Recent heightened interest in inhaled bacteriophage (phage) therapy for combating antibacterial resistance in pulmonary infections has led to the development of phage powder formulations. Although phages have been successfully bioengineered into inhalable powders with preserved bioactivity, the stabilization mechanism is yet unknown. This paper reports the first study investigating the stabilization mechanism for phages in these powders. Proteins and other biologics are known to be preserved in dry state within a glassy sugar matrix at storage temperatures (T _s) at least ~50°C below the glass transition temperature (T _g). This is because at (T _g − T _s) >50°C, molecules are sufficiently immobilized with reduced reactivity. We hypothesized that this glass stabilization mechanism may also be applicable to phages comprising mostly of proteins. In this study, spray dried powders of Pseudomonas phage PEV20 containing lactose and leucine as excipients were stored at 5, 25 or 50°C and 15 or 33% relative humidity (RH), followed by assessment of bioactivity (PEV20 stability) and physical properties. PEV20 was stable with negligible titer loss after storage at 5°C/15% RH for 250 days, while storage at 33% RH caused increased titer losses of 1 log₁₀ and 3 log₁₀ at 5 and 25°C, respectively. The plasticizing effect of water at 33% RH lowered the T _g by 30°C, thus narrowing the gap between T _s and T _g to 19–28°C, which was insufficient for glass stabilization. In contrast, the (T _g − T _s) values were higher (range, 46–65°C) under the drier condition of 15% RH, resulting in the improved stability which corroborated with the vitrification hypothesis. Furthermore, phage remained stable (≤1 log₁₀) when the (T _g − T _s) value lay between 26–48°C, but became inactivated as the value fell below 20°C. In conclusion, this study demonstrated that phage can be sufficiently stabilized in spray dried powders by keeping the (T _g − T _s) value above 46°C, thus supporting the vitrification hypothesis that phages are stabilized by immobilization inside a rigid glassy sugar matrix. These findings provide a guide to better manufacture and storage practices of inhaled phage powder products using for translational medicines.

中文翻译：

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可吸入噬菌体粉末：玻璃化转变温度和生物活性稳定性。


最近，人们对吸入噬菌体（噬菌体）疗法对抗肺部感染抗菌药物耐药性的兴趣日益浓厚，从而导致了噬菌体粉末制剂的开发。尽管噬菌体已成功地通过生物工程改造为保留生物活性的可吸入粉末，但其稳定机制尚不清楚。本文报告了第一项调查这些粉末中噬菌体稳定机制的研究。众所周知，蛋白质和其他生物制品在比玻璃化转变温度 ( T _g ) 至少低 50°C 的储存温度 ( T _s ) 下以干燥状态保存在玻璃状糖基质中。这是因为在 ( T _g – T _s ) >50°C 时，分子被充分固定，反应性降低。我们假设这种玻璃稳定机制也可能适用于主要由蛋白质组成的噬菌体。在本研究中，将含有乳糖和亮氨酸作为赋形剂的假单胞菌噬菌体PEV20喷雾干燥粉末储存在5、25或50°C和15或33%相对湿度（RH）下，然后评估生物活性（PEV20稳定性）和物理性质。 PEV20 在 5°C/15% RH 下储存 250 天后保持稳定，滴度损失可忽略不计，而在 33% RH 下储存则导致在 5°C 和 25°C 下滴度损失分别增加 1 log ₁₀和 3 log ₁₀ 。 33% RH 水的塑化作用使Tg降低了₃₀ °C，从而将Ts和_Tg之间的差距缩小到 19-28° _C ，这不足以稳定玻璃。 相比之下，在 15% RH 的干燥条件下，( T _g – T _s ) 值较高（范围为 46–65°C），从而提高了稳定性，这证实了玻璃化假设。此外，当 ( T _g – T _s ) 值在 26–48°C 之间时，噬菌体保持稳定 (≤1 log ₁₀ )，但当该值低于 20°C 时，噬菌体就会失活。总之，本研究证明，通过将 ( T _g – T _s ) 值保持在 46°C 以上，可以在喷雾干燥粉末中充分稳定噬菌体，从而支持噬菌体通过固定在刚性玻璃状糖基质内而稳定的玻璃化假说。这些发现为更好地制造和储存用于转化药物的吸入噬菌体粉末产品提供了指导。