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              <p>Even though mAbs have attracted the biggest interest in the development of therapeutic proteins, next generation of therapeutics such as single-domain antibodies (sdAb) are propelling an increasing at-tention 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 to their mAb counterparts. These proteins can be subjected to different bioconjugation processes to form single-domain antibody-drug conjugates (sdADC) and hence increase their therapeutic potency, and, akin to other therapeutic proteins, nano-bodies 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 on the extensive sequence characterization of a conjugated anti-EGFR 14 kDa sdADC by using state-of-the-art top-down mass spectrometry strategies in combi-nation with liquid chromatography (LC-TD-MS). Mass analysis revealed a highly homogeneous sam-ple with one conjugated molecule. Subsequently, the reduced sdADC was submitted to different frag-mentation techniques namely higher-energy collisional dissociation (HCD), electron-transfer dissoci-ation (ETD), and electron transfer higher-energy collision dissociation (EThcD) allowing to unam-biguously asses the conjugation site with 24 diagnostic fragment ions and 85% of global sequence coverage. The sequence coverage of the non-reduced protein was significantly lower (around 16%), however the analysis of the fragmentation spectra corroborated the presence of the intra-molecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the difficulties and challenges associated to the fragmentation of sdAb derived formats in the LC time scale due to their remarkable stability as a consequence of the intra-molecular disulfide bridge. However, the use of complementary activation techniques along with identification of specific ion fragments allow an improved sequence coverage, the characteriza-tion of the intra-molecular disulfide bond, and the unambiguous localization of the conjugation site.</p>
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