One-pot expression of an oligoclonal nanobody-based antivenom in growth-decoupled Escherichia coli.

Oligoclonal VHH mixtures targeting distinct epitopes offer therapeutic benefits in mitigating complex diseases, including cancer, infectious diseases, and snakebite envenoming. However, current production strategies rely on separate expression and purification of individual VHHs, followed by downstream mixing, resulting in high costs. We present a cocultivation approach for producing an experimental recombinant antivenom using growth-decoupled Escherichia coli strains. Six different cell lines were generated by transforming the same host with different VHH genes. The cell lines displayed high-yield extracellular secretion in microtiter plate and 1-l bioreactor cultivations, and cocultivation at defined inoculation ratios yielded oligoclonal VHH mixtures approaching target compositions. This strategy enables one-pot manufacturing with minimal downstream processing and predictable composition. A techno-economic assessment based on obtained titers and two-step purification indicates reduced per-treatment costs compared with Chinese Hamster Ovary cells produced IgG-based antivenoms. Our findings offer an alternative to conventional oligoclonal VHH production for multivalent therapeutics requiring broad neutralization, including recombinant antivenoms.