A non-affinity purification process for GMP production of prefusion-closed HIV-1 envelope trimers from clades A and C for clinical evaluation.
Metastable glycosylated immunogens present challenges for GMP manufacturing. The HIV-1 envelope (Env) glycoprotein trimer is covered by N-linked glycan comprising half its mass and requires both trimer assembly and subunit cleavage to fold into a prefusion-closed conformation. This conformation, the vaccine-desired antigenic state, is both metastable to structural rearrangement and labile to subunit dissociation. Prior reported GMP manufacturing for a soluble trimer stabilized in a near-native state by disulfide (SOS) and Ile-to-Pro (IP) mutations has employed affinity methods based on antibody 2G12, which recognizes only ~30% of circulating HIV strains. Here, we develop a scalable manufacturing process based on commercially available, non-affinity resins, and we apply the process to current GMP (cGMP) production of trimers from clades A and C, which have been found to boost cross-clade neutralizing responses in vaccine-test species. The clade A trimer, which we named "BG505 DS-SOSIP.664", contained an engineered disulfide (201C-433C; DS) within gp120, which further stabilized this trimer in a prefusion-closed conformation resistant to CD4-induced triggering. BG505 DS-SOSIP.664 was expressed in a CHO-DG44 stable cell line and purified with initial and final tangential flow filtration steps, three commercially available resin-based chromatography steps, and two orthogonal viral clearance steps. The non-affinity purification enabled efficient scale-up, with a 250 L-scale cGMP run yielding 9.6 g of purified BG505 DS-SOSIP.664. Antigenic analysis indicated retention of a prefusion-closed conformation, including recognition by apex-directed and fusion peptide-directed antibodies. The developed manufacturing process was suitable for 50 L-scale production of a second prefusion-stabilized Env trimer vaccine candidate, ConC-FP8v2 RnS-3mut-2G-SOSIP.664, yielding 7.8 g of this consensus clade C trimer. The successful process development and purification scale-up of HIV-1 Env trimers from different clades by using commercially available materials provide experimental demonstration for cGMP manufacturing of trimeric HIV-Env vaccine immunogens, in an antigenically desired conformation, without the use of costly affinity resins.
['Gulla|Krishana|K|', 'Cibelli|Nicole|N|', 'Cooper|Jonathan W|JW|', 'Fuller|Haley C|HC|', 'Schneiderman|Zachary|Z|', 'Witter|Sara|S|', 'Zhang|Yaqiu|Y|', 'Changela|Anita|A|', 'Geng|Hui|H|', 'Hatcher|Christian|C|', 'Narpala|Sandeep|S|', 'Tsybovsky|Yaroslav|Y|', 'Zhang|Baoshan|B|', 'Vrc Production Program|||', 'McDermott|Adrian B|AB|', 'Kwong|Peter D|PD|', 'Gowetski|Daniel B|DB|']
D016915:AIDS Vaccines D057134:Antibodies, Neutralizing D015483:HIV Antibodies D015488:HIV Antigens D015497:HIV-1 D055503:Protein Multimerization D054299:env Gene Products, Human Immunodeficiency Virus
Envelope trimer GMP manufacturing Glycoprotein Prefusion-closed conformation DS-SOSIP HIV-1 vaccine Non-affinity chromatography