High Yield Refolding and Purification
`Process for Recombinant Human
`Interleukin-6 Expressed in
`Escherichiacoli
`
`Daisuke Ejima, Mayumi Watanabe, Yutaka Sato, Masayo Date,
`Naoyuki Yamada, Yoshiyuki Takahara
`
`CentralResearchLaboratories,AjinomotoCompany,Inc.;1-1Suzuki-cho,
`Kawasaki-ku,Kawasaki210-8681,Japan;telephone:+81-44-244-7178;
`fax:+81-44-245-8584;e-mail:plr ejimad@te10.ajinomoto.co.jp
`
`Received3April1998;accepted24July1998
`
`Abstract: Recombinant human interleukin-6 (hIL-6), a
`pleiotropic cytokine containing two intramolecular disul-
`fide bonds, was expressed in Escherichia coli as an in-
`soluble inclusion body, before being refolded and puri-
`fied in high yield providing sufficient qualities for clinical
`use. Quantitative reconstitution of the native disulfide
`bonds of hIL-6 from the fully denatured E. coli extracts
`could be performed by glutathione-assisted oxidation in
`a completely denaturating condition (6M guanidinium
`chloride) at protein concentrations higher than 1 mg/mL,
`preventing aggregation of reduced hIL-6. Oxidation in
`6M guanidinium chloride (GdnHCl) required remarkably
`low concentrations of glutathione (reduced form, 0.01
`mM; oxidized form, 0.002 mM) to be added to the solu-
`bilized hIL-6 before the incubation at pH 8.5, and 22°C for
`16 h. After completion of refolding by rapid transfer of
`oxidized hIL-6 into acetate buffer by gel filtration chro-
`matography, residual contaminants including endotoxin
`and E.coliproteins were efficiently removed by succes-
`sive steps of chromatography. The amount of dimeric
`hIL-6s, thought to be purification artifacts, was decreased
`by optimizing the salt concentrations of the loading ma-
`terials in the ion-exchange chromatography, and gradu-
`ally removing organic solvents from the collected frac-
`tions of the preparative reverse-phase HPLC. These
`refolding and purification processes, which give an over-
`all yield as high as 17%, seem to be appropriate for the
`commercial scale production of hIL-6 for therapeutic
`use. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62:
`301–310, 1999.
`Keywords: interleukin-6; protein refolding; inclusion
`body; aggregation; purification
`
`INTRODUCTION
`
`Interleukin-6 (IL-6) is a cytokine, which is involved in di-
`verse biological activities such as proliferation, differentia-
`tion, and maturation events in host target cells (Hirano et al.,
`1985; Simpson et al., 1997). Clinical use of hIL-6 in cancer
`therapy has been of great interest and functional agonists
`and antagonists for therapeutic use in IL-6-associated dis-
`eases are now in development (Ciapponi et al., 1997;
`
`Correspondence to: Daisuke Ejima
`
`Sporeno et al., 1996). Some groups have already reported
`hIL-6 production systems in recombinant E. coli, which
`can produce large amounts of insoluble inclusion bodies
`(Brakenhoff et al., 1987; Rock et al., 1992; Tonouchi et al.,
`1988; Yasukawa et al., 1990). However, problems concern-
`ing the low yield in the refolding process owing to the
`aggregate-prone property of hIL-6 (Simpson et al., 1997;
`Ward et al., 1995) remain to be solved. This property also
`makes it rather difficult to design a high-yield purification
`process applicable to the commercial scale production of
`hIL-6, which can efficiently remove residual contaminants
`from host cells and degradation or modification impurities
`of hIL-6 from the purified product.
`Production of heterologous proteins as inclusion bodies
`in engineered E. coli is a common technique used to obtain
`valuable non-glycosylated proteins in large amounts. A re-
`cent study on refolding of denatured lysozyme (Hevehan et
`al., 1997) revealed that low concentrations of denaturants
`(guanidinium chloride or urea) can suppress the aggregation
`of refolding intermediates, even at high-protein concen-
`trations and thus, increase the yields of refolded lyso-
`zyme recovered. This study emphasized the availability of
`non-denaturating concentrations of guanidinium chloride
`(GdnHCl) for designing industrial-scale refolding pro-
`cesses. However, denaturant-induced equilibrium unfolding
`studies on murine IL-6 (Ward et al., 1995; Zhang et al.,
`1997) have demonstrated that natively disulfide-bonded
`IL-6 forms partially unfolded conformations, which have a
`high tendency to self-associate at low denaturant concen-
`trations. These studies also showed that fully disulfide-
`reduced IL-6 exhibits a higher tendency to self-associate
`than that of natively disulfide-bonded IL-6. Taken together,
`these results suggest that the refolding of hIL-6 by incubat-
`ing disulfide-reduced denatured forms in partially denatur-
`ating conditions, as in the case of refolding lysozyme
`(Hevehan et al., 1997), will in principle, fall into the low
`efficacy category.
