Anion exchange purification of plasmid DNA using expanded bed adsorption.

G N Ferreira, J M Cabral, D M Prazeres
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引用次数: 51

Abstract

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

扩展床吸附法纯化质粒DNA的阴离子交换。
最近在非病毒载体基因治疗和DNA疫苗接种方面的发展增加了对大量药用级质粒DNA的需求。工艺流的高粘度是质粒纯化的主要问题,因为它会在柱操作中引起高背压,从而限制了吞吐量。为了避免这些高背压,扩展床阴离子交换色谱被评估为固定床色谱的替代方法。流线型25柱填充100 ml流线型QXL培养基,在TE (10 mM Tris, 1 mM EDTA, pH = 8.0)缓冲液中以0.5 M NaCl平衡,向上流动300 cmh-1,将大肠杆菌裂解物(从多达3升发酵液中获得)注入柱中。洗掉未结合的物质后,让培养基沉淀,在TE缓冲液中用1 M NaCl以120 cmh-1的向下流速洗脱质粒。纯化因子为36 +/- 1倍,质粒纯度为26 +/- 0.4倍,质粒入样量小于1个沉淀柱体积时,产率接近100%。然而,当处理量较大时,回收率和纯度都急剧下降,当处理250 ml原料时,回收率和纯度分别为35 +/- 2和5 +/- 0.7。在这些情况下,观察到凝胶堵塞和膨胀塌陷。处理更大的体积,因此更大的质粒数量,只能通过在色谱步骤之前进行异丙醇沉淀步骤来实现。这一步导致了净化步骤的增强。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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