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细菌菌体制剂的可扩展净化方法

João P P Saavedra¹, A Rita Silva-Santos¹, Sofia O D Duarte¹

¹iBB- Institute of Bioengineering and Biosciences, Instituto Superior Técnico, Department of Bioengineering, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; Associate Laboratory i4HB-Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.

Journal of chromatography. A

|March 28, 2025

概括

对抗生素耐药性感染的菌体治疗净化使用性酸酶和离子交换染色学得到了改进. 这种可扩展的方法有效地去除内毒素,蛋白质和DNA,以便潜在的静脉注射.

科学领域:

生物技术是生物技术.
微生物学微生物学
生物制药制造业生物制药制造业

背景情况:

菌体疗法是抗生素治疗细菌感染的有希望的替代品,特别是那些对当前药物耐药的细菌感染.
可扩展和高效的净化方法对于菌体治疗的临床应用至关重要,特别是在静脉注射方面.
监管要求要求从菌体制剂中去除内毒素,蛋白质和宿主细胞DNA等杂质.

研究的目的:

开发一种可扩展的细菌的净化工作流程,使用离子交换色谱 (AEC).
研究用性酸酶 (AP) 进行酶性预处理以增强通过AEC的内毒素和菌体分离的疗效.
评估内毒素,蛋白质和宿主细胞DNA的去除,以满足菌体治疗的监管标准.

主要方法:

一个以离子交换色谱 (AEC) 为中心的净化工作流被开发为菌体T4和大肠杆菌Escherichia K12.
在AEC之前,在不同度 (20或200U/mL) 的性酸酶 (AP) 治疗被采用,以减少内毒素和菌体的负电荷.
评估了各种AEC配体,静止相和H键染色学. 测量了内毒素,蛋白质和DNA去除,并确定了菌体恢复和标位.

主要成果:

达到了1.26 × 10^11 PFU/mL的最大菌体标位和高达45.1%的整体恢复.
使用20U/mL的AP处理后,观察到最高的内毒素去除率为98.8%,其次是使用四级胺列的AEC.
所有测试的工作流程都有效地去除了几乎所有的蛋白质和宿主细胞DNA,一些制剂符合基于内毒素水平的静脉注射标准.

结论:

用酸酸酶与离子交换色谱相结合的酶治疗为菌体提供了一个有希望和可扩展的净化策略.
这种净化工作流有效地去除关键杂质,使菌体制剂适合临床应用,包括静脉注射.
开发的方法为现有的菌体净化技术提供了可行的替代方案,解决了菌体治疗大规模制造的关键挑战.

关键词:

染色体学染色体学是一种染色学.下游加工是下游加工.酶性处理是一种酶性处理.菌体疗法是一种菌体疗法.可扩展性可扩展性

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