`In this investigation, we have established a new refolding
`process for hIL-6, which consists of two steps. In the first
`
`© 1999 John Wiley & Sons, Inc.
`
`CCC 0006-3592/99/030301-10
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`APOTEX EX1031
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`step, high protein concentrations of natively disulfide-
`bonded hIL-6 were obtained from solubilized inclusion bod-
`ies under fully denaturating buffer conditions. In the second
`step, the native conformation of hIL-6 was obtained by
`rapidly removing the denaturant from the oxidized hIL-6
`solution without any dilution. Aggregation during the re-
`folding process was effectively suppressed. Refolded hIL-6
`was purified successively by ion-exchange, reverse-phase,
`and gel filtration chromatographies with aggregation con-
`trolled by unique techniques to give a good recovery and
`provide enough high quality material to be evaluated in vivo
`free of responses to residual endotoxin and immunogenic
`impurities.
`
`MATERIALS AND METHODS
`
`Materials
`
`Trifluoroacetic acid (spectroscopic grade), acetic acid, tris-
`hydroximethylaminomethane (Tris), reduced and oxidized
`glutathione (GSH and GSSG), ethylenediaminetetraacetic
`acid (EDTA), hydrochloric acid, sodium hydroxide, and
`Achromobacter protease I (lysylendopeptidase; EC
`3.4.21.50, 4.5 AU/mg) were purchased from Wako Pure
`Chemical Industries (Osaka, Japan). Guanidinium chloride
`(GdnHCl) and HPLC-grade solvents were purchased from
`Nakarai tesque (Kyoto, Japan). All buffers were prepared
`with deionized water purified by the Milli-Q SP/UF system
`(Millipore, Tokyo, Japan). All other chemicals were reagent
`grade.
`
`Recovery of Inclusion Bodies
`
`Escherichia coli strain HB101 with an expression plasmid
`containing the trp promoter followed by the mature hIL-6
`sequence was used as the expression host (Yasueda et al.,
`1990). The host cell was grown overnight in a 30 L fer-
`mentor and the cultured-broth (20 L) obtained was directly
`introduced into a high-pressure homogenizer (Type SHL-5;
`Alfa-Laval) for cell disruption. The cell homogenate was
`applied to a continuous centrifuge (type NO-U-5-HR;
`Kansai Centrifugal Separator Manufacturing, Osaka, Japan)
`at 7600g, and 1 L/min to recover the hIL-6 inclusion bodies.
`The collected inclusion bodies were suspended in 500 mL
`of washing buffer (20 mM Tris-HCl, 30 mM NaCl, pH 7.5),
`and then centrifuged at 8000g for 15 min. The pellet of
`inclusion bodies obtained was resuspended in 500 mL of 10
`mM EDTA, pH 5.5, and stored at −80°C until use.
`
`Solubilization of Inclusion Bodies
`and Air-Oxidation
`
`The pellet of inclusion bodies was solubilized in 6M
`GdnHCl (13.8 mg/mL), adjusted to pH 5.5 with HCl and
`allowed to stand for 2 h atroom temperature. Solubilized
`hIL-6 (1.5 mL) was rapidly diluted 10-fold to 1.38 mg/mL
`with 10 mM Tris-HCl, pH 8.5, containing varying concen-
`
`trations of GdnHCl (0.6 ~ 6M), and then incubated for 16 h
`at 22°C with gentle stirring. After incubation, each solution
`was centrifuged and 12 mL of the supernatant part was
`analyzed by reverse-phase HPLC for the yield of soluble
`oxidized hIL-6.
`
`Glutathione-Assisted Oxidation
`
`The solubilized hIL-6 from above (13.8 mg/mL, pH 5.5)
`was rapidly diluted 10-fold to 1.38 mg/mL in 6M GdnHCl.
`Varying concentrations of reduced and oxidized glutathione
`were then added to 15 mL of diluted hIL-6, keeping the
`reduced/oxidized glutathione ratio at 5/1. Each supple-
`mented solution was adjusted to pH 8.5 with 10 mM Tris-
`HCl and incubated for 16 h at 22°C with gentle stirring.
`Reverse-phase HPLC analysis for the yield of oxidized
`hIL-6 was performed in the same way as air-oxidation.
