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.

Strategies for developing a recombinant butyrylcholinesterase medical countermeasure for Organophosphorus poisoning.

Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.