Purification Strategies and Impact of Surfactant on Bacteriophage Stability and Aggregation

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However, their stability can be affected by purification methods and prolonged storage. A key factor contributing to viability loss is viral particle aggregation, which may be mitigated by surfactants such as Tween 20. This study investigated the relationship between viral aggregation and infectivity loss by purifying MS2 and PhiX174 bacteriophages using chloroform clarification and PEG concentration, with and without Tween 20, as well as Triton X-100 purification, over a 55-day period. The results showed that infectivity and aggregation profiles differed depending on the purification method. However, no direct correlation was observed between aggregation and infectivity. Moreover, MS2 and PhiX174 exhibited distinct responses to the same treatments, indicating that no single purification method is universally effective for all bacteriophages. The study concludes that viral aggregation is not directly associated with infectivity loss and emphasizes the need for virus-specific protocols to ensure stability during long-term storage. Bacteriophages aggregation stability surfactant storage purification Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Bacteriophages, also referred to as phages, are viruses exclusively adapted to infect and reproduce within a bacterial host. The term "bacteriophage", derived from Latin, translates to "bacteria eaters," reflecting their ability to infect and eliminate bacterial species (LEDERBERG et al., 1996). Discovered almost simultaneously by Frederick Twort and Felix d'Herelle, bacteriophages have long intrigued researchers due to antibacterial activity (D’Hérelle, 1917 ). As early as in the 1920s, d'Herelle used phages to combat cholera outbreaks in Egypt and India, paving the way to use phages to combat bacterial infections (KEEN et al., 2012; ALMEIDA et al., 2020). Although their use was eclipsed by the discovery of antibiotics, the growing threat of antibiotic resistance has renewed interest in bacteriophages as therapeutic agents. In some Western countries, including Belgium, phage therapy has already been approved for use in hospitals and pharmacies (PIRNAY et al., 2018). Nonetheless, barriers to widespread adoption remain, including concerns around the stability and reliability of preparations during long-term storage. Unlike antibiotics, phages are biologically complex and highly sensitive to environmental conditions such as temperature, which can lead to rapid loss of viability if not carefully controlled (SUMMERS et al., 2012). Even in Eastern Europe, where phage therapy has a long history in conventional medicine, there is limited understanding of the formulation and preservation conditions necessary to ensure therapy efficacy (MERABISHVILI et al., 2013). As a result, there is significant interest in improving bacteriophage preservation techniques to advance and enhance phage therapy without compromising product stability. Given the diverse nature of bacteriophages, no universal storage method can be applied to all. Instead, storage conditions must be tailored to the specific characteristics of each phage (ŁOBOCKA et al., 2017). This specificity means that bacteriophage formulations must be evaluated by the necessary physical and chemical conditions of each virus. A significant indicator of poor stability of phage formulations is viral aggregation, which can reduce the number of viable bacteriophages by promoting adsorption onto filters and membranes used in purification, thus compromising phage products which require long-term storage (JOŃCZYK et al., 2011). To address this, surfactants such as Tween 20 have been proposed as additives to reduce aggregation and surface adhesion. It has been shown that as little as 0.002% Tween 20 can be effective in reversing phage adhesion to polypropylene vials, enhancing their stability (RICHTER et al., 2021). Such results suggest that adding surfactants to phage-based products can enhance shelf life by preventing rapid product decay caused by viral adhesion and aggregation. The purity of preparations itself can also influence viral stability. Bacteriophages may adhere to bacterial debris present in solutions, causing steep declines in viability by several orders of magnitude (ŁOBOCKA et al., 2017). This indicates that purifying bacterial residues can lead to greater long-term viral stability, although the purification method must be carefully selected to avoid viral titre loss (ŁOBOCKA et al., 2017). This study investigates how different purification and recovery methods affect the stability and aggregation profiles of two model bacteriophages: MS2 and PhiX174 phages. The results aim to contribute to a better understanding of the conditions and factors required for maintaining phage stability during long-term storage, facilitating their application in biotechnological and therapeutic settings. 2. MATERIALS AND METHODS 2.1 ORIGIN OF THE BACTERIOPHAGES AND BACTERIA USED The bacteriophages MS2 (ATCC 15597-B1) and PhiX-174 (ATCC BAA-13706) were originally obtained from the American Type Culture Collection (ATCC, USA) and stored at 4°C until use. They are part of the virus collection of the Applied Virology Laboratory at UFSC. The bacterium E. coli (ATCC 25922), used for phage propagation, was also obtained from ATCC and stored at -80°C until use. 2.2 PROPAGATION OF BACTERIOPHAGES Briefly, 1 mL of each bacteriophage suspension was added separately to an Erlenmeyer flask containing 25 mL of LB medium (Luria Bertani) supplemented with 10 mM MgSO₄ and 10 mM CaCl₂, along with E. coli ATCC 25922 in exponential growth phase. The culture was incubated at 37°C for 6 to 12 hours, after which 1 mL of the bacterial culture was transferred to another Erlenmeyer flask containing fresh E. coli in exponential growth. This step was repeated twice, with three incubation cycles per bacteriophage. After propagation, the contents of the Erlenmeyer’s were centrifuged at 3,220 ×g for 10 minutes at room temperature using a model 5804R centrifuge (Eppendorf, Germany). The supernatant was then stored at 4°C for subsequent titration. 2.3 BACTERIOPHAGE ENUMERATION The quantification of bacteriophages was carried out using the double-layer agar method, adapted from Bichet et al. (2021). Briefly, 10 microliters of the propagated bacteriophage suspension were serially diluted at a 1:100 ratio in test tubes containing 1 mL of SM buffer (0.1 M NaCl, 0.0081 M MgSO₄ heptahydrate, and 0.2% gelatin). Then, 1 mL of E. coli ATCC 25922 in the exponential growth phase was added to each dilution. Samples were maintained at room temperature (15–30°C) for 15 to 30 minutes to allow phage infection of the bacteria. Simultaneously, LB semi-solid medium containing 0.75% bacteriological agar (Sigma-Aldrich, USA), supplemented with 10 mM MgSO₄ and 10 mM CaCl₂, was heated to near boiling. After that, 3 mL were transferred to test tubes maintained at 47°C in a heating block to prevent solidification, and 1 mL of each viral dilution was added. After a brief vortex homogenization, the solution was poured into Petri dishes containing solid LB agar (1.5% agar). The mixture was spread evenly, and plates were incubated at 37°C for 12 hours in a bacteriological incubator. Plaque-forming units (PFU) were then counted. The phage titer was calculated using the formula: PFU/mL = (number of plaques × dilution factor) / volume plated (mL). The experiment was performed in duplicate, and the final PFU/mL value was determined by averaging both counts. 