High-Throughput Developability Assays Enable Library-Scale Identification of Producible Protein Scaffold Variants

Abstract

Proteins require high developability - quantified by expression, solubility, and stability - for robust utility as therapeutics, diagnostics, and in other biotechnological applications. Measuring traditional developability metrics is low-throughput in nature, often slowing the developmental pipeline. We evaluated the ability of three high-throughput developability assays to predict the bacterial recombinant expression of paratope variants of the protein scaffold Gp2. Enabled by a phenotype/genotype linkage, assay performance for 105 variants was calculated via deep sequencing of populations sorted by proxied developability. We trained a random forest model that predicts expression from assay performance that is 35% closer to the experimental variance and trains 80% more efficiently than a model predicting from sequence information alone. Utilizing the predicted expression, we performed a sitewise analysis and predicted mutations consistent with enhanced developability. The validated assays offer the ability to identify developable proteins at unprecedented scales, reducing the bottleneck of protein commercialization.