Removing Endotoxin from rhsa-ifnα2b by Chromatography

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1 Chin J Biotech 2008, January 25; 24(1): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved. α2b 1, 2,3, 3, 3, 3, 3, 3 1, , , : α2b Blue-sepharose SOURCE 15 ISO Q Sepharose F.F.Sephadex G25 Coarse ; RP-HPLC, 1 EU/mg, 99.9%α2b :, α2b,, Removing Endotoxin from rhsa-ifnα2b by Chromatography Xiangrong Xu 1, Guochang Ma 2,3, Changmei Wang 3, Jianfeng Shan 3, Ling Sheng 3, Yanshan Huang 3, and Jinbao Zhou 3 1 Gynecological Hospital, School of Medicine Zhejiang University, Hangzhou , China 2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou , China 3 Hangzhou Jiuyuan Gene Engineering Co., Ltd, Hangzhou , China Abstract: We used chromatography to remove endotoxin during the purification of rhsa-ifnα2b (recombinant human serum albumin-interferon alpha 2b, rhsa- IFNα2b). Affinity chromatography of Blue-sepharose, hydrophobic interaction chromatography of SOURCE 15 ISO, ion exchange chromatography of Q Sepharose Fast Flow and gel filtration of sephadex G25 Coarse were applied consequently. The endotoxin levels were measured by Limulus Amebocyte Lysate gel-clot assay. Protein purity and concentration were determined by RP-HPLC. Up to 99.9% endotoxin of rhsa-ifnα2b was removed and to a concentration of 1 EU/mg. This process was effective to purify rhsa-ifnα2b and remove exdotoxin simultaneously. Key words: HSA, IFNα2b, column chromatography, endotoxin (Endotoxin)(Lipopolysac- charide, LPS),, Received: April 19, 2007; Accepted: June 4, 2007 Corresponding author: Xiangrong Xu. Tel: ; xxrd@163.com

2 160 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1 [1],,,,,,, [2],,,,, A,,,, [3], α2b (recom- binant human serum albumin-interferon alpha 2b, rhsa- IFNα2b) [4,5], [6,7], [8], rhsa-ifnα2b 10 EU( rhsa-ifnα2b 2 mg/, 5 EU/mg ) rhsa-ifnα2b EU/mL, rhsa-ifnα2b, [9 14], rhsa-ifnα2b, rhsa-ifnα2b rhsa-ifnα2b ( ); ( ); (); 1.2 Pharmacia AKTA explorer100 Pharmacia Blue-Sepharose; SOURCE 15 ISO; Q Sepharose F.F.; Sephadex G25 Coarse 1100 ; Grace Vydac C E RP-HPLC RP-HPLC C4 0.8mL/min; 214 nm; Solvent A0.1% TFA; Solvent B20% H 2 O-90% ACN-0.1% TFA; 0~35min0~90% B Blue-Sepharose HSA-IFNα2b, 20mmol/L (ph 6)() Blue-Sepharose,, 1.5mol/L NaCl A, 1 1 Blue-sepharose Fig. 1 Blue-sepharose affinity chromatograph SOURCE 15 ISO A (NH) 2 SO ms/cm, 110 ms/cm (NH) 2 SO 4 ( ), A,, 10%- 90% B, 2

3 : α2b Blue-Sepharose 1, Blue-Sepharose HSA-IFNα2b, HSA-IFNα2b Blue-Sepharose, 1 Blue-sepharose Table 1 The results of Blue-sepharose affinity chromatograph 2 SOURCE 15 ISO Fig. 2 SOURCE 15 ISO hydrophobic chromatography Q Sepharose F.F. B 5 ms/cm, Q Sepharose F.F.,, 0.5 mol/l NaCl C, 3 Detection item Fermentation broth Target peak A Endotoxin/(EU/mg) Purity/% SOURCE 15 ISO SOURCE 15 ISO,,,, 2 2 SOURCE 15 ISO Table 2 The results of SOURCE 15 ISO hydrophobic chromatography Detection item Target peak A Target peak B Endotoxin/(EU/mg) Purity/% Q Sepharose F.F. Fig. 3 Q Sepharose F.F. ion exchange chromatography Sephadex G25 Coarse C 10 mmol/l (ph= 6)() Sephadex G25 Coarse, D, Q Sepharose F.F.,, Q Sepharose F.F., ( 100 ), 3 3 Q Sepharose F.F. Table 3 The results of Q Sepharose F.F. ion exchange chromatography Detection item Target peak B Target peak C Endotoxin/(EU/mg) Purity/% Sephadex G25 Coarse Fig. 4 Sephadex G25 Coarse gel filtration chromatography 2.4 Sephadex G25 Coarse 4, Sephadex G25 Coarse

4 162 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1 4 Sephadex G25 Coarse Table 4 The results of Sephadex G25 Coarse filtration chromatography Detection item Target peak C Target peak D Endotoxin/(EU/mg) Purity/% ,, [15],,, (--- ),, Blue-Sepharose,,,, SOURCE 15 ISO,, ( ),,, Q Sepharose F.F.,, rhsa-ifnα2b Q Sepharose F.F.,, (1M ) (1M ),,,,,, Sephadex G25 Coarse, Sephadex G25 Coarse,, rhsa-ifnα2b, Sephadex G25 Coarse,, rhsa-ifnα2b rhsa-ifnα2b,,, REFERENCES [1] Hase S, Hofstad T, Rietschel ET. Chemical structure of lipid A component of lipopolysaccharides from Fusobacterium nucleatum. J Bacteriol. 1977, 129 (1): [2] Rietschel ET, Brade L, Brandenburg K, et al. Chemical structure and biologic activity of bacterical and synthetic lipid A. Rev Infect Dis. 1987, 9 Suppl 5: S [3] Hirayama C, Sakata M. Chromatographic removal of endotoxin from protein solutions by polymer particles. J Chromatogr Analyt Technol Biomed Life Sci. 2002, 781 (1-2): [4] Chang SH, Gong X, Yang ZY, et al. Expression in Pichia pastoris and properties of human serum albumin-interferon α2b chimera. Chinese Journal of Biotechnology, 2006, 22(2): ,,,. α2b., 2006, 22(2): [5] Huang YS, Chen Z, Yang ZY, et al. Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris. European Journal of Pharmaceutics and Biopharmaceutics (2007), doi: /j.ejpb [6] Gutterman JU. Cytokine therapeutics: lessons from interferon alpha. Proc Natl Acad Sci USA. 1994, 91(4): [7] Garwala SS, Kirkwood JM. Potential uses of interon alpha 2 as adjuvant therapy in cancer. Ann Surg Oncol, 1995, 2 (4): [8] Nation Pharmacopeia Committee ed. Pharmacopoeia of the

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