1111111111111111 IIIIII IIIII 11111 1111111111 111111111111111 111111111111111 1111111111 11111111
`US 20080260684Al
`
`c19) United States
`c12) Patent Application Publication
`Dietrich et al.
`
`c10) Pub. No.: US 2008/0260684 Al
`Oct. 23, 2008
`(43) Pub. Date:
`
`(54) METHOD FOR THE PURIFICATION OF
`G-CSF
`
`(30)
`
`Foreign Application Priority Data
`
`Jul. 15, 2005
`
`(DE) ...................... 10 2005 033 250.1
`
`(75)
`
`Inventors:
`
`Arndt Dietrich, Reichenbach (DE);
`Bernhard Janowski, Halle (DE);
`Jorg Schaffner, Salzmunde (DE);
`Ulrich Kurt Blaschke, Wiesbaden
`(DE)
`
`Correspondence Address:
`KNOBBE MARTENS OLSON & BEAR LLP
`2040 MAIN STREET, FOURTEENTH FLOOR
`IRVINE, CA 92614 (US)
`
`(73)
`
`Assignee:
`
`BIOCEUTICALS
`ARZNEIMITTEL AG, Bad Vilbel
`(DE)
`
`(21)
`
`Appl. No.:
`
`11/995,679
`
`(22)
`
`PCT Filed:
`
`Jul. 14, 2006
`
`(86)
`
`PCT No.:
`
`PCT /EP2006/064263
`
`§ 371 (c)(l),
`(2), ( 4) Date:
`
`Jun.27,2008
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 38/17
`C07K 1118
`
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl. ........................................ 424/85.1; 530/416
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a method for obtaining
`recombinant granulocyte-colony stimulating factor (G-CSF),
`comprising at least one cation exchange chromatography and
`at
`least one hydrophobic
`interaction chromatography,
`wherein said two chromatographic steps are immediately
`consecutive in optional order. In particular, the present inven(cid:173)
`tion relates to a method for purifying G-CSF from a mixture
`of G-CSF and other proteins, comprising two cation
`exchange chromatography steps which are conducted before
`and after a hydrophobic interaction chromatography, respec(cid:173)
`tively.
`
`1 of 7
`
`Fresenius Kabi
`Exhibit 1005
`
`

`

`US 2008/0260684 Al
`
`Oct. 23, 2008
`
`1
`
`METHOD FOR THE PURIFICATION OF
`G-CSF
`
`[0001] The present invention relates to a method for pro(cid:173)
`ducing recombinant granulocyte colony-stimulating factor
`(G-CSF), comprising at least one cation exchange chroma(cid:173)
`tography and at least one hydrophobic interaction chroma(cid:173)
`tography, wherein said two types of chromatographies imme(cid:173)
`diately follow each other in arbitrary order. In particular, the
`present invention relates to a method for purifying G-CSF
`from a mixture of G-CSF and other proteins, comprising two
`cation exchange chromatography steps which are performed
`before and after a hydrophobic interaction chromatography,
`respectively.
`[0002] G-CSF (granulocyte-colony stimulating factor) is a
`naturally occurring growth factor belonging in a broader
`sense to the family of cytokines and herein to the group of
`colony stimulating factors. G-CSF plays a decisive role in
`hematopoiesis and enhances the proliferation and differentia(cid:173)
`tion of hematopoietic precursor cells and the activation of
`neutrophiles. Due to said characteristics, G-CSF has come to
`be used in different medical fields, like for example in the
`reconstitution of normal blood cell populations subsequent to
`chemotherapy or irradiation or for stimulating the immune
`response to infectious pathogens. Thus, clinically speaking,
`G-CSF is mainly employed in anti-tumor therapy and in
`particular in the treatment of neutropenia as a consequence of
`chemotherapy and is furthermore used in bone marrow trans(cid:173)
`plantations and in the treatment of infectious diseases.