2.4 VIRAL CONCENTRATION AND PURIFICATION PROTOCOLS 2.4.1 Chloroform and Tween 20 Addition This protocol was adapted from Bichet et al . (2021) and Richter et al . (2021). After collecting the phage supernatant as previously described in the item 2.2, chloroform was added at 10% of the total volume. The mixture was centrifuged at 3,220 ×g for 10 minutes at 4°C, and the aqueous phase (supernatant) was carefully collected to avoid disturbing the chloroform layer containing impurities. This solution was filtered using a 0.22 µm cellulose acetate filter and stored at 4°C until use. In a parallel sample, 0.002% (v/v) Tween 20 was added after purification. The solution was manually agitated for homogenization and stored at 4°C. 2.4.2 Polyethylene Glycol (PEG) and Tween 20 Addition This protocol was adapted from Branston et al . (2015). Following purification as previously described in the item 2.4.1, the chloroform phage suspension was filtered through a 0.22 µm filter, and PEG 6000 was added at 2% (v/v). The solution was left to precipitate at room temperature for 10 minutes. It was then centrifuged at 15,000 ×g at 22°C using an AVANTI J301 centrifuge (Beckman Coulter, USA). The aqueous phase was discarded, and the pellet, although barely visible, was resuspended in 3 mL of Tris buffer (25 mM Tris, 100 mM NaCl, pH 8.0). The solution was stored at 4°C. In a parallel sample, 0.002% (v/v) Tween 20 was added after purification. The solution was manually homogenized and stored at 4°C. 2.4.3 Triton X-100 This protocol was adapted from Branston et al . (2015). Following PEG purification as previously described in the item 2.4.2, 2% PEG 6000 and 2% (v/v) of Triton X-100 (10% stock in Milli-Q water) were added to the sample. The solution was kept at room temperature for 10 minutes, then centrifuged at 15,000 ×g at 22°C in the AVANTI J301 centrifuge. This process was repeated three times, with the resulting pellet resuspended in 3 mL of Tris buffer (25 mM Tris, 100 mM NaCl, pH 8.0). 2.5 INFECTIVITY KINETICS ASSAY OF BACTERIOPHAGES The infectivity of the purified MS2 and PhiX174 bacteriophages was assessed using the double-layer agar assay in duplicate, as previously described in the item 2.3. Infectivity was monitored over 55 days following the purification protocols, using the same initial sample in a non-destructive assay. Kinetics were evaluated by tracking changes in PFU/mL and plaque size over time. The average diameter of 20 lysis plaques was measured using a caliper, following an adaptation of the method by Bichet et al . (2021). The pH of bacteriophage suspensions purified by the previously described methods, described in the 2.4 item, were measured on the same day of purification using a pH meter from the Acquacombo Colorimetric Kit / CD 1555 (Alfakit, Brazil). 2.6 AGGREGATION PROFILE IDENTIFICATION BY DYNAMIC LIGHT SCATTERING The aggregation profile of the bacteriophages was assessed by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, UK). One milliliter of each purified bacteriophage suspension was analyzed, according to the manufacturer's instructions, at 22°C on days 14, 35, and 55 post-purification, in a non-destructive manner using the same sample over time. 2.7 STATISTICAL ANALYSIS Statistical analyses were performed using GraphPad Prism version 8.2.1 (GraphPad Software, USA). The infectivity assay described in the item 2.5 was subjected to Two-way ANOVA, ordinary One-way ANOVA, and linear regression. Results were considered statistically significant when the probability value was p < 0.05. The aggregation profile analysis was evaluated using ordinary One-way ANOVA and Tukey’s test, with significance also set at p < 0.05. Shelf-stability analysis was conducted by calculating the average instability index, yielding a final stability score. 3. RESULTS 3.1 INFECTIVITY OF BACTERIOPHAGES AFTER PURIFICATION In order to verify the overall infectivity of the bacteriophages after purification methods described in section 2.4 , an estimated total decay analysis was conducted. The linear regression analysis, as shown in Fig. 1 , revealed a consistent and notable decay for MS2 treated with PEG and for PhiX174 purified with PEG and Tween 20. The coefficient of determination (R²) obtained from the linear regression indicated values of 74.10% for PEG-treated MS2 and 61.42% for PhiX174 purified with PEG and Tween 20. These results support the suitability of the regression model and suggest a trend of infectivity decay over time for these specific treatments. To evaluate how purification protocols may affect the phage’s infectious behavior, changes in plaque size post-treatment was chosen. Figure 2 displays the variation in average plaque size (PFUs) in millimeters over the 0–55 day period. Two-Way ANOVA revealed a significant change for PhiX174 purified with chloroform and Tween 20 ( p < 0.05), with no similar effect observed in other treatments. 3.2 AGGREGATION PROFILE OF BACTERIOPHAGES BY DYNAMIC LIGHT SCATTERING (DLS) To verify how purifications methods influence phage aggregation profiles (and consequently their infectivity), DLS analysis was employed. Figure 3 (A and B) shows the aggregation kinetics of bacteriophages as assessed by DLS over 55 days. One-Way ANOVA was used to calculate the percentage of particles ranging from 30 to 300 nm, a size range theorized to represent free particles (30 nm) through to aggregates of up to 10 viral particles (up to 300 nm). This parameter is based on Langlet et al. (2007), which identified 30 nm particles as corresponding to viable MS2 units and observed that infectivity decreased significantly as particle sizes deviated from this range. MS2 purified with chloroform exhibited a significant difference in aggregation ( p < 0.05) when compared to chloroform plus Tween 20 treatment, with the addition of Tween 20 drastically reducing the proportion of particles in the 30–300 nm range. Similarly, PhiX174 also displayed significant differences ( p < 0.05) between PEG and PEG plus Tween 20 treatments, with the presence of surfactant substantially improving the proportion of low-aggregation particles typically associated with infectious virions. 4. DISCUSSION The present results suggest that the stability indicators analysed vary not only with the purification method applied but also with the specific characteristics of each bacteriophage. MS2 and PhiX174 responded differently to identical treatments, indicating that the results may be phage-specific. The total decay analysis revealed that PEG-treated MS2 and PhiX174 purified with PEG and Tween 20 exhibited greater loss of infectivity over time. In contrast, other purification methods did not show a significant decay pattern, suggesting greater stability for those treatments during prolonged storage, as seen in Fig. 1 . These results highlight that different purification methods lead to distinct stability profiles, and that no single method can be applied to all phages, since a direct relationship between the purification methods in both strains could not be established. Variations in plaque size are linked to differences in phage adsorption to bacterial lawns. Shorter adsorption times are typically associated with smaller plaques, while prolonged adsorption times can produce smaller plaques with greater size variation, resulting in PFUs of differing sizes, as described by (ABEDON et al ., 2009; ADAMS, 1959). Similarly, our study could not establish a relationship between phage plaque size and purification method for either MS2 or PhiX174, as seen in Fig. 2 . Although a significant reduction in plaque size is often associated with lower infectivity, this relationship is not absolute, and individual phage infections may not be affected by plaque morphology (ABEDON et al ., 2009). Conversely, an increase in plaque size may arise from a low viral adsorption rate, in which unabsorbed viral particles can diffuse away from the plaque center for longer periods, forming larger PFUs (ABEDON et al. , 2009). Similar to our results, the significant reduction in PFU size, seen in Fig. 2 , was not accompanied by a proportional loss in viral quantity, as revealed in Fig. 1 . Therefore, it is difficult to determine whether this purification method significantly compromises phage infectivity over long-term storage. A crucial aspect for greater viral stability is the need to adjust the pH to the virus isoelectric point—i.e., the pH level at which an insoluble material has a neutral charge (KOZLOWSKI et al., 2021). For viruses, the isoelectric point refers to the pH level at which viral units have a neutral charge, preventing adhesion to other particles via electrostatic attraction, which can directly influence the bioavailability of viable viruses (MICHEN et al., 2010). In phage therapy, the isoelectric point affects the number of free particles and, consequently, the infectivity