An In Vivo Platform to Select and Evolve Aggregation-Resistant Protein Therapeutics

摘要

The production and formulation of biopharmaceuticals can be hindered by protein aggregation which can occur at every stage of the manufacturing process, ultimately jeopardising the successful development of promising candidates. Investigating protein aggregation and stability can be laborious and time consuming, due to the difficulties in both expression and purification for in vitro analysis and in determining the causes of aggregation. To address this, we have developed an in vivo platform to characterise the aggregation propensity of biopharmaceuticals that circumvents the need for recombinant expression and downstream analysis. The system, based on a split beta-lactamase enzyme assay, enables the identification of aggregation-prone sequences inserted between the two enzyme domains, whose function is necessary for the survival of the bacteria in which it is expressed. Applying the system to the antibody MEDI-1912 known to be aggregation prone, we demonstrate the system's ability to distinguish between aggregation and non-aggregation prone sequences, offering a powerful tool for assessing protein aggregation and stability earlier in the industrial pipeline. Furthermore, by developing a directed evolution methodology we show that this system can be used as a novel strategy to modulate the aggregation propensity of protein scaffolds enabling the identification of evolved variants with reduced aggregation propensity. Evolved scFvs have been grafted into IgGs for which the in vitro aggregation correlates with the split beta-lactamase assay. Importantly, IgGs still maintain affinity for their target.