Rapid Assessment Of Fab-VHH Expression Application Of The Pfast™ Program For Early-Stage Titer And Activity Screening

When developing a bi-specific antibody fragment fusion, getting expression conditions right early can save significant time and resources downstream. This case study details how a 10-day pooled expression screening program was used to evaluate 40 vector configurations across two operon orientations, two ribosome binding site strengths, and 23 periplasmic secretion leaders for a Fab-VHH construct.

The light chain-first orientation consistently outperformed its counterpart, with some conditions reaching the upper limit of the active Fab standard curve at 360 µg/mL and overall titers reaching up to 570 µg/mL. SDS-CGE confirmed correct subunit molecular weights for both chain orientations, and deconvoluted mass spectrometry verified fully assembled, full-length Fab-VHH at the expected mass of 62,083 Da across all 21 down-selected samples. Notably, outlier samples showed high binding activity despite lower total titer, underscoring the value of combining quantitative and functional analytics early in screening.

If you are interested in learning more, download the case study to see how a rapid, systematic screening approach can compress your timeline without sacrificing data quality.