PhD Thesis Defense: Andrew Chang

"A high-throughput solubility engineering platform and application to aggregation-prone model proteins"

Abstract

Recombinant biologics have a tendency to aggregate and form insoluble species that complicate manufacturing, reduce specific activity, and raise concerns regarding clinical safety (Roberts 2014). For example, interferon-β-1b (Betaseron) is an aggregation-prone biotherapeutic for which only 40% of the E. coli expressed recombinant product is monomeric (Runkel, Meier et al. 1998), and notably neutralizing antibodies develop in >22% of patients (Grossberg, Oger et al. 2011). Recombinant interleukin-2 (IL-2) is another biotherapeutic for which highly aggregated formulations are known to elicit anti-drug antibodies (ADAs) (Prummer 1997). More generally, there is a stronger need to consider solubility as a component of biotherapeutic developability, and more advanced tools and systems for addressing aggregation issues will be needed as the pipeline of biotherapeutic agents continues to expand.

Here, we detail development of an in vivo aggregation biosensor that enables high-throughput (HT) screening of biologics for improved solubility characteristics. We show that this biosensor readily distinguishes between soluble maltose-binding protein (MBP) and the insoluble variant MalE31, and we prospectively apply the system in engineering enhanced-solubility green fluorescent protein (GFP) variants via HT library screening. Several isolated variants outperformed the parental GFP molecule by numerous in vitro metrics, and one such variant was found to elicit reduced ADAs following immunization in mice. While this report describes application of the system to model proteins, we speculate that the biosensor could be used to engineer enhanced solubility variants of more practical biotherapeutic targets.

Thesis Committee

• Prof. Karl Griswold (Chair)
• Prof. Margie Ackerman
• Prof. Chris Bailey-Kellogg
• Prof. Navin Varadarajan

Contact

For more information, contact Theresa Fuller at theresa.d.fuller@dartmouth.edu.