of bacteriophages. In the present study, all purification conditions were maintained at pH 7.5, suggesting that the behavioral differences observed between phages and treatments were not due to variations in pH relative to the viruses’ isoelectric points. Furthermore, as the observed decay trends were not consistent across both phages, it can be concluded that stability depends on both the purification method and the phage in question. This also reveals that the pH resulting from the treatments did not interfere with the observed results of aggregation indicated in Fig. 3 , as phages tend to aggregate when the pH is equal to or below the isoelectric point, forming aggregates up to 222 times larger than the original virus size (LANGLET et al., 2007; ZUO et al., 2014). These aggregates reduce the number of viable bacteriophages as they favor adsorption onto filters and membranes used in purification, compromising phage products requiring long-term storage (JOŃCZYK et al., 2011). Thus, aggregate formation remains a significant indicator of poor bacteriophage viability and infectivity, with an increase in aggregates in the phage solution being a significant factor in their inactivation (FURIGA et al., 2011). However, in the present study, aggregate formation did not correlate directly with loss of viral infectivity. Although there were significant differences in aggregation profiles under different treatments (e.g., 100% of PhiX174 particles purified with PEG were > 300 nm), as seen in Fig. 3 , infectivity tests showed no direct correlation between aggregation level and the number of infectious particles observed in Fig. 1 . This suggests that, under the parameters analyzed, high aggregation levels does not necessarily impair phage infectivity, indicating potential viability for long-term storage even under aggregated conditions. However, further studies are required to determine whether these patterns persist over longer storage periods. In this study, Tween 20 did not universally promote disaggregation, in contrast to Richter et al . (2021) where 0.002% Tween 20 was effective in reversing phage adhesion to storage vials (promoting phage release and increasing detectable infectious particles),. In some cases in this study—such as with both phages treated with chloroform—aggregation increased. For PEG-purified MS2, the surfactant had no significant effect, while for PEG-purified PhiX174, Tween 20 considerably reduced aggregation. These differing outcomes may be due to interactions between Tween 20 and PEG, as described by Mahajan et al . (2004), who reported the formation of aggregates due to interactions between glycol oligomers present in both compounds. Similar results were observed by Hakami et al . (2015), who noted aggregation of PEG-concentrated phages after adding Tween 20. Both studies attributed this effect to PEG residues on the viral capsid forming strong hydrophilic bonds with Tween 20. It is possible that Tween 20 disrupted the interactions between phages and PEG in suspensions such as PhiX174’s, promoting disaggregation. However, Tween 20 has also been reported to form nonspecific interactions with proteins and other organic compounds, as reported by Jamur et al . (2009), which may explain unexpected aggregation when PEG is absent. It is plausible that the dynamics between Tween 20 and viral particles vary with exposure time and the compounds present in the solution. While Richter et al . (2021) observed disaggregation of phages from polypropylene surfaces with brief Tween 20 exposure, no subsequent reports assessed long-term aggregation profiles after extended contact with Tween 20, as conducted here. These findings also demonstrate that the interaction dynamics with Tween 20 may be phage-specific. This indicates that further studies involving Tween 20 and other phage species are needed before considering it a universal disaggregation supplement in phage suspensions. The dynamics between how Tween 20 addition may play in the increase or decrease of phage aggregation are summarized in Fig. 4 . This study successfully evaluated the stability of different bacteriophages subjected to different purification methods, aiming to identify approaches that promote greater stability and preservation of viral infectivity during both short- and long-term storage. The comparative analysis between viral infectivity and aggregation profiles demonstrated that aggregation is not a determining factor for phage viability—therefore, viral viability cannot be reliably assessed solely on aggregation index. The results reinforce that no universally effective purification method can be applied for all bacteriophages, since the preservation of viral activity depends on the specific characteristics of each phage. Overall, this work contributes valuable insights to the development of optimized preservation protocols, highlighting critical factors to be considered and appropriate analytical methods for evaluating phages intended for long-term storage. Declarations CONFLICTS OF INTEREST The authors declare no conflict of interest. FUNDING This work was funded a by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil. Author Contribution E.B.S. conceptualization, methodology, investigation, and writing of original draft, M.A.O. data curation, H.Y.C. methodology, investigation, A.R.P., and G.F. conceptualization, supervision, and review. References Stephen, A. B. E. D. O. N. (2009). T. Bacteriophage Plaques: theory and analysis. Methods in Molecular Biology, pp. 161–174, Humana Press. http://dx.doi.org/10.1007/978-1-60327-164-6_17 Hancock, A. D. A. M. S. M. (1959). Bacteriophages. New York: Interscience Publishers. de Freitas, A. L. M. E. I. D. A. G. M. (2020). may. The forgotten tale of Brazilian phage therapy. The Lancet Infectious Diseases, v. 20, n. 5, pp. 90–101, Elsevier BV. http://dx.doi.org/10.1016/s1473-3099(20)30060-8 Marion, B. I. C. H. E. T. C. et al. Protocols for studying bacteriophage interactions with in vitro epithelial cell layers. Star Protocols, v. 2, n. 3, p. 100697, set. 2021. Elsevier BV. http://dx.doi.org/10.1016/j.xpro.2021.100697 Steven, B. R. A. N. S. T. O. N. (2015). D. may. A non-chromatographic method for the removal of endotoxins from bacteriophages. Biotechnology and Bioengineering, v. 112, n. 8, pp. 1714–1719, 5 Wiley. http://dx.doi.org/10.1002/bit.25571 D’Hérelle, F. (1917). On an invisible microbe antagonistic to dysentery bacilli. C.R. Acad. Sci. Paris 165, 373–375 Research in Microbiology. Sep;158(7):553-4. http://dx.doi.org/10.1016/j.resmic.2007.07.005 Aurelie, F. U. R. I. G. A., et al. (jan. 2011). Effects of ionic strength on bacteriophage MS2 behavior and their implications for the assessment of virus retention by ultrafiltration membranes. Applied and Environmental Microbiology, v. 77, n. 1 (pp. 229–236). American Society for Microbiology. http://dx.doi.org/10.1128/aem.01075-10 Abdulrahim, H. A. K. A. M. I. (2015). R. sep. Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces. Journal of Virological Methods, v. 221, pp. 1–8, Elsevier BV. http://dx.doi.org/10.1016/j.jviromet.2015.04.023 Maria Célia, J. A. M. U. R., et al. (oct. 2009). Permeabilization of cell membranes (pp. 63–66). Humana. Methods in Molecular Biology http://dx.doi.org/10.1007/978-1-59745-324-0_9 JOńCZYK, E. (2011). may. The influence of external factors on bacteriophages—review. Folia Microbiologica, v. 56, n. 3, pp. 191–200, Springer Science and Business Media LLC. http://dx.doi.org/10.1007/s12223-011-0039-8 KEEN, Eric C. et al. Phage therapy: concept to cure. Frontiers in Microbiology, v. 3, p. 1, 2012. Frontiers Media SA . http://dx.doi.org/10.3389/fmicb.2012.00238 Lukasz Pawel, K. O. Z. L. O. W. S. K. I. (2021). IPC 2.0: prediction of isoelectric point and pka dissociation constants. Nucleic Acids Research, v. 49, n. 1. 27 apr (pp. 285–292). Oxford University Press (OUP. http://dx.doi.org/10.1093/nar/gkab295 LANGLET, J. (2007). Effects of pH on plaque forming unit counts and aggregation of MS2 bacteriophage. Journal of Applied Microbiology, v. 103, n. 5. 