康柏西普质量标准优化和提高

目的：为在更高水平上控制康柏西普质量，进一步确保产品的安全性和有效性，对其生物学活性、非还原SDS-PAGE纯度、唾液酸含量、糖基化项目的检测方法和不溶性微粒的质量标准进行提高。方法：建立和验证康柏西普3个质量控制方法，包括康柏西普生物学活性的荧光素酶报告基因分析（reporter gene assay，RGA）方法、唾液酸含量的反相高效液相色谱（reverse phase-high performance liquid chromatography，RP-HPLC）测定方法和糖谱的离子交换高效液相色谱（ion exchange-high performance liquid chromatography，IEX-HPLC）测定方法，并对非还原十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）纯度测定方法的凝胶浓度和上样量进行优化，按照2015年版《中华人民共和国药典》通则0903，采用光阻法测定不溶性微粒。结果：生物学活性测定中，RGA法在准确度、精密度和中间精密度等方面均优于人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVEC）增殖抑制法，且能够更好地反映康柏西普生物学活性的变化；非还原SDS-PAGE纯度检测中，4%~15%梯度胶较10%固定浓度凝胶对主带和杂带的分离度高，3 μg上样量时，能够更准确地测定康柏西普纯度；间苯二酚法测定唾液酸含量中，N-糖的非唾液酸单糖亦可与间苯二酚反应，产物在580 nm处有光吸收，从而影响检测结果，本研究建立的RP-HPLC法通过4，5-亚甲二氧基-1，2-邻苯二胺盐酸盐（4，5-methylenedioxy-1，2-phenylenediamine dihydrochloride，DMB）标记排除该影响，检测结果更准确；建立的IEX-HPLC糖谱测定方法可以有效控制影响康柏西普关键质量属性的N-糖基化类型；康柏西普成品的不溶性微粒检测结果能够满足USP<789>要求。结论：所建立/优化的质控方法和提高的质量标准可在更高水平上对康柏西普质量进行控制。

Objective:To improve the detection methods of the biological activity,the purity of non-reductive SDS-PAGE,the content of sialic acid,the glycosylation items and the specification of insoluble particles, to control the quality of Conbercept at a higher level and further ensure the safety and effectiveness of the product. Methods:Three quality control methods for Conbercept were established and validated,including reporter gene assay(RGA) for biological activity determination,reverse phase-high performance liquid chromatography(RP-HPLC) method for sialic acid content determination and ion exchange-high performance liquid chromatography(IEX-HPLC) method for glycan profile determination. The gel concentration and loading quantity of non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) were optimized for purity test. According to Chinese Pharmacopoeia (ChP) 2015 general chapter 0903,insoluble particles were determined by light blockage method. Results:Compared with human umbilical vein endothelial cells(HUVEC) proliferation inhibition assay,RGA showed a significant advantage in accuracy,precision and internal-precision. RGA could more accurately reflect the biological activity variation of the Conbercept products. Non reduced SDS-PAGE purity test using 4%-15% gradient gel concentration and 3 μg loading quantity showed improved resolution and better determination capacity for both main band and minor bands compared with those of 10% fixed gel concentration. In the sialic acid content determination,phenylenediol coloring method could be problematic since the non-sialic saccharides in N-linked oligosaccharides can react with phenylenediol resulting in a colored product that have absorbance at 580 nm which always leads to an over-estimated result. In this study,the RP-HPLC method established for sialic acid determination avoided the above interference and therefore it demonstrated better performance with improved accuracy. The established IEX-HPLC method can effectively control the type of N-glycosylation that affects the key quality properties of Conbercept,and the results of insoluble particle test of Conbercept could comply the requirements of USP<789>. Conclusion:The established/optimized quality control methods and improved specification in this study can facilitate more accurate and more effective quality control of Conbercept.

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