Therapeutic monoclonal antibodies (mAbs) make up a large portion of the rapidly growing drug market. Ensuring safety and efficacy through comprehensive understanding of these products’ critical quality attributes (CQAs), including charge heterogeneity, is a regulatory requirement. Various charge isoforms of mAbs can result from cell culture or production processes, potentially affecting the mAb structure and function. While imaged capillary isoelectric focusing (icIEF) is preferred for charge profiling, ion-exchange chromatography (IEC) is the major tool for mAb fractionation. However, IEC is not compatible with certain types of molecules, e.g., hydrophobic antibody drug conjugates (ADCs), and icIEF typically provides higher separation resolution. Moreover, an individual charge variant obtained from IEC fractionation may not be comparable to the peak in the icIEF profile. Therefore, there is an unmet need for IEF-based fractionation of charge variants for characterization. We have developed a novel icIEF fractionation solution for icIEF separation and collection of charge variants. This solution enables Maurice icIEF-based peak identification followed by downstream analysis. Here we report icIEF fractionation followed by ZipChip-based mass spectrometry characterization of the NIST mAb and XMT-1535 mAb. ZipChip (CE-ESI) was utilized to characterize the fractions due to its broad sample matrix compatibility, easy sample prep, and fast analysis time. Individual charge variants were successfully collected in less than 2 hours with purity > 80% using icIEF separation conditions with or without urea. Rapid analysis using ZipChip showed the mass spec identification of major and minor isoforms correlated well with reported mass spec data. Urea in icIEF separation did not affect the fractionation quality nor the mass spec result. Multiple fractionation runs of the NIST mAb suggested good system reproducibility. We believe this novel icIEF fractionation solution coupled with other analysis methods, such as mass spectrometry, will deliver a powerful charge variant characterization tool for biotherapeutic analytical tool kit.

Mass Spec 2021 poster - XS