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

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

Abstract:

The development of stable dry-state bacteriophage formulations is essential for expanding the therapeutic applicability of phage therapy and reducing cold-chain dependency. This study evaluates the long-term stability of spray-dried PEV1 bacteriophage formulated with polyvinylpyrrolidone (PVP) polymers of varying molecular weights (K15, K25, K40, K100). Formulations were stored for 180 days under controlled temperature (4, 22, and 40 °C) and relative humidity (15–53% RH) conditions. Differential scanning calorimetry demonstrated that glass transition temperature (Tg) increased with polymer molecular weight but decreased significantly with increasing humidity, consistent with water-induced plasticization. At low humidity (15% RH), all formulations maintained high stability (≤1 log10 titre loss) at 4 and 22 °C. At moderate humidity (33% RH), high-molecular-weight PVP (K40, K100) preserved long-term stability, maintaining large Tg–Ts offsets (ΔT ≥ 100 °C), whereas lower-molecular-weight grades showed substantial infectivity loss (2–3 log10). 

A critical water activity threshold (aw ≈ 0.43) was identified, above which degradation kinetics increased approximately tenfold. Arrhenius modeling confirmed thermally activated degradation behavior. Under combined high temperature and humidity stress (40 °C/≥43% RH), significant titre loss occurred despite large Tg–Ts offsets, indicating that Tg alone does not fully predict stability under plasticizing conditions. These findings demonstrate that high-molecular-weight PVP matrices enable ambient-stable phage formulations under controlled low-humidity conditions and provide a physicochemical framework for rational dry-state phage formulation design.

1) Introduction : 

The renewed clinical interest in bacteriophage therapy for multidrug-resistant bacterial infections has accelerated the development of dry powder formulations suitable for long-term storage and advanced delivery routes. Spray-drying has emerged as a scalable and translationally relevant manufacturing strategy; however, the process imposes significant thermal, osmotic, and shear stresses that may compromise phage structural integrity and infectivity. Consequently, excipient selection is a critical determinant of formulation success.

Solid-state stabilization of biologics is traditionally explained by two complementary mechanisms: water replacement and vitrification. The water replacement hypothesis posits that excipients form hydrogen bonds with biomolecular surfaces, compensating for dehydration-induced loss of structural water. This mechanism requires maintenance of the amorphous state, as crystallization reduces stabilizing intermolecular interactions. The vitrification hypothesis instead emphasizes kinetic stabilization: biologics are immobilized within a rigid glassy matrix whose protective capacity depends on the glass transition temperature (Tg). Above Tg, increased molecular mobility accelerates degradation processes. In protein systems, maintaining storage temperature at least 50 °C below Tg has been shown to significantly enhance stability by minimizing molecular motion.

Humidity represents a major destabilizing factor in amorphous systems. Water acts as a plasticizer, lowering Tg and increasing matrix mobility, even when storage temperature remains unchanged. In protein formulations, moisture-induced Tg depression correlates strongly with increased degradation kinetics. Despite extensive validation in protein therapeutics, systematic application of glass transition principles to phage formulations remains limited. The structural complexity, large size, and multicomponent architecture of bacteriophages suggest that stabilization requirements may differ from those of single-chain proteins, potentially necessitating larger thermal offsets or alternative excipient properties.

Previous work demonstrated that sugar-based matrices, such as lactose-leucine systems, preserve phage stability when the thermal offset (ΔT = Tg − Ts) exceeds approximately 50 °C. However, these studies were restricted to saccharide excipients. The present investigation extends this framework to polymer-based systems, focusing on polyvinylpyrrolidone (PVP), an excipient capable of forming stable amorphous matrices across a broad molecular weight range. Variations in PVP molecular weight (K15, K25, K40, K100) influence Tg and matrix rigidity, potentially altering stabilization performance. Additionally, PVP’s hydrogen-bonding capacity suggests possible contributions from both vitrification and water replacement mechanisms, though their relative roles in phage stabilization remain unclear.

