Figure 2 illustrates the amount of infectious (plaque-forming) virus observed at each time point PI. Note that at 140 hours post infection, virus titers reached 3 x 109 infectious units as determined by a plaque assay. Likewise, genomic equivalents (PCR) increased over a similar course and no detectable genetic mutation in HA was observed during the entire incubation period (data not shown). As with most influenza isolates, there are approximately 10–100 non-infectious particles for every infectious unit; thus, we believe that the total particle count was approximately 3 x 1010–3 x 1011.
While the Biovest HF Primer is useful for experimental needs, more sophisticated, automated, and higher production capacity units such as the AutovaxID (Figure 3) are needed for large-scale virus production. Since this system is functionally closed, yet automates mammalian cell processes, it is ideal for the production of viruses or cell-secreted proteins. A combination of pore-size selection and control of the fluid dynamics within the bioreactor encourages removal of harmful cytokines and virus-induced host shutoff proteins while maintaining virus stability.
Most importantly, the two-component design of the AutovaxID provides containment that minimizes the need for traditional cleanrooms, greatly reduces the risk of process contamination, and provides a small footprint that facilitates adaptation to existing BSL-2 or BLS-3 facilities.
AutovaxID is an example of how advanced hollow fiber technology can play an integral role in the manufacture of vaccines. In comparison to error-prone manual techniques or minimally customizable large-tank systems, the AutovaxID is:
- cGMP compliant and compact to minimize size requirements for manufacturing facilities,
- composed of disposable elements for all product contact components to maximize speed and manufacturing flexibility,
- self-contained, or functionally closed for avoidance of contamination and containment of infectious agents,
- automated to reduce operator intervention and expertise/skill levels, and
- designed to regulate perfusion rates for control of culture environments to enhance virus replication and stability.
Hollow-fiber bioreactors offer a compact, highly efficient, scalable, and economical method for virus production. The combination of an unlimited nutrient supply and the ability to de-bulk the culture through the cartridge ports allows the system to be maintained at relative equilibrium for extended periods.
This continuous production over long periods of time, rather than the batch-style approach of other systems, provides several benefits including consistency in culture conditions, dramatically increased production per unit footprint and culture volume, continuous or daily product harvest allowing timely and convenient stabilizing treatment for collection and storage, and selective dilution or removal of products from the culture that might be toxic or inhibitory to cells.