Process intensification for production of Adenovirus-Like Particles (ADDomer) as snake-bite therapy

Snakebite envenomation causes over 140,000 deaths annually, with the burden disproportionately affecting low-resource regions. Conventional antivenoms (AVs), derived from hyperimmunized equines, are expensive, have limited cross-species efficacy, and pose scalability challenges. Adenovirus-like particles (ADDomer) produced using insect cell-baculovirus vector system (IC-BEVS), emerged as a promising alternative platform for antivenom development. However, current ADDomer production relies on batch-mode processes, which inherently limit yield and scalability. In this study, we aimed to overcome these limitations through IC-BEVS process intensification. High Five and Sf-9 insect cells were infected at high cell densities (HCD; 5-40 × 10⁶ cell/mL) using a pseudo-perfusion strategy in shake-flask cultures to optimize medium exchange rates for maximal ADDomer expression. Cell-specific productivity was maintained up to 10 × 10⁶ cell/mL for High Five cultures and 20 × 10⁶ cell/mL for Sf-9 cultures, compared to standard low cell density production under batch operation mode (LCD, 1-2 × 10⁶ cell/mL). Implementation of the best-performing HCD strategy in a perfusion bioreactor allowed to increase volumetric ADDomer productivity by up to 8-fold compared to LCD. Space-time yield improved up to 33%, underscoring the cost effectiveness of process intensification for scalable ADDomer manufacturing. Physicochemical and structural characterization confirmed that ADDomer particles produced under intensified perfusion conditions maintained structural integrity and functionality. This work establishes a scalable, cost-effective platform for recombinant antivenom production, aligning with industry trends toward process intensification. This approach addresses critical bottlenecks of anti-venom availability and provides a pathway for meeting the urgent global demand for accessible, effective anti-venoms in resource-limited settings.