This study therefore aims to determine whether the established ΔT threshold derived from sugar systems applies to polymeric matrices, while elucidating the impact of polymer molecular weight, humidity-induced plasticization, and thermally activated degradation kinetics on long-term phage stability. By integrating thermodynamic and kinetic analyses, the work seeks to define rational design principles for durable phage powder formulations suitable for clinical deployment.

2) Materials and Methods 

2.1 Materials.
The study employed four grades of polyvinylpyrrolidone (PVP K15, K25, K40, and K100) as sole excipients, allowing direct evaluation of molecular weight–dependent effects without interference from secondary stabilizers. The lytic bacteriophage PEV1, active against Pseudomonas aeruginosa, was used as the model system at an initial concentration of approximately 10¹⁰ PFU/mL. The exclusive use of single-component polymer matrices strengthens mechanistic interpretation by isolating the contribution of PVP physicochemical properties.

2.2 Spray-dried phage powders.
Formulations were prepared by combining phage suspension with aqueous PVP solutions (25 mg/mL total solids), adjusted to pH 7.4. Spray-drying was conducted using a Büchi B-290 system under controlled parameters (inlet temperature 60 °C; outlet 40–41 °C; standard two-fluid nozzle). These conditions reflect moderate thermal exposure, designed to balance efficient drying with biological preservation. Notably, PVP was used at 100% w/w of total solids, meaning no disaccharides, amino acids, or other glass formers were included. This design enables direct assessment of polymer vitrification capacity and hydrogen-bonding contributions to stabilization.

Post-drying, powders were collected, aliquoted, and stored under defined environmental conditions. Phage recovery was determined after reconstitution in PBS, with titres quantified via plaque assay.

2.3 Phage stability assessment.
Stability was evaluated using a standard double-layer agar assay with P. aeruginosa PAV237 as host. Infectivity was quantified after 18 h incubation at 37 °C, and each condition was tested in triplicate. A formulation was operationally defined as “stable” when titre loss remained below 1 log₁₀. Statistical analysis was performed using two-way ANOVA with significance set at p < 0.05.

Methodological strengths.
The experimental design demonstrates strong internal validity:

• Use of single-excipient systems isolates polymer molecular weight effects.

• Controlled spray-drying parameters enhance reproducibility.

• Triplicate biological assays improve statistical robustness.

• Clear stability criterion (<1 log₁₀ loss) provides practical translational relevance.

Methodological considerations.
Several aspects could influence interpretation:

• Only one phage (PEV1) was evaluated, limiting generalizability across structurally diverse phages.

• No direct structural characterization (e.g., electron microscopy or capsid integrity assays) was included to differentiate between loss of infectivity and physical particle degradation.

• Residual moisture content immediately post-drying was not specified in this section, although it is critical in glassy systems.

• The inlet temperature of 60 °C, while common in spray-drying, may still impose sublethal thermal stress; clarification of immediate post-process titre loss would strengthen understanding of processing versus storage effects.

Overall, the methodology is well-structured to test the central hypothesis regarding Tg, molecular weight, and humidity-dependent stability in polymer-based phage formulations.

2.4 Storage conditions.
Phage powders (200 mg per vial) were stored under tightly controlled combinations of temperature (4, 22, 40 °C) and relative humidity (15%, 33%, 43%, 53% RH). A 7-day equilibration phase allowed moisture sorption to reach steady state prior to long-term monitoring (up to 180 days). Notably, high-stress conditions (40 °C with ≥43% RH) were discontinued after 7 days due to rapid and substantial infectivity loss, indicating early matrix destabilization under combined thermal and hygroscopic stress.

Humidity environments were generated using saturated salt systems (silica beads for 15% RH; magnesium chloride for 33% RH; potassium carbonate for 43% RH; magnesium nitrate for 53% RH), with verification via humidity sensors prior to sample introduction. Handling was performed in humidity-controlled enclosures to prevent unintended moisture uptake, reflecting careful control of environmental variables. Importantly, the selected conditions intentionally deviated from ICH stability guidelines in order to induce controlled Tg depression and mechanistically interrogate the relationship between humidity, glass transition, and phage stability.

