Optimized iCIEF Fractionation Enables Deep Profiling of Antibody Drug Conjugate Charge Heterogeneity

Antibody drug conjugates (ADCs) are a novel class of therapeutic drugs that combine selectivity of an antibody moiety with the cell killing potency of cytotoxic agent. ADCs are complex molecules, and their charge variants are one of the important critical quality attributes (CQAs) that need to be monitored throughout the drug development process. Imaged capillary isoelectric focusing (iCIEF) is considered a gold standard in charge variant analysis due to its short analysis time and ease of use. For comprehensive analysis, iCIEF fractionation is a newly developed technique that can be used to enable LC-MS characterization of charge variants. Here, we describe method development for an iCIEF fractionation system, as well as further characterization using LC-MS. Fractionation conditions were optimized to provide stabilized mobilization after separation. Fractions were collected with the optimized method, and native SEC-MS results showed that fractionation enabled detection of species previously undetectable. Further mass spectrometric analysis revealed the major posttranslational modifications (PTMs) and drug load distributions for both light and heavy chains. The results indicate that iCIEF fraction collection enables a deeper understanding of ADC charge variants and will be further useful for studying other complex biomolecules.