具创新分子结构的更优长效性重组人血清白蛋白/促红素融合蛋白的表达、制备和特性研究

摘要

Objective Studies on the compliance of recombinant human serum albumin/erythropoietin fusion protein (rHSA/EPO) manufacture process and on the physicochemical properties and propose as basis and technical indicators of quality as a long acting, bio-better innovative protein drug. Methods rHSA/EPO with long-acting and bio-better function was stably expressed in CHO cell line which constructed by genetically engineering technology. A manufacture process was established in a batch scale, suspension and growth in a serum-free medium. Then it was analyzed of the preparation process, detailed physicochemical properties and characteristics of technical quality indicators as a novel drug. Results The purity of rHSA/EPO protein reached more than 97%. Products of rHSA/EPO digested by 3 different binding sites of protease separately and was analyzed with peptide mass fingerprint by MALDI-TOF/TOF PMF and Nano LC-MS/MS with the overall amino acid sequence coverage of 100%. N-terminal amino acid sequence of the first 15 amino acids is identical to the theoretical sequence. The glycosylation sites of rHSA/EPO was positioned with its molecular weight (MW) in mass spectrometry as 93 kD, but the MW of the fusion protein without sugars was 84205 D similar with the theoretical MW of 84849.52 D. The fusion protein contained 1 free sulfhydryl groups, which indicated that there existed only 1 free cysteine in the structure. HSA and EPO on their two space conformation unchanged after a round of two color spectrum analysis. A plurality of charge isomers were in the range of pI 5.1 - 5.8 by a capillary electrophoresis with isoelectric focusing obtained (pI) as about pI 4.9. A typical protein spectrum was obtained by purple spectra. Residues of bacterial endotoxin, host protein, exogenous DNA were in line with the requirements of the regulations as drug raw materials. Immunological identification was positive with rhEPO which also expressed in CHO cells. Biological activity were determination with UT7 cellsin vitro and with micein vivo, but the activity were different. Thein vivo and in vitro bioassay method was used for different experimental purposes, and each also had a certain correlation coefficient. Conclusion The more comprehensive pharmacy data are obtained through studying on the physicochemical properties of the fusion protein. The results can be used as the guidance of the manufacture "Recombinant human serum albumin/erythropoietin fusion protein for injection" (CHO cells) on raw material medicine and preparation and show that the newly created molecular structure of rHSA/EPO (without a linker in between) is comply with the requirements as a bio-better novel protein drug.%目的：研究作为药物级别重组人血清白蛋白/促红素融合蛋白的生产制备工艺；开展详尽的融合蛋白理化特性研究；提出作为长效创新药物的质量基础和技术指标。方法利用基因工程技术构建1个能稳定表达全新分子结构和更优长效性的重组人血清白蛋白/促红素融合蛋白（rHSA/EPO）的基因工程 CHO细胞株；研究建立悬浮、无血清培养基，批次化、可线性放大的规模化生产工艺，并就分离纯化的融合蛋白进行详尽的理化特性分析。结果获得的 rHSA/EPO蛋白质单体纯度达到97%以上；经不同作用位点蛋白酶水解的 rHSA/EPO产物通过 MALDI-TOF/TOF质谱肽质量指纹谱和 Nano LC-MS/MS液质联用串联质谱检测得到总序列覆盖率100%；N-端氨基酸序列前15个氨基酸与理论序列相符；rHSA/EPO为糖基化蛋白，定位了糖基化位点；原液中融合蛋白的质谱分子量测定值为93 kD；酶深度脱糖后的质谱分子量测定结果为84205 D，与理论分子量84849.52 D值相近；融合蛋白含有1个自由巯基，表明仅有1个游离半胱氨酸存在；经圆二色光谱分析比对表明,HSA和 EPO各自的空间二级构象未变；毛细管电泳测定有多个电荷异构体范围在 pI 5.1~5.8，等电聚焦获得的等电点值（pI）约为 pI 4.9；紫外光谱呈现典型蛋白质光谱特征；原料药中的细菌内毒素、宿主蛋白质、外源性 DNA的残留量均符合药物原料药的要求。免疫学鉴别为阳性；小鼠体内生物比活性测定结果值为0.9×105 IU/mg；体外 UT7细胞法与小鼠体内生物活性测定较大差异，但体内与体外生物活性测定方法可用于不同试验目的，且相互间也具有一定的相关系数。结论理化特性研究获得较为全面的药学数据，其结果可作为指导“注射用重组人血清白蛋白/促红素融合蛋白（CHO细胞）”原料药的质量标准和制造检定规程的基础，并显示出原创的 rHSA/EPO（之间无连接肽）具有创新性，符合作为药物的更优分子结构要求。