Chang Rachel Yoon Kyung, Kwok Philip Chi Lip, Khanal Dipesh, Morales Sandra, Kutter Elizabeth, Li Jian, Chan Hak-Kim
Advanced Drug Delivery Group, School of Pharmacy The University of Sydney Sydney New South Wales Australia.
Phage Consulting Sydney New South Wales Australia.
Bioeng Transl Med. 2020 Apr 14;5(2):e10159. doi: 10.1002/btm2.10159. eCollection 2020 May.
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 ( ) at least ~50°C below the glass transition temperature ( ). This is because at ( - ) >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 log and 3 log at 5 and 25°C, respectively. The plasticizing effect of water at 33% RH lowered the by 30°C, thus narrowing the gap between and to 19-28°C, which was insufficient for glass stabilization. In contrast, the ( - ) 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 log) when the ( - ) 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 ( - ) 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.
近期，人们对吸入性噬菌体疗法用于对抗肺部感染中的抗菌耐药性的兴趣日益浓厚，这推动了噬菌体粉末制剂的研发。尽管噬菌体已成功通过生物工程制成具有生物活性的可吸入粉末，但其稳定机制尚不清楚。本文报告了第一项研究噬菌体在这些粉末中的稳定机制的研究。已知蛋白质和其他生物制剂在储存温度下（比玻璃化转变温度至少低约50°C）可在玻璃态糖基质中以干燥状态保存。这是因为在（储存温度 - 玻璃化转变温度）>50°C时，分子充分固定，反应性降低。我们假设这种玻璃态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，含有乳糖和亮氨酸作为辅料的铜绿假单胞菌噬菌体PEV20喷雾干燥粉末分别储存在5、25或50°C以及15%或33%相对湿度（RH）下，随后评估其生物活性（PEV20稳定性）和物理性质。PEV20在5°C/15%RH下储存250天后稳定，滴度损失可忽略不计，而在33%RH下储存时，在5°C和25°C时滴度损失分别增加1个对数和3个对数。33%RH下水分的增塑作用使玻璃化转变温度降低了30°C，从而使（储存温度 - 玻璃化转变温度）的差距缩小至19 - 28°C，这不足以实现玻璃态稳定。相比之下，在15%RH的干燥条件下，（储存温度 - 玻璃化转变温度）值更高（范围为46 - 65°C），稳定性提高，这与玻璃化假说相符。此外，当（储存温度 - 玻璃化转变温度）值在26 - 48°C之间时，噬菌体保持稳定（≤1个对数），但当该值降至20°C以下时会失活。总之，本研究表明，通过将（储存温度 - 玻璃化转变温度）值保持在46°C以上，噬菌体可在喷雾干燥粉末中充分稳定，从而支持了噬菌体通过固定在刚性玻璃态糖基质中而稳定的玻璃化假说。这些发现为吸入性噬菌体粉末产品用于转化医学的更好制造和储存实践提供了指导。
