Influenza Vaccine Production in Microcarrier Systems and Suspension Cells
Since several years the cultivation of animal cells to produce complex proteins and vaccines is being established in large scale culture. One aim of our work group is the development and optimization of integrated concepts to design and monitor vaccine production processes to ameliorate viral yields, to improve batch-to-batch consistency, and to increase efficacy, purity and safety of vaccines. With a process of influenza virus production as an example, we focus on the following steps: (a) Cultivation and scale-up of animal cells using adherent cell lines (microcarrier-systems) or suspension cell lines (b) Replication of different influenza viruses (equine, human, swine) (c) Variation of cultivation vessels (conventional vs. disposable) and process strategies (batch vs. continuous). An example for the various process options using animal cells is given in Fig. 1.
Aim of the project
For this concept, we develop mathematical models of different complexity, which describe the growth of adherent cells, virus replication as well as essential metabolism and regulation steps (unstructured & structured, non-segregated models). Together with experiments elucidating cell metabolism and virus replication in bioreactors or single-cell cultures and determination of on- and off-line values used in industrial processes, concepts for monitoring and control to facilitate optimization and validation of these processes are being developed.
Towards higher cell densities...
To accomplish this strategy many different parameters have to be analyzed experimentally and integrated into mathematical models (pH, pO2, glucose, lactate & ammonium concentration, cell growth rates, virus titer, ...). Furthermore, analytical methods such as flow cytometry to characterize the properties of cell populations, or HPLC methods to determine the concentration of intracellular metabolites and specific enzyme activities during cell growth and virus replication are being used.
Variations of cell culture medium (serum-free), bioreactors (disposable Wave bioreactor), virus subtypes (human influenza A and B) and host cells (Vero, AGE1.CR, etc.; Fig. 2) are carried out giving experimental data for different process conditions. Cultivation of suspension cells with perfusion could result in cell concentrations of more than 5 x 107 cells/mL. We currently focus on disposable hollow fiber technology (HFBR, ATF, TFF) to use perfusion aiming at constant cell specific virus productivity and a feasible downstream processing.
We further plan to look at intracellular metabolites, optimization of medium composition (serum-free, protein-free, chemically defined), high cell density cultures, higher virus production or options for continuous cultivations using suspension cells for detailed analysis of process conditions on cellular physiology. For model validation the use of real time PCR & 2D-DIGE technology should allow us to get additional insight into virus replication and cell metabolism.