`[0003] Human G-CSF in its naturally occurring form is a
`glycoprotein having a molecular weight of about 20,000 Dal(cid:173)
`ton and five cysteine residues. Four of these residues form two
`intramolecular disulfide bridges which are of essential impor(cid:173)
`tance for the activity of the protein. As G-CSF is available
`only in small amounts from its natural sources, recombinant
`forms ofG-CSF are mainly used for producing pharmaceu(cid:173)
`ticals, which can for example be obtained by means of expres(cid:173)
`sion in mammalian cells like CHO ( Chinese Hamster Ovary)
`cells or in prokaryotic cells like E. coli. The recombinant
`proteins expressed in mammalian cells differ from naturally
`occurring G-CSF in that they have a different glycosylation
`pattern, while in the proteins expressed in E. coli which can
`have an additional N-terminal methionine residue as a result
`of bacterial expression, glycosylation is not present at all.
`[0004] The recombinant production of G-CSF has been
`described in patent literature for the first time in 1987, in WO
`87/01132Al. The first commercially available G-CSF prepa(cid:173)
`ration on the basis of recombinant G-CSF was admitted in
`Germany in 1991 and is produced and distributed by Amgen
`under the trade name Neupogen®.
`[0005] While the production ofG-CSF in prokaryotic cells
`is preferred as compared to the production in mammalian
`cells, as the use of simpler expression systems and culture
`conditions is possible, a frequently occurring problem in the
`production of recombinant proteins in prokaryotic cells is,
`however, the formation of hardly soluble intracellular aggre(cid:173)
`gates of denatured forms of the protein expressed, the so(cid:173)
`called inclusion bodies, which partially have a secondary
`structure and can be found in the cytoplasm of the bacterial
`cells.
`[0006] The formation of said inclusion bodies leads to the
`necessity of solubilizing and renaturing the proteins subse(cid:173)
`quent to the isolation of the inclusion bodies by means of
`
`centrifugation at moderate speed with the aid of suitable
`means in order to maintain their active configuration. Herein,
`the competitive reaction between a transfer of the denatured
`protein into the right folding intermediate and an aggregation
`of several protein molecules is an essential factor limiting the
`yield ofrenatured protein.
`[0007]
`In the art, several patent documents deal with the
`aspect of solubilizing and renaturing the proteins obtained
`form inclusion bodies. In EP-A-0 719 860, for example, the
`isolation and purification of G-CSF including solubilization
`and refolding are described. General techniques relating to
`solubilization and renaturing of denatured proteins have been
`described in EP-A-0 512 097, EP-A-0 364 926, EP-A-0 219
`874 and WO 01/87925 and can furthermore be taken from
`scientific literature and standard works on protein chemistry.
`[0008] Subsequently, the refolded protein is purified by
`means of chromatographic methods, i.e. it is separated from
`other proteins and further impurities which are present after
`solubilizing and renaturing.
`[0009] WO 87/01132 Al already mentioned in the above,
`wherein the production of G-CSF inE. coli host cells has been
`described for the first time, also deals with chromatographic
`purification. Within the scope of the purification of the recom(cid:173)
`binant G-CSF, a cation exchange chromatography using a
`CM cellulose colunm is described in Example 7 of WO
`87/01132Al.
`[0010]
`In EP O 719 860 Al, the G-CSF is purified subse(cid:173)
`quently to solubilization and oxidation by means of Dow ex in
`order to remove the solubilizing agent, followed by an anion
`exchange chromatography and a cation exchange chromatog(cid:173)
`raphy. In EP O 719 860 Al, CM sepharose is also used for the
`cation exchange chromatography.
`[0011]
`In WO 03/051922 Al, a purification method for
`G-CSF is described, wherein a metal affinity chromatography
`is performed; more exactly, a chromatography on immobi(cid:173)
`lized metal (immobilized metal affinity chromatography,
`IMAC). Subsequently to the metal affinity chromatography, a
`cation exchange chromatography and/or a gel filtration may
`be performed according to WO 03/051922.
`[0012]
`In WO 01/04154 Al, a method for purifying G-CSF
`is described, wherein first a hydrophobic interaction chroma(cid:173)
`tography and a subsequent hydroxyapatite chromatography
`are conducted. Subsequently to the hydroxyapatite chroma(cid:173)
`tography, a cation exchange chromatography is performed.
