Explore the vast range of titles available.

NRC publication: Yes

Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 109IVP/mL demonstrating the scale-up potential of the process.

Background Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 10 6 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products. Results To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 10 6 cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 10 11 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 10 15 IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles. Conclusions These results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.

NRC publication: Yes

Background: Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results: We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion: Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

•An adherent Vero cell line was adapted to grow in suspension in a serum-free medium.•The suspension adapted cells retained their genetic stability and were not found to be tumorigenic.•The adapted cells were tested for high yield production of a virus in different suspension culture processes. Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 106 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 106 cells/mL.

•Extracellular vesicles (EVs) are co-produced with influenza virus-like particles (VLPs) in HEK293 cells.•RNA, DNA, total proteins are quantified in VLP production samples and EV control samples.•nanoLC-MS/MS analyses allow characterization of protein content of VLPs and EVs.•Specific proteins were uniquely identified in different preparations of VLPs and EVs. One of the concerns associated with the use of influenza virus-like particles (VLPs) as vaccine candidate or delivery system is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out not only the viral antigenic proteins but also host proteins. In addition, the intrinsic nature of cells to produce membrane derived vesicles or extracellular vesicles (EVs), which have similar size to the VLPs, makes VLP purification process challenging. To further characterize these particles and identify proteins that are unique to each population, comparative proteomic analyses were completed to ultimately provide guidance for rational design of separation protocols. The VLPs were produced in suspension and serum free media by transient transfection of an inducible clone of a Human Embryonic Kidney (HEK-293SF) cells expressing HA and NA (H1N1/A/Puerto Rico/8/34), with a plasmid containing the gag gene of HIV-1 fused to GFP. EVs were produced independently from the non-transformed HEK-293SF cell line as a control for comparative studies. Both preparations were characterized for total nucleic acids and protein concentrations and extensively analyzed by nanoLC-MS/MS for their protein compositions. The proteomic analyses showed that aside from the recombinant VLP proteins, nucleolin was the most abundant host cell protein uniquely identified within VLPs (considering the MASCOT score value) while lactotransferrin and heat shock protein 90 were the most abundant proteins in EVs. Overall, this comparative study identifies potential target proteins as specific markers to guide VLP purification and discusses the biogenesis of enveloped particles released in HEK-293 cell suspension cultures emphasizing on the biological functions of host cell proteins identified.

•Development of stable cell line expressing hemagglutinin and neuraminidase (293HA-NA).•Gag HIV-1 protein is more efficient than M1 to induce VLPs production in 293HA-NA cells.•Influenza VLPs production using Gag as scaffold was evaluated in a 3-L bioreactor and purified.•Characterization of VLPs showed typical size and morphology of HIV-1 immature particles.•The immunogenicity and protective efficacy of the VLPs was demonstrated in mice. Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150–200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.

•Generation of inducible cell line stably expressing hemagglutinin, neuraminidase and Gag-GFP.•Process optimized to increase the cell density and achieve high VLPs productivity.•A 3-L perfusion bioreactor operated at 14 million cells/ml increased VLPs production by 60-fold.•The 9.5L harvest was clarified and concentrated by TFF and the VLPs were extensively characterized.•Electron micrographs of VLPs final retentate revealed the expected Gag-HA-NA VLP morphology. Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14×106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54×1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100–150nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.

Mammalian-inducible expression systems are increasingly available and offer an attractive platform for the production of recombinant proteins. In this work, we have conducted process development for a cumate-inducible GS-CHO cell-line-expressing rituximab. To cope with the limitations encountered in batch when inducing at high cell densities, we have explored the use of fed-batch, sequential medium replacements, and continuous perfusion strategies applied during the pre-induction (growth) phase to enhance process performance in terms of product yield and quality. In shake flask, a fed-batch mode and a complete medium exchange at the time of induction were shown to significantly increase the integral of viable cell concentration and antibody titer compared to batch culture. Further enhancement of product yield was achieved by combining bolus concentrated feed additions with sequential medium replacement, but product galactosylation was reduced compared to fed-batch mode, as a result of the extended culture duration. In bioreactor, combining continuous perfusion of the basal medium with bolus daily feeding during the pre-induction period and harvesting earlier during the production phase is shown to provide a good trade-off between antibody titer and product galactosylation. Overall, our results demonstrate the importance of selecting a suitable operating mode and harvest time when carrying out high-cell-density induction to balance between culture productivity and product quality.