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Received August 31, 2004
Accepted February 20, 2005
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유전자재조합 단백질 회수 공정에서의 고체상 재접힘 기술: 여러 바이오의약 단백질에의 적용 사례
Solid-Phase Refolding Technology in Recombinant Proteins Recovery: Application Examples to Various Biopharmaceutical Proteins
Min Young Kim
Chang Woo Suh1
Chang Sung Kim2
Tae Hoon Jo3
Sang Joong Park4
Won Chan Choi5
Eun Kyu Lee†
한양대학교 화학공학과 생물공정연구실, 426-791 경기도 안산시 사1동 1271 1(주)대웅 생명과학연구소, 449-814 경기도 용인시 포곡면 심계리 503-1 2듀폰, 135-080 서울시 강남구 역삼동 726 3Univ. of Southern California Dept. of Chem. Eng., Los Angeles, CA 4(주)종근당 바이오 중앙연구소, 425-100 경기도 안산시 단원구 목내동 454 5(주)녹십자, 449-900 경기도 용인시 기흡읍 구갈리 272
Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, 1271, Sa-1-dong, Ansan, Gyeonggi-do 426-791, Korea 1Biotechnology Research Center, Daewoong Corp., 503-1, Samgye-ri, Pogok-myon, Yongin, Gyunggi-do 426-791, Korea 2Dupont Korea Ltd., 726, Yeoksam-dong, Gangnam-gu, Seoul 135-080, Korea 3Department of Chem. Eng., Univ. of Southern California, Los Angeles, CA, USA 4CKD Bio Research Institute, CKD Bio Corp., 454, Mongnae-dong, Danwon-gu, Ansan, Gyunggi-do 425-100, Korea 5Quality Control Team, Green Cross Plasma Derivatives, Corp., 272, Gugal-ri, Giheung-eup, Yongin, Gyeonggi-do 449-900, Korea
Korean Chemical Engineering Research, April 2005, 43(2), 187-201(15), NONE Epub 9 May 2005
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Abstract
최근 전통적인 액체상 공정을 대체하는 기술로서 고체 담체와 단백질 사이의 ‘생물인식’ 기능을 이용하는 새로운 생물공정기술이 개발되고 있다. 통상 고체 담체로는 표면에 특정한 기능기가 노출되어 있는 크로마토그래피용 담체를 사용한다. 단백질의 반응이나 상호작용이 단백질이 담체 표면에 부착되어 있는 상태에서 일어나기 때문에 이 ‘고체상 기술’은 액체상 기술에 비해 뚜렷한 장점을 갖고 있다. 고체상 재접힘은 변성제에 의해 용해된 내포체 형태의 재조합 단백질을 이온교환수지 표면에 흡착시켜 시작한다. 변성제를 단백질 주위로부터 서서히 제거시키면서 고유의 3차 구조로 재접힘시킨다. 재접힘이 완료되면 염 구배와 같은 전통적인 방법에 의해 재접힘된 단백질을 정제된 상태로 용출시킨다. 이 개념은 ‘확장층 흡착 재접힘’에도 연장 적용된다. 세포파쇄액에 변성제를 첨가하여 용해한 내포체 단백질은 확장층 흡착 크로마토그래피용 Streamline 담체에 흡착되고 세포찌꺼기와 불순 단백질들은 확장층 사이로 빠져 칼럼 밖으로 제거된다. 흡착된 목적 단백질은 고체상 재접힘 방법에 의해 재접힘 된 후 용출된다. 수년간 연구·발전되어 온 이 새로운 재접힘 기술은 정제수율 향상, 공정 단계 감축, 공정 시간 및 부피 감소에 따라 생물의약공정의 경제성을 크게 향상시킬 수 있는 것으로 증명되고 있다. 본 논문에서는 실험실에서 수행한 여러 생물의약용 단백질들을 대상으로 한 연구 실험 자료를 바탕으로 고체상 재접힘 기술의 적용 사례를 서술하였다.
. Bioprocessing technologies utilizing ‘biorecognition’ between a solid matrix and a protein is being widely experimented as a means to replacing the conventional, solution-based technology. Frequently the matrices are chromatographic resins with specific functional groups exposed outside. Since the reactions of and interactions with the proteins occur as they are attached to the solid matrix, this ‘solid-phase’ processing has distinct advantages over the solution-phase technology. Solid-phase refolding of inclusion body proteins uses ion exchange resins to adsorb denaturant-dissolved inclusion body. As the denaturant is slowly removed from the micromoiety around the protein, it is refolded into a native, three-dimensional structure. Once the refolding is complete, the folded protein can be eluted by a conventional elution technique such as the salt-gradient. This concept was successfully extended to ‘EBA (expanded bed adsorption)-mediated refolding,’ in which the denaturant-dissolved inclusion body in whole cell homogenate is adsorbed to a Streamline resin while cell debris and other impurity proteins are removed by the EBA action. The adsorbed protein follows the same refolding steps. This solid-phase refolding process shows the potential to improve the refolding yield, reduce the number of processing steps and the processing volume and time, and thus improve the overall process economics significantly. In this paper, the experimental results of the solid-phase refolding technology applied to several biopharmaceutical proteins of various types are presented.