`[0013]
`It is a problem underlying the present invention to
`disclose a method for purifying biologically active recombi(cid:173)
`nant human G-CSF, by means of which it is possible to obtain
`G-CSF with satisfactory purity and yield. Herein, the method
`should be as simple and straightforward in conduction as
`possible. Desirable is a purification method that can be con(cid:173)
`ducted with as few chromatographic steps as possible in order
`to keep technical complexity and costs on a low level and to
`avoid high losses of protein.
`[0014] This and further problems are solved by means of
`the method given in claim 1. Preferred embodiments are
`described in the dependent patent claims.
`[0015]
`It has been found that it is possible in the chromato(cid:173)
`graphic purification of renatured G-CSF by means of a cation
`exchange chromatography and a hydrophobic interaction
`chromatography to achieve acceptable purity of the recombi(cid:173)
`nant biologically active G-CSF with a satisfactory yield.
`Purity can be further increased by means of a second cation
`exchange chromatography step.
`
`2 of 7
`
`Fresenius Kabi
`Exhibit 1005
`
`

`

`US 2008/0260684 Al
`
`Oct. 23, 2008
`
`2
`
`[0016] Thus, the present invention relates to a method for
`purifying recombinantly produced biologically active human
`G-CSF in which at least one cation exchange chromatogra(cid:173)
`phy and at least one hydrophobic interaction chromatography
`are conducted, wherein said chromatographic steps are per(cid:173)
`formed in arbitrary order, provided that there is not performed
`any other chromatographic step or any other purification step
`between said steps. Thus, cation exchange chromatography
`and hydrophobic interaction chromatography are immedi(cid:173)
`ately consecutive.
`[0017] According to the present invention, the term "bio(cid:173)
`logically active human G-CSF" is understood to denote that
`G-CSF which has been purified by means of the method
`according to the present invention is capable of enhancing the
`differentiation and proliferation of hematopoietic precursor
`cells and of causing the activation of mature cells of the
`hematopoietic system. Thus, the G-CSF obtained by means
`of the method according to the present invention is suitable
`for treating indications in case of which the administration of
`G-CSF is advantageous. It is understood, that the term "bio(cid:173)
`logically active human G-CSF" also includes mutants and
`modifications of G-CSF, whose amino acid sequence is
`altered as compared to the wild type sequence, but which have
`a similar biological activity as the wild type G-CSF like those,
`for example, that are described in WO 01/87925 and EP 0 456
`200. The same applies to G-CSF conjugates. Preferably, the
`G-CSF to be purified is human Met-G-CSF produced in E.
`coli cells.
`[0018]
`In one embodiment of the present invention, the
`method for purifying G-CSF comprises two cation exchange
`chromatography steps which are conducted before and after
`the hydrophobic interaction chromatography, respectively.
`[0019]
`In a further embodiment of the present invention, the
`method comprises a tangential flow filtration subsequent to
`the only or-in case more than one cation exchange chroma(cid:173)
`tography steps are conducted-the last cation exchange chro(cid:173)
`matography.
`[0020]
`In a further embodiment, conducting an anion
`exchange chromatography is omitted in the method for puri(cid:173)
`fying G-CSF.
`[0021]
`In a further embodiment, the purification method
`according to the present invention is sufficient without gel
`filtration chromatography.
`[0022]
`In a further embodiment of the present invention,
`conducting a preparative HPLC is omitted. The same applies
`to reversed phase chromatography, which is to be distin(cid:173)
`guished from the hydrophobic interaction chromatography
`according to the present invention and which is possibly also
`omitted in the preparation. rpHPLC is only employed for
`analytical purposes.
`[0023]
`In a further embodiment, no affinity chromatogra(cid:173)
`phy, in particular no dye, metal or immunoglobulin affinity
`chromatography, is conducted within the scope of the
`method.
`[0024]
`In a further embodiment, conducting a hydroxyapa(cid:173)
`tite chromatography is omitted within the scope of the puri(cid:173)
`fication method.
