Flex Your mAbs: LC-MS Characterization of Charge Variants from MauriceFlex™

Monoclonal antibodies (mAbs) are complex biotherapeutic proteins, typically produced in a mammalian cell line to achieve sufficient yield but importantly also to decorate the antibody with necessary post-translational modifications (e.g. glycosylation) to ensure safety and efficacy.

As a result, the antibody can have significant charge heterogeneity, which can change from batch to batch and can influence the therapeutic index of the molecule.

In this presentation, we will discuss a workflow using the new MauriceFlex™ instrument to fractionate mAb charge variants with downstream LC-MS analysis. Antibodies are large molecules with a lot of inherent heterogeneity. While some differences in the fractioned charge variants were detectable at the intact level, unambiguous peak annotations were not always possible.

In order to analyze the fractioned charge variants in more detail a subunit approach based on FabRICATOR (Genovis AB) was chosen. This enzyme cuts mAbs at on specific site just below the hinge which – together with a reduction of disulfides – generates subunits 23-25kDa in size. These smaller, more manageable subunits are easier to analyze by LC-MS, leading to cleaner, high-quality spectra that allow for much more detailed analysis. With minimal sample prep, this provided a much clearer picture of the subtletie of mAb assembly, glycosylation, and other PTMs.

Presenter: Chris Heger (Director, Application Science, Bio-Techne)

Presenter: Andreas Nägeli (Principal Scientist, Genovis)

Presenter: Dr. Birgit Foltas (Scientific Editor, Wiley, Moderator)