目的 建立重组细胞毒性T淋巴细胞相关抗原4(CTLA4)-Fc融合蛋白的质控方法。方法 应用基于CTLA4/IL-2-Luciferace/Jurkat细胞(简称Jurkat细胞)的报告基因法检测重组CTLA4-Fc融合蛋白的生物学活性;表面等离子体共振(surface plasmon resonance,SPR)测定重组CTLA4-Fc融合蛋白与B7的结合活性测定法;分子排阻高效液相色谱(size exclusion high performance liquid chromatography,SEC-HPLC)、毛细管凝胶电泳(capillary electrophoresis sodium dodecyl sulfate,CE-SDS)进行纯度分析;等电聚焦电泳(isoelectric focusing,IEF)进行电荷异质性分析;肽图法和毛细管胶束电动色谱(micellar electrokinetic chromatography, MEKC)进行鉴别试验;反相高效液相色谱(reversed phase-high performance liquid chromatography,RP-HPLC)进行唾液酸含量测定;亲水高效液相色谱(hydrophilic high performance liquid chromatography,HILIC-HPLC)进行N糖型含量测定等,对重组CTLA4-Fc融合蛋白进行了质量研究。结果 重组CTLA4-Fc融合蛋白的生物学活性EC50值为(0.40±0.08) μg·mL-1,RSD为20.00%;BIAcore测定与B7相对结合活性为参比品的(91.72±0.12)%,RSD为0.14%;SEC-HPLC分析的纯度为(99.09±0.01)%,RSD为0.01%;还原CE-SDS分析的纯度为(100.00±0.00)%,非还原CE-SDS单体为(98.50±0.17)%,RSD为0.18%;IEF等电点主区带pI范围为4.6~5.9;肽图法中供试品与参比品一致;MEKC可对重组CTLA4-Fc融合蛋白及其变体进行有效鉴别;唾液酸含量为(10.16±0.41)mol/mol蛋白,RSD为4.07%;N糖主要糖型的含量:G0F含量为7.5%,G1F含量为15.0%,G2F含量为17.1%,G1FS1含量为4.2%,G2FS1含量为19.5%,G2FS2含量为9.6%。结论 建立了重组CTLA4-Fc融合蛋白的关键质量属性评价方法,上述方法为抗体融合蛋白类生物制品的质量控制提供参考。
Abstract
OBJECTIVE To develop the quality control methods of a recombinant CTLA4-Fc fusion protein. METHODS Mehtods for quality research of CTLA4-Fc fusion protein were established, such as reporter gene assay for bioassay based on CTLA4/IL-2-Luciferace/Jurkat cells, binding activity with B7 by SPR, purity by SEC-HPLC and CE-SDS, charge heterogeneity analysis by IEF, identity by peptide mapping and MECK, determination of sialic acid by RP-HPLC and content analysis of N-glycan by HILIC-HPLC. RESULTS The bioactivity EC50 of CTLA4-Fc fusion protein was (0.40±0.08) μg·mL-1, and the RSD was 20.00%. The relative binding activity with B7 by BIAcore test was (91.72±0.12)%,and the RSD was 0.14%. The purity determined by SEC-HPLC was(99.09±0.01)%,and the RSD was 0.01%. The purity determined by reduced CE-SDS was(100±0.00)%. The purity determined by non-reduced CE-SDS was(98.50±0.17)%, and the RSD was 0.18%. The pI of its major zone ranged from 4.6 to 5.9, which was consistent with the reference substance in peptide mapping. CTLA4-Fc fusion protein and its variant could be identified by MECK. The content of sialic acid determined by RP-HPLC was (10.16±0.41)mol/mol protein and the RSD was 4.07%. The contents of major N-glycoforms were as follows:G0F was 7.5%,G1F was 15.0%,G2F was 17.1%,G1FS1 was 4.2%,G2FS1 was 19.5% and G2FS2 was 9.6%. CONCLUSION The evaluation methods for critical quality attribute of CTLA4-Fc fusion protein were been initially established and provide reference for the quality control of this product.
关键词
融合蛋白 /
细胞毒性T淋巴细胞相关抗原4 /
质量控制 /
生物学活性
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Key words
fusion protein /
CTLA-4 /
quality control /
bioassay
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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
国家重点研发计划课题资助项目(2016YCF1200904)
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