`[0025] Thus, in a preferred embodiment, the purification
`method according to the present invention utilizes only two
`different chromatographic separation methods, namely the
`method of ion exchange on the basis of competitive interac(cid:173)
`tion of charged ions and the method of hydrophobic interac(cid:173)
`tion, which is characterized in that the nonpolar surface
`
`regions of a protein adsorb to the weakly hydrophobic ligands
`of a stationary phase at high salt concentrations.
`[0026] To be distinguished therefrom is the chromato(cid:173)
`graphic separation principle of affinity which is based on the
`specific and reversible adsorption of a molecule to an indi(cid:173)
`vidual matrix-bound bonding partner. The hydroxyapatite
`chromatography, which is based on the use of inorganic
`hydroxyapatite crystals, is a further separation method which
`differs from the ion exchange chromatography in form of
`cation exchange chromatography and hydrophobic interac(cid:173)
`tion chromatography.
`[0027] Said chromatographic principles mentioned are also
`correspondingly distinguished among experts (see, for
`example, Bioanalytik, F. Lottspeich, H. Zorbas (ed.), Heidel(cid:173)
`berg, Berlin, Germany, SpektrumAkad. Verlag 1998).
`[0028]
`In a preferred embodiment, the chromatographic
`purification does not comprise more than three chromato(cid:173)
`graphic steps, in which only two different chromatographic
`separation methods are employed.
`[0029] Renatured G-CSF, which is supposed to be trans(cid:173)
`ferred to achieve a purity that allows its use in the form of a
`pharmaceutical preparation, is employed as starting material
`for chromatographic purification.
`[0030] Herein, solubilizing and refolding the protein can be
`conducted according to the methods known in the art, for
`example as described in EP-A-1 630 173.
`[0031] The refolded G-CSF can be prepared subsequent to
`refolding and previous to the first chromatographic step, for
`example by means of filtration, concentration, precipitation,
`acidification and/or dialysis.
`[0032]
`In many cases it will be advantageous to purify the
`folding setup previous to the first chromatographic step, i.e. to
`remove high-molecular particles, which are mostly protein
`aggregates that have been formed in folding. Said purification
`can be conducted by means of depth filtration, wherein a
`granulate bulk material serves as filter means. The solid par(cid:173)
`ticles are larger than the pores of the filter means or are held
`back by absorption at the inner surface of the bulk.
`[0033]
`In depth filtration, the use of cellulose ester fibers as
`filter means is preferred. Suitable filter means as well as
`corresponding instructions for use are, for example, available
`from Millipore under the trade names Millistak Plus C0HC
`and Millistak+BlHC.
`[0034] Preferably, the folding setup is acidified previous to
`the depth filtration, so that the filtrate can be immediately
`employed for the cation exchange chromatography in a par(cid:173)
`ticularly efficient manner. Herein, the ph value of the folding
`setup is preferably set to below 4.0, particularly preferably to
`3.2.
`[0035] For the cation exchange chromatography, conven(cid:173)
`tional commercially available matrices can be employed.
`Herein, the G-CSF binds to the cation exchange matrix within
`a specific pH range due to its positive total charge, while most
`of the contaminating substances
`like nucleic acids,
`lipopolysaccharides and proteins originating from host cells
`as well as ionic isomers ofG-CSF and altered forms ofG-CSF
`having different pH values are not capable of binding or of
`being removed by means of washing.
`[0036] Suitable cation exchange matrices include, but are
`not limited to, carboxymethyl (CM) cellulose, AG 50 W,
`Bio-Rex 70, carboxymethyl (CM) Sephadex, sulfopropyl
`(SP) Sephadex, carboxymethyl (CM) sepharose CL-6B, CM
`sepharose HP, Hyper D-S ceramic (Biosepra) and sulfonate
`(S) sepharose, SP sepharose FF, SP sepharose HP, SP
`
`3 of 7
`
`Fresenius Kabi
`Exhibit 1005
`
`

`

`US 2008/0260684 Al
`
`Oct. 23, 2008
`
`3
`
`sepharose XL, CM sepharose FF, TSK gel SP 5PW, TSK gel
`SP-5PW-HR, Toyopearl SP-650M, Toyopearl SP-650S,
`Toyopearl SP-650C, Toyopearl CM-650M, Toyopearl
`CM-650S, Macro-Prep High S Support, Macro-Prep S Sup(cid:173)
`port, Macro-Prep CM Support etc.