19 jun (pp. 1632–1638). Oxford University Press (OUP. http://dx.doi.org/10.1111/j.1365-2672.2007.03396.x LEDERBERG, J. (1996). apr. Smaller fleas … ad infinitum: therapeutic bacteriophage redux. Proceedings of The National Academy of Sciences, v. 93, n. 8, pp. 3167–3168, 16 Proceedings of the National Academy of Sciences. http://dx.doi.org/10.1073/pnas.93.8.3167 ŁOBOCKA, Małgorzata, B. (2017). nov. Methods for bacteriophage preservation. Methods in Molecular Biology, pp. 219–230, 9 Springer New York. http://dx.doi.org/10.1007/978-1-4939-7395-8_17 Rakesh Kumar, M. A. H. A. J. A. N. Effects of monomeric and polymeric glycol additives on micellar properties of Tween non-ionic surfactants as studied by cyclic voltammetry. Colloids and Surfaces A: Physicochemical and Engineering Aspects, v. 237, n. 1–3, pp. 119–124, apr. 2004. Elsevier BV. http://dx.doi.org/10.1016/j.colsurfa.2004.02.013 Maia, M. E. R. A. B. I. S. H. V. I. L. I. (2013). jul. Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization). Plos One, v. 8, n. 7, p. 68797, 2 Public Library of Science (PLoS). http://dx.doi.org/10.1371/journal.pone.0068797 MICHEN, B. (2010). jan. Isoelectric points of viruses. Journal of Applied Microbiology, v. 109, n. 2, pp. 388–397, 22 Oxford University Press (OUP). http://dx.doi.org/10.1111/j.1365-2672.2010.04663.x Jean-Paul, P. I. R. N. A. Y. (2018). feb. The magistral phage. Viruses, v. 10, n. 2, p. 64, 6 MDPI. http://dx.doi.org/10.3390/v10020064 RICHTER, Łukasz, et al. (apr. 2021). Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research. Scientific Reports, v. 11, n. 1 (1., pp. 1–11). Springer Science and Business Media LLC. http://dx.doi.org/10.1038/s41598-021-86571-x William, S. U. M. M. E. R. S. (2012). C. apr. The strange history of phage therapy. Bacteriophage, v. 2, n. 2, pp. 130–133, Informa UK Limited. http://dx.doi.org/10.4161/bact.20757 Zhili, Z. U. O., et al. (may 2014). Survival of airborne MS2 bacteriophage generated from human saliva, artificial saliva, and cell culture medium. Applied and Environmental Microbiology, v. 80 (Vol. 9n., pp. 2796–2803). American Society for Microbiology. http://dx.doi.org/10.1128/aem.00056-14 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7273821","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502494216,"identity":"e4427b8a-327b-4468-9a77-985b9569cb67","order_by":0,"name":"Estêvão Brasiliense Souza","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Estêvão","middleName":"Brasiliense","lastName":"Souza","suffix":""},{"id":502494217,"identity":"b7f2517c-8be8-442d-bbfa-f584a823f81d","order_by":1,"name":"Miguel Abreu Oliveira","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Abreu","lastName":"Oliveira","suffix":""},{"id":502494220,"identity":"b1410b19-7524-42c8-ab06-1dd6763fc8bb","order_by":2,"name":"Helena Yurevna Caio","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Helena","middleName":"Yurevna","lastName":"Caio","suffix":""},{"id":502494222,"identity":"faada519-be5e-4c2c-ac36-24d69edcdc9f","order_by":3,"name":"Aguinaldo R. Pinto","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Aguinaldo","middleName":"R.","lastName":"Pinto","suffix":""},{"id":502494223,"identity":"e59273c2-80a0-4b2a-9801-c8c67799d27f","order_by":4,"name":"Gislaine Fongaro","email":"data:image/png;base64,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","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":true,"prefix":"","firstName":"Gislaine","middleName":"","lastName":"Fongaro","suffix":""}],"badges":[],"createdAt":"2025-08-01 18:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7273821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7273821/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89676095,"identity":"5d3e792e-7ef2-46eb-84dd-ebc08408e433","added_by":"auto","created_at":"2025-08-22 13:40:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40756,"visible":true,"origin":"","legend":"\u003cp\u003eProjected decay for total loss of PFUs of bacteriophages following different purification protocols described in section 2.4, based on linear regression analysis. PFU data were obtained according to the method described above. Asterisks indicate a significant coefficient of determination, indicating treatments that express a constant decay of infectious particles over time.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7273821/v1/e7735b181a03b1df50d657fc.jpg"},{"id":89675234,"identity":"156e2513-2c1a-4db0-8865-a9be119c0d1d","added_by":"auto","created_at":"2025-08-22 13:32:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36977,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in infectivity based on the average size of lysis plaques following viral purification processes described in section 2.4. Bars represent the variation in millimeters of the average plaque diameter between day 0 and day 55. Asterisks indicate \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, expressing a significant reduction of PFU size after treatments over time.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7273821/v1/13485ae24e5bfb559e21f2f0.jpg"},{"id":89676471,"identity":"3052e2c0-f78b-46f3-9a40-6be986b819c8","added_by":"auto","created_at":"2025-08-22 13:48:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64913,"visible":true,"origin":"","legend":"\u003cp\u003eAggregation profile of bacteriophages identified by DLS, discussed in the item 2.6, after purification methods, described in section 2.4. \u003cstrong\u003eA.\u003c/strong\u003e Percentage of particles ranging from 30 to 300 nm, indicating free viral particles, obtained from different purification methods of MS2 phage over 55 days, represented by data points in the graph. \u003cstrong\u003eB.\u003c/strong\u003e Percentage of particles ranging from 30 to 300 nm obtained from different purification methods of PhiX174 phage over 55 days. Asterisks indicate \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, expressing significance difference of the aggregation profile over time or between treatments.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7273821/v1/1163132b6a71873d6704e14d.jpg"},{"id":89675239,"identity":"a45e2c52-cab0-4599-b50f-9ac840a1967d","added_by":"auto","created_at":"2025-08-22 13:32:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76015,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the proposed mechanism of Tween 20 and its influence on phage aggregation. \u003cstrong\u003eA.\u003c/strong\u003e When the strength of the bound between PEG and Tween 20 is higher than the bound between PEG and the phage, Tween 20 is able to promote disaggregation by removing the PEG bonded unto phage particles. \u003cstrong\u003eB.\u003c/strong\u003e When the bond between PEG and phage is stronger than PEG and Tween 20, instead, Tween 20 is unable to separate PEG molecules and consequently reduce viral aggregation. \u003cstrong\u003eC.\u003c/strong\u003e When Tween 20 is left in exposure with phage particles for too long, unspecific binding of Tween 20 may promote aggregation between phages.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7273821/v1/90bbf1084197de121cc46564.jpg"},{"id":90117165,"identity":"a9a38090-3c80-42fe-8046-220916119011","added_by":"auto","created_at":"2025-08-28 16:31:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":741814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7273821/v1/f74ec7c2-bf78-48d4-bd9c-c36f85c5f31d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Purification Strategies and Impact of Surfactant on Bacteriophage Stability and Aggregation","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eBacteriophages, also referred to as phages, are viruses exclusively adapted to infect and reproduce within a bacterial host. The term \"bacteriophage\", derived from Latin, translates to \"bacteria eaters,\" reflecting their ability to infect and eliminate bacterial species (LEDERBERG et al., 1996). Discovered almost simultaneously by Frederick Twort and Felix d'Herelle, bacteriophages have long intrigued researchers due to antibacterial activity (D\u0026rsquo;H\u0026eacute;relle, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1917\u003c/span\u003e). As early as in the 1920s, d'Herelle used phages to combat cholera outbreaks in Egypt and India, paving the way to use phages to combat bacterial infections (KEEN et al., 2012; ALMEIDA et al., 2020).