Streamlined purification of ADDomer nanoparticles for scalable biomanufacturing.

Current lab-scale purification methods for ADDomer are labour-intensive, time-consuming and poorly scalable. The intracellular nature of ADDomer production further complicates downstream processing, requiring robust, scalable solutions for cell lysis and clarification. In the present work we focus on developing a scalable, GMP-compliant process for ADDomer purification. The workflow combines tangential microfiltration (TMF) using a 0.4 μm hollow fiber for cell retention, chemical lysis with 0.06 % Deviron^® C16, and in-line Benzonase^® treatment for DNA removal. Several capture approaches were explored, including anion and cation exchange, hydrophobic interaction and multimodal chromatography. Among these, the Sartobind^® Q anion exchange membrane demonstrated superior performance, achieving ADDomer purity of ~ 83 % and recovery yields > 85 %. Ultrafiltration membranes of different chemistries (regenerated cellulose, RC, and polyethersulfone, PES) and cut-off (300 kDa, 500 kDa, 1 MDa) were evaluated for polishing and buffer exchange. The 1 MDa RC membrane enabled a recovery of ~ 87 % and purity > 97%. Final sterile filtration with a PES membrane preserved particle integrity, purity and achieved > 80 % recovery. Overall, the purification process herein established yielded ~ 47 mg of ADDomer particles per L of culture volume while removing > 97 % and > 99 % of total protein and dsDNA, respectively. In summary, this study showcases the implementation of a scalable and GMP-compliant purification platform for ADDomer, paving the way for the development of next-generation ADDomer-based vaccines and antivenoms.