WO2005054287A1

WO2005054287A1 - Process for producing human interferon alpha

Info

Publication number: WO2005054287A1
Application number: PCT/KR2004/003172
Authority: WO
Inventors: Jong-Sang Chung; Min-Ji Baek; Jee-Won Ahn; Ki-Wan Kim; Hyung-Ki Park; Dong-Eok Lee; Hyune-Soo Lee; Hyung-Kon Koh; Myung-Suk Oh
Original assignee: Cj Corporation
Priority date: 2003-12-03
Filing date: 2004-12-03
Publication date: 2005-06-16
Also published as: KR100531670B1; KR20050054005A

Abstract

Provided is a process for producing a human interferon alpha, which includes: (a) lysing E. coli cells isolated from a human interferon alpha-producing recombinant E. coli culture, followed by denaturation of interferon alpha and aging; (b) concentrating a solution obtained in operation (a), followed by antibody affinity column chromatography; (c) refolding an eluted solution obtained in operation (b) using an urea-containing buffer solution; (d) performing cation exchange column chromatography of a solution obtained in operation (c); and (e) performing gel-filtration column chromatography of an eluted solution obtained in operation (d). Therefore, an active human interferon alpha can be produced in high yield and purity.

Description

PROCESS FOR PRODUCING HUMAN INTERFERON ALPHA

Technical Field The present invention relates to a process for producing an active interferon alpha using a human interferon alpha-producing recombinant E. coli culture in high purity and yield.

Background Art Interferons in a broad meaning are extracellular messengers mediating reactivity of hosts and evolutionally conserved protein families that are released in a relatively small size from cells. Interferons are released from interferon-producing cells in response to stimulation by viruses, double-stranded RNAs, various microorganisms, or cytokines such as TNF or IL1 , and then bind to surfaces of neighboring cells with interferon receptors. Thereafter, interferons induce synthesis of various proteins so that reactivity and homeostasis of hosts are maintained by consecutive signaling in the cells.

Therefore, interferons act as antiviral, antiproliferative, and immune signaling proteins in the bodies and have direct antiproliferation effects on cancer cells, and thus, have received much attention as therapeutic agents [Postka S., Langer J. A. and Zoon K. C.

(1987) Interferons and their actions, Annu. Rev. Biochem. 56:727-777]. Interferons belong to the class of helical, physiologically active substances.

According to physicochemical characteristics and functionalities, there are two classes of interferons: type 1 and 2. Interferon- alpha, -beta, -tau, and -epsilon are members of the type 1 interferon [Weissman C. and Weber H. (1986) The Interferon genes, Prog.

Nucleic Acid Res. Mol. Biol. 33:251-300] and interferon gamma is a member of the type

2 interferon. Among them, interferon alphas belonging to the type 1 interferon are proteins that exhibit species specificity. Interferon alphas bind to the same receptors of cell surfaces with interferon betas belonging to the type 1 interferon, and then induce transcription of antiviral factors in response to a consecutive cell signaling system. Interferon alphas are also called as leukocyte interferons considering their sources. Interferon alphas are single-chain proteins consisting of 165 amino acids and having two disulfide bonds. However, with respect to interferon alphas produced in E.coli systems, after monoclonal antibody affinity chromatography, there are observed mixed interferon alphas comprising interferon alphas having incorrect disulfide bonds, one disulfide bond and two cysteine residues, or no disulfide bonds [S. Pestka and S. J. Tamowski, Purification of the interferons. Pharma. Theraphy 29;299-319, 1986]. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, interferon alphas with correct two disulfide bonds (also called as fast-moving monomers (FMMs), hereinafter) show predicted bands. However, interferon alphas with incorrect disulfide bonds or incompletely refolded interferon alphas (also called as slow-moving monomers (SMMs), hereinafter) show higher bands than the predicted bands, i.e., slow-moving bands. SMMs are structurally unstable. For this reason, as a purification process proceeds, SMMs continuously form oligomers or are detected as impurities upon HPLC analysis. Furthermore, residual SMMs after termination of a purification process tend to have low biological activity, relative to FMMs [H. Moorehead et al., Roles of the 29-138 disulfide bond of subtype A of human α interferon in its antiviral activity and conformational stability, Biochemistry 23; 2500-07, 1984]. There have been reported numerous documents that disclose a technique of separating FMMs and SMMs of interferon alpha. That is, there have been known removal of polymers and SMMs of interferon alpha at a relatively high temperature and acidic pH, modified metal-chelate chromatography, conversion of oligomers to monomers with a redox agent, removal of SMMs by reversed-phase high-performance liquid chromatography (RP-HPLC) and cation exchange chromatography column, and the like [EP108585, A. M. Felix et al., Analysis of different forms of recombinant human leukocyte interferons by high performance liquid chromatography, Processs in enzymology, 119; 242-48, 1986, US4432895, US4765903]. These techniques are focused on separation of SMMs and FMMs. However, there arises a problem of yield reduction in spite of slight purity increase. Recently, efforts were made to increase the yield of interferon alphas by single-column purification after refolding of interferon alphas produced in E. coli. However, with respect to dye-affinity chromatography, in spite of yield increase, SMMs are still contained in a finally purified solution, thereby lowering a purity [S. Swaminathan and N. Khanna, Affinity Purification of Recombinant Interferon-α on a Mimetic Ligand Adsorbent, Protein Expression and Purification, 15;236-242, 1999]. Q-Sepharose column purification provides a low yield of about 7.5% [K. R. Babu, S. Swaminathan, S. Marten, N. Khanna and U. Rinas, Production of Interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins, Appl. Microbiol. Biotechnol., 53; 655-660, 2000].

