is preferably produced by recombinant methods
`in bacterial expression systems. The G-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 grown by fermentation under suitable
`conditions that promote the maximum expression of the desired protein.
`The isolation and purification process ft)r G-CSF .involves lysing the said cells
`by high-pressure homogenization or sonication and isolating the 1B pellets by
`centrifugation. The G-CSF present in the lB is solubilized by using a chaotrope like
`urea or guanidinium chloride in the concentration range of 2.0 to 4.0 Mand in a buffer
`of high pH. The protein is rel'oldecl at low pH, preferably in the range ol'4.U to 6.0
`and the relc.1Ided protein is loaded on a cation exchange chromatography column at a
`low pH. Increasing the salt concentration in the buffer effects the elution or the protein
`and Curther puriricntion is attempted by a hydrophobic chromatography step. In a
`prefeITed embodiment of this invention, although G-CSF is tn a large extent purified
`by using a single ion-exchange chromatography step. but a combination or ion
`exchange with hydrophobic column ensures lot-to-lot reproducibility with recd streams
`that can have minor alterations when carried out at industrial scale.
`The process described in the present invention can be applied for industrial
`scale puril'ication or recombinant G-CSF to homogeneity and nl'thcrapeutic grade
`quality. The purified G-CSF protein has similar physico-chemical clrnractcristics as the
`nati\'e protein.
`
`CLONING AND EXPRESSION
`A cDNA library is constructed from a hunrnn urinary bladder carci1wnrn cell
`line. Appropriate oligonuclcotide primers specific Cor the mature coding portion nr
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`G-CSF gene are synthesized and used to amplify the gene by RT-PCR. This is then
`cloned into the Nde I - EcoR I sites of the expression vector pTCF- 01, suitably placed
`downstream of the lac-based promoter (Fig 1 ). Restriction mapping and DNA
`sequencing is used to confo111 the DNA sequence of the cloned fragment (Fig 2). This
`plasmid construct is then used to transform the expression host (a strain of£. coli).
`The expression host harboring the plasmid construct expresses G-CSF protein at high
`levels when induced with IPTG or lactose. The microbial host strain used for
`production of recombinant G-CSF is one in which G-CSF is produced in inclusion
`bodies. Standard procedures as described by Sambrook et. al. (Molecular Cloning. A
`Laboratory Mnnual. Cold Spring Harbor Laboratory Press. 1989) and Pomn~Is el. al
`(Cloning Vectors: /\ Laboratory Manual, Elsevier. N.Y .. 1985) are used in the design
`c11.1d use nr clnning strntc·gies and cxpressinn \·cc!L)rs.
`PURIFICATION
`Fermentation oCthe recombinant E coli strains containing the G-CSF gene is
`done under conditions optimized ror maximum expression. The cells are hal"\'ested
`after the desired cell density is achieved and stored frozen al temperatures betwee11-l 0
`to -20 degrees Centigrade or processed immedintely for purification.
`Purificntion of G-CSF from hnr\'cstcd E. coli cells is carried out by 8 two-step
`chromatogrnphy procedure or the refolded protein. The inclusion bodies containing the
`G-CSF protein are soluhilised in 2.0 - 4.0 fvl Urea or guanidinium hydrochlnride and
`desalted (in the case ol'Gdn HCI) and refolded at an acidic pH so as to be suitable ror
`direct loading on a cation c.\clrnngc column. The matrix used ror cation exchange
`chromatography can have Carboxy Methyl, Sulpho Propyl or Sulphonate functional
`groups attached to resins made or eellulo:,e. ngarnse or their derivatives.
`The overall methodology involn.:s lysing bacterial cells, isolating inclusion
`hndies and purifying the pn•tcin hy inn exchange and hydrophobic chromatogrnphy.
`The frozen bacterial cell paste is suspended in lysis buffer, at a pellet to buffer ratio in
`the range of lgm: 5ml to !gm: 10ml. The lysis buffer is composed or 50ml\1 Tris HCI
`buffer, at pH8.0, lmM EDT/\ and 1 rniVI phenyl methyl sulJ'onyl fluoride (P1'vlSF). The
`CL:!! suspension is lysed by snnicalinn or high pressure homogenization using multiple
`pnsscs in the homogenizer. The cell lys8lc is centrifuged and the inclusion bodies arc
`isolated l'rom the pellet fraction.
