A single column multimodal cation exchange process for removal of half antibody, homodimer and light chain mispaired product-related impurities from a bispecific antibody.

During synthesis of IgG-like bispecific antibodies (bsAbs), product-related impurity formation via the mispairing of heavy chains and light chains (LCs) is common despite protein engineering efforts to mitigate these species. These mispaired impurities have similar biophysical characteristics to the target bsAb and are often difficult to remove with conventional platform processes. In this paper we demonstrate the utility of Capto MMC ImpRes for removing ½ IgG, homodimer and LC-mispair product-related impurities from an asymmetric IgG-like bsAb. Screening experiments from pH 5.0 to 9.5 showed that the ½ IgG and homodimer impurities eluted at pH 9.5, while the LC-mispair could be removed from the bsAb at pH 5.5 using a linear salt gradient. These results were then employed as a guide to propose a single column, multi-pH step gradient process for the removal of all product-related impurities. The final process included loading at pH 7.5 followed by elution of the homodimer and ½ IgG impurities from the column with a pH 9.5 wash. The column pH was then reduced to 5.5 and the LC-mispair impurity was subsequently removed during a 1 M NaCl step. Finally, a pH 8.0 with 1 M NaCl eluent was employed to recover the bound bsAb. This one step MMCEX polishing process resulted in an increased purity of the bsAb from 67.3% to 94.5% with a 72% overall recovery of the bsAb. These results demonstrate the utility of single column multimodal chromatographic processes when operated at multiple pHs for the removal of challenging product-related impurities from bsAbs.