This design is mechanistically rigorous: by systematically modulating relative humidity, the study directly probes water-induced plasticization and its impact on the thermal offset (ΔT = Tg − Ts). The early exclusion of extreme stress conditions underscores the sensitivity of phage infectivity to combined temperature–moisture exposure and suggests the presence of kinetic thresholds beyond simple Tg considerations.

A limitation is that real-world pharmaceutical storage typically involves sealed packaging systems rather than uncapped storage. While the open-vial design enhances mechanistic clarity, additional studies incorporating packaging simulations would strengthen translational relevance.

2.5 X-ray powder diffraction (XRPD).
XRPD analysis was conducted to determine the crystallinity of the spray-dried matrices. Diffraction patterns recorded across 3°–50° 2θ confirmed the solid-state nature of the formulations. This step is critical, as crystallization would disrupt hydrogen-bonding networks and compromise both water replacement and vitrification mechanisms. Maintaining an amorphous matrix is central to the study’s Tg-based stabilization hypothesis.

2.6 Scanning electron microscopy (SEM).
Particle morphology was examined via SEM following gold sputter-coating. Morphological characterization provides insight into particle surface structure, potential porosity, and geometric uniformity—all factors influencing moisture uptake kinetics and surface-mediated degradation. While SEM does not directly measure internal glass properties, it supports interpretation of humidity sensitivity and equilibration behavior.

Overall assessment of this section.
The storage methodology is experimentally robust and specifically tailored to dissect humidity-induced Tg modulation. Integration of XRPD confirms maintenance of the amorphous state, a prerequisite for vitrification-based stabilization. However, inclusion of residual moisture quantification (e.g., Karl Fischer titration) and sorption isotherm analysis would further strengthen interpretation of plasticization thresholds and water activity–dependent degradation kinetics.

This section effectively establishes the environmental and physicochemical framework necessary to interpret subsequent stability results in the context of polymer molecular weight and glass transition behavior.

2.7 Differential Scanning Calorimetry (Tg)

The glass transition temperatures (Tg) of different PVP grades were determined using modulated differential scanning calorimetry (MDSC) on a DSC 823e instrument (Mettler Toledo, Greifensee, Switzerland). Each formulation underwent a 7-day equilibration period under its specific storage conditions to allow complete Tg adjustment in response to environmental temperature and relative humidity (RH). Samples of 5 ± 1 mg were prepared in sealed aluminum pans and heated from 30 to 200 °C at a heating rate of 2 °C/min, employing a modulation amplitude of ±0.5 °C and a modulation period of 60 s. Each sample was analyzed in duplicate to ensure reproducibility.

The results demonstrated that Tg values were sensitive to both the molecular weight of PVP and the ambient humidity. Higher molecular weight PVP generally yielded higher Tg values, indicating greater structural rigidity. Exposure to elevated humidity caused systematic Tg depression, illustrating the critical influence of water content on thermal properties. These measurements provide a quantitative basis for predicting the physical stability of amorphous phage formulations under different storage conditions.

2.8 Phage Degradation Kinetics

Phage stability was evaluated by monitoring the change in viable phage titre (PFU/mg) over time under various storage conditions. An apparent first-order kinetic model was applied, with the degradation rate constant (k, day⁻¹) calculated using:

$$\ln \frac{N}{N_0} = -k t$$

where $N$ is the phage titre at time $t$, and $N_0$ is the initial titre.

The temperature dependence of phage degradation was assessed at 4, 22, and 40 °C under 15% and 33% RH over a 30-day period, with rate constants fitted to the Arrhenius equation:

$$\ln k = \ln A - \frac{E_a}{R T}$$

where $A$ is the pre-exponential factor, $E_a$ is the activation energy (J/mol), $R = 8.314$ J/mol·K is the universal gas constant, and $T$ is absolute temperature (K).