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`The IB pellet is so.lubilized using a combination of a suitable denaturant (urea
`
`or guanidinium chloride) at alkaline pH in the range of 8.0 to 11.0. Refolding of the
`
`protein is carried out at room temperature for 6 - 16 hrs at acidic pH. The pH of the
`
`refolded protein solution is maintained in the range of 3.5 to 5.5 usirig any appropriate
`
`buffer suitable for maintaining pH in the acidic range.
`
`A chromatography column is packed with a cation exchange matrix, \\"hich is
`
`equilibrated with a suitable buffer that can maintain the pH at an acidic range. Buffers
`
`of phosphate and acetate are preferred although citrate salts can also be used. Low
`
`ionic strengths are preferred for equilibration, with values ranging from 5m!VI to
`
`50rnM of the buffer salt and a pH range of 3.5 to 5.5. The refolded protein solution in
`
`the pH range or 3.5 to 5.5 is loaded on an ion exchange column and washed \\·ith
`
`equilibration buffer till the optical density value at 280nm returns lo baseline. G-CSF.
`
`is eluted from this column using a gradient of an ionic salt like chloride, citrate or
`sulphate in the range oro.05 i'\'l to 0.25M. An improved recovery or G-CSF ,,·as
`obtained under these elution conditions and the protein was found to be hornogenous
`
`with minimum amount of aggregates. The G-CSF eluate form this column is directly
`
`loaded onto a column packed \\'ith a hydrophobic matrix having butyl, octyl ,,r phenyl
`
`functional groups attached to a resin derived from eel lulose, agarose, dextran.
`
`synthetic polymers or their derivatives. The column is equilibrated at a pH bclL,w 7.0
`
`in a suitable buffer containing 0.5 M ammonium sulphate. The bound G-CSF protein is
`
`eluted in the same buffer by gradient elution from 0.5M - 0.0 M ammonium sulphate.
`
`The G-CSF protein after this step can be buffer exchanged with the final storage buffer
`
`and stored as a liquid solution at:?. to 8 degrees Centigrade without loss or activity.
`
`EXAMPLE I
`The following example illustrates the simplified process for solubilization nr
`inclusion bodies and refolding or the protein ,11 acidic pH. This example relates to the
`use of a combination of sub-denaturing conccntrntions or lirea or guanicliniurn chloride
`in the concentration range or 2.0 to -LO M with allrnlinc pH for the solubilizatinn nr
`
`G-CSF from the inclusion bodies. In a prclcrred crnboclirnent 01· this inventiL,n. :?..OM
`
`to 6.0M urea ()r guanidinium hyclrochlnricle in \\',1tcr is added to the 18 at a ratiP ur
`
`I 0% to 20%, \\', ,·. the pH of the solution is held constant in the range or 8.0 tl, I 1.0
`
`depending on the clarity of the solubilizcd solution. ror a brief' period of I 5 w .30
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`minutes. The pH of this solution is shifted directly to an acidic pH in the range of 3.5
`to 5.5 and left at room temperature for 6 to 16 hrs for refolding.
`EXAMPLE2
`
`This example relates to the ion exchange chromatography step that is used to
`purify the G-CSF protein solubilised and refolded from inclusion bodies. The rc!'olded
`G-CSF is loaded onto a cation exchange column (carboxymethyl, sulphonyl or
`sulphopropyl functional groups) in pH range 3.5 to 5.5, preferably at pH 4.0 tc) 5.0 in
`anionic buffers that can provide buffering in this pH range for example citrate.
