Focal Molography Enables Rapid Evaluation of Miniprotein Plasma Protein Binding

About the talk

Recorded at the 3rd focal Molography User Meeting on September 9, 2026. Alyssa Anderson (AI Proteins) explains why plasma protein binding (PPB) drives the pharmacokinetics of a drug, why separation-based methods do not work for miniproteins, and how a label-free plasma titration on focal molography measures binding directly in plasma.

Topics covered

• Why standard methods fall short for miniproteins. In rapid equilibrium dialysis the 40 to 60 amino acid miniproteins cannot cross the membrane, and in ultracentrifugation they partly sediment, so the free fraction is underestimated. Separate quantification also limits throughput to a few proteins per day.
• Plasma binding measured on the chip. Strep-tagged miniproteins are captured via Strep-Tactin oligo conjugates by DNA-directed immobilization (DDI), plasma is titrated from 0.0006% to 100%, and the apparent affinity is read as the plasma percentage that gives half-maximal binding.
• Validation against ultracentrifugation. 15 miniproteins with four replicates each on one 64-plex chip, from albumin binders to sticky and unbound controls. The ranking agreed with ultracentrifugation; the single outlier turned out to be a protein that forms aggregates.
• Kinetics on top of affinity. Two proteins with very similar apparent affinity for plasma differed sevenfold in their dissociation rate, a difference in residence time that matters for in vivo distribution.

About the speaker

Alyssa Anderson is Head of Protein Characterization at AI Proteins in Boston, a platform company that designs miniprotein therapeutics de novo. The work presented was a collaboration with the lino Biotech team.

The recording is available on demand behind a short registration form.