Keywords
References
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Bowden GA, Paredes AM, Georgiou G, Bioresour. Technol., 9, 725 (1991)
Gekko K, Timasheff SN, Biochemistry, 20(16), 4667 (1981)
Badcoe I, Smith C, Wood S, Halsall D, Holbrook J, Lund P, Clarke A, Biochemistry, 30(38), 9195 (1991)
Martin J, Langer Y, Boteva R, Schramal A, Horwich A, Hartl F, Nature, 352, 36 (1991)
Bam NB, Cleland JL, Randolph TW, Biotechnol. Prog., 12(6), 801 (1996)
Brems DN, Biochemistry, 27(12), 4541 (1988)
Defellippis MR, Alter LA, Pekar AH, Havel HA, Brems DN, Biochemistry, 32(6), 1555 (1993)
Cleland JL, Wang DIC, Biochemistry, 29(50), 11072 (1990)
Cleland JL, Hedgepeth C, Wang DIC, J. Biol. Chem., 267(19), 13327 (1992)
Cleland JL, Randolph TW, J. Biol. Chem., 267(5), 3147 (1992)
Kim YH, "Direct Quantification of Interferon-α Inclusion body in Recombinant E. coli. Fermentor Beer Using Reversed-phase HPLC", M.S. Thesis, Dept. of Chem. Eng., Hanyang Univ., Seoul, Korea (1995)
Hemann R, "Protein Folding, in EPO Applied Technology Series", 12(7), European Patent Office, Netherlands (1993)
Fischer B, Summer I, Goodenough P, Biotechnol. Bioeng., 41(1), 3 (1993)
Kiefhaber T, Rudolph R, Kohler HH, Buchner J, Bioresour. Technol., 9, 825 (1991)
Zettlmeissl G, Rudolph R, Jaenicke R, Biochemistry, 18(25), 5567 (1979)
Buchner J, Rudolph R, Bioresour. Technol., 9, 157 (1991)
Cleland JL, Wang DIC, Bioresour. Technol., 274 (1990)
Creighton TE, "Process for the Production of a Protein", US Patent No. 4,977,248 (1990)
Stempfer G, Neugebauer BH, Rudolph R, Nat. Biotechnol., 14(3), 329 (1996)
Lee EK, Cho TH, Suh CW, Korean J. Biotechnol. Bioeng., 16(2), 146 (2001)
Psarras K, Ueda M, Yamamura T, Ozawa S, Kitajima M, Aiso S, Komatsu S, Seno M, Protein Eng., 11(12), 1285 (1998)
Yoon JM, Han SH, Kowon OB, Kim SH, Park MH, Kim BK, Life Sci., 16, 1435 (1999)
Hirata Y, Orth AN, J. Clin. Endocrinol. Metab., 48, 673 (1979)
Shapiro R, Riordan JF, Ballee BL, Biochemistry, 25(12), 3527 (1986)
Wu YN, Saxena SK, Ardelt W, Gadina M, Mikulshki S, Lorenzo CD, Alessio GD, Youle J, J. Biol. Chem., 270(29), 17476 (1995)
Soncin F, Strydom DJ, Shapiro R, J. Biol. Chem., 272(15), 9818 (1997)
Tice PA, Mazsaroff I, Line NT, Reginer FE, J. Chromatography, 410, 43 (1987)
Lee YS, "Fermentation and Purification Processes for Fusion Protein of Human Epidermal Growth Factor and Angiogenin from Recombinant E. coli", M.S. Thesis. Hanyang University, Dept. of Chemical Engineering, Korea (1999)
Lali A, Kaul R, Yu I, Mattiason B, Isolation and Purification, 2, 289 (1997)
Thommes J, Bader A, Halfar M, Karau A, Kula MR, J. Chromatography, 752, 111 (1996)
Smith MP, Bulmer M, Hjorth R, Titchener-Hooker NJ, "A Comparative Engineering Study of the Use of Expanded Bed and Packed Bed Routes for the Recovery of Labile Proteins from Crude Feedstocks", Proceedings of 5th World Congress of Chemical Engineering, 2, 565 (1996)
Chang YK, Chase HA, "Expanded Bed Adsorption for the Direct Extraction of Proteins", Separation for Biotechnology, 3, 106 (1994)
Kim CS, Lee EK, Process Biochem., 36, 111 (2000)
Carmeliet P, Jain RK, Nature, 407, 249 (2000)
Folkman J, Shing Y, J. Biol. Chem., 267(16), 10931 (1992)
Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Sohndel S, McCance SG, OReilly MS, Llinas M, Folkman J, J. Biol. Chem., 271(46), 29461 (1996)
Angles-Cano E, Rojas G, Biol. Chem., 383, 93 (2002)
Trieu VN, Uckun FM, Biochem. Biophys. Res. Commun., 257(3), 714 (1999)
Jang JH, Kim JS, Park EJ, Yeum JS, Jung SI, Korean Patent No. 10-2002-7003512 (2002)
Cho TH, Ahn SJ, Lee EK, Bioseparation, 10, 189 (2002)
MacDonald NJ, Murad AC, Fogler WE, Lu Y, Sim KL, Biochem. Biophys. Res. Commun., 264(2), 469 (1999)
Ellman GL, Arch. Biochem. Biophys., 82(1), 70 (1959)
Buchner J, Rudolph R, Bioresour. Technol., 9, 157 (1991)
Tang W, Sun ZY, Pannell R, Gurewich V, Liu JN, Protein Exp. Purif., 11(3), 279 (1997)
Park SJ, Kang R, Suh CW, Chai YG, Kwon OB, Park SK, Lee EK, Biotechnol. Bioprocess Eng, 7(1), 1 (2002)
Feuser J, Walter J, Kula MR, Thommes J, Bioseparation, 8, 99 (1999)