`
`Large-Scale Refolding by Glutathione-Assisted
`Oxidation and Desalting Chromatography
`
`The pellet of inclusion bodies containing 11 g of reduced
`hIL-6 was solubilized in 12 L of 6M GdnHCl, pH 5.5, and
`allowed to stand for 2 h at22°C. Reduced and oxidized
`glutathione were added into the solubilized hIL-6 at the
`final concentrations of 0.01 mM and 0.002 mM, respec-
`tively. The supplemented solution was adjusted to pH 8.5
`with 10 mM Tris-HCl and incubated for 16 h at 22°C. After
`the oxidation yield was confirmed by reverse-phase HPLC
`analysis, the solution was directly applied to a gel filtration
`column (Sephadex G-25 M, 44 cm internal diameter × 30
`cm; Amersham Pharmacia Biotech, Tokyo, Japan) equili-
`brated with 10 mM sodium acetate, pH 5.0, and the protein
`was eluted with the same buffer at a flow-rate of 750 mL/
`min. Eluted materials were detected by UV absorbance at
`280 nm in all chromatography steps.
`
`Purification of Oxidized hIL-6
`
`Refolded hIL-6 (10 g) in 15 L of 10 mM sodium acetate, pH
`5.0 was adjusted to 250 mM sodium acetate, pH 5.0 and
`applied to a cation-exchange column (CM-Sepharose FF, 26
`cm internal diameter × 10 cm; Amersham Pharmacia Bio-
`tech, Tokyo, Japan) equilibrated with 10 mM sodium ac-
`etate, pH 5.0. After the column was washed with 1 column
`volume of the same buffer, hIL-6 was eluted with a linear
`gradient of sodium acetate from 10 mM to 500 mM and a
`pH gradient from 5.0 to 5.5 (final elution buffer) over 10
`column volumes at a flow-rate of 0.5 L/min. Fractions of
`500 mL were collected and assayed by analytical cation-
`exchange HPLC by injecting 100 mL of each fraction.
`Pooled hIL-6 fractions (1.2 g) in the cation-exchange
`chromatography were adjusted to pH 4.0 with formic acid
`and applied to a preparative reverse-phase HPLC column
`(Vydac 214TPB10, 5 cm internal diameter × 25 cm, The
`Separations Group, Hesperia, CA, USA) equilibrated with
`0.1M sodium formate, pH 4.0. After the column was washed
`
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`with 1 column volume of the same buffer, hIL-6 was eluted
`with a linear gradient of 0–60% acetonitrile in the elution
`buffer (0.1M sodium formate, pH 4.0) over six column vol-
`umes at a flow-rate of 50 mL/min. Five fractions were col-
`lected, and assayed by the analytical reverse-phase HPLC
`by injecting 50 mg of hIL-6 from each fraction. Four runs of
`this chromatography were performed. Pooled hIL-6 frac-
`tions (800 mL) were applied to a gel filtration column
`(Sephadex G-25 M, 18 cm internal diameter × 18 cm, Am-
`ersham Pharmacia Biotech, Tokyo, Japan) equilibrated with
`20 mM acetic acid and 10% acetonitrile, and proteins were
`eluted with the same solvent at a flow rate of 120 mL/min.
`The hIL-6 (1760 mL) collected was kept at 22°C for 1 h and
`then applied to another Sephadex G-25 M column (30 cm
`internal diameter × 18 cm) equilibrated with 5 mM sodium
`acetate, pH 4.5, and run with the same buffer at a flow-rate
`of 350 mL/min.
`Prior to the final purification step, hIL-6 was concen-
`trated to greater than 8 mg/mL by cation-exchange chroma-
`tography. One gram of solvent-free hIL-6 (5 mM sodium
`acetate, pH 4.5) was adjusted to 175 mM sodium acetate, pH
`5.0 and applied to a CM-Sepharose FF column (5 cm in-
`ternal diameter × 2.5 cm) equilibrated with the 10 mM
`sodium acetate, pH 5.0. Adsorbed hIL-6 was eluted step-
`wise with 10 mM sodium citrate, pH 6.5 containing 50 mM
`NaCl at a flow rate of 30 mL/min. Three runs of this chro-
`matography were performed. Finally, 50 mL of concen-
`trated hIL-6 was applied to a preparative gel filtration
`HPLC column (Superdex-75 HR 60/600, 6 cm internal di-
`ameter × 60 cm, Amersham Pharmacia Biotech, Tokyo,
`Japan) equilibrated with 10 mM sodium citrate, pH 6.0 and
`the proteins were eluted with the same buffer at a flow-rate
`of 24 mL/min. Monomeric hIL-6 fractions were pooled,
`filter sterilized (Millex GVHD-25020, Millipore, Tokyo, Ja-
`pan) and stored at 5°C for characterization.
