Effective parallel evaluation of molecular design, expression and bioactivity of novel recombinant butyrylcholinesterase medical countermeasures

Current medical countermeasures (MCMs) for nerve agent poisoning have limited efficacy, and can cause serious adverse effects, prompting the requirement for new broad-spectrum therapeutics. Human plasma-derived butyrylcholinseterase (huBChE) is a promising novel bioscavenger MCM which has shown potential in animal studies, however, is economically prohibitive to manufacture at scale. This study addresses current challenges for the economical production of a bioactive and long-acting recombinant huBChE (rBChE) in mammalian cells by being the first to directly compare novel rBChE design strategies. These include co-expression of a proline rich attachment domain (PRAD) and fusion of BChE with a protein partner. Additionally, a pre-purification screening method developed in this study enables parallel comparison of the expression efficiency, activity and broad-spectrum binding to nerve agents for ten novel rBChE molecular designs. All designed rBChE demonstrated functionality to act as broad-spectrum MCMs to G, V and A series nerve agents. Expression using the ExpiCHO™ Max protocol provided greatest expression levels and activity for all constructs, with most rBChE expressing poorly in Expi293™. Fc- or hSA-fused rBChE significantly outperformed constructs designed to mimic huBChE, including PRAD-BChE, and proved an effective strategy to significantly improve enzyme activity and expression. Choice of protein partner, directionality and the addition of a linker also impacted fusion rBChE activity and expression. Overall, hSA fused rBChE provided greatest expression yield and activity, with BChE-hSA the best performing construct. The purified and characterised BChE-hSA demonstrated similar functionality to huBChE to be inhibited by GD, VX and A-234, supporting the findings of the pre-screening study and validating its capacity to assess and streamline the selection process for rBChE constructs in a cost-effective manner. Collectively, these outcomes contribute to risk mitigation in early-stage development, providing a systematic method to compare rBChE designs and a focus for future development.