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