Disclosure of the Invention While searching for a process for producing an active interferon alpha using a human interferon alpha-producing recombinant E. coli culture in high purity and yield, the present inventors developed a process for producing an active interferon alpha in high yield and purity by converting a slow-moving monomer (SMM), i.e., an inactive interferon alpha with no or incorrect disulfide bonds, to a fast-moving monomer (FMM), i.e., an active interferon alpha with correct disulfide bonds, and completed the present invention. Therefore, the present invention provides a process for producing an active interferon alpha using a human interferon alpha-producing recombinant E. coli culture. According to an aspect of the present invention, there is provided a process for producing a human interferon alpha, which includes: (a) lysing E. coli cells isolated from a human interferon alpha-producing recombinant E. coli culture, followed by denaturation of interferon alpha and aging; (b) concentrating a solution obtained in operation (a) and performing antibody affinity column chromatography; (c) refolding an eluted solution obtained in operation (b) using an urea-containing buffer solution; (d) performing cation exchange resin column chromatography of a solution obtained in operation (c); and (e) performing gel-filtration column chromatography of an eluted solution obtained in operation (d). According to the production process of the present invention, SMMs are converted to FMMs, i.e., inactive interferon alphas with no or incorrect disulfide bonds are converted to active interferon alphas with correct disulfide bonds, thereby increasing both the purity and yield of final products. In conventional production processes for interferon alphas, SMMs are removed from various types interferon alpha pools isolated from monoclonal antibodies using various columns and chemical methods, thereby leading to slight purity increase. However, as the production processes proceed, there arises a problem of consecutive reduction of yield. Such a problem can be overcome by the production process of the present invention. The production process of the present invention includes lysis and denaturation. That is, most interferon alphas are expressed as inactive inclusion bodies in E. coli cells due to no disulfide bonds and some interferon alphas are expressed as an inactive form dissolved in cytoplasms. In this regard, interferon alphas isolated from lysed E. coli cells are exposed to an aqueous solution and denaturated. The lysis of E. coli cells may be performed by a common lysis method such as using a bead beater. The denaturation of interferon alphas may be performed by dissolution in a buffer solution for a predetermined time and removal of precipitates. Preferably, the lysis and denaturation of E. coli cells may be simultaneously performed using a buffer solution of pH 7-7.5, preferably about pH 7.4, containing 5-7M, preferably about 6M guanidine hydrochloride. Preferably, an interferon alpha solution that has been subjected to the lysis and denaturation is aged in a buffer solution of pH 7-7.5, preferably about pH 7.4, containing 0.5-1.5M, preferably about 1 M guanidine hydrochloride, for about 12-24 hours. The aging may be performed at about 2-8 J . A solution obtained after the aging is filtered and concentrated using, for example, a Millipore Pellicon system with about 100K MWCO Millipore filter, and then loaded on an antibody affinity chromatography column to perform antibody affinity column chromatography. In the antibody affinity column chromatography, interferon alpha-containing fractions may be eluted with a buffer solution of pH 2-3, preferably about pH 2.0, containing 100-500 mM, preferably about 300 mM salt (e.g., NaCI). The production process of the present invention includes conversion of SMMs to FMMs, i.e., conversion of inactive interferon alphas with no or incorrect disulfide bonds to active interferon alphas. That is, interferon alphas composed of about 75±7% SMMs and about 25±7% FMMs obtained by the antibody affinity column chromatography are converted to interferon alphas composed of about 75+10% FMMs. In the refolding, an elution solution from the antibody affinity column chromatography undergoes replacement with a fresh buffer solution by an appropriate method (e.g., diafiltration), followed by addition of a glycine buffer solution of pH 9-10.5, preferably about pH 10, containing 1.5-2.5M, preferably about 2M urea. The refolding may be performed with slightly stirring for about 16-20 hours and may be stopped at pH 4 or less. According to the production process of the present invention, residual SMMs and oligomers are removed by cation exchange column chromatography. The cation exchange column chromatography is performed as follows: a solution obtained by the refolding is loaded on a cation exchange resin column at pH 3-6 after a salt concentration of the solution is reduced to 100 mM or less, preferably 50 mM or less, followed by washing the column with a 10-100 mM, preferably about 50 mM acetate buffer solution of pH 2-4.5, preferably pH 3-3.5, and elution with a 30-100 mM, preferably about 50 mM acetate buffer solution of pH 5-5.5 containing 20-70 mM, preferably 35-50 mM salt (e.g., NaCI). The resultant eluted solution is analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) to thereby obtain FMM fractions satisfying a desired purity grade. The cation exchange resin column may be a CM Sepharose column or a Q Sepharose column. The salt concentration reduction can be accomplished by diafiltration using a Millipore Pellicon system or by dilution. An eluted solution from the cation exchange column chromatography is subjected to gel-filtration column chromatography for buffer exchange and final purification. In the gel-filtration column chromatography, the eluted solution from the cation exchange column chromatography is concentrated and loaded on a Sephadex column, followed by elution with an ammonium acetate buffer solution of pH 4-5.5, preferably about pH 5, containing 50-200 mM, preferably 120 mM salt (e.g., NaCI). A flowchart illustrating sequential purification processes for producing interferon alpha according to the present invention is shown in FIG. 1.