`
`phosphate or acetate. The buffers are generally in the molarity range of 5mM to 50mM
`preferably IOm{\1 to 25111M. Washing of the column is done with the same buffer till
`the optical density at 280nm comes to baseline. Elution of the protein from the
`column is done by a linear gradient of ionic salts c1.1ntaining chloride. citrate l)r
`sulphate in the Cl1ncentrntion range orO.UM to 0. S\1 in the equilibration bufft.'r l)ra
`pH range 4.0 ll) 6.0. The G-CSF protein is reuwcrccl with good yields nnd ,1 nrn1imum
`
`amount of aggrcgnted protein.
`
`EXAMPLE 3
`This exam pk describes the use or a hydn-.plwbic chronrntogrnphy column as a
`polishing step for the therapeutic grade purilicati,)n or G-CSF. The cation exchange
`column eluate is buffer exchanged with the cquilibrnlion buffer or the hydrl1phnbic
`column containing ammonium sulphate in lhc nwlarity range ur0.25 to 1.0;\I rnure
`preferably around (l . ..i to ().(1 M. The cquilibratit)ll buffer is in the acidic pH in the r,111ge
`of 4.0 to 7.0: more preferably in the range or 4.0 w 5.0. Elution rrom this column is
`effected by reducing the molarity of ammonium sulphate in the buffer in a continuous
`linear gradient elution. The protein very ol"ten clutcs towards the end of the gr,1dicnt
`with improved recoveries seen when a small amount or ethanol is added to the eluting
`buffer prcl'erably in the range ol"2°/ci to 20 '1;1. The hydrophobic matrix chost:n can be.
`o!'butyl, octyl L)r phenyl functional gwups mo1T prckrnbly butyl or octyl aw1chcd to a
`resin deri\·ed ['i·,1111 cellulose, agarosc. dcxtrnn. synthetic polymers ,)r lhcir dcri\·atives.
`The G-CSF pn.)tcin arter this step can be bu!Tt::r exchanged with the linal Stl1rngc buffer
`and stored as a liquid solution at 2 to S degrees Centigrade vvithllul l,)SS oracti\·ity.
`
`l)
`
`11 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`We claim:
`
`PCT /IN2006/000090
`
`l.
`
`A method for large scale purification of therapeutic grade recombinant human
`
`G-CSF, said method comprising the steps of:
`
`isolating inclusion bodies containing G-CSF from microbial cells
`
`solubilizing said G-CSF proteins from isolated inclusion bodies
`
`refolding the said solubilized G-CSF proteins to obtain active refolded
`
`protein
`
`subjecting the said refolded G-CSF protein to two step clu-omatography
`
`\\'herein the said refolded G-CSF protein is first subjected to cation
`
`exchange chromatography followed by hydrophobic interaction
`
`c hn)rnatography
`
`to obtain puriCied therapeutic grade G-CSF protein
`
`2.
`
`A method as claimed in claim 1, wherein the said G-CSF isolated from
`
`inclusion bodies is solubilized in a concentration of urea or guanidinium
`hydrochloride at alkaline pH and refolded at an acidic pH for 6 to 16 hrs at
`room temperature.
`
`3.
`
`A method as claimed in claim 1, wherein said refolded protein is bound to a
`
`sulphonate. carboxymethyl or sulphopropyl functional group containing
`
`chrnmatt1graphy matrices.
`
`4.
`
`A method ;:is claimed in claim l, wherein the ion exchange column is run in the
`
`pH range or 3.5 to 5.5 using buffers of citrnte. phosphate or acetate snits in the
`
`molarity range of 5mM to 50mM.
`
`5.
`
`A method as claimed in claim 1, wherein the said protein from the ion
`
`6.
`
`7.
`
`exchange group is eluted by increasing the ionic strength of the buffer by the
`addition Pr chloride. citrate or sulphate s;:ilts in the pH range of 4.0 to 6.0.
`J\ method as claimed in claim 1 wherein the G-CSF containing protein solution
`
`eluted from the cation exchange column is purified using hydrophobic
`
`interaction chromatography on resins having butyl, octyl or phenyl functional
`
`groups.
`
`/\ metlwd as claimed in claim 6, wherein the said column is equilibrated \\'ith
`buffers in the pH range nr 4.0 to 7.0 and ,,·ith ammonium sulphate salts in the
`molarity ra,ngc of0.25 M to 1.0 M.
