摘要
利用分子排阻高效液相色谱(SEC-HPLC)、非还原毛细管电泳(CE-SDS)并结合液质联用(LC-MS)、抗体依赖细胞介导的细胞毒活性检测(ADCC)等技术对重组人源化单克隆抗体(rhumAb1)在高温(40℃)条件下产生的分子大小变异体进行研究。本研究鉴定了rhumAb1在SEC-HPLC分析中的4个分子大小变异体(SEC-1~SEC-4)和非还原CE-SDS中的7个主要变异体(NR-1~NR-7)的组成和结构。发现主要的低分子量变异体(片段)是由于重链的铰链区断裂所致,断裂位于铰链的Ser221-Cys-Asp-Lys-Thr-His-Thr-Cys228区域,其中C222-D223和H226-T227为主要断裂位点。ADCC活性检测表明,rhumAb1分子大小变异体(二聚体和片段)显著降低了产品的活性。该研究为rhumAb1产品的质量标准和相应的质控策略的制定奠定了基础,也为其他抗体药物的质量控制提供了参考。
The composition and potency of the high temperature(40 ℃) stress induced size variants of a recombinant humanized monoclonal antibody(rhumAb1) were characterized by means of SEC-HPLC, non- reduced CE-SDS, liquid chromatography coupled with mass spectrometry(LC-MS) and antibody dependent cell-mediated cytotoxicity(ADCC) assay. The molecular masses of the four size variants(SEC-1-SEC-4) separated by SEC-HPLC and seven size variants(NR-1-NR-7) detected by non-reduced CE-SDS were all characterized by LC-MS. The major low molecular weight variants were generated due to the hinge region fragmentation of heavy chain. The hinge region cleavage was found mainly in the Ser221-Cys-Asp-Lys-Thr- His-Thr-Cys228 sequence, in which C222-D223 and H226-T227 were the major cleavage sites. The size variants of rhumAb1, namely dimer and fragments, have significantly reduced ADCC activity in comparison with the intact rhumAb1 drug product. This study provided insights into the stability profiling for rhumAb1 drug product. The study protocols presented here may be applicable to the analytical characterization of other monoclonal antibody-based therapeutic products.
引文
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