Development of Top-Down Mass Spectrometry Strategies in the Chromatographic Time Scale (LC-TD-MS) for the Extended Characterization of an Anti-EGFR Single-Domain Antibody-Drug Conjugate in Both Reduced and Nonreduced Forms.

Even though mAbs have attracted the biggest interest in the development of therapeutic proteins, next-generation therapeutics such as single-domain antibodies (sdAb) are propelling increasing attention as new alternatives with appealing applications in different clinical areas. These constructs are small therapeutic proteins formed by a variable domain of the heavy chain of an antibody with multiple therapeutic and production benefits compared with their mAb counterparts. These proteins can be subjected to different bioconjugation processes to form single-domain antibody-drug conjugates (sdADCs) and hence increase their therapeutic potency, and akin to other therapeutic proteins, nanobodies and related products require dedicated analytical strategies to fully characterize their primary structure prior to their release to the market. In this study, we report for the first time the extensive sequence characterization of a conjugated anti-EGFR 14 kDa sdADC by using state-of-the-art top-down mass spectrometry strategies in combination with liquid chromatography (LC-TD-MS). Mass analysis revealed a highly homogeneous sample with one conjugated molecule. Subsequently, the reduced sdADC was submitted to different fragmentation techniques, namely, higher-energy collisional dissociation, electron-transfer dissociation, and electron-transfer higher-energy collision dissociation, allowing to unambiguously assess the conjugation site with 24 diagnostic fragment ions and 85% of global sequence coverage. The sequence coverage of the nonreduced protein was significantly lower (around 16%); however, the analysis of the fragmentation spectra corroborated the presence of the intramolecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the difficulties and challenges associated with the fragmentation of sdAb-derived formats in the LC time scale due to their remarkable stability as a consequence of the intramolecular disulfide bridge. However, the use of complementary activation techniques along with the identification of specific ion fragments allows an improved sequence coverage, the characterization of the intramolecular disulfide bond, and the unambiguous localization of the conjugation site.