Moisture effects were further evaluated by plotting k as a function of water activity (a_w), estimated from equilibrium RH using $a_w \approx \text{RH}/100$. RH values of 15%, 33%, 43%, and 53% corresponded to $a_w \approx 0.15, 0.33, 0.43,$ and $0.53$, respectively. This approach allowed a quantitative assessment of both temperature and moisture impact on phage stability, forming a basis for rational formulation and storage design.

3) Results 

3.1 Physicochemical Characterization of Spray-Dried Phage Formulations

The solid-state and morphological properties of spray-dried phage formulations containing PVP of varying molecular weights (K15, K25, K40, K100) were systematically evaluated under different temperature and relative humidity (RH) conditions. X-ray powder diffraction (XRPD) analysis (Figure 1) confirmed that all formulations remained fully amorphous after a 7-day equilibration period, as no crystalline peaks were detected across temperatures of 4, 22, and 40 °C and RH levels of 15%, 33%, 43%, and 53%. This indicates that the spray-drying process successfully produced stable amorphous formulations under all tested environmental conditions.

Scanning electron microscopy (SEM, Figure 2) revealed that particle morphology was strongly influenced by both PVP molecular weight and storage conditions. Formulations with PVP K15 exhibited dimpled and irregular particle surfaces at 4–22 °C with 15–33% RH, and showed extensive particle fusion at 40 °C/53% RH. PVP K25 formulations generally displayed smooth and spherical particles under lower temperature and RH conditions but began to show signs of fusion at 40 °C/53% RH. In contrast, PVP K40 and K100 formulations maintained well-defined, discrete spherical particles under all tested conditions. Notably, PVP K100 particles stored at 40 °C/53% RH were exceptionally smooth and uniform, exhibiting fewer surface irregularities or fusion compared to lower molecular weight formulations, highlighting superior morphological stability under stress.

The glass transition temperature (Tg) of the formulations under dry conditions (15% RH) increased proportionally with PVP molecular weight, ranging from 124 °C for K15 to 171 °C for K100. Exposure to elevated humidity systematically decreased Tg: increasing RH from 15% to 33% reduced Tg by approximately 40 °C, while a further increase to 43% RH caused an additional 25–30 °C reduction. These results demonstrate that moisture has a pronounced destabilizing effect on the thermal properties of amorphous phage formulations, with higher molecular weight PVP providing greater resistance to humidity-induced Tg depression.

Overall, the study highlights that PVP molecular weight is a critical determinant of both particle morphology and thermal stability, with K100 offering the most robust performance under extreme temperature and humidity conditions, making it the most promising excipient for long-term phage storage.

The results (Figures 1 and 2) clearly demonstrate that higher molecular weight PVP (K40, K100) enhances both particle morphology and thermal stability of spray-dried phage formulations. PVP K100 maintained smooth, discrete spherical particles even at 40 °C/53% RH, while lower molecular weight excipients (K15, K25) showed surface irregularities and fusion under the same conditions. Tg values increased with PVP molecular weight, from 124 °C for K15 to 171 °C for K100, but were highly sensitive to humidity, decreasing by up to 70 °C at higher RH.

Critically, although the study demonstrates the protective role of high molecular weight PVP, longer-term equilibration and testing under dynamic or cyclic humidity conditions would better simulate real storage scenarios. Additionally, linking particle morphology directly to phage viability would confirm that the observed physical stability translates into biological effectiveness. Despite these limitations, the data highlight K100 as the most promising excipient for robust, long-term phage storage.

3.2 | Phage viability following processing and storage

3.2.1 Initial Processing and Short-Term Stability (7 Days)

The spray-drying process caused minimal loss of phage viability, with all formulations retaining titre losses below 1 log10 immediately after processing. Specifically, losses were 0.20 log10 for PVP K15, 0.45 log10 for K25, 0.38 log10 for K40, and 0.58 log10 for K100 (Figure 3), indicating that the process is generally well-tolerated by the phages across all excipient grades.

