Rapid Assessment of Fab-VHH Expression Application of the Pfast™ Program for Early-Stage Titer and Activity Screening 

by | Aug 24, 2026 | Case Studies

Rapid Assessment of Fab-VHH Expression

Application of the Pfast™ Program for Early-Stage Titer and Activity Screening

Pfenex Expression Technology® | Primrose Bio

Challenge

  • Development of a bi-specific antibody derivative: a Fab–VHH fusion
  • Assess Pfenex Toolbox and optimize heavy and light chain orientation on a single operon for correct folding and assembly using the Pfast™ 10-day assessment program
  • Confirm binding activity of the new Fab-VHH construct

Expression Strategy

  • Synthesize two operons with heavy chain and light chain are co-transcribed in each orientation (HC-LC and LC-HC)
    • Optimize coding sequence
    • Ligate synthesized coding sequences into 40 toolbox expression vectors with strong IPTG inducible promoter
    • Evaluate two ribosome binding sites (RBS) /translation initiation strengths (High/Medium), and 23 periplasmic secretion leaders (SL)
  • Pfast™ pooled ligation strategy transformed into single host strain to evaluate expression strategies in 10 days
  • Analyze Expression Strategies. Harvest cultures, generate lysates by sonication, evaluate qualitative and quantitative by SDSCGE and indirect ELISA

Key Results

  • LC-HC orientation Pfast™ screen delivered up to 500 mcg/mL soluble, active, intact Fab-VHH within 10 days for further development.
  • Assembled Fab-VHH seen in all 21 down-selected samples; correct mass observed, confirming leader sequence processed

Results & Takeaways

  • The Pfast screening platform identified optimal expression conditions for Fab-VHH, yielding up to 570 μg/mL within 10 days
  • Light chain-first gene orientation outperformed heavy chainfirst, providing a scalable foundation for further development
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