\u003c/p\u003e\u003cp\u003eAlthough their use was eclipsed by the discovery of antibiotics, the growing threat of antibiotic resistance has renewed interest in bacteriophages as therapeutic agents. In some Western countries, including Belgium, phage therapy has already been approved for use in hospitals and pharmacies (PIRNAY et al., 2018). Nonetheless, barriers to widespread adoption remain, including concerns around the stability and reliability of preparations during long-term storage. Unlike antibiotics, phages are biologically complex and highly sensitive to environmental conditions such as temperature, which can lead to rapid loss of viability if not carefully controlled (SUMMERS et al., 2012). Even in Eastern Europe, where phage therapy has a long history in conventional medicine, there is limited understanding of the formulation and preservation conditions necessary to ensure therapy efficacy (MERABISHVILI et al., 2013). As a result, there is significant interest in improving bacteriophage preservation techniques to advance and enhance phage therapy without compromising product stability.\u003c/p\u003e\u003cp\u003eGiven the diverse nature of bacteriophages, no universal storage method can be applied to all. Instead, storage conditions must be tailored to the specific characteristics of each phage (ŁOBOCKA et al., 2017). This specificity means that bacteriophage formulations must be evaluated by the necessary physical and chemical conditions of each virus.\u003c/p\u003e\u003cp\u003eA significant indicator of poor stability of phage formulations is viral aggregation, which can reduce the number of viable bacteriophages by promoting adsorption onto filters and membranes used in purification, thus compromising phage products which require long-term storage (JOŃCZYK et al., 2011). To address this, surfactants such as Tween 20 have been proposed as additives to reduce aggregation and surface adhesion. It has been shown that as little as 0.002% Tween 20 can be effective in reversing phage adhesion to polypropylene vials, enhancing their stability (RICHTER et al., 2021). Such results suggest that adding surfactants to phage-based products can enhance shelf life by preventing rapid product decay caused by viral adhesion and aggregation.\u003c/p\u003e\u003cp\u003eThe purity of preparations itself can also influence viral stability. Bacteriophages may adhere to bacterial debris present in solutions, causing steep declines in viability by several orders of magnitude (ŁOBOCKA et al., 2017). This indicates that purifying bacterial residues can lead to greater long-term viral stability, although the purification method must be carefully selected to avoid viral titre loss (ŁOBOCKA et al., 2017).\u003c/p\u003e\u003cp\u003eThis study investigates how different purification and recovery methods affect the stability and aggregation profiles of two model bacteriophages: MS2 and PhiX174 phages. The results aim to contribute to a better understanding of the conditions and factors required for maintaining phage stability during long-term storage, facilitating their application in biotechnological and therapeutic settings.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 ORIGIN OF THE BACTERIOPHAGES AND BACTERIA USED\u003c/h2\u003e\u003cp\u003eThe bacteriophages MS2 (ATCC 15597-B1) and PhiX-174 (ATCC BAA-13706) were originally obtained from the American Type Culture Collection (ATCC, USA) and stored at 4\u0026deg;C until use. They are part of the virus collection of the Applied Virology Laboratory at UFSC. The bacterium \u003cem\u003eE. coli\u003c/em\u003e (ATCC 25922), used for phage propagation, was also obtained from ATCC and stored at -80\u0026deg;C until use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 PROPAGATION OF BACTERIOPHAGES\u003c/h2\u003e\u003cp\u003eBriefly, 1 mL of each bacteriophage suspension was added separately to an Erlenmeyer flask containing 25 mL of LB medium (Luria Bertani) supplemented with 10 mM MgSO₄ and 10 mM CaCl₂, along with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in exponential growth phase. The culture was incubated at 37\u0026deg;C for 6 to 12 hours, after which 1 mL of the bacterial culture was transferred to another Erlenmeyer flask containing fresh \u003cem\u003eE. coli\u003c/em\u003e in exponential growth. This step was repeated twice, with three incubation cycles per bacteriophage. After propagation, the contents of the Erlenmeyer\u0026rsquo;s were centrifuged at 3,220 \u0026times;g for 10 minutes at room temperature using a model 5804R centrifuge (Eppendorf, Germany). The supernatant was then stored at 4\u0026deg;C for subsequent titration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 BACTERIOPHAGE ENUMERATION\u003c/h2\u003e\u003cp\u003eThe quantification of bacteriophages was carried out using the double-layer agar method, adapted from Bichet \u003cem\u003eet al.\u003c/em\u003e (2021). Briefly, 10 microliters of the propagated bacteriophage suspension were serially diluted at a 1:100 ratio in test tubes containing 1 mL of SM buffer (0.1 M NaCl, 0.0081 M MgSO₄ heptahydrate, and 0.2% gelatin). Then, 1 mL of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in the exponential growth phase was added to each dilution. Samples were maintained at room temperature (15\u0026ndash;30\u0026deg;C) for 15 to 30 minutes to allow phage infection of the bacteria.\u003c/p\u003e\u003cp\u003eSimultaneously, LB semi-solid medium containing 0.75% bacteriological agar (Sigma-Aldrich, USA), supplemented with 10 mM MgSO₄ and 10 mM CaCl₂, was heated to near boiling. After that, 3 mL were transferred to test tubes maintained at 47\u0026deg;C in a heating block to prevent solidification, and 1 mL of each viral dilution was added. After a brief vortex homogenization, the solution was poured into Petri dishes containing solid LB agar (1.5% agar). The mixture was spread evenly, and plates were incubated at 37\u0026deg;C for 12 hours in a bacteriological incubator. Plaque-forming units (PFU) were then counted. The phage titer was calculated using the formula:\u003c/p\u003e\u003cp\u003ePFU/mL = (number of plaques \u0026times; dilution factor) / volume plated (mL).\u003c/p\u003e\u003cp\u003eThe experiment was performed in duplicate, and the final PFU/mL value was determined by averaging both counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 VIRAL CONCENTRATION AND PURIFICATION PROTOCOLS\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Chloroform and Tween 20 Addition\u003c/h2\u003e\u003cp\u003eThis protocol was adapted from Bichet \u003cem\u003eet al\u003c/em\u003e. (2021) and Richter \u003cem\u003eet al\u003c/em\u003e. (2021). After collecting the phage supernatant as previously described in the item 2.2, chloroform was added at 10% of the total volume. The mixture was centrifuged at 3,220 \u0026times;g for 10 minutes at 4\u0026deg;C, and the aqueous phase (supernatant) was carefully collected to avoid disturbing the chloroform layer containing impurities. This solution was filtered using a 0.22 \u0026micro;m cellulose acetate filter and stored at 4\u0026deg;C until use. In a parallel sample, 0.002% (v/v) Tween 20 was added after purification. The solution was manually agitated for homogenization and stored at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Polyethylene Glycol (PEG) and Tween 20 Addition\u003c/h2\u003e\u003cp\u003eThis protocol was adapted from Branston \u003cem\u003eet al\u003c/em\u003e. (2015). Following purification as previously described in the item 2.4.1, the chloroform phage suspension was filtered through a 0.22 \u0026micro;m filter, and PEG 6000 was added at 2% (v/v). The solution was left to precipitate at room temperature for 10 minutes. It was then centrifuged at 15,000 \u0026times;g at 22\u0026deg;C using an AVANTI J301 centrifuge (Beckman Coulter, USA). The aqueous phase was discarded, and the pellet, although barely visible, was resuspended in 3 mL of Tris buffer (25 mM Tris, 100 mM NaCl, pH 8.0). The solution was stored at 4\u0026deg;C. In a parallel sample, 0.002% (v/v) Tween 20 was added after purification. The solution was manually homogenized and stored at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Triton X-100\u003c/h2\u003e\u003cp\u003eThis protocol was adapted from Branston \u003cem\u003eet al\u003c/em\u003e. (2015). Following PEG purification as previously described in the item 2.4.2, 2% PEG 6000 and 2% (v/v) of Triton X-100 (10% stock in Milli-Q water) were added to the sample. The solution was kept at room temperature for 10 minutes, then centrifuged at 15,000 \u0026times;g at 22\u0026deg;C in the AVANTI J301 centrifuge. This process was repeated three times, with the resulting pellet resuspended in 3 mL of Tris buffer (25 mM Tris, 100 mM NaCl, pH 8.0).