Translating Innovation to Clinic: End-to-End Bioprocess Development and cGMP Manufacturing of N332-GT5 HIV Vaccine...

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 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 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 with preserved 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 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.