重组CTLA4-Fc融合蛋白的质量研究

发布时间：2018-05-10 22:26

本文选题：融合蛋白 + 细胞毒性T淋巴细胞相关抗原　；参考：《中国药学杂志》2017年20期

【摘要】：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EC_(50)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p I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 establish a method for quality control of recombinant cytotoxic T lymphocyte associated antigen (4(CTLA4)-Fc) fusion protein. Methods the biological activity of recombinant CTLA4-Fc fusion protein was detected by reporter gene method based on CTLA4/IL-2-Luciferace/Jurkat cells (Jurkat cells), and the binding activity of recombinant CTLA4-Fc fusion protein to B7 was determined by surface plasmon resonance assay (SPR). The purity was analyzed by capillary electrophoresis sodium dodecyl gel electrophoresis (Capillary electrophoresis sodium dodecyl sulfate CE-SDS), the charge heterogeneity was analyzed by isoelectric focusing electrophoresis (isoelectric focusing IEF), the identification test was carried out by peptide map method and micellar electrokinetic chromatographyMEKC by capillary micellar electrokinetic chromatography (MEC). The quality of recombinant CTLA4-Fc fusion protein was studied by reversed-phase high performance liquid chromatography (RP-HPLC) and hydrophilic high performance liquid chromatographyHILIC-HPLC. the content of sialic acid was determined by reversed-phase high performance liquid chromatography (RP-HPLC). Results the biological activity of the recombinant CTLA4-Fc fusion protein was 0.40 卤0.08 渭 g / mL, RSD = 20.00%. The relative binding activity of B7 to B7 was determined by BIAcore. The RSD was 0.14 卤0.12. The purity of CE-SDS was 0.01; the purity of CE-SDS was 100.00 卤0.005; the purity of non-reducing CE-SDS was 98.50 卤0.17; the purity of CE-SDS was 0.1850 卤0.17; the purity of CE-SDS was 100.00 卤0.005; the purity of non-reducing CE-SDS was 98.50 卤0.17; the purity of CE-SDS was 0.01; the purity of reductive CE-SDS was 100.00 卤0.005; and the purity of non-reducing CE-SDS was 98.50 卤0.17. The pI range of the main zone was 4.6 ~ 5.9.The recombinant CTLA4-Fc fusion protein and its variants could be effectively identified by MEKC in the same way as the reference protein in the peptide map method. The content of sialic acid was 10.16 卤0.41)mol/mol protein and the content of main sugar forms was 7.5G0F was 7.5. G1F was 15.0and G2F was 17.1G1FS1 was 4.2G2FS1 was 19.5G2FS2 was 9.6. Conclusion A method for evaluating the key quality attributes of recombinant CTLA4-Fc fusion protein is established, which provides a reference for the quality control of antibody fusion protein biological products.
【作者单位】：中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室;
【基金】：国家重点研发计划课题资助项目(2016YCF1200904)
【分类号】：R392-33

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