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Standardized bacteriophage purification for personalized phage therapy.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-07-24 , DOI: 10.1038/s41596-020-0346-0
Tiffany Luong 1 , Ann-Charlott Salabarria 1 , Robert A Edwards 1, 2 , Dwayne R Roach 1, 2
Affiliation  

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward—by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 1011 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 109 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 106-fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.



中文翻译:

用于个性化噬菌体疗法的标准化噬菌体纯化。

世界正处于后抗生素时代的风口浪尖,但研究人员和医生已经找到了一条前进的道路——回顾抗生素出现之前如何治疗感染,即使用噬菌体疗法。尽管噬菌体(噬菌体)在西方医学中仍然缺乏药物批准,但越来越多的患者正在接受扩大准入的紧急研究新药治疗。为了简化高质量和临床安全的噬菌体制剂的生产,我们开发了用于药用噬菌体分离、升规模培养、浓缩和纯化的系统程序。本协议中描述的 16 至 21 天程序结合了改进的经典技术、现代膜过滤工艺和无有机溶剂,平均产生 23 mL 的 10 11测试的假单胞菌克雷伯氏菌沙雷氏菌噬菌体每毫升的噬菌斑形成单位 (PFU) 。因此,单次生产运行可以在 10 9 PFU下产生多达 64,000 次治疗剂量,这对于大多数扩展访问噬菌体治疗案例和潜在的临床 I/II 期应用来说已经足够了。该方案侧重于通过多次低速离心、微滤和错流超滤尽早去除内毒素,将内毒素减少多达 10 6-折叠噬菌体制剂。在参与噬菌体生产的研究实验室中实施标准化噬菌体培养和纯化,以扩大噬菌体疗法的使用范围,可能是将噬菌体疗法重新引入西医的关键。

更新日期:2020-07-24
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