`
`Analytical Methods
`
`Analytical reverse-phase HPLC was performed using a
`Vydac C4 column (214TP54, 4.6 mm internal diameter ×
`250 mm, The Separations Group, Hesperia, CA, USA). The
`column was equilibrated with 32% acetonitrile and 0.1%
`trifluoroacetic acid/water, and the proteins were eluted with
`a linear gradient of up to 60% acentonitrile and 0.1% tri-
`fluoroacetic acid/water over 28 min at a flow rate of 1
`mL/min at ambient temperature. The eluted materials were
`detected by UV absorbance at 280 nm (all analytical HPLC
`of hIL-6).
`Analytical cation-exchange HPLC was performed using a
`SP-NPR column (35 mm internal diameter × 30 mm, Tosoh
`Corporation, Tokyo, Japan) equilibrated with 10 mM so-
`dium acetate, pH 5.0, and the proteins were eluted with a
`linear gradient of up to 0.5M sodium acetate, pH 5.5 over 5
`min at a flow rate of 1 mL/min.
`Analytical gel filtration was performed using a Superdex
`75 HR 10/30 (10 mm internal diameter × 300 mm, Amer-
`sham Pharmacia Biotech, Tokyo, Japan) equilibrated with
`
`10 mM sodium citrate, 8.7 mM sodium phosphate, pH 7.0
`and the proteins were eluted with the same buffer at a flow
`rate of 0.8 mL/min.
`Sodium dodecylsulfate polyacrylamide gel electrophore-
`sis (SDS-PAGE) was performed using the Phastsystem and
`20% homogeneous separating gels (Amersham Pharmacia
`Biotech, Tokyo, Japan) under non-reducing conditions ac-
`cording to the manufacturer’s instruction. For GdnHCl-
`containing samples, GdnHCl was removed using a small
`prepacked desalting column (PD-10, Amersham Pharmacia
`Biotech, Tokyo, Japan) equilibrated with 10 mM sodium
`acetate, pH 5.0 according to the manufacturer’s instruction.
`Gels were visualized by silver staining (diamine method).
`Phosphorylase b (94 kDa), bovine serum albumin (67 kDa),
`ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean
`trypsin inhibitor (20 kDa), and alpha-lactalbumin (14.4
`kDa) were used as molecular weight markers.
`Peptide mapping was performed by enzymatic digestion
`(S/E 4 100/1 in molar ratio) and analytical reverse-phase
`HPLC. Purified hIL-6 (1 nanomole/21 mg in 10 mM sodium
`citrate, pH 6.0) was diluted into 0.1 mL of the digestion
`buffer (10 mM sodium phosphate, pH 7.0). Ten picomoles
`Achromobacter protease I enzyme (Wako Pure Chemical
`Industries, Osaka, Japan) dissolved in 0.01 mL of the di-
`gestion buffer was added to the hIL-6 solution, and the
`mixture was incubated for 12 h at ambient temperature. The
`whole solution was then injected directly into a Vydac C4
`(214TP54, 4.6 mm internal diameter × 250 mm, The Sepa-
`rations Group, Hesperia, CA, USA) equilibrated with 0.1%
`trifluoroacetic acid/water, and the peptides were eluted with
`a linear gradient of 0–60% acetonitrile with 0.1% trifluoro-
`acetic acid/water over 75 min at a flow rate of 1 mL/min.
`The eluted peptide fragments were detected by UV absor-
`bance at 215 nm. Amino acid sequences of detected pep-
`tides were assigned by FABMS (JMS-HX110/HX110,
`JEOL, Tokyo, Japan) and automatic Edman degradation
`(model 470A, Applied Biosystems, Tokyo, Japan) of the
`collected peptide peaks.
`The concentration of purified hIL-6 was determined by
`UV spectroscopy at 280 nm utilizing an extinction coeffi-
`cient for hIL-6 of 0.47 mg−1 cm2 at 280 nm, which was
`obtained experimentally using an hIL-6 protein standard
`measured by quantitative amino acid analysis. The concen-
`trations of crude hIL-6 were determined by analytical re-
`verse-phase HPLC (above) using purified hIL-6 as a protein
`standard.
`
`Biological Assay
`The hIL-6 titer was determined by an IgM-inducing assay
`using SKW6-CL4 cells, as previously described (Hirano et
`al., 1985).
`
`Endotoxin and Escherichiacoli Protein Contents
`The amount of endotoxin in the hIL-6 samples was deter-
`mined using the Lymulus amoebocyte lysate assay (Toxi-
`color system, Seikagaku Kogyo Inc., Tokyo, Japan) accord-
`ing to the manufacturer’s instruction.
