Intensified Influenza Virus Production in Suspension HEK293SF Cell Cultures Operated in Fed-Batch or Perfusion with Continuous Harvest

Abstract

Major efforts in the intensification of cell culture-based viral vaccine manufacturing focus on the development of high-cell-density (HCD) processes, often operated in perfusion. While perfusion operations allow for higher viable cell densities and volumetric productivities, the high perfusion rates (PR) normally adopted—typically between 2 and 4 vessel volumes per day (VVD)—dramatically increase media consumption, resulting in a higher burden on the cell retention device and raising challenges for the handling and disposal of high volumes of media. In this study, we explore high inoculum fed-batch (HIFB) and low-PR perfusion operations to intensify a cell culture-based process for influenza virus production while minimizing media consumption. To reduce product retention time in the bioreactor, produced viral particles were continuously harvested using a tangential flow depth filtration (TFDF) system as a cell retention device and harvest unit. The feeding strategies developed—a hybrid fed-batch with continuous harvest and a low-PR perfusion—allowed for infections in the range of 8–10 × 106 cells/mL while maintaining cell-specific productivity comparable to the batch control, resulting in a global increase in the process productivity. Overall, our work demonstrates that feeding strategies that minimize media consumption are suitable for large-scale influenza vaccine production.

Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

“…With a TFDF fed batch/perfusion setup for the production of IAV in HEK293SF cells, Silva et al could recently show virus production and efficient harvest through the membrane without facing issues with virus retention, membrane clogging, or increased shear stress. 97 We also obtained promising results for IAV production in MDCK cells using this technology in our lab; however, data are not published yet. TFDF modules are scalable and commercially available from pilot to 2,000 L scale and could push process development for vaccines toward integrated continuous downstream processing.…”

“…In fed-batch mode, the production of A/PR/8/34 (H1N1) in HEK293SF cells resulted in a 2.7-fold higher space-time-yield (STY). 97 However, in a study involving PBG.PK2.1 cells, applying a fed-batch strategy could not outcompete results from a batch process ( Table 3 ). 38 Yet, regardless of the dilution step, operation in fed-batch mode can still result in the accumulation of metabolic side products such as lactate and ammonia to inhibitory levels, especially when concentrated feeds are used and thereby hinder virus production.…”

Digital Twin for Continuous Production of Virus-like Particles toward Autonomous Operation

“…For cell retention in the production of virus particles, the acoustic separator and an innovative combination of depth filter and hollow fiber module have proven successful. ,,,,− To accelerate cell settling in density-based separation, the g-force can be increased by an acoustic resonance field. , The acoustic separator not only enables separation of the product but also removal of dead cells, host cell proteins, and double-stranded RNA, leading to higher cell densities. ,, However, for scale-up and higher throughputs, increased input power is required to maintain separation efficiency. , This can result in higher temperatures in the separator, necessitating efficient temperature control. In addition, the decreasing concentration of dissolved oxygen in the acoustic separator can affect cell viability. ,, …”

“…A macroporous tubular filter is preferred for size-dependent separation, as it ensures efficient cell retention and good product permeability. ,,,,,− In contrast to the acoustic separator, a complete recirculation of the cells into the bioreactor is also possible since they do not pass the filter, and thus no cell loss occurs. In addition, the recirculation time is shorter, since no settling of the cells is required for cell separation. ,, …”