利用超滤技术分离完整和空腺病毒外壳的新方法。

IF 3.3 4区 工程技术 Q2 CHEMISTRY, PHYSICAL Membranes Pub Date : 2024-09-12 DOI:10.3390/membranes14090194
Deepraj Sarmah, Scott M Husson
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引用次数: 0

摘要

腺相关病毒载体(AAV)是基因治疗应用的主要病毒载体。获得有效剂量的一个重大挑战是去除缺乏 DNA 货物的非治疗性空病毒外壳。目前分离全(含基因)和空囊壳的方法在规模化方面具有挑战性,产品产量低、速度慢,而且难以在连续生物制造中操作。这篇论文展示了通过超滤分离全壳和空壳的可行性。通过使用 ELISA、qPCR 和基于绿色荧光蛋白表达的活细胞成像的感染性检测法测量完整和空白囊壳的筛分系数,对分离性能进行了量化。我们证明,孔径为 30 nm 的聚碳酸酯履带蚀刻膜可选择性地将空包囊渗透到完整包囊中,完整包囊的回收率很高(89%)。通过 ELISA/qPCR 计算得到的完整和空囊壳的平均筛分系数分别为 0.25 和 0.49,表明空囊壳的渗透性约为完整囊壳的两倍。将超滤确立为分离完整和空 AAV 胶囊的可行单元操作方法,对开发无规模的 AAV 连续纯化技术具有重要意义。
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A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs.

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来源期刊
Membranes
Membranes Chemical Engineering-Filtration and Separation
CiteScore
6.10
自引率
16.70%
发文量
1071
审稿时长
11 weeks
期刊介绍: Membranes (ISSN 2077-0375) is an international, peer-reviewed open access journal of separation science and technology. It publishes reviews, research articles, communications and technical notes. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. Full experimental and/or methodical details must be provided.
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