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Trileucine and Pullulan Improve Anti-Campylobacter Bacteriophage Stability in Engineered Spray-Dried Microparticles

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Abstract

Spray drying biologics into a powder can increase thermal stability and shelf-life relative to liquid formulations, potentially eliminating the need for cold chain infrastructure for distribution in developing countries. In this study, process modelling, microparticle engineering, and a supplemented phase diagram were used to design physically stable fully amorphous spray-dried powder capable of stabilizing biological material. A greater proportion of anti-Campylobacter bacteriophage CP30A remained biologically active after spray drying using excipient formulations containing trehalose and a high glass transition temperature amorphous shell former, either trileucine or pullulan, as compared to the commonly used crystalline shell former, leucine, or a low glass transition temperature amorphous shell former, pluronic F-68. Particle formation models suggest that the stabilization was achieved by protecting the bacteriophages against the main inactivating stress, desiccation, at the surface. The most promising formulation contained a combination of trileucine and trehalose for which the combined effects of feedstock preparation, spray drying, and 1-month dry room temperature storage resulted in a titer reduction of only 0.6 ± 0.1 log10(PFU mL−1). The proposed high glass transition temperature amorphous formulation platform may be advantageous for stabilizing biologics in other spray drying applications in the biomedical engineering industry.

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Notes

  1. A droplet emitted from the atomizer is assumed to initially be well-mixed in terms of the excipients being evenly distributed within the droplet, while the phages are assumed to be randomly distributed. “A”: trileucine is surface-active and near saturation initially and forms an amorphous shell with small amounts of trehalose early, before all phages are present at the surface. The mainly trileucine shell folds, potentially since it is thin and rubbery; the mainly trehalose interior has yet to solidify. The shell potentially maintains the same surface area upon folding and prevents phages from reaching the surface. “B”: the crumpled appearance is controlled by the folded shell. The interior later solidifies into a glass from a highly viscous solution due to further desiccation. “C”: pullulan enriches at the surface due to high molecular mass and forms a viscous amorphous shell that contains small amounts of trehalose; the shell contracts, causing phages to recede with the surface, and desiccates until a glassy mixture of pullulan and trehalose is present at the surface. “D”: the surface is mainly pullulan, with some trehalose that may have prevented shell deformation. The interior is mainly trehalose with some pullulan. “E”: nucleation at the surface results in small leucine crystals that eventually become close enough and large enough to form a shell. Trehalose and remaining leucine eventually solidify. “F”: phages could be expelled from crystals, be damaged by inter-crystal forces, or inhibit crystallization. Small crystals are at the surface of the microparticles. The interior is primarily trehalose and may contain voids. “G”: pullulan enriches near the surface and solidifies there. The thin shell is moderately rigid due to a high glass transition temperature and may easily deform and contract as it is not hindered by a trehalose interior. “H”: there is no trehalose glass stabilizer or void space in the interior. “I”: surfactant may form a film that recedes but does not adequately stabilize the phages. “J”: small cohesive spherical microparticle with phages near or on the surface where they are not adequately stabilized by the low glass transition temperature surfactant. “K”: small cohesive solid spherical microparticle formed. There is a high chance that phages reside on the surface. “L”: same as “K” except that a very small microparticle results and there is a very high chance that phages reside on surface. Void space is not shown in the schematic and may be possible in many cases. Note that there is likely a radial distribution of each excipient within the drying droplet rather than complete separation of excipients.

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Acknowledgments

NC thanks the Killam Trusts, the Natural Sciences and Engineering Research Council of Canada, Alberta Innovates, and the University of Alberta for scholarship funding. This work was financially supported by the Biotechnology and Biological Sciences Research Council [Grant Number BB/P02355X/1] (United Kingdom). The funding source had no role in study design, collection, analysis, or interpretation of data, writing the article, or in decision to submit the article for publication.

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Authors and Affiliations

  1. Department of Mechanical Engineering, University of Alberta, Edmonton, Canada

    Nicholas B. Carrigy & Hui Wang

  2. School of Biosciences, University of Nottingham, Loughborough, UK

    Lu Liang & Ian F. Connerton

  3. Centre for Microbiology Research, Kenyan Medical Research Institute, Nairobi, Kenya

    Samuel Kariuki

  4. Phages for Global Health, Oakland, USA

    Tobi E. Nagel

  5. Department of Mechanical Engineering, University of Alberta, 10-203 Donadeo Innovation Centre for Engineering, 9211 116th Street NW, Edmonton, AB, T6G 1H9, Canada

    Reinhard Vehring

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  1. Nicholas B. Carrigy
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Correspondence to Reinhard Vehring.

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Carrigy, N.B., Liang, L., Wang, H. et al. Trileucine and Pullulan Improve Anti-Campylobacter Bacteriophage Stability in Engineered Spray-Dried Microparticles. Ann Biomed Eng 48, 1169–1180 (2020). https://doi.org/10.1007/s10439-019-02435-6

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