Monoclonal antibodies (mAbs) are complex biotherapeutic proteins, commonly generated in a mammalian cell line to attain adequate yield and, importantly, to decorate the antibody with essential post-translational modifications (such as glycosylation) to guarantee safety and effectiveness.
Consequently, the antibody may exhibit substantial charge heterogeneity, which can vary from one batch to another and can impact the therapeutic index of the molecule.
This webinar will explore a workflow using the new MauriceFlex™ instrument to separate mAb charge variants, followed by downstream LC-MS analysis. Antibodies are large molecules with a considerable degree of inherent heterogeneity.
Although some distinctions in the separated charge variants were noticeable at the intact level, unambiguous peak observations were not always feasible.
To analyze the fractioned charge variants more comprehensively, a subunit approach based on FabRICATOR (Genovis AB) was selected. This enzyme precisely cuts mAbs just below the hinge at a specific site which, together with a decrease in disulfides, produces subunits 23-25 kDa in size.
These smaller, more controllable subunits are simpler to analyze via LC-MS, producing cleaner, high-quality spectra that enable a much more thorough analysis. With nominal sample preparation, this approach provides a clearer insight into the subtleties of mAb assembly, glycosylation, and other PTMs.
Speakers
Chris Heger
Director, Application Science | Bio-Techne
Andreas Nägeli
Principal Scientist, Genovis
Moderator
Dr. Birgit Foltas
Scientific Editor, Wiley