Two-stage, self-cycling process for the production of bacteriophages

Abstract

BackgroundA two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF) conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER) as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI) with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage). This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle.ResultsIt was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.). Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF) not only maintained the volumetric productivity (phages per volume) but also led to higher specific productivity (phage per cell per hour) in the second stage (SCI).ConclusionsProduction of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in the second stage while maintaining the volumetric productivity. These results demonstrate the significant potential of this approach for both upstream and downstream process optimization.

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

“…The research presented here has shown that by narrowing the conditions used and focussing on those that positively influence phage infection, the titer achieved is reliable and validated at small scale and in a scale‐up system which focuses on larger volumes, automation, and controllable parameters, that is, pH and DO 2 . Previous studies have shown a greater variance in titer achieved compared to our work as no optimization of the bioprocess was performed (Bourdin et al, ; Sauvageau & Cooper, ). Improving the phage bioprocess in shake flasks allows more rapid experiments to be completed which can then be moved into a stirred tank system.…”

“…Previous studies have shown a greater variance in titer achieved compared to our work as no optimization of the bioprocess was performed (Bourdin et al, 2014;Sauvageau & Cooper, 2010).…”

High Throughput Manufacturing of Bacteriophages Using Continuous Stirred Tank Bioreactors Connected in Series to Ensure Optimum Host Bacteria Physiology for Phage Production

“…Phages are typically produced in batch fermenters e.g., shaken flasks, and more recently in disposable wave bags that are used for tissue cell culture; there are no real issues with regard to residence times and complex control strategies [ 23 , 24 ]. The downsides for industrial scale batch fermentation include higher capital costs, large process footprints, labour-intensive operation, that the proportion of downtime compared with production time can be high, a lack of process control, and variability of product quality [ 24 ]. The continuous upstream production of phages using chemostat systems has heretofore received little attention in the published literature, which instead has focused on using such systems for studying coevolution processes [ 25 , 26 , 27 ].…”

“…Decoupling the bacterial host propagation from phage production removes the selection pressure for bacteria mutation and should allow stable long-term steady-state operation of the process [ 28 ]. Semi-continuous production approaches using such a strategy include a two-stage self-cycling process for the production of phages [ 24 , 29 , 30 ]. Synchronous host populations at high cell concentrations are produced in the first reactor operated in batch mode.…”

Impact of the cell life-cycle on bacteriophage T4 infection

“…The authors hypothesized that the larger cellular protein synthesizing system of older cells, not simply a larger cell volume, is the more important determinant of infection outcome. In addition, a study on the use of synchronized cultures of Escherichia coli in bacteriophage production found evidence that phage productivity was considerably larger for a synchronized culture than an asynchronous one, even at slower growth rates (Sauvageau & Cooper, ).…”