RESUMO
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, nonreduced 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.