目的 为了对产品质量进行有效的控制,根据《中国药典》要求,对重组人白介素-15理化对照品进行分析鉴定。方法 参照2010年版《中国药典》三部,测定该样品的生物学活性、蛋白含量、纯度、等电点等指标;通过测定N-末端氨基酸序列、质谱相对分子质量与液质肽图,确证其一级结构。结果 该样品的比活性为1.03×107 IU·mg-1;蛋白含量为(0.879±0.065) mg·mL-1;非还原SDS-PAGE纯度为100.0%;分子排阻色谱纯度为100.0%;等电点测定结果为5.2;以上结果均符合质量标准要求。实测质谱相对分子质量(12 900.80)与理论值(12 900.71)一致;氨基酸序列与理论一致,氨基酸覆盖率100%;二硫键连接方式为Cys36-Cys86、Cys43-Cys89,与文献报道相符。结论 该理化对照品质量合格、氨基酸序列正确,可用于白介素-15的常规质量控制。
Abstract
OBJECTIVE To evaluate the reference standard of recombinant human interleukin-15 (IL-15) for the effective quality control of IL-15 products according to the requirements of Chinese Pharmacopeia.METHODS The biological activity, concentration, purity, and isoelectric point of IL-15 were tested according to Chinese Pharmacopeia (volume Ⅲ, 2010 edition). The primary structure was confirmed by analyzing the N-terminal amino acid sequence and relative molecular mass and peptide mass mapping. RESULTS The measured biological activity of IL-15 was 1.03×107 IU·mg-1, the content was (0.879±0.065) mg·mL-1, the purities tested by SDS-PAGE and SEC-HPLC were all 100%, and the isoelectric point was 5.2, which all met the criteria specified in the quality standard. The observed relative molecular mass, 12 900.80, was consistent with theoretical value (12 900.71). Its amino acid sequence was verified with coverage of 100%. The disulfide bonds were identified to to be Cys36-Cys86/Cys43-Cys89, which was in accordance with the published papers. CONCLUSION This reference standard, which is qualified and has correct amino acid sequence, can be used for the routine quality control of IL-15.
关键词
白介素-15 /
理化对照品 /
氨基酸序列 /
二硫键
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Key words
interleukin 15 /
reference standard /
amino acid sequence /
disulfide bond
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中图分类号：
R917
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参考文献
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脚注
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基金
十三五科技重大专项课题资助项目(2015ZX09501008)
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