Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

Abstract

a b s t r a c tA variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype -transgene combination, requiring extensive further optimization for each new vaccine.There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell -promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo Ò HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 Â 10 13 -5 Â 10 13 purified virus particles per litre of culture, such that a 2-4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

Lentiviral Vector Bioprocessing

“…For some depth filters, the media bears a charge for electrostatic retention of host cell proteins and DNA [ 184 , 185 ], with diatomaceous earth showing improved feed capacity although at high concentration it can lower LV titre [ 186 ]. In some small-scale studies, the use of multiple depth filter media, including cellulose-based and synthetic media (such as polyacrylic fibre with silica filter aids), has shown 90% turbidity reductions whilst maintaining high recovery of non-enveloped vectors, such as simian adenovirus based vectors [ 187 ]. In LV applications, depth filters without filter aids such as diatomaceous earth have recovered >95% of titre while reducing >85% HCP at 50 L scale [ 188 ].…”

The Science is There: Key Considerations for Stabilizing Viral Vector-Based Covid-19 Vaccines

“…Because of their structural complexity and the associated challenges to manufacture viral products at large scale, these preparations contain a mixture of infectious and noninfectious viruses (e.g., full and empty viral capsids). 8 Methods to monitor the potency of a live virus-based vaccine can include determination of viral particles or viral genomes, viral infectivity in cell-based assays, or immune responses in animals. Such assays are the cornerstone of live virus-based vaccine stability assessments.…”

Process-related impurities in the ChAdOx1 nCov-19 vaccine

“…ChAdOx1 nCov-19 is produced in the T-REx-293 cell system to prevent expression of the SARS-CoV-2 spike protein during vector production. The vaccine is purified by a combination of filtration steps and Anion-Exchange Chromatography (AEX) 7,8 . Previously, it has been shown, that simian adenovirus vectors including ChAdOx1 can be purified at high yield and purity using such technology for purification 8 .…”

“…The vaccine is purified by a combination of filtration steps and Anion-Exchange Chromatography (AEX) 7,8 . Previously, it has been shown, that simian adenovirus vectors including ChAdOx1 can be purified at high yield and purity using such technology for purification 8 . Although in our experiment the same number of particles was loaded, the staining patterns looked very different (Fig.…”