Spray-dried anti-Campylobacter bacteriophage CP30A powder suitable for global distribution without cold chain infrastructure

Abstract

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p < 0.05) titer reduction than spray drying using a vibrating mesh 2 nebulizer. The use of centrifugation and dilution of filtered bacteriophage lysate in the formulation step resulted in a significantly greater (p < 0.05) proportion of bacteriophages remaining active relative to use of no centrifugation and dilution. The spray-dried bacteriophage powder generated using leucine and trehalose as excipients was flowable, non-cohesive, and exhibited a high manufacturing yield. The powder retained its titer with no significant differences (p > 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 kilometers, including with a 38°C temperature excursion.The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure.

Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport

“…The lyophilized product showed a typical moisture content of 13% after 24 hours of lyophilization, measured by TGA. The excipient formulation of leucine and trehalose described in a previous spray-drying study [26] showed a 1.22 ± 0.07 log10 PFU mL-1 titer reduction following lyophilization (Figure 2A). We further examined alternative excipients to maintain the phage titer more effectively during lyophilization.…”

“…To our knowledge, this is the first study to report the stability of lyophilized Campylobacter phage and where the small titer loss upon lyophilization is favorable compared to other studies of bacteria-specific phages [34]. Future work could explore alternative excipients, especially the formulation of trileucine and pullulan that have enhanced the performance of the spray-drying technique to stabilize phage titers [26]. Further study could also focus on the application of secondary drying at warmer temperatures to desorb any bound water present after primary drying to reduce the residual moisture content.…”

“…We have investigated the formulation of leucine and trehalose to lyophilize CP30A phage, which showed a 1.22 log 10 PFU mL −1 titer drop postlyophilization. Although this titer reduction was significantly less than that of a spray-drying procedure using a similar formulation [26], we aimed to further reduce the fall in titer by analyzing other suitable excipients. Among the substances tested, tryptone and casamino acids additions showed the best performance in terms of stabilizing phages from inactivation during lyophilization.…”

“…To realize the promise of phage therapy to reduce the burden of pathogenic bacteria, studies have focused on making dry powder preparations for phages to maintain titer stability, extend product shelf-life and transport range, provide ease of handling and, optionally, to enable application as an inhalable product [23][24][25][26]. There are two common drying processes: lyophilization, which removes ice from frozen samples by sublimation under vacuum, and spray-drying, which evaporates water from an atomized liquid sample in a hot drying gas.…”

Engineered Bacteriophages for Practical Applications

“…87) A similar phenomenon is seen with endolysins; however, a recent report indicates that the engineering of an endolysin in order to add an albumin-binding domain extended the half-life of the endolysin. 88) Another area that could become relevant, if phage therapy advances toward widespread use, is the study of storage conditions and administration routes. For example, even though phages are considered relatively stable, being able to unequivocally guarantee their stability under different conditions would be useful.…”