Purification Process Study And Quality Analysis Of Recombinant Human IFN-β1a

Interferon-β is the multifunctional protein of high activity, it is induced andsynthesized by the biological cells in response to the interferon inducer. It isinvolved in antiviral, antiproliferative and immunomodulatory responses, acting toboth maintain homeostasis and in host defense. It has been found, they of basic andclinical research of cytokines. In this study, the human Interferon β1a was producedby gene-recombination CHO(China hamster ovary) cells cultured on suspendingmicro-carriers in the15L bioreactor, the process of protein purification andpurification methods were set up for rhIFN-β1a and quality analysis.The cell culture harvest was concentrated by ultrafiltration to5times, and therecombinant protein was purified by blue sepharose6Fast Flow affinitychromatography compared with metal chelate affinity chromatography for theprimary purification. We explored the optimal conditions of blue sepharose6FastFlow to purify the rhIFN-β1a by investigating the salt concentration and polarityreductant concentration of elution buffer. The results showed that the optimalpurification conditions were: eluent Ⅰ:20mmol/L Tris-HCl (pH7.2),2mol/L NaCl,eluent Ⅱ:20mmol/ml Tris-HCl (pH7.2),2mol/L NaCl,50%propylene glycol. Weexamined the imidazole concentration of zinc chelate affinity chromatography tooptimize the purification conditions, the results showed the optimal purificationcondition was: the eluent was composed of20mmol/L sodium phosphate (pH7.4),30mmol/L imidazole-0.5mol/L NaCl. The SDS-PAGE indicated that the bluesepharose FF affinity chromatography was better than the zinc chelate affinitychromatography to purify the rhIFN-β1a. Then the rhIFN-β1a was purified by sephadex G-25and ion exchange chromatography, the eluent of CM cation exchangechromatography was20mmol/L citric acid-sodium citrate (pH5.0),1mol/L NaCl,SDS-PAGE indicated that the purity was about95%, the average specific activity ofrhIFN-β1a was1.11×108IU/mg, and the average activity recovery was17.94%. Thescale purification process was magnified20times to the pilot scale, the resultsshowed that the blue sepharose FF affinity chromatography was reproducible andbasically consistent within the small test results. We changed the Q anion exchangechromatography as the refine purification with Tris-HCl (pH8.0) buffer, SDS-PAGEindicated that the purity was about95%, the average specific activity of rhIFN-β1awas4.08×107IU/mg, and the average activity recovery was39.7%.The analytical methods included bioactivity determined by VSV virusinhibition method, protein concentration determined by LOWRY, isoelectric pointdetermined by isoelectric focusing electrophoresis, purity determined by SDS-PAGEand HPLC, immunocompetence assayed by Western blot, molecular massdetermined by mass spectra. Western blot showed that the specificity of rhIFN-β1aexpressed by CHO was consistent with the natural product. The molecular mass was20.4kDa, the isoelectric point was pH6.6, which met the quality standards. Theprocess of protein purification and purification methods were set up for rhIFN-β1a.All of these laid the foundation to establish the large-scale and purification processof rhIFN-β1a and to obtain rhIFN-β1a which meet the quality standards.