Glycoform engineering of a mammalian platform to sculpt a humanized recombinant bioscavenger

This study assessed three mammalian expression platforms-human embryonic kidney (HEK293), Chinese hamster ovary (CHO), and HepG2 cells-for their ability to produce recombinant human butyrylcholinesterase (rHuBChE), a promising bioscavenger for organophosphate (OP) poisoning treatment. Key challenges remain in producing rHuBChE with a glycosylation profile similar to that of the native human plasma enzyme butyrylcholinesterase (HuBChE), which is critical for its stability and in vivo half-life. HEK293 cells showed the most promising results, prompting extensive glycoengineering targeting over ten glycosylation-related genes to optimize the glycan profile. This involved knocking out genes associated with fucosylation, enhancing sialylation, and reducing glycan branching. Glycan analysis revealed a substantial reduction in aberrant glycan structures, improved sialylation, and a glycoprofile closely resembling that of plasma-derived HuBChE. These comprehensive modifications to glycosyltransferases and nucleotide sugar donor levels are expected to enhance pharmacokinetics and reduce immunogenicity, positioning rHuBChE as a viable candidate for further preclinical and clinical development against OP poisoning and other medical emergencies. A record of this paper's transparent peer review process is included in the supplemental information.