After a 7-day storage period, phage viability became increasingly dependent on the interaction between PVP molecular weight, storage temperature, and relative humidity (RH) (Figure 4). Under low humidity (15% RH), titre losses remained below 1 log10 for all formulations at 4 and 22 °C, demonstrating good short-term stability under mild conditions. However, at 40 °C, the protective effect of PVP was more pronounced: the K100 formulation exhibited only a 0.95 log10 loss, whereas lower molecular weight grades (K15–K40) suffered 2.1–2.23 log10 losses, highlighting the importance of polymer molecular weight for thermal protection.

At elevated humidity (53% RH) and 4 °C, a clear inverse relationship between PVP molecular weight and titre loss was observed: PVP K100 lost 1.76 log10, while K15 lost 9.45 log10, demonstrating the critical role of high molecular weight excipients in mitigating moisture-induced phage inactivation. Under the most extreme conditions tested (40 °C and ≥43% RH), all formulations experienced titre losses greater than 9 log10, indicating that high temperature combined with high humidity overwhelms the protective effect of PVP, regardless of molecular weight.

These results confirm that PVP molecular weight significantly influences short-term phage stability, with K100 providing the strongest protection against temperature- and moisture-induced inactivation, while lower molecular weight excipients are more susceptible to rapid titre loss under stressed conditions.

The data (Figures 3 and 4) show that spray-drying caused minimal immediate phage loss (<1 log10), with n=3 for Figure 3 measurements. Specifically, K15 lost 0.20 log10 and K100 lost 0.58 log10, indicating good process tolerance. After 7 days, phage stability was strongly influenced by PVP molecular weight, temperature, and RH. At 15% RH, all formulations remained below 1 log10 loss at 4–22 °C, but at 40 °C, K100 outperformed lower grades (0.95 log10 vs 2.1–2.23 log10). At 53% RH/4 °C, titre losses ranged from 1.76 log10 (K100) to 9.45 log10 (K15). Data in Figure 4 were analyzed by two-way ANOVA, confirming statistically significant effects of PVP molecular weight and environmental conditions. Under extreme conditions (40 °C, ≥43% RH), all formulations lost >9 log10.

Critically, while K100 clearly offers superior short-term protection, the findings highlight the limitations of lower molecular weight PVP under high humidity or temperature. Future studies including intermediate RH levels or slightly longer storage periods would provide a more nuanced understanding of moisture-driven instability. Additionally, correlating these physical titre losses with functional phage activity would strengthen their predictive relevance for real-world storage.

3.2.2 Long-Term Stability (180 Days)

The long-term stability data (Figure 5) show that all formulations maintained high phage titres (~10⁸ PFU/mg) for 180 days at 4 °C under both 15% and 33% RH. At 22 °C and 33% RH, PVP K100 retained titres of 10⁸ PFU/mg, while PVP K40 ranged from 10⁸ to 10⁹ PFU/mg. In contrast, lower molecular weight excipients were highly susceptible under stress: PVP K25 dropped to <1 PFU/mg by 180 days at 4 °C/43% RH, and PVP K15 reached <1 PFU/mg by Day 90.

Critically, these findings reinforce that high molecular weight PVP, particularly K100, provides robust long-term protection, while lower molecular weight excipients are prone to rapid degradation under elevated humidity. Incorporating intermediate RH levels or slightly higher storage temperatures in future studies would allow a more detailed understanding of formulation limits and improve predictive guidance for real-world storage.

3.3 Correlation of Stability with Physicochemical Parameters

The relationship between the physical state of the PVP matrix and phage stability was evaluated by correlating phage titre loss with the thermal offset ($\Delta T = T_g - T_s$), where $T_g$ is the glass transition temperature and $T_s$ is the storage temperature (Figure 6, Table 2). A clear inverse correlation was observed: formulations with higher ΔT values showed lower titre losses, confirming that the thermal margin between Tg and storage temperature is a critical determinant of stability.

