Charge Variants Analysis of a Bispecific Antibody Using a Fully Automated One-step Capillary Isoelectric Focusing - Mass Spectrometry Method

Background: Bispecific antibody (BsAb) therapeutics have emerged as the nextgeneration immuno-oncology therapy. The architecture of bsabs is inherently more complex than that of mAb therapeutics. As a result, prior knowledge of critical quality attributes (CQAs) assessment of mAbs is no longer inclusive for bsabs. Objective: The main objective of this work is to develop a fully automated one-step capillary isoelectric focusing - mass spectrometry (cIEF-MS) workflow for the charge variant analysis of a bispecific antibody molecule. Methods: A number of critical factors for the method development are investigated: the performance of two commonly used ampholytes compared; the impact of protein concentration for the cIEF-MS assay is examined; as for sample preparation, off-line and on-line desalting are compared; various combinations of Pharmalyte® 3-10 and 8-10.5 are considered. Results: In this fully automated workflow, the charge variants of this BsAb molecule are clearly separated and accurately identified. Based on six repeat injections, RSDs of the migration time of the identified charge variants are between 3 and 6%. The identified masses of each charge variant show a variation between 0.48 and 1.40 Da. The delta masses of the basic and acidic variants are from the most basic to the most acidic, -58.59, 162.26, 453.44, -907.47, 1,563.60, and 1,566.98 Da, respectively. Conclusion: Overall, the separation resolution, system sensitivity, robustness, and reproducibility of this fully automated cIEF-MS workflow, as demonstrated using this BsAb example, proves it a powerful assay for the quality assessment of recombinant protein therapeutics.