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目的建立重组人血管内皮生长因子(vascular endothelial growth factor,VEGF)抑制剂理化对照品质控方法及质量标准。方法利用HEK293细胞增殖抑制法测定重组人VEGF抑制剂理化对照品生物学活性;紫外分光光度法测定蛋白含量;质谱法进行相对分子质量测定、液质肽图及糖基化位点分析、二硫键分析、N-寡糖糖型及比例分析;SDS-PAGE及SEC-HPLC法分析纯度;等点聚焦法进行电荷异质性分析;Edman降解法进行N-末端氨基酸序列分析。结果重组人VEGF抑制剂理化对照品各项检定指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》三部(2015版)相关要求。结论建立的质控方法和质量标准符合理化对照品的相关规定,具有保证该理化对照品结构正确、质量可控的特点。检定合格的理化对照品可用于该类产品的常规检定。
Objective To establish a method and quality standard for quality control of recombinant human vascular endothelial growth factor (VEGF) inhibitor. Methods HEK293 cell proliferation inhibition assay was used to determine the biological activity of recombinant human VEGF inhibitor. The UV-Vis spectrophotometry was used to determine the protein content. The relative molecular mass was determined by mass spectrometry. The peptide map and glycosylation site analysis, Key analysis, N-oligosaccharide glycoforms and ratio analysis; SDS-PAGE and SEC-HPLC analysis of purity; isoelectric focusing method of charge heterogeneity analysis; Edman degradation method N-terminal amino acid sequence analysis. Results The calibration indexes of the recombinant human VEGF inhibitor physical and chemical reference substance all met the relevant requirements of the Guiding Principles for Quality Control of Human Recombinant DNA Products and the Third Edition of Chinese Pharmacopoeia (2015 Edition). Conclusion The established quality control methods and quality standards are in line with the relevant provisions of physical and chemical reference materials, with the characteristics of ensuring that the physical and chemical reference materials have the correct structure and quality control. Qualified physical and chemical control can be used for routine testing of such products.