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5 INFECTIVITY KINETICS ASSAY OF BACTERIOPHAGES\u003c/h2\u003e\u003cp\u003eThe infectivity of the purified MS2 and PhiX174 bacteriophages was assessed using the double-layer agar assay in duplicate, as previously described in the item 2.3. Infectivity was monitored over 55 days following the purification protocols, using the same initial sample in a non-destructive assay. Kinetics were evaluated by tracking changes in PFU/mL and plaque size over time. The average diameter of 20 lysis plaques was measured using a caliper, following an adaptation of the method by Bichet \u003cem\u003eet al\u003c/em\u003e. (2021). The pH of bacteriophage suspensions purified by the previously described methods, described in the 2.4 item, were measured on the same day of purification using a pH meter from the Acquacombo Colorimetric Kit / CD 1555 (Alfakit, Brazil).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6 AGGREGATION PROFILE IDENTIFICATION BY DYNAMIC LIGHT SCATTERING\u003c/h2\u003e\u003cp\u003eThe aggregation profile of the bacteriophages was assessed by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, UK). One milliliter of each purified bacteriophage suspension was analyzed, according to the manufacturer's instructions, at 22\u0026deg;C on days 14, 35, and 55 post-purification, in a non-destructive manner using the same sample over time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7 STATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism version 8.2.1 (GraphPad Software, USA). The infectivity assay described in the item 2.5 was subjected to Two-way ANOVA, ordinary One-way ANOVA, and linear regression. Results were considered statistically significant when the probability value was p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The aggregation profile analysis was evaluated using ordinary One-way ANOVA and Tukey\u0026rsquo;s test, with significance also set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Shelf-stability analysis was conducted by calculating the average instability index, yielding a final stability score.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 INFECTIVITY OF BACTERIOPHAGES AFTER PURIFICATION\u003c/h2\u003e\u003cp\u003eIn order to verify the overall infectivity of the bacteriophages after purification methods described in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e, an estimated total decay analysis was conducted. The linear regression analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, revealed a consistent and notable decay for MS2 treated with PEG and for PhiX174 purified with PEG and Tween 20. The coefficient of determination (R\u0026sup2;) obtained from the linear regression indicated values of 74.10% for PEG-treated MS2 and 61.42% for PhiX174 purified with PEG and Tween 20. These results support the suitability of the regression model and suggest a trend of infectivity decay over time for these specific treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate how purification protocols may affect the phage\u0026rsquo;s infectious behavior, changes in plaque size post-treatment was chosen. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the variation in average plaque size (PFUs) in millimeters over the 0\u0026ndash;55 day period. Two-Way ANOVA revealed a significant change for PhiX174 purified with chloroform and Tween 20 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no similar effect observed in other treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 AGGREGATION PROFILE OF BACTERIOPHAGES BY DYNAMIC LIGHT SCATTERING (DLS)\u003c/h2\u003e\u003cp\u003eTo verify how purifications methods influence phage aggregation profiles (and consequently their infectivity), DLS analysis was employed. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A and B) shows the aggregation kinetics of bacteriophages as assessed by DLS over 55 days. One-Way ANOVA was used to calculate the percentage of particles ranging from 30 to 300 nm, a size range theorized to represent free particles (30 nm) through to aggregates of up to 10 viral particles (up to 300 nm). This parameter is based on Langlet \u003cem\u003eet al.\u003c/em\u003e (2007), which identified 30 nm particles as corresponding to viable MS2 units and observed that infectivity decreased significantly as particle sizes deviated from this range.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMS2 purified with chloroform exhibited a significant difference in aggregation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to chloroform plus Tween 20 treatment, with the addition of Tween 20 drastically reducing the proportion of particles in the 30\u0026ndash;300 nm range. Similarly, PhiX174 also displayed significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between PEG and PEG plus Tween 20 treatments, with the presence of surfactant substantially improving the proportion of low-aggregation particles typically associated with infectious virions.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe present results suggest that the stability indicators analysed vary not only with the purification method applied but also with the specific characteristics of each bacteriophage. MS2 and PhiX174 responded differently to identical treatments, indicating that the results may be phage-specific. The total decay analysis revealed that PEG-treated MS2 and PhiX174 purified with PEG and Tween 20 exhibited greater loss of infectivity over time. In contrast, other purification methods did not show a significant decay pattern, suggesting greater stability for those treatments during prolonged storage, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These results highlight that different purification methods lead to distinct stability profiles, and that no single method can be applied to all phages, since a direct relationship between the purification methods in both strains could not be established.\u003c/p\u003e\u003cp\u003eVariations in plaque size are linked to differences in phage adsorption to bacterial lawns. Shorter adsorption times are typically associated with smaller plaques, while prolonged adsorption times can produce smaller plaques with greater size variation, resulting in PFUs of differing sizes, as described by (ABEDON \u003cem\u003eet al\u003c/em\u003e., 2009; ADAMS, 1959). Similarly, our study could not establish a relationship between phage plaque size and purification method for either MS2 or PhiX174, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Although a significant reduction in plaque size is often associated with lower infectivity, this relationship is not absolute, and individual phage infections may not be affected by plaque morphology (ABEDON \u003cem\u003eet al\u003c/em\u003e., 2009). Conversely, an increase in plaque size may arise from a low viral adsorption rate, in which unabsorbed viral particles can diffuse away from the plaque center for longer periods, forming larger PFUs (ABEDON \u003cem\u003eet al.