Tell us about your project and how SmartEnzymes enhanced it:
In our project we focused on glycan-mediated conjugation technology to create homogeneous and site-specific ADC products. The aim was to develop an analytical platform that can quickly and accurately monitor this conjugation process.
First, we used the innovative GlyCLICK-technology to couple the commonly used maytansine payload DM1 to trastuzumab in a site-specific manner. The glycan-mediated technology allowed to not only control the DAR, but also the drug load distribution (DLD) of the ADC product. Then, to monitor these important CQAs and the conjugation process, we used a middle-up LC/HRMS approach to compare the GlyCLICK product with unconjugated trastuzumab. The subunits were generated by using FabRICATOR and FabALACTICA enzymes and were analyzed in both RPLC- and HILIC-mode. A significant shift in retention of the crystallizable fragment (Fc/2) was observed as result of the lipophilic drug payload conjugation, confirming the site-specific conjugation process. At last, MS detection confirmed the accurate DAR ratio of 2.0 and the absence of randomly conjugated payloads.
Therefore, we believe that the GlyCLICK procedure to create novel ADCs combined with the middle-up analysis to monitor important CQAs related to the conjugation process holds a great potential in the field of ADC development.
Any ideas for new SmartEnzymes and what they could be called?
Tell us more: For middle-up analysis of complex protein therapeutics, with both N- and O-glycans it would be interesting to have an enzyme that enables the removal of the O-glycans without prior removal of the sialic acids. This would be useful when you aim to keep the N-glycans intact during the analysis. Another option would be a sialidase that only removes the sialic acids from O-glycans while keeping the N-glycans intact. This would be useful when analyzing, e.g., highly glycosylated fusion proteins like etanercept, where a judicious combination of enzymatic digestions can provide highly interesting information on the glycoform heterogeneity.
