Enzymatic removal of sialic acid enables iCIEF stability monitoring of charge variants of a highly sialylated bispecific antibody

Antibody-based therapeutic proteins have highly complex molecular structures. The final therapeutic protein product may contain a wide range of charge variants. Accurate analysis of this charge variant composition is critical to determine manufacturing process consistency and protein stability and ultimately helps to ensure that patients receive a safe and efficacious product. Here, a highly sialylated bispecific antibody (bsAb-1) challenged the ability to monitor stability by imaged capillary isoelectric focusing (iCIEF). This challenge was overcome by optimization of the iCIEF master mix buffer (adjustment of urea concentration, addition of l-arginine) and enzymatic removal of sialic acid. The method was qualified by assessing linearity, precision, LOD, LOQ, accuracy, and robustness in accordance with ICH guidance. Main species loss detectability increased up to approximately fivefold compared to the iCIEF method without desialylation when monitoring changes in stressed samples. Importantly, the results of the iCIEF method with desialylation correlated with results obtained through LC-MS tryptic peptide mapping and enabled analysis of formulation development stability samples. Finally, this analytical method shows the potential to assess low-concentration formulation development samples down to a sample concentration of 0.1 mg/ml.

Automated summary

Antibody-based therapeutic proteins have complex structures with charge variants, and accurate analysis of these variants is important for manufacturing process consistency and protein stability. By optimizing the iCIEF master mix buffer and removing sialic acid, the challenge of monitoring stability for a highly sialylated bispecific antibody was overcome. The method was validated for linearity, precision, LOD, LOQ, accuracy, and robustness, and correlated with LC-MS tryptic peptide mapping results.