\u003c/em\u003e, 2009). Similar to our results, the significant reduction in PFU size, seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, was not accompanied by a proportional loss in viral quantity, as revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Therefore, it is difficult to determine whether this purification method significantly compromises phage infectivity over long-term storage. A crucial aspect for greater viral stability is the need to adjust the pH to the virus isoelectric point\u0026mdash;i.e., the pH level at which an insoluble material has a neutral charge (KOZLOWSKI et al., 2021). For viruses, the isoelectric point refers to the pH level at which viral units have a neutral charge, preventing adhesion to other particles via electrostatic attraction, which can directly influence the bioavailability of viable viruses (MICHEN et al., 2010). In phage therapy, the isoelectric point affects the number of free particles and, consequently, the infectivity of bacteriophages. In the present study, all purification conditions were maintained at pH 7.5, suggesting that the behavioral differences observed between phages and treatments were not due to variations in pH relative to the viruses\u0026rsquo; isoelectric points. Furthermore, as the observed decay trends were not consistent across both phages, it can be concluded that stability depends on both the purification method and the phage in question. This also reveals that the pH resulting from the treatments did not interfere with the observed results of aggregation indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, as phages tend to aggregate when the pH is equal to or below the isoelectric point, forming aggregates up to 222 times larger than the original virus size (LANGLET et al., 2007; ZUO et al., 2014). These aggregates reduce the number of viable bacteriophages as they favor adsorption onto filters and membranes used in purification, compromising phage products requiring long-term storage (JOŃCZYK et al., 2011). Thus, aggregate formation remains a significant indicator of poor bacteriophage viability and infectivity, with an increase in aggregates in the phage solution being a significant factor in their inactivation (FURIGA et al., 2011).\u003c/p\u003e\u003cp\u003eHowever, in the present study, aggregate formation did not correlate directly with loss of viral infectivity. Although there were significant differences in aggregation profiles under different treatments (e.g., 100% of PhiX174 particles purified with PEG were \u0026gt;\u0026thinsp;300 nm), as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, infectivity tests showed no direct correlation between aggregation level and the number of infectious particles observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This suggests that, under the parameters analyzed, high aggregation levels does not necessarily impair phage infectivity, indicating potential viability for long-term storage even under aggregated conditions. However, further studies are required to determine whether these patterns persist over longer storage periods.\u003c/p\u003e\u003cp\u003eIn this study, Tween 20 did not universally promote disaggregation, in contrast to Richter \u003cem\u003eet al\u003c/em\u003e. (2021) where 0.002% Tween 20 was effective in reversing phage adhesion to storage vials (promoting phage release and increasing detectable infectious particles),. In some cases in this study\u0026mdash;such as with both phages treated with chloroform\u0026mdash;aggregation increased. For PEG-purified MS2, the surfactant had no significant effect, while for PEG-purified PhiX174, Tween 20 considerably reduced aggregation. These differing outcomes may be due to interactions between Tween 20 and PEG, as described by Mahajan \u003cem\u003eet al\u003c/em\u003e. (2004), who reported the formation of aggregates due to interactions between glycol oligomers present in both compounds. Similar results were observed by Hakami \u003cem\u003eet al\u003c/em\u003e. (2015), who noted aggregation of PEG-concentrated phages after adding Tween 20. Both studies attributed this effect to PEG residues on the viral capsid forming strong hydrophilic bonds with Tween 20.\u003c/p\u003e\u003cp\u003eIt is possible that Tween 20 disrupted the interactions between phages and PEG in suspensions such as PhiX174\u0026rsquo;s, promoting disaggregation. However, Tween 20 has also been reported to form nonspecific interactions with proteins and other organic compounds, as reported by Jamur \u003cem\u003eet al\u003c/em\u003e. (2009), which may explain unexpected aggregation when PEG is absent. It is plausible that the dynamics between Tween 20 and viral particles vary with exposure time and the compounds present in the solution. While Richter \u003cem\u003eet al\u003c/em\u003e. (2021) observed disaggregation of phages from polypropylene surfaces with brief Tween 20 exposure, no subsequent reports assessed long-term aggregation profiles after extended contact with Tween 20, as conducted here.\u003c/p\u003e\u003cp\u003eThese findings also demonstrate that the interaction dynamics with Tween 20 may be phage-specific. This indicates that further studies involving Tween 20 and other phage species are needed before considering it a universal disaggregation supplement in phage suspensions. The dynamics between how Tween 20 addition may play in the increase or decrease of phage aggregation are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis study successfully evaluated the stability of different bacteriophages subjected to different purification methods, aiming to identify approaches that promote greater stability and preservation of viral infectivity during both short- and long-term storage. The comparative analysis between viral infectivity and aggregation profiles demonstrated that aggregation is not a determining factor for phage viability\u0026mdash;therefore, viral viability cannot be reliably assessed solely on aggregation index. The results reinforce that no universally effective purification method can be applied for all bacteriophages, since the preservation of viral activity depends on the specific characteristics of each phage. Overall, this work contributes valuable insights to the development of optimized preservation protocols, highlighting critical factors to be considered and appropriate analytical methods for evaluating phages intended for long-term storage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis work was funded a by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Brazil.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.B.S. conceptualization, methodology, investigation, and writing of original draft, M.A.O. data curation, H.Y.C. methodology, investigation, A.R.P., and G.F. conceptualization, supervision, and review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStephen, A. B. E. D. O. N. (2009). T. Bacteriophage Plaques: theory and analysis. Methods in Molecular Biology, pp. 161\u0026ndash;174, Humana Press. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1007/978-1-60327-164-6_17\u003c/span\u003e\u003cspan address=\"10.1007/978-1-60327-164-6_17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHancock, A. D. A. M. S. M. (1959). Bacteriophages. New York: Interscience Publishers.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Freitas, A. L. M. E. I. D. A. G. M. (2020). may. The forgotten tale of Brazilian phage therapy. The Lancet Infectious Diseases, v. 20, n. 5, pp. 90\u0026ndash;101, Elsevier BV. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/s1473-3099(20)30060-8\u003c/span\u003e\u003cspan address=\"10.1016/s1473-3099(20)30060-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarion, B. I. C. H. E. T. C. et al. Protocols for studying bacteriophage interactions with in vitro epithelial cell layers. Star Protocols, v. 2, n. 3, p. 100697, set. 2021. Elsevier BV. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.xpro.2021.100697\u003c/span\u003e\u003cspan address=\"10.1016/j.xpro.2021.100697\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteven, B. R. A. N. S. T. O. N. (2015). D. may. A non-chromatographic method for the removal of endotoxins from bacteriophages. Biotechnology and Bioengineering, v. 112, n. 8, pp. 1714\u0026ndash;1719, 5 Wiley. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1002/bit.25571\u003c/span\u003e\u003cspan address=\"10.1002/bit.25571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;H\u0026eacute;relle, F. (1917). On an invisible microbe antagonistic to dysentery bacilli. C.R. Acad. Sci. Paris 165, 373\u0026ndash;375 Research in Microbiology. Sep;158(7):553-4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.resmic.2007.07.005\u003c/span\u003e\u003cspan address=\"10.1016/j.resmic.2007.