Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production.

In the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (qP), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the qP with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan "hole" naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.