`
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`The E. coli protein (ECP) content of hIL-6 samples was
`determined by ELISA using polyclonal anti-ECP antibod-
`ies. The recombinant host cell was constructed by trans-
`forming E. coli with a vector identical to the production
`system but lacking the gene insert coding the product. They
`were then cultured in a fermentor (3 L), disrupted, and
`centrifuged in the same manner as the production. The pellet
`obtained was solubilized, purified by desalting and cation-
`exchange chromatographies using the same conditions for
`hIL-6 except for the scales to yield the semi-purified ECP
`mixtures. Rabbit and mouse polyclonal anti-ECP antibodies
`were obtained using these ECP mixtures and a highly sen-
`sitive sandwich ELISA system for ECP was constructed
`using anti-ECP antibodies, biotin-labeled alkaliphosphatase
`kit (AK 5001, Vectastain Vector Lab, Inc., Burlingame, CA,
`USA) and BCIP/NBT phosphatase substrate system (Kap-
`pel, Funakoshi, Tokyo, Japan). The detection limit of this
`ELISA for ECP was 1 ng/mL.
`
`RESULTS
`
`Production and Refolding of hIL-6
`
`Recombinant hIL-6 was produced as insoluble inclusion
`bodies in the engineered E. coli strain HB101 containing an
`expression plasmid for mature hIL-6 sequence, as previ-
`ously reported (Yasueda et al., 1990). After completion of
`the fermentation, the cultured broth was directly introduced
`into a high pressure homogenizer, and insoluble inclusion
`bodies recovered by continuous centrifugation. The pellet
`collected was resuspended in Tris-HCl buffer, centrifuged
`again, and the washed inclusion bodies finally suspended in
`10 mM EDTA, pH 6.0 before being stored at −80°C until
`use.
`Frozen inclusion bodies were thawed, and then solubi-
`lized in 6M GdnHCl, pH 5.5 for 2 h atambient temperature
`without any disulfide reducing agents. Formation of the
`disulfide bonds at a concentration of 1.38 mg/mL denatured
`hIL-6 under several GdnHCl concentrations was examined
`in 15 mL volumes by air-oxidation. Each solution was cen-
`trifuged and obtained supernatant part was assayed by ana-
`lytical reverse phase HPLC (Figs. 1,2). As shown in Figure
`1B, most of extracted hIL-6 had reduced disulfide bonds
`before oxidation. It was found that the yields of oxidized
`hIL-6 after a 16 h incubation at 22°C were remarkably
`dependent on the GdnHCl concentrations used (Fig. 2). As
`the GdnHCl concentration increased from 0.6M, the yield of
`oxidized hIL-6 first decreased to give the minimum yield at
`3M (12%), and then increased to give the maximum yield at
`6M (87%). Solutions with GdnHCl below 4M became tur-
`bid after incubation for 16 h due to the presence of aggre-
`gation. The oxidation yield with 1M GdnHCl (62%) was
`distinctly lower than the maximum yield (87% in 6M), but
`the purity of the oxidized material as judged by reverse
`phase HPLC (Fig. 1F) was much better than that of the
`maximum yield (Fig. 2). Partially denaturating GdnHCl
`concentrations (2 ~ 4M), which are commonly used in oxi-
`
`Figure 1. Air oxidation of denatured hIL-6. HIL-6 extracted in 6M
`GdnHCl (13.8 mg/mL) was diluted to 1.38 mg/mL in 10 mM Tris-HCl, pH
`8.5 containing each concentration of GdnHCl. The solutions (15 mL) were
`incubated at room temperature for 16 h, and 12 mL of each was assayed by
`reverse-phase HPLC. Arrows and a dotted arrow show oxidized and re-
`duced hIL-6, respectively.
`
`dative refolding processes of many kinds of denatured pro-
`teins (Fischer et al., 1993), were not effective for refolding
`of reduced hIL-6.
`Applications of thiol/disulfide redox buffer systems (glu-
`tathione system) in 6M GdnHCl were examined in the same
`15 mL volumes (Fig. 3). Combinations of reduced and oxi-
`dized glutathione (GSH and GSSG) were added to the oxi-
`dation solutions, keeping the molar ratio of GSH/GSSG at
`5/1, and the mixtures incubated for 16 h at 22°C, similar to
`the air-oxidation. The highest yield was obtained at 0.01
`mM GSH/0.002 mM GSSG (105%, Fig. 3B), and this ma-
`terial showed a molecular weight equivalent to that of pu-
`rified hIL-6 on SDS-PAGE (Fig. 4, lane 3). The reason why
`the yield was above 100% may be due to unresolved impu-
`rities in the oxidized hIL-6 peak. The combination of 1 mM
`GSH/0.2 mM GSSG, which is one of the popular conditions
`used in oxidative refolding of denatured proteins (Fischer et
`al., 1993), increased the yield a little (95%, Fig. 3C) as
`compared with that of air-oxidation (91%, Fig. 3A). How-
`ever, the oxidized hIL-6 obtained under this condition
`
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`Figure 2. Yield of soluble oxidized hIL-6 in varying concentrations of
`GdnHCl (Fig. 1).