At 15% RH, ΔT values were high (102–171 °C), and corresponding titre losses were limited to <2.3 log10 across all formulations. In contrast, at 33% RH, ΔT values were lower (69–131 °C), and measured titre losses increased substantially, ranging from 0.50–3.79 log10. Notably, for comparable ΔT values, PVP K100 provided superior protection (0.50 log10 loss at ΔT = 131 °C) compared to lower molecular weight grades (3.18–3.79 log10 loss), highlighting the importance of high molecular weight excipients in maintaining phage viability under stress.

Temperature-dependent degradation was further quantified by calculating degradation rate constants (k) at 15% and 33% RH. The data followed first-order kinetics, as indicated by linear Arrhenius plots of ln(k) versus 1/T (R² = 0.60–0.99, Figure 7). The influence of water activity (a_w) was assessed at 4 and 22 °C (Figure 8). Across both temperatures, k increased with a_w from 0.15 to 0.43. Between a_w 0.15–0.33, ln(k) values showed minimal variation, while at a_w = 0.43, ln(k) increased markedly, especially at 22 °C, approaching –1.1 for PVP K15–K40 and –1.9 for PVP K100, indicating that moisture strongly accelerates degradation, even in high molecular weight formulations.

Overall, these results confirm that phage stability is governed by the combination of thermal margin (ΔT) and water activity, with PVP K100 providing both a larger ΔT and enhanced resistance to moisture, making it the most effective excipient for maintaining long-term phage viability.

The data (Figures 6–8, Table 2) show a strong inverse correlation between thermal offset (ΔT = Tg – Ts) and phage titre loss, confirming that a larger thermal margin protects against short-term degradation. At 15% RH, high ΔT values (102–171 °C) corresponded to losses <2.3 log10, while at 33% RH, lower ΔT (69–131 °C) led to losses of 0.50–3.79 log10. PVP K100 consistently provided superior protection (0.50 log10 loss at ΔT = 131 °C) compared to lower molecular weight excipients.

Critically, the results highlight that both thermal stability and water activity are key predictors of phage viability. Although PVP K100 shows robust protection, the sharp increase in degradation at a_w = 0.43 suggests that formulations remain sensitive to elevated moisture, emphasizing the need for controlled RH in storage and the potential benefit of combining high molecular weight PVP with additional moisture-stabilizing excipients.

4. Comprehensive Evaluation and Critical Analysis of the Study

The article presents a comprehensive study on the stabilization of spray-dried phage formulations using polyvinylpyrrolidone (PVP)-based polymeric matrices, with a particular focus on the influence of polymer molecular weight, relative humidity, and storage temperature on phage viability. The experimental approach is methodically structured, combining physicochemical characterization, including X-ray powder diffraction (XRPD) and scanning electron microscopy (SEM), with systematic stability assessments over both short-term and long-term storage periods. The study is notable for its breadth, examining four PVP grades (K15, K25, K40, K100) across a range of temperatures from 4 °C to 40 °C and relative humidity levels from 15% to 53%, providing a robust dataset that captures a wide spectrum of environmental stresses relevant to phage formulation.

One of the strengths of the study lies in its clear demonstration of the relationship between polymer molecular weight and both thermal and morphological stability. High-molecular-weight PVP (K100) consistently outperformed lower-molecular-weight variants in preserving phage viability under challenging conditions, which is convincingly supported by both the glass transition temperature (Tg) data and the SEM images showing particle morphology. The study also effectively highlights the mechanistic underpinnings of stabilization, attributing the enhanced performance of high-molecular-weight polymers to increased chain entanglement, higher Tg, more tortuous diffusion pathways, and potential hydrogen bonding interactions with phage proteins. These explanations are grounded in established principles of polymer physics and protein stabilization, lending credibility to the conclusions drawn.

The experimental design is rigorous, incorporating multiple humidity and temperature conditions, short- and long-term storage studies, and quantitative analysis of phage titre losses, which strengthens the generalizability of the findings. The use of thermal offset (ΔT) as a predictive parameter for stability is well justified and aligns with prior literature on protein and phage stabilization in glassy matrices. Furthermore, the inclusion of kinetic analyses and Arrhenius modeling adds an important quantitative dimension, providing insight into the rates of degradation under different environmental stresses. The study also situates its findings in the broader context of saccharide-based stabilization systems, providing meaningful comparisons that highlight the advantages of polymeric matrices in mitigating moisture-induced destabilization.

