Optimizing Peptide Permeability Assays

Optimizing Peptide Permeability Assays:
Insights from our GRC 2025 Poster Presentation

The bidirectional permeability assay is a widely used in vitro model to assess the transcellular transport properties of drug candidates across epithelial barriers, such as the intestinal epithelium or the blood-brain barrier. In this system, MDCK (Madin-Darby Canine Kidney) cells overexpressing human MDR1 (also known as P-glycoprotein 1, P-gp) are employed to evaluate whether a compound is a substrate or inhibitor of P-gp, a key efflux transporter involved in limiting drug absorption and distribution.

Our work, presented at GRC 2025, focuses on overcoming the analytical and biological hurdles associated with peptide permeability testing. The MDCK-MDR1 cell model is a well-established system for evaluating P-glycoprotein (P-gp)-mediated transport, commonly used for small molecules. But peptides, due to their high molecular weight, lipophilicity, and strong intracellular binding, require a more nuanced approach.

Key Methodological Enhancements

To improve the accuracy and reproducibility of the assay, we introduced several critical optimizations:

• Lowering incubation concentrations: Peptides tend to aggregate in aqueous solutions, which can distort permeability measurements. By reducing the concentration, we minimized aggregation and improved solubility.

• Extending preincubation times: This allows the unbound intracellular concentration of peptides to reach a steady state, which is essential for reliable transport data.

• Adding bovine serum albumin (BSA) to the receiver compartment: Peptides often bind non-specifically to plastic surfaces and proteins. BSA helps reduce this binding, significantly improving recovery rates.

• Using highly sensitive LC-MS/MS methods: Peptides typically produce weak analytical signals due to low permeability and degradation. Our method achieves a detection limit of 1–2 ng/mL, enabling precise quantification even at low concentrations.

We validated our optimized protocol using model peptides such as Emodepside, Cyclosporin A, and peptide analog Rapamycin. Results show significant improvements in reproducibility, sensitivity, and recovery – especially for challenging cyclic peptides. The results also showed strong agreement with published literature, confirming the reliability of our approach. We also included well-characterized positive controls like propranolol to benchmark passive diffusion and a low-permeability peptide analog to represent challenging compounds. These controls helped ensure the robustness of our assay across a range of permeability profiles.

Impact on Drug Development

This optimized assay provides a more accurate and reproducible platform for evaluating peptide permeability and P-gp-mediated efflux. It supports better decision-making in early drug development, particularly for compounds that fall outside the traditional “Rule of 5” parameters.

By addressing key limitations in peptide transport assays, our work contributes to a growing toolkit for peptide drug development – enabling researchers to better understand and predict in vivo behavior from in vitro models.

Download the poster below and contact us if you would like to discuss in more detail.