The aggregation of protein-therapeutics is a major hindrance to the development of successful drug candidates. The propensity to aggregate can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. This project will seek to establish and implement a very high-throughput platform for the directed evolution of therapeutic proteins (TPs) towards increased thermal stability and lowered aggregation propensity. TPs have become one of the fastest growing classes of approved pharmaceutical products, and yet pose significant challenges in their manufacture and formulation due to molecular instability and the formation of aggregates. This can lead to adverse immunogenicity and life threatening situations, or limited efficacy.
This is important for society because this action will introduce novel technologies and new ideas, and enable new modalities of biopharmaceuticals to reach the market, including engineered mAbs, fusion proteins, and bispecifics. Thermostable TPs will facilitate their efficient functioning inside body, improve their long-term storage stability, and decrease their aggregation tendency resulting in less immunogenicity. After a large number of variants screening to produce a stable TP, it will benefit the production of a potentially safer drug with a lower treatment costs.
The overall objective of this project:
The project will establish a novel high-throughput directed evolution platform for evaluating large libraries of TP variants for increased thermostability, slower global or local unfolding rates, and minimised aggregation propensity. DE can be carried out by encapsulating single plasmids, each encoding a unique protein variant, into droplets of an emulsion, then using cell free expression to generate the protein in each droplet. This project will extend this approach to incorporate non-natural amino-acid fluorophores into the protein to create a FRET-based signal that would give a direct report on protein expression levels. Such a platform would be used to directly evolve TPs with increased thermostability, and then slower local unfolding, which will combine to decrease aggregation propensity. An enzymatic system will be used initially to establish and validate the set-up, taking advantage of the simple fluorescent assay for enzyme activity. Once established, the set-up will then be applied to therapeutic antibody fragment proteins, using FRET to screen for retention of folded structure upon heating.