Despite these strengths, several limitations temper the overall impact of the work. First, the study focuses exclusively on a single phage (PEV1), leaving open the question of whether the observed stabilization trends are generalizable to other phages with different capsid structures, genome types, or thermal sensitivities. Second, while the role of ΔT and polymer molecular weight is convincingly demonstrated, the study does not directly investigate the molecular interactions between phage proteins and PVP chains, leaving mechanistic hypotheses somewhat inferential. Advanced analytical techniques such as solid-state NMR or AFM-IR could provide more direct evidence of these interactions. Third, while relative humidity and temperature were systematically varied, hygroscopicity and water uptake kinetics of the formulations were not directly measured, limiting insight into the dynamic processes that may influence long-term stability under fluctuating environmental conditions. Similarly, the study’s long-term stability assessment is restricted to 180 days, which may not fully capture the shelf-life requirements for clinical or commercial applications.

Additionally, some inconsistencies in the data warrant closer examination. For instance, certain formulations, such as PVP K40 at 22 °C/33% RH, showed unexpected titre losses despite having substantial ΔT values, suggesting that factors beyond bulk polymer Tg, such as local microenvironmental heterogeneity, polymer-phage surface interactions, or phase separation, may play a role. The study also emphasizes ΔT as a primary predictor of stability, yet observations at high humidity or elevated temperatures indicate that ΔT alone is insufficient, highlighting the need for more integrated models that account for polymer morphology, moisture plasticization, and virion-specific properties.

In terms of presentation, the article is dense and data-rich, which is both a strength and a limitation. While the text thoroughly describes the experiments and findings, some sections are highly technical and repetitive, making it challenging for non-specialist readers to extract the key conclusions without careful analysis. Figures and tables are informative, but the discussion could be improved by more explicitly connecting individual datasets to overarching mechanistic interpretations, rather than presenting them sequentially. Minor issues, such as occasional typographical inconsistencies and complex sentences, slightly detract from readability but do not compromise scientific validity.

Overall, the article makes a meaningful contribution to the field of phage formulation by systematically demonstrating that high-molecular-weight PVP provides superior stabilization under diverse environmental conditions, supporting the development of polymer-based dry powder therapeutics. It offers mechanistic insights, practical guidance for excipient selection, and a foundation for future studies exploring other polymers or phage types. To enhance its impact, future work should extend the analysis to multiple phage species, directly probe polymer-virion interactions, evaluate moisture uptake kinetics, and explore storage stability beyond 180 days to better inform clinical translation. Despite these limitations, the study establishes a compelling framework for understanding how polymer matrix properties influence the stability of spray-dried phage formulations and provides clear evidence that polymer molecular weight and glass transition behavior are critical determinants of long-term viability.

5 | CONCLUSIONS 

This study presents the first systematic investigation of PVP-based polymer matrices for phage stabilization, establishing novel formulation principles distinct from saccharide-based systems. All PVP formulations preserved phage viability within 1 log10 loss over 180 days when stored at 4 and 22
C under 15% RH. At higher humidity (33% RH), long-term stability was successfully achieved at ambient temperatures (≤22
C) using high-molecular-weight PVPs (K40, K100), which provided the necessary thermal offsets (ΔT ≥ 100
C) to maintain a protective glassy state. These findings introduce humidity-tolerant design parameters for phage formulation in PVP that allow long-term phage stability storage and reduce cold-chain dependency.

Reference :

Li M, Cao Y, Chan H-K. Stability of bacteriophages in spray-dried polymeric formulations: Effect of excipient polyvinylpyrrolidone glass transition temperature and molecular weight. Bioeng Transl Med. 2025;e70096. doi:10.1002/btm2.70096

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2025 The Author(s). Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers