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

Abstract

Future industrial demand for large quantities of bacteriophages e.g., for phage therapy, necessitates the development of scalable Good Manufacturing Practice compliant (cGMP) production platforms. The continuous production of high titres of E coli T3 phages (1011 PFU mL−1) was achieved using two continuous stirred tank bioreactors connected in series, and a third bioreactor was used as a final holding tank operated in semi-batch mode to finish the infection process. The first bioreactor allowed the steady-state propagation of host bacteria using a fully synthetic medium with glucose as the limiting substrate. Host bacterial growth was decoupled from the phage production reactor downstream of it to suppress the production of phage-resistant mutants, thereby allowing stable operation over a period of several days. The novelty of this process is that the manipulation of the host reactor dilution rates (range 0.1–0.6 hr−1) allows control over the physiological state of the bacterial population. This results in bacteria with considerably higher intracellular phage production capability whilst operating at high dilution rates yielding significantly higher overall phage process productivity. Using a pilot-scale chemostat system allowed optimisation of the upstream phage amplification conditions conducive for high intracellular phage production in the host bacteria. The effect of the host reactor dilution rates on the phage burst size, lag time, and adsorption rate were evaluated. The host bacterium physiology was found to influence phage burst size, thereby affecting the productivity of the overall process. Mathematical modelling of the dynamics of the process allowed parameter sensitivity evaluation and provided valuable insights into the factors affecting the phage production process. The approach presented here may be used at an industrial scale to significantly improve process control, increase productivity via process intensification, and reduce process manufacturing costs through process footprint reduction.

A Rapid Method for Performing a Multivariate Optimization of Phage Production Using the RCCD Approach

“…With an additional 1.5 hours of incubation, the number of phages produced by Treatment 3 became statistically equal to Treatment 1. These results are similar to other studies involving phage production and show that MOI is an essential factor to be considered at the beginning of large-scale phage production since it is this variable that will control the number of infection cycles and the batch time [ 33 , 39 , 83 ]. The worst conditions predicted by the model were also tested (Treatment 4; Figure 4 ) and resulted in the lowest viral progeny of all the conditions evaluated.…”

“…The multiplicity of infection (MOI) is probably one of the most critical cultivation variables for phage production in bioreactors. It will determine the dynamics of infection between the virus and host, and consequently, the batch time until the maximum number of viral particles can be obtained [ 33 , 39 , 83 ]. When a higher number of phage particles was used (MOI of 1 or more), the equilibrium state was reached quickly since it is statistically likely that all cells present in the bioreactor will be infected, and there will not be enough cells left for subsequent rounds.…”

Bacteriophage Therapy of Bacterial Infections: The Rediscovered Frontier

“…Each system has brought about its distinct benefits and drawbacks, discussed earlier by Merabishvili et al [36]. Mancuso et al [37] developed a production process that makes it possible to obtain high titers of E. coli T3 phages at high concentrations (10 11 PFU mL −1 ) using two continuous stirred tank bioreactors. The first bioreactor is just for propagation of the host bacteria at a steady-state growth rate by using controllable dilution rates and growth-limiting substrate (glucose).…”

Bacteriophage Production Models: An Overview

“…Continuous culture has higher scalability when optimizing bacterial dilution rate via inlet and outlet flux modification. Besides, regulating the dilution rate will allow direct control over the bacterial growth rate, which has a direct influence on infection parameters like burst size, adsorption constant and latent period (Mancuso et al, 2018; Nabergoj et al, 2018b). Dilution rate can also be used to increase the productivity of the system, as was shown by Nabergoj et al (2018a) where a maximum phage productivity of 10 9 phages mL –1 h –1 was achieved with a low dilution rate of 2 h –1 in a 1 L cellstat system.…”