`
`showed a contaminating band with a molecular weight a
`little higher than that of natively disulfide-bonded hIL-6 on
`SDS-PAGE (Fig. 4, lane 4). The amount of this contaminant
`band increased (Fig. 4, lane 5) and the oxidation yield de-
`creased to 81% (Fig. 3D) with the combination of 5 mM
`GSH/1 mM GSSG. Peptide-mapping and Electrospray Ion-
`ization mass spectrometry analyses of this contaminant
`band revealed that it was probably due to an oxidation ar-
`tifact which had one mixed disulfide bond with glutathione
`at Cys(44) or Cys(50) (data not shown). Consequently, the
`optimal oxidation condition was obtained using fully dena-
`turating buffer containing 6M GdnHCl, 0.01 mM GSH, and
`0.002 mM GSSG, at pH 8.5.
`Large scale oxidation (12 L) was carried out almost quan-
`titatively and the oxidized hIL-6 (0.9 mg/mL) was directly
`applied to a Sephadex G-25 desalting column equilibrated
`with 10 mM sodium acetate buffer, pH 5.0 (Fig. 5). The
`fraction (15 L) indicated by the arrow was collected and
`analyzed by reverse-phase HPLC (inserted chromatogram).
`Almost 95% of hIL-6 loaded was recovered (0.68 mg/mL)
`as one major peak with trace impurities as shown by ana-
`lytical reverse-phase HPLC, and it had comparable bioac-
`tivity to purified hIL-6 in the IgM inducing assay (data not
`shown). Therefore, it was concluded that refolding of hIL-6
`from inclusion bodies had been complete.
`
`Purification and Characterization of
`Refolded hIL-6
`
`Cation-ExchangeChromatography
`
`Refolded hIL-6 (10 g in 10 mM sodium acetate buffer, pH
`5.0) was adjusted to 250 mM sodium acetate, pH 5.0 and
`applied to the CM-Sepharose FF column equilibrated with
`10 mM sodium acetate buffer, pH 5.0. Adsorbed hIL-6 was
`
`Figure 3. Glutathione-assisted oxidation of reduced hIL-6. Oxidation
`conditions were similar to those of air-oxidation (Fig. 1) except additions
`of reduced and oxidized glutathione (GSH and GSSG). Solutions were
`incubated for 16 h at 22°C. The yield of oxidized hIL-6 was assayed by
`analytical reverse-phase HPLC, shown inserted in each chromatogram.
`GSH/GSSG: A, air-oxidation; B, 0.01 mM/0.002 mM; C, 1.0 mM/0.2 mM;
`D, 5 mM/1 mM.
`
`eluted with a linear gradient of up to 0.5 M sodium acetate,
`pH 5.5 (Fig. 6). Forty-eight percent of the loaded hIL-6 was
`recovered (4.8 g), and it showed a homogeneous peak in the
`analytical ion-exchange HPLC (inserted A in Fig. 6) with a
`more than 1000-fold decrease in the residual endotoxin con-
`tent (Table I). When refolded hIL-6 (dissolved in 10 mM
`acetate, pH 5.0) was directly added to the CM-Sepharose FF
`column and eluted under the same conditions, dimeric hIL-6
`appeared as a purification artifact and the yield of hIL-6
`decreased to less than 35% (inserted B in Fig. 6).
`
`Reverse-PhaseHPLC
`
`The hIL-6 collected (1200 mg) from ion-exchange chroma-
`tography was adjusted to pH 4.0 with formic acid, and
`loaded on a 50 mm internal diameter reverse-phase column
`equilibrated with 0.1M sodium formate, pH 4.0. Adsorbed
`hIL-6 was eluted with a linear gradient of 0–60% acetoni-
`trile in 0.1M sodium formate, pH 4.0 (Fig. 7), and then
`assayed by analytical reverse-phase HPLC (inserted in Fig.
`7). Fraction 3, which showed an almost homogeneous hIL-6
`
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`Figure 4. SDS-PAGE analysis of glutathione-assisted oxidation of
`hIL-6. Oxidized hIL-6 (Fig. 3) were desalted by PD-10 (Materials and
`Methods) and 400 pg of each were analyzed using Phastsystem and 20%
`homogeneous separating gels (Amersham Pharmacia Biotech) under non-
`reducing conditions. Gels were visualized with silver staining (diamine
`method). Arrows indicate the mixed disulfide form. Lane 1: Low molecular
`weight markers; Lane 2: air-oxidation. Lane 3: GSH/GSSG, 0.01 mM/
`0.002 mM; Lane 4: GSH/GSSG, 1 mM/0.2 mM; Lane 5: GSH/GSSG, 5
`mM/1 mM; Lane 6: Purified hIL-6.
