Binder discovery using Enzene’s phage display platform

Enzene executed a structured, multi-stage phage display workflow designed to maximize library diversity, eliminate non-specific binders, and enrich the selection for high-affinity candidates.

The team used a cDNA repertoire to generate a VH-based phagemid library (>1E7 diversity). To ensure true diversity, Enzene used primers with single degenerate positions and performed amplification across each VH subfamily, avoiding primer pools in favor of individually tailored primers. This strategy ensured broad representation and minimized amplification bias.

During biopanning, Enzene implemented a smart deselection strategy to eliminate non-specific and unwanted tag-specific binders. Two unrelated similarly tagged proteins were incorporated into the workflow to prevent enrichment of undesired clones. Stringency was carefully tuned across rounds of selection and ELISA screening to drive enrichment toward true antigen-specific binders.

Despite limited availability of the target protein, Enzene optimized antigen usage by gradually reducing target concentration across rounds and fine-tuning capture concentrations for screening ELISAs. This enabled efficient enrichment without compromising binder diversity.

Across three rounds of biopanning, the enrichment index (EI) improved ~200-fold (see Figure 1), and screening of 180 monoclones yielded an ~88% hit frequency using an OD₄₅₀ cutoff of 0.575 (5x above non-specific signal (see Figure 2)). Representative screening of 90 clones confirmed strong enrichment and specificity.

Subsequent clone funnelling identified:

• 92 ELISA‑positive hits
• 52 unique binder sequences
• 28 sequenced hits
• 11 diverse germlines

These results exceeded the client’s design goals and demonstrated the robustness of Enzene’s discovery platform.

Enzene successfully met, and exceeded, all design goals for the program:

• A validated VH‑based phagemid library >1E7 was generated.
• More than 20 diverse binders were identified.
• Binders represented multiple germlines, supporting downstream engineering flexibility.

The biopanning campaign achieved ~200‑fold enrichment and ~88% hit frequency, demonstrating strong selection efficiency.

The client received a high-quality panel of diverse, sequence confirmed binders suitable for downstream optimization, engineering, and developability assessment. The success of this project reinforced Enzene’s position as a trusted discovery partner capable of delivering high value antibody leads through advanced display technologies.