Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

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 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 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, 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 on the complete sequence characterization of a conjugated anti-EGFR 15 kDa sdADC by using cutting-edge 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 (HCD), electron-transfer dissociation (ETD), and ultraviolet photo-dissociation (UVPD) allowing to unambiguously asses the conjugation site with 16 diagnostic fragment ions and more than 85% of global sequence coverage. The sequence coverage of the non-reduced protein was significantly lower (around 33%), however the thorough analysis of the fragmentation spectra and the inclusion of the internal fragments corroborated the presence of the intra-molecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the complementary of the different fragmentation techniques to provide a thorough analytical characterization of sdAbs-formats even in the chromatographic time-scale, which allows a high-throughput and streamlined analysis of this kind of therapeutic proteins facilitating the implementation on dedicated R&D laboratories.