Brief Description of the Drawings FIG.1 is a flowchart illustrating a production process of interferon alpha according to the present invention. FIGS. 2A and 2B are reversed-phase high-performance liquid chromatography (RP-HPLC) analysis results before and after refolding using urea, respectively. FIGS. 3A and 3B are respectively an RP-HPLC analysis result of a gel-filtration chromatography buffer solution and an RP-HPLC analysis result of an eluted solution after gel-filtration chromatography.

Best mode for carrying out the Invention Hereinafter, the present invention will be described more specifically by Examples. However, the following Examples are provided only for illustrations and thus the present invention is not limited to or by them. Example 1 After centrifugation of a human interferon alpha-producing recombinant E. coli

(KFCC 10237) culture, only E. coli cells were collected and stored at -70 TJ . The E. coli cells were appropriately thawed at room temperature, followed by cell lysis by exposure to a 25 mM phosphate buffer solution (pH 7.4) containing 6M guanidine hydrochloride for denaturation of interferon alphas. Then, the resultant solution was aged overnight at 4^°C in a 25 mM phosphate buffer solution (pH 7.4) containing 1 M guanidine hydrochloride with slightly stirring. Example 2 An interferon alpha-containing solution obtained in Example 1 was filtered and concentrated using a Millipore Pellicon system with 100K MWCO Millipore filter and loaded on an interferon alpha monoclonal antibody column prepared by attaching an interferon alpha monoclonal antibody (R&D Systems 21 101-1 ) to a CNBr-activated Sepharose CL4B resin. The column was washed with 3 or more column volumes of a 25 mM phosphate buffer solution (pH 7.4) containing 125 mM NaCI and then eluted with a 100mM citrate buffer solution (pH 2.0) containing 300 mM NaCI. Example 3 An eluted sample from Example 2 underwent replacement with a fresh buffer solution by diafiltration and then slightly stirred in a 50 mM glycine buffer solution (pH