`
`peak, was pooled and stored below 5°C. Product-related
`impurities were effectively removed to side streams (frac-
`tions 1, 2, 4, and 5) and residual endotoxin content in the
`pooled fractions was below the detection limit (<0.006 EU/
`mL). Four runs were performed with an average yield of
`75% resulting in recovery of 3.6 g of hIL-6 from the 4.8 g
`loaded (cation-exchange chromatography).
`
`RemovalofSolvents
`
`The removal of acetonitrile and sodium formate used in
`reverse-phase HPLC was performed by a two-step gel fil-
`tration chromatography. HPLC fractions containing 3.6 g
`hIL-6 were first applied on a Sephadex G-25 column equili-
`brated with 20 mM acetic acid and 10% acetonitrile (Fig.
`8A). HIL-6 (3.2 g) was eluted as a non-symmetrical peak.
`The hIL-6 collected was kept at 22°C for 1 h, and then
`introduced to the second Sephadex G-25 column equili-
`brated with 5 mM sodium acetate buffer, pH 4.5 (Fig. 8B).
`The amount of hIL-6 was 2900 mg, and it almost all showed
`the monomeric form on analytical gel filtration HPLC (Fig.
`8C). When reverse-phase HPLC fractions were directly
`added to the second Sephadex G-25 column using 5 mM
`sodium acetate buffer, almost half of the collected hIL-6
`turned to be dimers (inserted in Fig. 8C).
`
`GelFiltrationHPLC
`
`Before being applied to the final purification step, hIL-6
`was concentrated to greater than 8 mg/mL by cation-
`exchange chromatography. Fifty milliliters of concentrated
`hIL-6 (almost 400 mg/50 mL) were loaded on to the pre-
`parative gel filtration HPLC column (Superdex-75 HR 60/
`600) equilibrated with 10 mM sodium citrate, pH 6.0 as a
`final purification step (Fig. 9). Monomeric hIL-6 (fraction
`4) was separated completely from the hIL-6 dimers, which
`were mainly formed in the two steps of the gel filtration for
`removing HPLC solvents and the following concentration
`steps. The hIL-6 fragment [Pro(1) − Asp(140)] formed by
`acid-hydrolysis between Asp(140) and Pro(141) during the
`storage of reverse phase HPLC fractions was eluted be-
`tween the hIL-6 dimers and hIL-6 in fraction 2, suggesting
`that the cleaved hIL-6 had formed dimers (data not shown).
`Contaminating E. coli proteins (ECP) were eluted in the
`fractions just before monomeric hIL-6 (inserted values in
`Fig. 9). Six runs were performed, and a total of 1870 mg of
`purified monomeric hIL-6 was recovered. All of these pu-
`rifications are summarized in Table I.
`
`CharacterizationsofPurifiedhIL-6
`
`The homogeneity of the purified hIL-6 was confirmed by
`high loaded reverse-phase HPLC (Fig. 10A) and SDS-
`PAGE (Fig. 10B). Its primary structure was confirmed by
`peptide mapping using Achromobacter protease I as a di-
`gestion enzyme (Fig. 11). Purified hIL-6 containing very
`low amounts of endotoxin and ECP (<0.006 EU/mg hIL-6
`and 3ng/mg hIL-6, respectively; Table I) showed a biologi-
`cal activity of 5 U/ng hIL-6 in the IgM inducing assay.
`
`Figure 5. Desalting chromatography of large-scale oxidation. Oxidized
`hIL-6 (12L) was directly introduced to a Sephadex G-25M column (44 cm
`internal diameter × 30 cm) equilibrated with 10 mM sodium acetate buffer,
`pH 5.0 and eluted with the same buffer. Fraction (15L) indicated by the
`horizontal bar was collected and analyzed by reverse-phase HPLC (in-
`serted chromatogram).
`
`DISCUSSION
`
`Refolding of hIL-6
`
`Low recovery yields in protein refolding processes are often
`due to aggregation of unfolded or partially folded proteins
`
`306
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`
`Page 6
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`

`
`Table I. Summary of hIL-6 refolding and purification. Purity was determined by analytical reverse-phase HPLC. All analytical conditions are described
`in Materials and Methods.
`
`Purification step
`
`HIL-6 (g)
`
`Yield (%)
`
`Purity (%)
`
`Endotoxin (EU/mL)
`
`ECP (mg/g hIL-6)
`
`Solubilization
`Oxidation
`Removal of GdnHCl
`Cation-exchange chromatography
`Reverse-phase HPLC
`Removal of solvents
`Concentration
`Gel filtration HPLC
`
`N.D. 4 not determined.
`
`11
`10.8
`10.2
`4.8
`3.6
`2.9
`2.4
`1.87
`
`100
`98
`93
`44
`33
`26
`22
`17
`
`~ 60
`~ 60
`90
`97
`>99
`>99
`>99
`>99
`
`>100000
`N.D.
