ABSTRACT:
A platform-based approach leveraging the Pfenex Expression Technology® was developed for implementing screening through lead optimization of novel ADCs
As an example of the workflow, an Anti-GPCR VHH therapeutic candidate modified to include a single ncAA (pAzF) substitution was developed
The site of ncAA incorporation was determined through rationale design using hydrogen deuterium exchange (HDX) mass spectrometry
pAzF feed strategies were evaluated at the 2L fermentation scale, identifying a continuous feed strategy that achieved very similar titers to the canonical VHH (~15 g/ L)
Higher pAzF concentrations were shown to be toxic, likely due to off-target amber suppression
Peptide mapping of Y84pAzF VHH showed 100% pAzF incorporation. Intact mass spectrometry showed 100% click reaction conversion
Binding activity was not affected by conjugation