重组人源化兔抗VEGF单克隆抗体质控方法与质量标准研究

目的: 建立重组人源化兔抗VEGF单抗的质控方法和质量标准。 方法: 利用HUVEC细胞增殖抑制试验测定重组人源化兔抗VEGF单抗的生物学活性;SDS-PAGE和SEC-HPLC测定纯度;胰酶酶切,HPLC测定其肽图;实时定量PCR检测CHO宿主DNA残留量;采用ELISA法分别测定VEGF结合力、残留ProA含量和残留菌体蛋白含量;水平等电聚焦电泳法测定等电点;其余检测项目按中国药典2010年版三部规定进行。 结果: 用建立的方法对重组人源化兔抗VEGF单抗的原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》、《人用单克隆抗体质量控制技术指导原则》和中国药典2010年版三部的要求。 结论: 建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于该类产品的常规检定。

Objective: To establish a quality control method and standard for recombinant humanized rabbit anti-VEGF mAb. Methods: Bioactivity of recombinant humanized rabbit anti-VEGF mAb was evaluated by determining its inhibitory effect on HUVEC proliferation.Purity of recombinant humanized rabbit anti-VEGF mAb was analyzed by SDS-PAGE and SEC-HPLC.The peptide map of recombinant humanized rabbit anti-VEGF mAb was obtained by trypsin digestion and HPLC assay.CHO residual DNA was detected by real-time PCR.The binding activity of recombinant humanized rabbit anti-VEGF Mab to human VEGF,residual protein A content and residual hose protein content were measured by ELISA.In addition,isoelectric focusing electrophoresis method was used for the determination of pI.Routine tests were all carried out according to Pharmacopoeia of Peoples' Republic of China(volume Ⅲ,2010 edition). Results: The stock solution and final product of recombinant humanized rabbit anti-VEGF mAb were evaluated by the established quality control method and standard.The results of all tests complied with the requirements of "guideline for quality control of recombinant DNA products for human use","guideline for quality control of monoclonal antibodies for human use" and "Pharmacopoeia of People's Republic of China"(volumeⅢ,2010 edition). Conclusion: The established methods and standards are safe and effective,which can be used for routine quality control of recombinant humanized rabbit anti-VEGF mAb.

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