An Integrated Workflow for Intact and Subunits of Monoclonal Antibody Accurate Mass Measurements

Monoclonal antibody (mAb) based entities represent a rapidly growing class of
biologics that require extensive characterization to obtain approval for clinical trials and
subsequent market release. Accurate mass measurement is a challenging step in the
analytical characterization of antibodies because of their large size and the presence of
post-translational modifications such as glycosylation. These characteristics also make
determining the location of modifications more complex.
To overcome the challenges associated with antibody mass measurement, a number of
complementary approaches are typically used. Antibodies can be treated with PNGase F
to remove the N-Glycans, digested with proteases such as IdeS to generate antibody
fragments, or reduced to generate light and heavy chains prior to mass measurement.
These techniques can be used in various combinations. Sample preparation can be
laborious, time-consuming, and have limited reproducibility. This Application Note
demonstrates how these approaches can be streamlined by automation on the Agilent
AssayMAP Bravo to reduce the probability of human error, increase reproducibility, and
create more walk-away time (Figures 1 and 2).

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