`[0037] Suitable matrices and protocols for conducting the
`cation exchange chromatography can be taken from the prod(cid:173)
`uct information of suppliers like Amersham Biosciences
`(http://www.amershambiosciences.com, now GE Health(cid:173)
`care) or Bio-Rad (http://www.bio-rad.com) by the person
`skilled in the art.
`[0038] Sulfopropyl matrices, in particular the products SP
`Sepharose XL and SP Sepharose FF (Fast Flow), available by
`Amersham Biosciences, Freiburg, Germany (now GE
`Healthcare), are preferably used as matrix for the cation
`exchange chromatography.
`[0039]
`In a preferred embodiment of the present invention,
`in which two cation exchange chromatographies are con(cid:173)
`ducted, namely before and after the hydrophobic interaction
`chromatography, respectively, a sulfopropyl matrix
`is
`employed in both cases, particularly preferably SP Sepharose
`XL in the first cation exchange chromatography and SP
`Sepharose FF in the second cation exchange chromatography.
`[0040] Suitable buffers for the cation exchange chromatog(cid:173)
`raphy include maleate, malonate, citrate, lactate, acetate,
`phosphate, HEPES and Bicin buffers. Preferably, the concen(cid:173)
`tration of the buffer lies between 10 and 100 mM, preferably
`between 20 mM and 50 mM. For purifying the G-CSF, the pH
`value of the buffer should possibly not be higher than 7 .0,
`preferably not higher than 6.5.
`[0041]
`In a preferred embodiment, 20 mM sodium acetate,
`pH 5.0, which is employed for equilibrating and washing, is
`used for the cation exchange chromatography.
`[0042]
`In case a second cation exchange chromatography is
`conducted, 50 mM sodium phosphate, pH 5 .4, is herein pref(cid:173)
`erably used for equilibrating and washing.
`[0043] Subsequently to washing, the G-CSF can be eluted
`from the colunm by means of an alteration, in case of the
`cation exchange chromatography by means of an increase in
`pH value or an increase in ionic strength.
`[0044] Preferably, the elution is effected by means of
`increasing the ionic strength. In case 20 mM sodium acetate,
`pH 5.0, is used as buffer, a solution of20 mM sodium acetate,
`pH 5.0, and 200 mM NaCl is, for example, suitable for the
`elution.
`[0045] Further suitable conditions for the cation exchange
`chromatography can be taken from the relevant literature, like
`for example from the manual "Ion Exchange Chromatogra(cid:173)
`phy-Principles and Methods" by Amersham Biosciences,
`Freiburg, Germany (now GE Healthcare), 2002.
`[0046] The salt concentration in the charging buffer for the
`cation exchange chromatography should be sufficiently low
`in order to allow binding to the matrix, wherein binding also
`depends on the pH value of the solution.
`[0047] Within the scope of the cation exchange chromatog(cid:173)
`raphy, different buffers can be employed for charging and
`binding to the matrix, for example buffers selected from the
`group consisting of acetate, citrate, Tris/HCl, Tris/acetate,
`phosphate, succinate, malonate, 2-(N-morpholinoethane(cid:173)
`sulfonate) (MES) and other buffers.
`[0048] After charging the colunm, the colunm is washed
`and subsequently the proteins are eluted from the colunm.
`Herein, the elution can be conducted by means of increasing
`the ionic strength, which is effected by means of increasing
`
`the salt concentration in the buffer solution. Alternatively, an
`increase in pH value is suitable. Herein, discontinuous step
`gradients, linear gradients or a suitable combination of such
`gradients can be employed.
`[0049] Elution buffers suitable for washing and for the elu(cid:173)
`tion can be selected from acetate, citrate, Tris/HCl, Tris/
`acetate, phosphate, succinate, malonate, MES and other suit(cid:173)
`able buffers with the addition of salts like NaCl or KC!. The
`ionic strength and the salt concentration, by means of which
`the elution is achieved, are dependent on the pH value of the
`buffer solution. The higher the pH value of the buffer, the
`lower is the ionic strength that is required for the elution of the
`proteins from the column.
