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Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-07-25 , DOI: 10.1021/acs.analchem.4c02150 Daniel Ngoc Nguyen 1 , Peggy Ko 2 , Brian Roper 3 , Gary Console 4 , Yuan Gao 2 , David Shaw 2 , Christopher Yu 3 , Peter Yehl 1 , Kelly Zhang 1 , Alexandre Goyon 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-07-25 , DOI: 10.1021/acs.analchem.4c02150 Daniel Ngoc Nguyen 1 , Peggy Ko 2 , Brian Roper 3 , Gary Console 4 , Yuan Gao 2 , David Shaw 2 , Christopher Yu 3 , Peter Yehl 1 , Kelly Zhang 1 , Alexandre Goyon 1
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Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.
中文翻译：
质粒 DNA 拓扑形式和多聚体的分析和功能表征
质粒 DNA (pDNA) 是基因工程中的重要工具，在细胞和基因治疗中得到了广泛应用。质粒以超螺旋 (SC)、开环 (OC) 和线性形式存在。质粒多聚化也可以在制造过程中发生。尽管 SC 形式被认为可提供最佳的敲入 (KI) 效率，但对于拓扑形式和多聚体对功能活性的影响尚未达成强烈共识。此外，传统 pDNA (>5 kbp) 获得的结果不一定适用于较小的 pDNA (∼3 kbp)。在本研究中，开发了用于 pDNA 拓扑形式和多聚体的分析和功能表征的工作流程。首先开发了阴离子交换色谱（AEC）方法来量化拓扑形式和多聚体。最初对四种 AEC 色谱柱进行了比较，发现其中一种具有优异的色谱性能。系统研究了流动相pH、各种盐、柱温、乙腈含量对分离性能的影响。评估了方法性能，包括精密度和准确度。通过分析几个 pDNA 序列和批次，将最终的 AEC 方法与毛细管凝胶电泳 (CGE) 进行比较。强制降解研究显示 SC 形式的降解程度出人意料地高。最后，比较了 SC 和 OC 形式以及多聚体的 KI 效率。
更新日期：2024-07-25
中文翻译：
质粒 DNA 拓扑形式和多聚体的分析和功能表征
质粒 DNA (pDNA) 是基因工程中的重要工具，在细胞和基因治疗中得到了广泛应用。质粒以超螺旋 (SC)、开环 (OC) 和线性形式存在。质粒多聚化也可以在制造过程中发生。尽管 SC 形式被认为可提供最佳的敲入 (KI) 效率，但对于拓扑形式和多聚体对功能活性的影响尚未达成强烈共识。此外，传统 pDNA (>5 kbp) 获得的结果不一定适用于较小的 pDNA (∼3 kbp)。在本研究中，开发了用于 pDNA 拓扑形式和多聚体的分析和功能表征的工作流程。首先开发了阴离子交换色谱（AEC）方法来量化拓扑形式和多聚体。最初对四种 AEC 色谱柱进行了比较，发现其中一种具有优异的色谱性能。系统研究了流动相pH、各种盐、柱温、乙腈含量对分离性能的影响。评估了方法性能，包括精密度和准确度。通过分析几个 pDNA 序列和批次，将最终的 AEC 方法与毛细管凝胶电泳 (CGE) 进行比较。强制降解研究显示 SC 形式的降解程度出人意料地高。最后，比较了 SC 和 OC 形式以及多聚体的 KI 效率。
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