The hydrophobic interaction chromatography (HIC) process employs 50 mM Tris-HCl, 2 M ammonium sulfate, pH 8.0 for equilibration and wash, and 50 mM Tris-HCl, 1.45 M ammonium sulfate, pH 8.0 for elution. 2.7. trimer, which we named BG505 Rabbit Polyclonal to CDC25A 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 Ivabradine HCl (Procoralan) 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 9.57 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. Keywords: GMP manufacturing, DS-SOSIP, envelope trimer, glycoprotein, HIV-1 vaccine, non-affinity chromatography, prefusion-closed conformation 1.?Introduction The HIV-1 envelope (Env) trimer is the only viral antigen to protrude through the virion-lipid bilayer and is thus the sole viral target of virus-neutralizing antibodies [reviewed in 1,2, 3]. Vaccine elicitation of such antibodies, however, has been hampered by the fragile nature of the trimer, which is metastable in conformation [4], labile to subunit dissociation [5], and covered by a dense shield of N-linked glycans comprising half of its mass [6C9]. The Env precursor gp160 is expressed as a single polypeptide, cleaved to gp120 and gp41 subunits in the Golgi, releasing at the newly cleaved N terminus of gp41 the fusion peptide that is critical for membrane fusion, and subsequently assembled via noncovalent interactions into a gp120-gp41 heterodimer. Three such moieties assemble to form Ivabradine HCl (Procoralan) the surface Env, a type 1 transmembrane quaternary homotrimer of heterodimers. Structural and functional studies have informed the development of soluble, native-like Env trimers, stabilized in a prefusion state that displays broadly neutralizing epitopes [10C24]. Vaccine studies with prefusion-stabilized Env trimers in multiple animal models have demonstrated the ability of these trimers to induce autologous neutralizing responses, capable of neutralzing only strains closely related to the vaccine immunogen [25, 26]. However, by using fusion peptide (FP)-carrier Ivabradine HCl (Procoralan) protein conjugates as priming immunogens to induce and expand immune responses targeting the FP-site of HIV-1 vulnerability [27, 28], Env trimer boosts could mature cross-clade neutralizing responses in mice, guinea pigs, and non-human primates [28], with FP-directed antibodies of up to almost 60% neutralization breadth being elicited in rhesus macaques [29]. Additional boosting with a heterologous trimer could increase the neutralization breadth and potency of fusion-peptide directed immune responses in guinea pigs [15, 30]. These findings suggest the potential utility of Env trimer immunogens in an effective HIV vaccine regimen, and several of these Env trimer immunogens are under development as vaccine candidates for phase I clinical trials. To date, soluble HIV-1 Env trimers for research or clinical trials are predominantly produced in CHO- or HEK-based expression systems and are subsequently purified by methodologies including immunoaffinity, lectin, or Strep-Tactin affinity chromatography and size-exclusion chromatography (SEC) [30C38]. Commonly used immunoaffinity chromatography has utlilized antibodies like 2G12, PGT145, PGT151, VRC01, or a cocktail of V3-directed/CD4-induced antibodies to select for or against a specific conformational Env state. The cGMP production of trimeric HIV-1 Env immunogens has relied on the use of antibodies, such as the 2G12 antibody [39C42] used in the manufacture of the BG505 SOSIP.664 HIV-1 envelope glycoprotein trimer [34]. Immunoaffinity resins, while specific to well-formed, antigenically-pure trimer populations, require production or purchase of cGMP lots of each selector antibody as well as custom resin conjugation in order to use in a cGMP facility. Additionally, a Protein A flow-through step to remove any leached antibody (process-related impurity) is commonly employed, necessitating another costly affinity chromatography step. Size-exclusion chromatography, while commercially available, requires an Ivabradine HCl (Procoralan) ultrafiltration step prior Ivabradine HCl (Procoralan) to operation to meet load volume and process time restrictions. In-short, these existing strategies are complex, expensive, and difficult to scale-up for cGMP production. Here we report a scalable, cost effective, cGMP-suitable manufacturing process using commercial, non-affinity purification methods to produce two HIV-1 Env trimer immunogens from different clades. We describe the cGMP production.