Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 10(11) total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10-20-liter bioreactor scale.

Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field toward developing suspension processes that are conducive to large-scale production. This study describes a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a green fluorescent protein (GFP)-expressing lentiviral vector in the 106 transduction units (TU)/mL range without optimization. The stable producer cell line, called clone 92, was derived by stable transfection from a packaging cell line with a plasmid encoding the transgene GFP. The packaging cell line expresses all the other necessary components to produce LV upon induction with cumate and doxycycline. First, the study demonstrated that LV production using clone 92 is scalable from 20 mL shake flasks to 3 L bioreactors. Next, two strategies were developed for high-yield LV production in perfusion mode using acoustic cell filter technology in 1–3 L bioreactors. The first approach uses a basal commercial medium and perfusion mode both pre- and post-induction for increasing cell density and LV recovery. The second approach makes use of a fortified medium formulation to achieve target cell density for induction in batch mode, followed by perfusion mode after induction. Using these perfusion-based strategies, the titer was improved to 3.2 × 107 TU/mL. As a result, cumulative functional LV titers were increased by up to 15-fold compared to batch mode, reaching a cumulative total yield of 8 × 1010 TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing.

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.

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Lab and pilot scale batch cultivations of a CHO K1/dhfr À host cell line were conducted to evaluate on-line multifrequency permittivity measurements as a process monitoring tool. The b-dispersion parameters such as the characteristic frequency (f C ) and the permittivity increment (De max ) were calculated on-line from the permittivity spectra. The dual-frequency permittivity signal correlated well with the off-line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off-line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C M ), and effects caused by medium conductivity (r m ) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f C as a result of a fall in intracellular conductivity (r i ) was identified as responsible for the observed changes in the dual-frequency permittivity signal. It is hypothesized that the b-dispersion parameter f C is indicative of changes in nutrient availability that have an impact on intracellular conductivity r i . On-line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control.

Two infected Sf-9 cell cultures were monitored on-line by multi-frequency permittivity measurements using the Fogale BIOMASS SYSTEM((R)) and by applying different off-line methods (CASY((R))1, Vi-CELLtrade mark, packed cell volume) to measure the biovolume and the mean diameter of the cell population. During the growth phase and the early infection phase the measured permittivity at the working frequency correlated well with the different off-line methods for the biovolume. We found a value of 0.67 pF cm(-1) permittivity per unit of total biovolume (CASY) (muL mL(-1)). After the maximum value in the permittivity was reached, i.e. when the viability of the cultures decreased significantly, we observed different time courses for the biovolume depending on the applied method. The differences were compared and could be explained by the underlying measurement principles. Furthermore, the characteristic frequency (f(C)) was calculated from the on-line scanning permittivity measurements. The f(C) may provide an indication of changes in cell diameter and membrane properties especially after infection and could also be an indicator for the onset of the virus production phase. The changes in f(C) were qualitatively explained by the underlying equation that is correlating f(C) and the properties of the cell population (cell diameter, intracellular conductivity and capacitance per membrane area).