`[0050] The hydrophobic interaction chromatography can
`also be conducted with conventional matrices. Suitable are
`matrices like butyl, phenyl or octyl sepharose (Amersham
`Biosciences, now GE Healthcare), Makro-Prep-methyl or
`t-butyl (Bio-Rad) and Fractogel EMD with propyl or phenyl
`ligands (Merck).
`[0051] Preferably, the hydrophobic ligands are butyl, phe(cid:173)
`nyl or octyl groups, particularly preferably they are phenyl
`groups. Herein, the products by Amersham Biosciences (now
`GE Healthcare) can be employed.
`[0052] Suitable matrices and protocols for conducting the
`hydrophobic interaction chromatography can be taken from
`the product information of suppliers like Amersham Bio(cid:173)
`sciences (http://www.amershambiosciences.com, now GE
`Healthcare) or Bio-Rad (http://www.bio-rad.com) by the per(cid:173)
`son skilled in the art.
`[0053] Preferably, the matrix is Phenyl Sepharose HP
`(High Performance), available by Amersham Biosciences
`(now GE Healthcare).
`[0054] Conventional buffers, which are also employed in
`other types of chromatography, are suitable as buffers for the
`hydrophobic interaction chromatography. In a preferred
`embodiment, a citrate buffer is used. Advantageously, the
`elution is conducted by means of increasing the pH value. A
`pH gradient from about pH 3.0 to about 6.0 has proven to be
`particularly suitable.
`[0055] Further conditions suitable for the hydrophobic
`interaction chromatography can be taken from the relevant
`literature, like for example from the manual "Hydrophobic
`Interaction Chromatography-Principles and Methods" by
`Amersham Biosciences (now GE Healthcare), Freiburg, Ger(cid:173)
`many, 2002.
`[0056]
`In general, the person skilled in the art is familiar
`with the chromatographic principles utilized in the method
`according to the present invention; in any case, they are
`described in detail in established manuals or protocols by the
`suppliers of chromatography matrices, colunms and other
`means.
`[0057] The tangential flow filtration (TFF), which is con(cid:173)
`ducted within the scope of one embodiment of the present
`invention subsequently to the chromatic purification, in par(cid:173)
`ticular subsequently to the only or the last cation exchange
`chromatography, can be conducted by means of conventional
`TFF systems and protocols, like for example supplied by the
`companies Millipore and Pall Corporation. The TFF is a
`filtration as an additional purification step in contrast to the
`previous purification steps of the cation exchange chroma(cid:173)
`tography and the hydrophobic interaction chromatography.
`[0058] The G-CSF purified within the scope of the present
`invention is expressed in host cells by means of conventional
`gene-technological methods. Preferably, it is human G-CSF.
`
`4 of 7
`
`Fresenius Kabi
`Exhibit 1005
`
`

`

`US 2008/0260684 Al
`
`Oct. 23, 2008
`
`4
`
`Various expression systems for the expression in E. coli cells
`are commercially available. Suitable is, for example, the
`expression ofhuman G-CSF under the control of an inducible
`promoter, for example an IPTG-inducible promoter, see for
`example Sambrook and Russel, Molecular Cloning-A
`Laboratory Manual, 3rd edition 2001, Cold Spring Harbor
`Laboratory Press, Cold Spring Harbor, N.Y., USA, chapter
`15, or established manufacturers' protocols, for example by
`Proinega or Stratagene.
`[0059] Fermentation is conducted according to standard
`protocols, like they are described in patent and scientific
`literature, for example in a two-step process consisting of a
`batch cultivation and a fed batch cultivation.
`[0060] Harvesting the so-called inclusion bodies contain(cid:173)
`ing the G-CSF overexpressed in E. coli and the lysis of said
`inclusion bodies have partly been described in the patent
`literature discussed in the above. However, suitable protocols
`can also be found in standard works on protein chemistry as
`well as in laboratory manuals. The same applies for solubi(cid:173)
`lizing and refolding, which are objects of various patent docu(cid:173)
`ments, as has been discussed in the above.
