Sven Ansorge

Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures

Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long‐term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large‐scale LV production remains an important challenge for the translation of LV‐based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed.

Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing

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 10(9)IVP/mL demonstrating the scale-up potential of the process.

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

BackgroundCell 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 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.ResultsTo 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 × 106 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 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 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.ConclusionsThese 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.

New protocol for lentiviral vector mass production.

Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

BackgroundHemophilia 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.ResultsWe 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.ConclusionSerum-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.

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

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.

Critical phases of viral production processes monitored by capacitance

Over the last decade industrial manufacturing of viral vaccines and viral vectors for prophylactic and therapeutic applications is experiencing a remarkable growth. Currently, the quality attributes of viral derived products are assessed only at the end-point of the production process, essentially because in-process monitoring tools are not available or not implemented at industrial scale. However, to demonstrate process reproducibility and robustness, manufacturers are strongly advised by regulatory agencies to adopt more on-line process monitoring and control. Dielectric spectroscopy has been successfully used as an excellent indicator of the cell culture state in mammalian and yeast cell systems. We previously reported the use of this technique for monitoring influenza and lentiviral productions in HEK293 cell cultures. For both viruses, multi-frequency capacitance measurements allowed not only the on-line monitoring of the production kinetics, but also the identification of the viral release time from the cells. The present study demonstrates that the same approach can be successfully exploited for the on-line monitoring of different enveloped and non-enveloped virus production kinetics in cell culture processes. The on-line monitoring multi-frequency capacitance method was assessed in human HEK293 and Sf9 insect cells expression systems, with viral productions initiated by either infection or transfection. The comparative analyses of all the data acquired indicate that the characteristic capacitance signals were highly correlated with the occurrence of viral replication phases. Furthermore the evolution of the cell dielectric properties (intracellular conductivity and membrane capacitance) were indicative of each main replication steps. In conclusion, multi-frequency capacitance has a great potential for on-line monitoring, supervision and control of viral vector production in cell culture processes.

Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

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.

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza

Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015–2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains.

Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures

Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.