Translating Innovation to Clinic: End‐to‐End Bioprocess Development and cGMP Manufacturing of N332‐GT5 HIV Vaccine Candidate for First‐In‐Human Trials (HVTN144)

The successful translation of rationally designed HIV‐1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical‐grade quality standards. We developed and scaled up a robust, cGMP‐compliant process for N332‐GT5 gp140, a germline‐targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first‐in‐human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap‐In® transposon technology, we established a production clone exhibiting high‐titer expression (> 200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled up efficiently from Ambr® 250 miniature bioreactors to 200‐L single‐use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three‐step purification strategy—affinity capture, multimodal polishing, and viral clearance —yielded > 99% trimeric purity while preserving quaternary structure and native‐like antigenicity. Orthogonal LC‐MS analyses confirmed site‐specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18‐log and 11‐log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in clinical trial HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV‐1 envelope immunogens, advancing the field toward rational vaccine design based on germline‐targeting principles.