10) containing 2M urea for about 18 hours. After evaluation of refolding efficiency by reversed-phase high-performance liquid chromatography (RP-HPLC), pH of the resultant solution was reduced to 4 or less so that the refolding reaction was stopped. A sample obtained in Example 2 and a sample obtained in Example 3 were analyzed by RP-HPLC according to analysis of related proteins by LC in an interferon alpha-2 analysis method of the European Pharmacopoeia. That is, small quantity of each sample was loaded on a C18 column (Vydac 218TP54, 4.6mm in inner diameter x 25cm in length, 5 m in particle size, 300 A in pore size) at a flow rate of 1 ml/min. Then, a mobile phase, which consists of solution A (30% acetonitrile containing 0.2% trifluoroacetic acid) and solution B (80% acetonitrile containing 0.2% trifluoroacetic acid), was allowed to flow through the C18 column at a rate of 1 ml/min as follows: isocratic solution of solution A 72% and solution B 28% for 1 minute, linear gradient of solution B from 28 to 33% (solution A from 72 to 67%) for 4 minutes, linear gradient of solution B from 33 to 37% (solution A from 67 to 63%) for 15 minutes, linear gradient of solution B from 37 to 43% (solution A from 63 to 57%) for 10 minutes, linear gradient of solution B from 43 to 60% (solution A from 57 to 40%) for 10 minutes, isocratic solution of solution A 40% and solution B 60% for 2 minutes, linear gradient of solution B from 60 to 28% (solution A from 40 to 72%) for 8 minutes, and isocratic solution of solution A 72% and solution B 28% for 10 minutes. Chromatogram patterns detected using a 210 nm absorption spectrophotometer were analyzed. The RP-HPLC analysis results of the samples of Examples 2 and 3 are shown in FIGS. 2A and 2B. Example 4 A sample obtained in Example 3 was diluted until a salt concentration reached 50 mM and then loaded on a CM Sepharose column at pH 4. The column was washed with a 50 mM acetate buffer solution (pH 3) and then a 50mM acetate buffer solution (pH 5.4) and then eluted with a 50mM acetate buffer solution (pH 5.4) containing 40mM NaCI. An eluted solution was analyzed by RP-HPLC to yield FMM fractions satisfying a desired purity grade. Example 5 The FMM fractions obtained in Example 4 were concentrated to about 10 mg/ml and then loaded on a Sephadex-G50 column. Then, elution was performed with a 25 mM ammonium acetate buffer solution (pH 5.0) containing 120 mM NaCI. An eluted sample was analyzed by RP-HPLC and the analysis results are shown in FIG. 3. FIGS. 3A and 3B show respectively an RP-HPLC analysis result of the gel-filtration buffer solution and an RP-HPLC analysis result of the eluted sample after gel-filtration chromatography. At this time, the RP-HPLC analysis was performed in the same manner as in Example 3.

Industrial Applicability According to the interferon alpha production process of the present invention, stable interferon alphas of FMMs (fast-moving monomers) can be produced from a human interferon alpha-producing recombinant E. coli culture with a high purification efficiency of about 30 to 50%.

Claims

CLAIMS 1. A process for producing a human interferon alpha, which comprises: (a) lysing E. coli cells isolated from a human interferon alpha-producing recombinant E. coli culture, followed by denaturation of interferon alpha and aging; (b) concentrating a solution obtained in operation (a), followed by antibody affinity column chromatography; (c) refolding an eluted solution obtained in operation (b) using an urea-containing buffer solution; (d) performing cation exchange column chromatography of a solution obtained in operation (c); and (e) performing gel-filtration column chromatography of an eluted solution obtained in operation (d).

2. The process of claim 1 , wherein the refolding is performed in a buffer solution of pH 9-10.5 containing 1.5-2.5M urea.

3. The process of claim 1 or 2, wherein the lysis and denaturation of the E. coli cells are performed in a buffer solution of pH 7-7.5 containing 5-7M guanidine hydrochloride.

4. The process of claim 1 or 2, wherein the aging is performed in a buffer solution of pH 7-7.5 containing 0.5-1.5 M guanidine hydrochloride.

5. The process of claim 1 or 2, wherein the cation exchange column chromatography, the solution obtained in operation (c) is loaded on a cation exchange resin column at pH 3-6 after a salt concentration of the solution is reduced to 100mM or less, the cation exchange resin column is washed with a 10-100 mM acetate buffer solution of pH 2-4.5 and eluted with a 30-100 mM acetate buffer solution of pH 5-5.5 containing a 20-70 mM salt.