`[0061] The invention also relates to pharmaceutical prepa(cid:173)
`rations containing the G-CSF obtained according to the
`present invention. The G-CSF obtained can either be stored in
`the form of a lyophilisate or in liquid form. It is administered
`either subcutaneously or intravenously. Suitable adjuvants in
`the formulations of the recombinantly expressed G-CSF are,
`for example, stabilizers like sugar and sugar alcohols, amino
`acids and tensides like for example polysorbate 20/80 as well
`as suitable buffer substances. Examples for formulations are
`described in EP O 674 525, EP O 373 679 and EP O 306 824,
`see also the trade products Neupogen® and Granocyte in the
`"ROTE LISTE 2004".
`
`EXAMPLES
`
`[0062] The following examples are intended to illustrate
`the present invention without limiting the scope thereof.
`[0063] The human G-CSF was expressed under the control
`of an IPTG-inducible promoter inE. coli cells. Examples for
`suitable expression systems can be taken, for example, from
`the laboratory manual Sambrook and Russell, Molecular
`Cloning-A Laboratory Manual, 3rd edition, 2001, Cold
`Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
`USA, chapter 15, or from established manufacturers' proto(cid:173)
`cols, for example by Promega or Stratagene.
`[0064] The fermentation was conducted according to stan(cid:173)
`dard protocols, like they are described in patent and scientific
`literature, in a two-step process comprising a batch cultiva(cid:173)
`tion and a fed batch cultivation. The bacteria were cultivated
`for 17 to 18 hours before they were stimulated by means of
`adding 1 mM IPTG to form the recombinant G-CSF. The
`induction period was 4.0 hours.
`[0065] Harvesting the bacteria was conducted by means of
`beaker centrifugation for 20 min at 5,000 g and 4 ° C. After the
`centrifugation, the supernatant was discarded and the cells
`were filled up again with buffer (20 mM sodium phosphate,
`pH 7.0; 1 mM EDTA) to the fermentation volume before they
`were lysed by means of three passages at 800 bar. Subse(cid:173)
`quently, the lysate was purified by means of separation
`(CSAl-Separator, Westfalia, Oelde, Germany).
`
`Concentrating the Inclusion Bodies
`
`[0066]
`In principle, the suspension of the inclusion bodies
`that has been obtained by harvesting can immediately be used
`for the subsequent solubilization. In this case, however, the
`maximum achievable protein concentration in the solubili(cid:173)
`sate is strongly limited, which may lead to limitations during
`folding. Thus, the suspension of the inclusion bodies should
`be concentrated by means of centrifugation subsequently to
`harvesting and washing in order to achieve a high protein
`concentration in the solubilisate.
`[0067] The suspension of the inclusion bodies was centri(cid:173)
`fuged for 20 minutes at 10,000 gin a beaker centrifuge. The
`paste of inclusion bodies that is obtained by means of cen(cid:173)
`trifugation can be stored at -20° C. for at least 12 weeks.
`
`Solubilization
`
`[0068]
`In order to allow effective solubilization of larger
`pellets of inclusion bodies in a relatively short time, mechani(cid:173)
`cal crushing of said pellets, for example by means of Ultra
`Turrax treatment, is furthermore required. The paste of the
`inclusion bodies that was obtained by means of centrifugation
`was weighed, mixed with 9.0 ml solubilizing buffer (30 mM
`Tris, 1 mM EDTA, 6.0 M guanidine-HCl, 100 mM GSH, pH
`8.0) per gram inclusion bodies and crushed by means ofU!tra
`Turrax treatment. The setup was thoroughly vortexed and was
`then incubated on a roller mixer or a magnetic stirrer at room
`temperature for about 2 hours.
`
`Refolding
`
`[0069] The protein concentration in the solubilisate was
`determined by means of the method according to Bradford
`using BSA as standard protein. For folding, the amount nec(cid:173)
`essary to achieve a protein concentration of 700 µg/ml in a
`desired amount ofbufferwas added to the refolding buffer (30
`mM Tris, 2 mM GSSG, 2 mM GSH, 3M urea, pH 7 .5; 4 ° C.).
