Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

Electron transfer dissociation (ETD) is an analytically useful tool for primary structure interrogation of intact proteins, but its utility is limited by higher-order reactions with the products. To inhibit these higher-order reactions, first-generation fragment ions are kinetically excited by applying an experimentally tailored parallel ion parking waveform during ETD (ETD-PIP). In combination with subsequent ion/ion proton transfer reactions, precursor-to-product conversion was maximized as evidenced by the consumption of more than 90% of the 21 kDa Protein G precursor to form ETD product ions. The employment of ETD-PIP increased sequence coverage to 90% from 80% with standard ETD. Additionally, the inhibition of sequential electron transfers was reflected in the high number of complementary ion pairs from ETD-PIP (90%) compared to standard ETD (39%).

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Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.