美妥珠单抗(HcHAb18)质量标准的建立及理化对照品结构确证

Development of standardized methods for quality control of humanized monoclonal antibody HcHAb18 and structure confirmation of its reference substance for physicochemical analysis

目的:建立美妥珠单抗的质控方法和质量标准。方法:利用乳酸脱氢酶释放试验检查外周血分离的单核细胞对人肺腺癌A549细胞的杀伤效应测定美妥珠单抗的生物学活性;非还原CE-SDS和SECHPLC测定纯度;胰酶酶切后HPLC测定其肽图;实时定量PCR检测CHO宿主DNA残留量;采用ELISA法分别测定抗原CD147结合力、残留ProA含量和残留宿主蛋白含量;CE-SDS法测定等电点;液质联用技术进行相对分子质量、肽图及糖型分析;其余检测项目按《中华人民共和国药典》2010年版三部规定进行。结果:用建立的方法对美妥珠单抗的成品进行了检定,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《中华人民共和国药典》2010年版三部的要求;对理化对照品进行了一级结构确证,相对分子质量、氨基酸序列和糖型分析结果均与理论预测相符。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 the quality control system for the humanized monoclonal antibody HcHAb18. Methods： The potency of HcHAb18 was determined by ADCC（ antibody dependent cell-mediated cytotoxicity） assay with PBMC（ peripheral blood mononuclear cell） and A549 cells. The purity of the fusion protein was analyzed by SEC-HPLC and CE-SDS,respectively. The peptide mapping was analyzed by HPLC with trypsin digestion. The residue host cell DNA content of CHO cells was determined real-time PCR. The binding activity to antigen CD147,the residue protein A content and host cell protein content of CHO were detected by ELISA,respectively. The PI（ isoelectric point） was obtained by CE-SDS. The molecular mass,peptide mass spectra and the glycoform were analyzed by LC-ESI-MS technique. Other control tests were performed according to the requirements in Chinese Pharmacopoeia（ Volume III,2010 edition）. Results： Control tests were performed on one lot of final products of HcHAb18 monoclonal antibody by the developed methods. The results showed that all the indexes met the requirements in Guideline for Quality Control of Recombinant DNA Products for Human Use and Chinese Phar-macopoeia（ Volume III,2010 edition）. Primary structure confirmation of the reference substance for physicochemical analysis showed that the molecular mass,amino acid sequence and the glycoform modification are consistent with the theoretical predictions. Conclusion： The developed methods and standard may assure the safety,effectiveness and controllability of the humanized monoclonal antibody HcHAb18,which might be used for the routine quality control of products of the same kind.