Chiara Nosengo , Desiree Bozza , Giulio Lievore , Sebastian Vogg , Martina Catani , Alberto Cavazzini , Thomas Müller-Späth , Chiara De Luca , Simona Felletti
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引用次数: 0
Abstract
Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps. For oligonucleotides, and biomolecules in general, preparative liquid chromatography is the technique of choice to perform large scale purifications, typically in batch mode, i.e. using a single column. Specifically, since ONs are mainly large, hydrophilic and charged molecules, Anion Exchange chromatography (AEX) and Ion Pair Reversed Phase chromatography (IP-RP) are the preferred chromatographic modes for their downstream processing. Nevertheless, these approaches suffer from a purity-yield trade-off, and for this reason, more than one purification step is usually required. The two chromatographic modes can therefore be used consequently to remove different groups of impurities, thanks to their orthogonality.
In this work, a multidimensional and orthogonal approach on a (semi)preparative scale, namely “Integrated Batch process”, was applied for the purification of a single-stranded DNA oligonucleotide. This process combines two chromatographic steps without any hold step, operator intervention or sampling of the first step. The performance parameters of the Integrated Batch were compared to those obtained in the single batch runs under different experimental conditions (chromatographic mode, eluent systems), showing the potential of this integrated approach. This proof-of-concept study illustrates how this technique can considerably reduce overall production time and how it allows to increase the robustness and reproducibility of the method, since the process is highly automated.
治疗性寡核苷酸具有高度特异性的基因表达调控能力,是制药业的最新突破。在这方面,寡核苷酸可以治疗多种疾病。然而,由于寡核苷酸用于治疗目的,活性药物成分(API)必须满足严格的纯度要求,这就需要密集的纯化步骤。对于寡核苷酸和一般生物大分子而言,制备型液相色谱法是进行大规模纯化的首选技术,通常采用批处理模式,即使用单一色谱柱。具体来说,由于 ONs 主要是大分子、亲水性分子和带电分子,因此阴离子交换色谱(AEX)和离子对反相色谱(IP-RP)是其下游处理的首选色谱模式。然而,这些方法在纯度-产量之间存在权衡,因此通常需要一个以上的纯化步骤。在这项工作中,我们采用了一种(半)制备规模的多维正交方法,即 "集成批次工艺",用于纯化单链 DNA 寡核苷酸。该工艺将两个色谱步骤结合在一起,第一步没有任何保持步骤、操作员干预或取样。在不同的实验条件(色谱模式、洗脱液系统)下,集成批次的性能参数与单批次运行的性能参数进行了比较,显示了这种集成方法的潜力。这项概念验证研究说明了该技术如何大大缩短整体生产时间,以及如何通过高度自动化的流程提高方法的稳健性和可重复性。
期刊介绍:
The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.