Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody-Drug Conjugates

Monoclonal antibodies (mAbs) andantibody-drug conjugates(ADCs) are two of the most important therapeutic drug classes thatrequire extensive characterization, whereas their large size and structuralcomplexity make them challenging to characterize and demand the useof advanced analytical methods. Top-down mass spectrometry (TD-MS)is an emerging technique that minimizes sample preparation and preservesendogenous post-translational modifications (PTMs); however, TD-MSof large proteins suffers from low fragmentation efficiency, limitingthe sequence and structure information that can be obtained. Here,we show that including the assignment of internal fragments in nativeTD-MS of an intact mAb and an ADC can improve their molecular characterization.For the NIST mAb, internal fragments can access the sequence regionconstrained by disulfide bonds to increase the TD-MS sequence coverageto over 75%. Important PTM information, including intrachain disulfideconnectivity and N-glycosylation sites, can be revealed after includinginternal fragments. For a heterogeneous lysine-linked ADC, we showthat assigning internal fragments improves the identification of drugconjugation sites to achieve a coverage of 58% of all putative conjugationsites. This proof-of-principle study demonstrates the potential valueof including internal fragments in native TD-MS of intact mAbs andADCs, and this analytical strategy can be extended to bottom-up andmiddle-down MS approaches to achieve even more comprehensive characterizationof important therapeutic molecules.

Automated summary

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are difficult to characterize due to their large size and structural complexity. Top-down mass spectrometry (TD-MS) is a promising technique for their characterization, but suffers from low fragmentation efficiency. This study demonstrates the value of including internal fragments in native TD-MS for improved molecular characterization of mAbs and ADCs.