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目的对两种长效干扰素α融合蛋白原料药的理化特性多项指标开展分析和进行比较。方法利用基因工程技术分别构建了可高效表达重组人血清白蛋白/干扰素α2a融合蛋白(rHSA/IFNα2a)或重组人血清白蛋白/干扰素α2b融合蛋白(rHSA/IFNα2b)的毕赤酵母工程菌。融合蛋白直接分泌到组分简单的无机盐培养基中,并经特别建立的高效分离纯化工艺进行纯化,随后对两个融合蛋白进行详尽的理化特性分析。结果获得的融合蛋白纯度在98%以上;N端前15个氨基酸序列与理论序列相同;质谱分子量分别为85640.8D和85781.5D;圆二色光谱分析比对蛋白空间构象未变;等电点pI约在5.1左右;为非糖基化蛋白;紫外光谱呈典型蛋白质光谱;自由巯基含量测定值为1.4;肽图批次间一致;肽质量指纹图谱可匹配分别达83%和82%;对制备的2个融合蛋白原料药中的细菌内毒素、宿主蛋白质、外源性DNA、甲醇和甘油的残留量检测符合标准要求;融合蛋白的免疫学鉴别为阳性;体外细胞生物比活性约为2.5×105IU/mg,并且2个融合蛋白生物比活性测定值无不同,由此,获得了符合临床用药标准的原料药。结论研究所获得的结果可以作为指导《注射用重组人血清白蛋白/干扰素α2a融合蛋白》和《注射用重组人血清白蛋白/干扰素α2b融合蛋白》2个新药的原料药生产质量标准的建立和检定方法的确定。
Objective To analyze and compare the physical and chemical properties of two long-acting interferon alpha fusion protein APIs. Methods Recombinant human serum albumin / interferon α2a fusion protein (rHSA / IFNα2a) or recombinant human serum albumin / interferon α2b fusion protein (rHSA / IFNα2b) . The fusion protein is directly secreted into the simple inorganic salt medium and purified by a specially established high efficient separation and purification process. The detailed analysis of the physical and chemical properties of the two fusion proteins is carried out. The results showed that the purity of fusion protein was over 98%. The first 15 amino acid sequences of N-terminal were the same as theoretical ones. The molecular weight of the fusion protein was 85640.8D and 85781.5D, respectively. The conformational space conformation of circular dichroism spectra was unchanged. About 5.1 or so; for non-glycosylated protein; UV spectrum showed a typical protein spectrum; free thiol content was determined to be 1.4; peptide batch batch consistency; peptide mass fingerprinting matched 83% and 82% respectively; Of the two fusion protein drug substance in the bacterial endotoxin, host protein, exogenous DNA, methanol and glycerol residues in line with the requirements of the standard; immunological identification of fusion protein was positive; in vitro cell biological activity of about 2.5 × 105IU / mg, and the two fusion protein biological activity measured no different values, thus obtaining the drug standards in line with clinical drug use. Conclusions The results obtained in this study can be used as a guide for the quality standards of APIs for the two new drugs, “recombinant human serum albumin / interferon alpha 2a fusion protein for injection” and “recombinant human serum albumin / interferon alpha 2b fusion protein for injection” Establishment and verification method determination.