Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions

ABSTRACTWe report a case study in which liquid-liquid phase separation (LLPS) negatively impacted the downstream manufacturability of a therapeutic mAb. Process parameter optimization partially mitigated the LLPS, but limitations remained for large-scale manufacturing. Electrostatic interaction driven self-associations and the resulting formation of high-order complexes are established critical properties that led to LLPS. Through chain swapping substitutions with a well-behaved antibody and subsequent study of their solution behaviors, we found the self-association interactions between the light chains (LCs) of this mAb are responsible for the LLPS behavior. With the aid of in silico homology modeling and charged-patch analysis, seven charged residues in the LC complementarity-determining regions (CDRs) were selected for mutagenesis, then evaluated for self-association and LLPS properties. Two charged residues in the light chain (K30 and D50) were identified as the most significant to the LLPS behaviors ...