Chin J Biotech 2008, January 25; 24(1): 159-163 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved. α2b 1, 2,3, 3, 3, 3, 3, 3 1, 310006 2, 310031 3, 310018 : 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 310006, China 2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, China 3 Hangzhou Jiuyuan Gene Engineering Co., Ltd, Hangzhou 310018, 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: +86-571-87061501; E-mail: xxrd@163.com
160 ISSN1000-3061 CN11-1998/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 100000 EU/mL, rhsa-ifnα2b, [9 14], rhsa-ifnα2b, rhsa-ifnα2b 1 1.1 rhsa-ifnα2b ( ); ( ); (); 1.2 Pharmacia AKTA explorer100 Pharmacia Blue-Sepharose; SOURCE 15 ISO; Q Sepharose F.F.; Sephadex G25 Coarse 1100 ; Grace Vydac C 4 1.3 1.3.1 2005 E 1.3.2 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 1.4 1.4.1 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 1.4.2 SOURCE 15 ISO A (NH) 2 SO 4 110 ms/cm, 110 ms/cm (NH) 2 SO 4 ( ), A,, 10%- 90% B, 2
: α2b 161 2 2.1 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 1.4.3 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) 86530 10547 Purity/% 62.1 86.2 2.2 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) 10547 597 Purity/% 86.2 95.7 3 Q Sepharose F.F. Fig. 3 Q Sepharose F.F. ion exchange chromatography 1.4.4 Sephadex G25 Coarse C 10 mmol/l (ph= 6)() Sephadex G25 Coarse, D, 4 2.3 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) 597 5 Purity/% 95.7 97.2 4 Sephadex G25 Coarse Fig. 4 Sephadex G25 Coarse gel filtration chromatography 2.4 Sephadex G25 Coarse 4, Sephadex G25 Coarse
162 ISSN1000-3061 CN11-1998/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) 5 1 3 Purity/% 97.2 98.1,, [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): 9 14. [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: S527 536. [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): 419 432. [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): 173 178.,,,. α2b., 2006, 22(2): 173 178. [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: 10.1016 /j.ejpb.2007.02. 015. [6] Gutterman JU. Cytokine therapeutics: lessons from interferon alpha. Proc Natl Acad Sci USA. 1994, 91(4): 1198 1205. [7] Garwala SS, Kirkwood JM. Potential uses of interon alpha 2 as adjuvant therapy in cancer. Ann Surg Oncol, 1995, 2 (4): 365 371. [8] Nation Pharmacopeia Committee ed. Pharmacopoeia of the
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