新型肝靶向干扰素融合蛋白的质量标准研究

Study on the quality control standards of recombinant interferon-circumsporozoite protein

目的建立新型肝靶向干扰素(interferon-circumsporozoite protein,IFN-CSP)的质量标准。方法采用细胞病变抑制法进行生物学活性测定;RP-HPLC法及非还原SDS-PAGE法进行纯度分析;BCA法、ELISA法、抗生素微生物学检定法、凝胶法分别测定蛋白质含量、宿主菌蛋白质残留量、氨苄青霉素残留量和细菌内毒素含量;紫外光谱法确定吸收峰位置;液质联用技术进行肽指纹图谱分析;Edman降解法检测N-端氨基酸序列。结果 IFN-CSP的生物学活性、纯度、蛋白质含量、宿主菌蛋白质残留量、氨苄青霉素残留量及细菌内毒素的检验结果均在质量标准控制范围内,符合《人用重组DNA制品质量控制技术指导原则》和2015年版《中国药典》对重组干扰素α2b质量标准的要求。紫外光谱检测吸收峰约在225 nm处,液质联用分析肽指纹图谱覆盖率达80%,N-端氨基酸序列结果显示其N端前15个氨基酸与天然蛋白质一致。结论 IFN-CSP符合基因工程药物质量标准的要求,为其进一步应用开发提供了依据。

Objective To establish quality control standards for the recombinant interferon-circumsporozoite protein（ IFN-CSP）. Methods Cytopathic effect inhibition was used to analyze the biological activity of the IFN-CSP. The protein content was determined by BCA assay. The purity was analyzed by RP-HPLC and SDS-PAGE. ELISA was used to analyze the residual protein of host bacteria. Ampicillin residues were detected by microbiological assay. The content of endotoxin Limulus gel was detected by the Limulus reagent gel method. The absorption peak position was determined by ultraviolet spectrum. The peptide mass spectra were analyzed by LC-ESI-MS technique. The IFN-CSP amino acid sequence was detected by Edman degradation method. Results According to the requirement of interferon α2b in the Chinese Pharmacopoeia,the quality standard of IFN-CSP was studied. The biological activity, protein content, specific activity,purity, the host protein residue, ampicillin residual and the bacterial endotoxin of IFN-CSP met the requirements in the Guideline for Quality Control of Recombinant DNA Products for Human Use and Chinese Pharmacopoeia（ Volume Ⅲ,2015 edition）. The UV absorption peak was at about 225 nm. The LC-MS analysis of peptide fingerprint coverage rate was 80%. The N-terminal amino acid sequence of the first 15 amino acids was consistent with the native protein. The results showed that all the indexes met the Chinese Pharmacopoeia requirements. Conclusion The developed standard may assure the safety,effectiveness and controllability of the IFN-CSP,which might be used for the routine quality control.