Optimization of lytic phage manufacturing in bioreactor using monolithic supports

Abstract

A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.

Application of monoliths for bioparticle isolation

“…To optimize downstream processing, upstream processing has to be monitored to reproducibly produce high bacteriophage titers with minimal amount of impurities, which would coelute with bacteriophage from a chromatographic column. An extensive study of lytic bacteriophages was performed by Smrekar and co‐workers . Lytic bacteriophages are released from bacterial cell by cell lyses.…”

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

“…However results obtained from HPLC analytics are accurate and precise when A c c e p t e d M a n u s c r i p t 18 The effective use of HPLC analytics in the up-stream production of bacteriophages was reported by Smrekar et al [44] as they demonstrated the importance of up-stream optimization for further downstream processing of bacteriophages. An indirect analytical method for on-line monitoring of phage titer was developed and applied for the termination of lytic phage cultivation.…”

“…The incorporation of DNA degradation step into the downstream process of phages was studied by Smrekar et al [44]. Optimized SEC method can result in high virus yields and it is very efficient in simultaneous removal of residual impurities and buffer exchange [13].…”

“…The appearance of DNA fragments affected the phage DSP, since DNA fragments co-eluted with the phage fraction and as a consequence the final phage product was contaminated with fragmented host cell DNA.Cultivation monitoring and determination of cultivation termination using an HPLC analytical Downloaded by [University of Saskatchewan Library] at 03:essential for achieving high phage quantity and pure final phage product. The same analytical method was applied for measuring the purity of the final phage product as well[44].HPLC analytical methods can also be used as fingerprinting methods during DSP development providing rapid information on the efficiency of a particular process step and greatly contributing to faster DSP development. DSP process consists of several purification steps.…”

Monoliths in Bioprocess Technology

“…Our research group is currently conducting experiments to implement monolith technology as an impurity monitoring process analytical technology (PAT) tool across unit operations. In a similar approach, Smrekar et al already implemented the monolith technology as a PAT tool to monitor a virus production process [97].…”