Overview
Biomolecular charge variant characterization has been a challenge that involves a cumbersome and time-consuming process. In this study, we demonstrate the application of the MauriceFlex™ and Alto™ platforms to fractionate and characterize binding kinetics of charge variants from a therapeutic bispecific antibody (BsAb) Mosunetuzumab and a research-grade biosimilar. First, the charge heterogeneity of both molecules was analyzed using imaged capillary isoelectric focusing (icIEF) on the MauriceFlex system, followed by the collection of individual charge variant fractions on the same system. Next, the collected fractions were tested for their ability to bind to antigens CD3 and CD20, which are the intended targets of Mosunetuzumab. To accomplish this, surface plasmon resonance (SPR) with the Alto System was used to measure the binding of each fraction and ligand, requiring only 2 µL of each. Alto offers a key advantage over other SPR platforms due to its low sample volume requirements and higher throughput for fractionated samples. The binding data obtained from SPR correlated well with the structural information obtained from LC-MS analysis of the charge variant fractions, revealing a significantly weaker binding of the acidic fraction of the biosimilar to CD20. Overall, this poster highlights the streamlined fractionation and characterization of charge variants in biotherapeutics. This will help identify critical charge species that impact binding potency to inform the control steps needed in the process development and manufacturing of a biotherapeutic.