人源化抗表皮生长因子受体单克隆抗体质控方法的建立

目的建立人源化抗表皮生长因子受体(Epithelial growth factor receptor,EGFR)单克隆抗体的质控方法。方法采用体外细胞生长抑制试验测定人源化抗EGFR单抗的生物学活性;质谱法和还原型SDS-PAGE法测定其准确相对分子质量;去封闭后用Edman降解法测定其N-末端氨基酸序列;SDS-PAGE和分子排阻高效液相色谱(SEC-HPLC)法测定其纯度;反向液相色谱(RP-HPLC)法测定其肽图;定量PCR法和狭缝杂交法测定其外源性DNA残留量;其余项目按《中国药典》三部(2010版)要求进行。结果人源化抗EGFR单抗原液和成品的相对生物学活性分别为(100±20)%和(94±14)%;质谱法测得该单抗参考品轻、重链的相对分子质量分别为23 370.75和50 341.00,相对误差分别为0.005%和0.006%;还原型SDS-PAGE测得该单抗轻、重链的相对分子质量为27 000和53 400;轻、重链N-末端氨基酸序列均与理论一致;单抗原液的还原型SDS-PAGE纯度为98.5%;SEC-HPLC原液纯度为(98.39±0.05)%和成品SEC-HPLC纯度为(98.17±0.04)%;肽图图谱与理化参考品一致;定量PCR测得其外源DNA残留量小于100 pg/剂量;其他各项指标均符合《中国药典》三部(2010版)要求及其他相关要求。结论初步建立了人源化抗表皮生长因子受体单克隆抗体的质控方法,可用于该制品的常规质量控制。

Development of a method for quality control of humanized monoclonal antibody against epithelial growth factor receptor

Objective To develop a method for quality control of humanized monoclonal antibody(McAb) against epithelial growth factor receptor(EGFR).Methods Humanized McAb against EGFR was determined for biological activity by in vitro cell growth inhibition test,and for relative molecular mass by mass spectrometry and reduced SDS-PAGE.After deblocking by Edman degradation,the amino acids at N-terminus of the McAb were sequenced,while the peptide map was analyzed by RP-HPLC,and the residual exogenous DNA was determined by quantitative PCR and slot hybridization.Other tests were carried out according to requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).Results The relative biological activities of bulk and final product of McAb against EGFR were(100 ± 20) % and(94 ± 14) % respectively.The relative molecular masses of light and heavy chains of the McAb reference measured by ESI-MS were 23 370.75 and 50 341.00,with relative errors of 0.005% and 0.006%,while those measured by reduced SDS-PAGE were 27 000 and 53 400,respectively.All the amino acid sequences at N-terminuses of light and heavy chains were consistent with those in theory.The purity of bulk of the McAb were 98.5% by reduced SDS-PAGE and(98.39 ± 0.05)% by SEC-HPLC.However,the purity of final product of the McAb determined by SEC-HPLC was(98.17 ± 0.04)%.The peptide map was consistent with that of reference.The residual exogenous DNA content determined by quantitative PCR was less than 100 pg / dose.Other quality indexes met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition) and other relevant requirements.Conclusion A method for quality control of McAb against EGFR was successfully developed,which might be used for the routine quality control of the product.