`The corresponding amount of solubilisate was added slowly
`and steadily while stirring with a magnetic stirrer in order to
`avoid locally increased concentrations of solubilisate or pro(cid:173)
`tein. The afflux speed and the mode of mixing can be adjusted
`to the respectively employed volume of solubilizing setup.
`After the addition of the solubilisate had been completed, the
`setup was incubated for at least 12 hours at 4 ° C. During this
`period no further mixing was required.
`
`Depth Filtration
`
`[0070] Subsequently to refolding, the refolding setup is
`filtrated before the first chromatographic step is conducted.
`Herein, for example, a depth filter can be used for the filtra(cid:173)
`tion, for example a suitable filter by Millipore, Schwalbach,
`Germany. Previously to the filtration, the pH value is adjusted
`to pH 3.2 by means of2 M citric acid.
`
`Conducting the First Cation Exchange
`Chromatography
`
`[0071] The first chromatographic step serves for capturing
`the target protein and separates refolding agents like urea,
`GSH, GSSG, as far as those are present in the folding setup,
`from the target protein. In said step, incorrectly folded protein
`species and host cell proteins are also separated. Here, a
`cation exchange chromatography is employed. SP Sepharose
`XL by Amersham Biosciences (now GE Healthcare) is used
`as matrix. The chromatography is conducted at pH 5.0.
`
`5 of 7
`
`Fresenius Kabi
`Exhibit 1005
`
`

`

`US 2008/0260684 Al
`
`Oct. 23, 2008
`
`5
`
`[0072] The SP Sepharose XL matrix was equilibrated with
`1.5 column volumes of 20 mM sodium acetate, pH 5.0. The
`filtered refolding setup was loaded onto the column and was
`subsequently washed with 1.5 colunm volumes washing
`buffer (20 mM sodium acetate, pH 5.0). Subsequently, the
`G-CSF was eluted from the colunm with 3 colunm volumes
`elution buffer (20 mM sodium acetate, 200 mM NaCl, pH
`5.0). The purity of the eluted G-CSF was determined by
`means of rpHPLC; it was higher than 80%. As related to the
`filtered folding setup, the yield was also higher than 80%.
`
`Conducting the Hydrophobic Interaction
`Chromatography
`
`[0073]
`In the second chromatographic step, a further puri(cid:173)
`fication of the G-CSF is conducted on the basis of the eluate
`of the SP Sepharose XL. In particular the product-related
`contaminations are substantially depleted. Here, a hydropho(cid:173)
`bic interaction chromatography with Phenyl Sepharose HP
`by Amersham Biosciences (now GE Healthcare) is con(cid:173)
`ducted.
`[0074] The Phenyl Sepharose HP colunm was first equili(cid:173)
`brated with 2 column volumes of 12% buffer B, 88% buffer A
`(buffer B: 20 mM sodium citrate, pH 6.7; buffer A: 20 mM
`sodium citrate, pH 2. 7, 110 mM Na Cl). Then, the eluate of the
`SP Sepharose XL colunm, which had previously been diluted
`with 5 volumes of buffer A (20 mM sodium citrate, pH 2.7,
`110 mM NaCl), was applied onto the colunm. Subsequently,
`the colunm was washed with 2 colunm volumes of 12% buffer
`13, 88% buffer A and a linear gradient from 12% to 90%
`buffer B was run in 5-8 colunm volumes. The elution occurred
`within the scope of said linear pH gradient from about pH 3 .0
`to about 6.0. Finally, the colunm was rinsed with 3 column
`volumes of 90% buffer B, 10% buffer A.
`[0075] The elution fractions were tested for their purity by
`means of rpHPLC and fractions having a purity higher than
`95% were combined.
`[0076] The G-CSF obtained after the hydrophobic interac(cid:173)
`tion chromatography had a purity of more than 96%. The
`yield from the HIC step was almost 80%.
`
`Conducting the Second Cation Exchange
`Chromatography
`
`[0077]
`In the third chromatographic step, a further purifi(cid:173)
`cation of the G-CSF to a purity of more than 99% is conducted
`on the basis of the eluate of the Phenyl Sepharose HP. In
`particular the product-related contaminations are substan(cid:173)
`tially depleted. Here, a cation