`
`10
`
`12 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`8.
`
`A method as claimed in claim 6 wherein the said column is eluted by
`decreasing the concentration of ammonium sulphate salt in the buffer and by
`optionally increasing the concentration of ethanol from 2 to 20% for enhanced
`recovenes.
`
`I I
`
`13 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`Figure 1 is a restriction map of E.coli expression vector containing the mature coding
`sequence of human G-CSF
`
`Transcriptional
`Fl rnigin
`~ n n i n a t o r
`. J c oR I
`
`/
`
`/
`
`:\lllp
`
`pTCF-01
`
`Coding sequence
`for mature
`hG-CSF
`
`~p,,,,,
`
`Originof
`replication
`
`\
`
`I PTG/Lactosc
`lnducihlc promoter
`
`14 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`Figure 2 is the nucleotide sequence of the G-CSF gene in the cloned fragment
`along with the derived amino acid sequence
`
`ATG ACA CCA TT A GGA CCT GCC AGC TCC CTG CCC CAG AGC TTC CTG CTC AAG TGC TT A GAG CAA
`MT PLG PASS L PQS FLL KCLE Q
`
`GTG AGG AAG ATC CAG GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT
`V
`R K I Q GD GAAL Q EK LC
`
`GCC ACC TAC AAG ere; TGC CAC CCC GAG GAG CTG GTG CTG ere GGA CAC TCT
`L C
`A
`T Y
`K
`11
`P
`E
`E
`L
`\.
`L
`[.
`1-1
`S
`(i
`
`CTG GGC ATC CCC TGG GCT CCC CTG AGC AGC TGC CCC ACiC C AG GCC CTG CA.Ci
`c:;
`S Q
`:\
`L
`I
`P W A
`P
`L
`S
`S
`P
`L
`()
`C
`
`era GCA GGC TGC TTG AGC CAA CTC CAT J\GC GGC err TTC CTC TAC C,,\(i GGG
`Ci
`L A
`G
`C
`L
`S
`L
`I f
`S
`G
`L
`F
`I . Y
`Q
`<)
`
`CTC CTG CAG GCC CTG GAA GGG ATC TCC CCC GAG TTG GGT CCC ACC TTG GAC
`Q A
`L
`L
`L
`E
`G
`S
`P
`E
`L G
`P
`T
`L D
`
`ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG
`D VA D FAT T I W
`TL Q
`L
`Q QM
`
`GAAGAJ\ CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT CiCC ATG CCu
`L Q
`T Q
`E
`E
`L G M A
`P A
`P
`.-\
`!\.1
`P
`Ci
`
`GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG C.iTT C ;cc TCC
`Ci
`A
`F
`S
`F Q
`R
`R
`Ci
`V
`I, V A
`S
`A
`A
`A.
`
`CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC'CGC GTT CTA CGC CAC en GCC
`H
`L Q SF LEV SY RV LR H LA
`
`CAG CCCTGA
`*
`p
`Q
`
`2
`
`15 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`Figure 3 is the chromatographic separation profile of refolded G-CSF on ion exchange
`
`column
`
`•,t,1 ··1.~• 1 '" A'
`
`, 1 "' ••'',l h
`
`G-CSF ..
`
`I
`
`; 1,,
`
`16 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`Figure 4 is the chromatographic separation profile of CM - eluate of G-CSF on
`hydrophobic column
`
`1i1i\ll
`
`H)/)
`
`'
`
`,.,
`
`G-CSF-
`
`,0
`
`100
`
`150
`
`:co
`
`150
`
`4
`
`17 of 18
`
`Fresenius Kabi
`Exhibit 1006
`
`

`

`WO 2006/097944
`
`PCT /IN2006/000090
`
`Figure 5 is the SDS-PAGE profile of purified G-CSF after chromatography steps
`
`Lanes
`
`Lane 1: G-CSF Reducing conditions
`
`Lane 2: G-CSF Non Reducing
`
`Lane 3: Molecular weight markers
`
`5
`
`18 of 18
`
`Fresenius Kabi
`Ex