Stability of bacteriophages in spray-dried polymeric formulations: Effect of excipient polyvinylpyrrolidone glass transition temperature and molecular weight

Abstract

Effective dry-state stabilization of bacteriophages is crucial for expanding their therapeutic use. This study builds on previous findings that saccharide-based formulations maintain phage stability when the glass transition temperature (Tg) exceeds the storage temperature (Ts) by approximately 50°C. We investigated polymer-based matrices for long-term stabilization by spray-drying PEV1 phage with polyvinylpyrrolidone (PVP) of varying molecular weights (K15, K25, K40, K100) and storing for 180 days at temperatures (4, 22, and 40°C) and relative humidity (15%, 33%, 43%, and 53% RH). All formulations achieved minimal titre losses (≤1 log₁₀) at 4 and 22°C under 15% RH. Differential scanning calorimetry (DSC) demonstrated Tg increased with PVP molecular weight but decreased substantially with humidity (30–50°C reduction per 20% RH increase). At 33% RH, long-term stability was achieved with high-molecular-weight PVPs (K40, K100), which maintained thermal offsets between Tg and Ts (ΔT ≥ 100°C), while lower-weight (K15, K25) showed 2–3 log₁₀ titre loss. Water activity (aw) analysis revealed a critical threshold at aw 0.43, above which degradation kinetics increased by approximately a tenfold rate. Arrhenius analysis confirmed that phage degradation rates increased with temperature, consistent with thermally activated destabilization mechanisms. Under higher stress conditions (40°C/≥43% RH), water absorption plasticized the PVP matrix and depressed the Tg while elevated temperature simultaneously accelerated degradation kinetics, resulting in substantial titre losses even when ΔT exceeded 100°C. In conclusion, at mild humidity (aw ≤ 0.33) and ambient temperature (≤22°C), high-molecular-weight PVP-based formulations can offer enhanced storage flexibility and reduced cold-chain dependency for optimal therapeutic viability.

Introduction

Recent advances in bacteriophage (phage) therapy for antibiotic-resistant bacterial infections have driven the development of phage powder formulations.1, 2 Spray-drying has emerged as a viable approach for producing phage powders, with demonstrated feasibility both in vitro and in vivo.3, 4 The critical challenge in phage powder development lies in preserving biological activity during processing and storage. The spray-drying process exposes phages to thermal, osmotic, and shear stresses that can cause structural damage and viability loss.5 Protective excipients are essential to maintain phage infectivity in the dry state. While disaccharides such as lactose have shown promise in phage formulations,6 alternative polymeric stabilizers like polyvinylpyrrolidone (PVP) offer unique advantages including superior processing characteristics and matrix-forming properties.7, 8

The mechanisms of biologic stabilization in solid formulations are generally explained by two established hypotheses: water replacement and vitrification.9, 10 In the water replacement hypothesis, excipient molecules form hydrogen bonds with proteins to replace water interactions lost during dehydration, maintaining the protein structure.11 This mechanism requires the excipient to remain in an amorphous state, as crystallization reduces molecular interactions between excipient and protein, leading to structural damage.12, 13 The vitrification hypothesis proposes that biologics are immobilized within a rigid, glassy matrix formed by the excipient. The protective capacity of this glassy state is characterized by the glass transition temperature (Tg), above which the matrix transitions from rigid glass to mobile rubber, potentially compromising stability.9 Studies on protein formulations have established that maintaining storage temperature at least 50°C below glass transition temperature (Tg) ensures adequate protection through reduced molecular mobility.14, 15

Environmental factors, particularly humidity, can significantly affect matrix properties through plasticization effects. Increased humidity reduces Tg values, potentially compromising the protective glassy state even when storage temperature remains constant.16 This humidity dependence has been demonstrated in protein formulations, where moisture-induced Tg depression correlates with increased degradation rates.17 Despite the success of using glass state in protein stabilization, its systematic application to phage formulations remains largely unexplored. Protein stability in amorphous solids depends on the specific structural and physicochemical features of each protein, which can influence their susceptibility to desiccation-induced damage.18 The larger size and structural complexity of phages may necessitate higher thermal offsets or different excipient properties for adequate protection.

Limited studies have investigated the relationship between Tg and phage stability in powder formulations. Vandenheuvel et al. demonstrated that recrystallization of trehalose-based phage powders at elevated humidity resulted in up to 3 log10 titre reduction, highlighting the importance of maintaining amorphous matrices.19 More recently, our previous work on phage powder stabilization established the fundamental relationship between glass transition temperature and phage bioactivity of lactose-leucine formulations.20 This study demonstrated that maintaining storage temperature below Tg was critical for phage stability, with optimal protection achieved when thermal offsets ΔT exceeded 50°C. However, the investigation focused on sugar-based excipient systems only.

Building upon these foundational findings, the present study extends the glass transition approach to polymer-based formulations to explore the influence of polymer molecular weight on phage stabilization. Polyvinylpyrrolidone (PVP) offers advantages for phage formulation due to its ability to form stable amorphous matrices across a range of molecular weights. Different PVP molecular weights (K15, K25, K40, K100) exhibit varying Tg values and matrix properties, potentially providing tenable stabilization characteristics.21 The hydrogen-bonding capacity of PVP may contribute to both water replacement and vitrification mechanisms,22 though the relative importance of these effects in phage stabilization remains unclear.

The present study investigated the stability of PEV1 phages in spray-dried formulations containing PVP excipients of varying molecular weights. We systematically evaluated the validity of established ΔT thresholds for phage stability while characterizing molecular weight-dependent effects on matrix properties and long-term stability. The research integrated thermodynamic analysis of glass transition behavior with kinetic evaluation of degradation processes to establish design principles for phage powder formulations. Our approach examined whether the ~50°C thermal offset proven effective for phage stabilization in sugar-based formulations applies to polymeric systems, while identifying critical humidity thresholds and molecular weight effects that govern formulation performance. The findings provided insights into phage-polymer relationships and established a framework for developing stable phage therapeutics with extended shelf-life suitable for clinical applications.

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Materials

Polyvinylpyrrolidone excipients of varying molecular weights (PVP K15, K25, K40, and K100) were purchased from Sigma-Aldrich (St. Louis, MO). Agar and nutrient broth were supplied by Amyl Media Pty Ltd. The lytic phage PEV1, specific against Pseudomonas aeruginosa, was sourced from AmpliPhi Biosciences AU (Australia) with an initial concentration of approximately 10¹⁰ PFU/mL.

Mengyu Li, Yue Cao, Hak-Kim Chan, Stability of bacteriophages in spray-dried polymeric formulations: Effect of excipient polyvinylpyrrolidone glass transition temperature and molecular weight, Received: 23 June 2025 Revised: 25 October 2025 Accepted: 31 October 2025, DOI: 10.1002/btm2.70096, https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.70096