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAurelie, F. U. R. I. G. A., et al. (jan. 2011). \u003cem\u003eEffects of ionic strength on bacteriophage MS2 behavior and their implications for the assessment of virus retention by ultrafiltration membranes. Applied and Environmental Microbiology, v. 77, n. 1\u003c/em\u003e (pp. 229\u0026ndash;236). American Society for Microbiology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1128/aem.01075-10\u003c/span\u003e\u003cspan address=\"10.1128/aem.01075-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdulrahim, H. A. K. A. M. I. (2015). R. sep. Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces. Journal of Virological Methods, v. 221, pp. 1\u0026ndash;8, Elsevier BV. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.jviromet.2015.04.023\u003c/span\u003e\u003cspan address=\"10.1016/j.jviromet.2015.04.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaria C\u0026eacute;lia, J. A. M. U. R., et al. (oct. 2009). \u003cem\u003ePermeabilization of cell membranes\u003c/em\u003e (pp. 63\u0026ndash;66). Humana. Methods in Molecular Biology\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1007/978-1-59745-324-0_9\u003c/span\u003e\u003cspan address=\"10.1007/978-1-59745-324-0_9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJOńCZYK, E. (2011). may. The influence of external factors on bacteriophages\u0026mdash;review. Folia Microbiologica, v. 56, n. 3, pp. 191\u0026ndash;200, Springer Science and Business Media LLC. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1007/s12223-011-0039-8\u003c/span\u003e\u003cspan address=\"10.1007/s12223-011-0039-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKEEN, Eric C. et al. Phage therapy: concept to cure. Frontiers in Microbiology, v. 3, p. 1, 2012. \u003cem\u003eFrontiers Media SA\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.3389/fmicb.2012.00238\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2012.00238\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLukasz Pawel, K. O. Z. L. O. W. S. K. I. (2021). IPC 2.0: prediction of isoelectric point and pka dissociation constants. Nucleic Acids Research, v. 49, n. 1. \u003cem\u003e27 apr\u003c/em\u003e (pp. 285\u0026ndash;292). Oxford University Press (OUP. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1093/nar/gkab295\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkab295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLANGLET, J. (2007). Effects of pH on plaque forming unit counts and aggregation of MS2 bacteriophage. Journal of Applied Microbiology, v. 103, n. 5. \u003cem\u003e19 jun\u003c/em\u003e (pp. 1632\u0026ndash;1638). Oxford University Press (OUP. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1111/j.1365-2672.2007.03396.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.2007.03396.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLEDERBERG, J. (1996). apr. Smaller fleas \u0026hellip; ad infinitum: therapeutic bacteriophage redux. Proceedings of The National Academy of Sciences, v. 93, n. 8, pp. 3167\u0026ndash;3168, 16 Proceedings of the National Academy of Sciences. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1073/pnas.93.8.3167\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.8.3167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eŁOBOCKA, Małgorzata, B. (2017). nov. Methods for bacteriophage preservation. Methods in Molecular Biology, pp. 219\u0026ndash;230, 9 Springer New York. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1007/978-1-4939-7395-8_17\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-7395-8_17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRakesh Kumar, M. A. H. A. J. A. N. Effects of monomeric and polymeric glycol additives on micellar properties of Tween non-ionic surfactants as studied by cyclic voltammetry. Colloids and Surfaces A: Physicochemical and Engineering Aspects, v. 237, n. 1\u0026ndash;3, pp. 119\u0026ndash;124, apr. 2004. Elsevier BV. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.colsurfa.2004.02.013\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfa.2004.02.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaia, M. E. R. A. B. I. S. H. V. I. L. I. (2013). jul. Stability of Staphylococcus aureus phage ISP after freeze-drying (lyophilization). Plos One, v. 8, n. 7, p. 68797, 2 Public Library of Science (PLoS). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1371/journal.pone.0068797\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0068797\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMICHEN, B. (2010). jan. Isoelectric points of viruses. Journal of Applied Microbiology, v. 109, n. 2, pp. 388\u0026ndash;397, 22 Oxford University Press (OUP). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1111/j.1365-2672.2010.04663.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.2010.04663.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJean-Paul, P. I. R. N. A. Y. (2018). feb. The magistral phage. Viruses, v. 10, n. 2, p. 64, 6 MDPI. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.3390/v10020064\u003c/span\u003e\u003cspan address=\"10.3390/v10020064\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRICHTER, Łukasz, et al. (apr. 2021). \u003cem\u003eAdsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research. Scientific Reports, v. 11, n. 1\u003c/em\u003e (1., pp. 1\u0026ndash;11). Springer Science and Business Media LLC. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1038/s41598-021-86571-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-86571-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilliam, S. U. M. M. E. R. S. (2012). C. apr. The strange history of phage therapy. Bacteriophage, v. 2, n. 2, pp. 130\u0026ndash;133, Informa UK Limited. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.4161/bact.20757\u003c/span\u003e\u003cspan address=\"10.4161/bact.20757\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhili, Z. U. O., et al. (may 2014). \u003cem\u003eSurvival of airborne MS2 bacteriophage generated from human saliva, artificial saliva, and cell culture medium. Applied and Environmental Microbiology, v. 80\u003c/em\u003e (Vol. 9n., pp. 2796\u0026ndash;2803). American Society for Microbiology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1128/aem.00056-14\u003c/span\u003e\u003cspan address=\"10.1128/aem.00056-14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacteriophages, aggregation, stability, surfactant, storage, purification","lastPublishedDoi":"10.21203/rs.3.rs-7273821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7273821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteriophages are promising bactericidal agents extensively studied, particularly for their potential in infection control. However, their stability can be affected by purification methods and prolonged storage. A key factor contributing to viability loss is viral particle aggregation, which may be mitigated by surfactants such as Tween 20. This study investigated the relationship between viral aggregation and infectivity loss by purifying MS2 and PhiX174 bacteriophages using chloroform clarification and PEG concentration, with and without Tween 20, as well as Triton X-100 purification, over a 55-day period. The results showed that infectivity and aggregation profiles differed depending on the purification method. However, no direct correlation was observed between aggregation and infectivity. Moreover, MS2 and PhiX174 exhibited distinct responses to the same treatments, indicating that no single purification method is universally effective for all bacteriophages. The study concludes that viral aggregation is not directly associated with infectivity loss and emphasizes the need for virus-specific protocols to ensure stability during long-term storage.\u003c/p\u003e","manuscriptTitle":"Purification Strategies and Impact of Surfactant on Bacteriophage Stability and Aggregation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 13:24:26","doi":"10.21203/rs.3.rs-7273821/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0454df52-0808-4355-afd0-4fa99907b4d7","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-28T16:23:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 13:24:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7273821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7273821","identity":"rs-7273821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}