Lentiviral Vector Purification Using Nanofiber Ion-Exchange Chromatography.
Lentiviral Vector Purification Using Nanofiber Ion-Exchange Chromatography.
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使用纳米纤维离子交换色谱法纯化慢病毒载体。
DOI：
10.1016/j.omtm.2019.08.007
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Ruscic J
中科院分区：
文献类型：
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作者：
Ruscic J
慢病毒载体（LVs）被用于细胞和基因治疗，因为它们能够转导分裂和非分裂细胞，同时携带相对较大的遗传载荷，并通过基因整合提供长期的基因表达。目前的培养方法产生的滴度为105 - 107转导单位(TU)/mL；因此，有必要浓缩lv以及去除与工艺和产品相关的杂质。在这项工作中，我们使用了一个包装细胞系WinPac-RD-HV进行低压生产，以简化上游加工。建立了一种基于离子交换色谱和纤维素纳米纤维的直接捕集法用于LV的浓缩和纯化。这种新型的可扩展固定相提供了LV可访问的高表面积，因此与传统的基于珠状支架相比，具有高容量操作的潜力。我们能够浓缩lv 100倍，同时实现宿主细胞蛋白的两对数去除，并保持高达90%的功能载体产量。
Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105–107transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector.