(191ap) A High-Throughput Platform Technology for Engineering Enhanced-Solubility in Biotherapeutics

Biomolecular aggregation is a ubiquitous challenge encountered in the development of biotherapeutics. For manufacturers, protein aggregation can complicate biotherapeutic production, purification, formulation and storage, while for patients, biotherapeutic aggregates can induce unwanted immune responses. Enhanced solubility is therefore a desirable property for biotherapeutic engineering programs. Directed evolution using large combinatorial libraries can be a powerful strategy for solubilizing aggregation-prone biomolecules, but such an approach requires a robust and generalizable HT-screening method for protein aggregation. Here, we describe a platform technology for ultra-high throughput screening of protein solubility in E. coli expression hosts. Using high speed flow cytometry, the aggregation propensity of arbitrary protein libraries can be screened at rates of 10,000-30,000 clones per second, enabling the rapid isolation of highly soluble variants. By integrating deep sequencing into the experimental workflow, we have constructed a high-resolution map of the sequence-solubility-function space for a GFP model system, and we further demonstrate the utility of the platform with biotherapeutics of practical interest. This powerful aggregation screening method is quantitative, applicable to a vast array of protein targets, and enables rapid sorting of massive recombinant libraries. We anticipate that this technology will provide novel solutions to the aggregation problems suffered by many biotherapeutic agents.