`420
`0.25
`<0.006
`<0.006
`<0.006
`<0.006
`
`N.D.*
`N.D.
`N.D.
`3100
`110
`88
`94
`3
`
`(Goldberg et al., 1991; Thatcher and Hitchcock, 1994). It is
`known to be very important to suppress aggregation, espe-
`cially in the early phase of the refolding process, to generate
`high yields. In the preliminary refolding experiments of
`hIL-6, we tried to recover the natively oxidized form by
`rapidly diluting the denatured, reduced form into partially
`denaturating buffers containing glutathione systems. How-
`ever, aggregation of the materials seemed to take place dur-
`ing the dilution and following incubation in all cases, and
`the yields were very low (data not shown). Therefore, in this
`study we first investigated the effects of a wide range of
`denaturant concentrations (up to a completely denaturating
`condition) on the air-oxidation of reduced hIL-6. Almost all
`
`of the Cys residues in crude hIL-6 extracted from inclusion
`bodies have free SH (Fig. 1B), and these reduced hIL-6 tend
`to aggregate to give low yields on oxidative refolding using
`the non-denaturating concentrations of GdnHCl (Figure 1E
`~ G). It was suggested that non-denaturating concentrations
`of GdnHCl (2M ~ 4M), which are known to be effective for
`suppressing aggregation and promoting oxidative refolding
`of many proteins (Fischer et al., 1993; Hevehan and Clark,
`1997), may produce particular conformation in reduced
`hIL-6. These aggregation properties with varying GdnHCl
`
`Figure 6. Cation-exchange chromatography of hIL-6. Refolded hIL-6
`(10 g) in 10 mM sodium acetate, pH 5.0 was adjusted to 250 mM sodium
`acetate, pH 5.0 and applied to a cation-exchange column (CM-Sepharose
`FF, 26 cm internal diameter × 10 cm) equilibrated with 10 mM sodium
`acetate, pH 5.0. HIL-6 was eluted with a linear gradient to the elution
`buffer (500 mM sodium acetate buffer, pH 5.5) over 10 column volume at
`a flow rate of 0.5 L/min. The horizontal bar shows pooled hIL-6 fractions.
`Inserted Figure A, cation-exchange HPLC analysis of pooled hIL-6 frac-
`tions; inserted Figure B, cation-exchange chromatography when refolded
`hIL-6 was directly applied to the column without addition of sodium ac-
`etate.
`
`Figure 7. Preparative reverse-phase HPLC of hIL-6. Twelve hundred
`milligrams of hIL-6 pooled in ion-exchange chromatography were adjusted
`to pH 4.0 with formic acid, applied to a 50 mm internal diameter reverse-
`phase column equilibrated with 0.1M sodium formate, pH 4.0. HIL-6 was
`eluted with a linear gradient to 0.1M sodium formate, pH 4.0 containing
`60% acetonitrile. Five fractions (horizontal bar) were collected and assayed
`by the analytical reverse-phase HPLC (inserted figure).
`
`EJIMA ET AL.: REFOLDING AND PURIFICATION OF HUMAN INTERLEUKIN-6
`
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`Page 7
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`

`
`Figure 8. Removal of solvents used in reverse-phase HPLC. HPLC frac-
`tions containing hIL-6 (Fig. 7) were combined and introduced to the first
`Sephadex G-25 column equilibrated with 20 mM acetic acid and 10%
`acetonitrile (A). Collected hIL-6 (horizontal bar) was kept at 22°C for 1 h
`and introduced to the second Sephadex G-25 column equilibrated with 5
`mM sodium acetate buffer, pH 4.5 (B). Recovered hIL-6 (bar) was ana-
`lyzed by analytical gel filtration HPLC (C). In Figure 8C, monomeric and
`dimeric hIL-6 peaks were indicated by arrow and dotted arrow, respec-
`tively. Inserted figure of C shows solvent-free hIL-6 obtained by one-step
`gel filtration of HPLC fractions to 5 mM sodium acetate buffer.
`
`concentrations (Fig. 2) seem to be consistent with that of the
`unfolding intermediates of murine IL-6 reported by Ward et
`al. (1995) and Zhang et al. (1997). With equilibrium dena-
`turation studies, they observed that unfolding intermediates
`are formed under partially denaturating conditions and these
`tend to self-associate or aggregate. They also found that
`unfolding intermediates derived from disulfide-reduced
`form have a higher tendency to self-associate than that of
`the oxidized form of murine IL-6. Intermediates prone to
`self-association or aggregation may be a common feature of
`IL-6s from some species. As shown in Figure 2, reduced
`hIL-6 can be efficiently oxidize