开发新型脂质体配制方法,改进慢病毒载体的大规模瞬时转染生产

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Thomas Williams-Fegredo, Lee Davies, Carol Knevelman, Kyriacos Mitrophanous, James Miskin, Qasim A. Rafiq
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引用次数: 0

摘要

在过去 20 年中,大规模瞬时转染技术取得了长足的进步,使包括病毒载体在内的各种生物制药产品的有效生产成为可能。然而,与转染试剂稳定性和转染复合物制备时间有关的一些挑战依然存在。要确保基于瞬时基因表达的生物工艺能满足对病毒载体日益增长的需求,就需要新的发展和改进的转染技术。在本文中,我们证明了阳离子脂质体脂质体的生长是许多阳离子脂质体转染工艺中必不可少的一步,可通过采用低 pH 值(pH 值为 6.40 至 6.75)和低盐浓度(0.2× PBS)配方来控制,从而改善对纳米粒子生长动力学的控制并提高粒子的稳定性。与标准制备方法相比,这种复合物能长时间保持高效转染能力。这些发现对大规模生产慢病毒载体具有重要的工业应用价值,主要原因有两个。首先,替代制备策略可延长脂质体的孵育时间,从而有助于在良好生产规范环境下进行有效控制。其次,颗粒稳定性的提高有助于设定更宽的工艺操作范围,这将大大提高工艺的稳健性,最大限度地实现批次间的控制和产品一致性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of novel lipoplex formulation methodologies to improve large-scale transient transfection for lentiviral vector manufacture
Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.
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来源期刊
Molecular Therapy-Methods & Clinical Development
Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.90
自引率
4.30%
发文量
163
审稿时长
12 weeks
期刊介绍: The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.
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