Investigating the role of excipients in mitigating surface adsorption, aggregation and structural degradation in liquid phage formulations

Abstract

The race to produce therapeutically acceptable phages is driven by their rapid loss of infectivity, placing strain on laboratories, healthcare infrastructure and patients. Enhancing product shelf-life will alleviate this barrier to their upscaling and clinical application. We systematically investigated the physicochemical drivers of phage instability using the myovirus, Pae7, under clinically relevant conditions: temperature (4–37 °C), storage materials, NaCl salt and preparation purity. We show that phage instability is not primarily impacted by storage temperature (4–37 °C). Rather, virions adsorbed to container surfaces as a major contributor to titre loss, with borosilicate vials preserving infectivity more effectively than Eppendorf tubes, particularly in 0.9% NaCl solution. Loss of infectivity also occurred concurrently with changes to native state surface charge, structural integrity, aggregation and fragmentation. Notably, crude lysates retained infectivity; meanwhile, purified, patient-grade samples experienced substantial titre loss. These findings indicate that phages behave like colloidal particles and proteins, which directed stabilisation strategies. We characterised phage-excipient interactions and demonstrated the steric stabilisation of patient-grade phages with the biocompatible surfactant, Tween-20, and Reversible Addition-Fragmentation chain Transfer (RAFT) polymers, poly-oligo-(ethylene glycol methyl ether acrylate)- block -poly(3-vinyl-benzaldehyde) (POEGA- b -PVBA) and POEGA as previously unreported excipients, resulting in the maintenance of their infectivity over 56 days. This work has significantly furthered insights into phage destabilising phenomena to establish a rational approach to formulating shelf-stable therapeutic phages.