Stoll Dwight R, Ghimire Ajit, Sorensen Matthew J, Maloney Todd D
Department of Chemistry, Gustavus Adolphus College, Saint Peter, Minnesota, 56082, USA.
Department of Chemistry, Gustavus Adolphus College, Saint Peter, Minnesota, 56082, USA.
J Chromatogr A. 2025 Mar 15;1744:465687. doi: 10.1016/j.chroma.2025.465687. Epub 2025 Jan 16.
As the importance of therapeutic oligonucleotides (ONs) continues to grow in the pharmaceutical industry, the importance of high performing analytical methods needed to characterize them also grows. The characteristics of these molecules (e.g., highly charged phosphate backbone, and small but important modifications such as methylation and fluorination) make them difficult to analyze thoroughly using conventional liquid chromatography (LC) conditions. Recently, other research groups have been emphasizing the utility of ultra-short (<< 50 mm) columns for proteins and other large biomolecules, and have remarked that long columns only add unnecessary peak dispersion without providing additional resolution over short columns. These statements naturally call into question the long-established theory for small molecule LC separations that asserts that separation performance is maximized by working at the highest available operating pressure, and then choosing the longest column possible while working at the van Deemter optimum flow rate. This apparent contradiction in turn raises the question - for which types of large biomolecule does the established chromatographic theory no longer apply? In this study we have carried out experiments and calculations aimed at answering this question for ion-pairing reversed-phase separations of therapeutic ONs with masses on the order of 6 kDa. This included measuring isocratic plate heights for these molecules after establishing an empirical relationship between retention, mobile phase composition, and flow rate, because retention of the ONs is extremely sensitive to pressure (20 % increase in k per bar pressure drop), and thus retention varies with flow rate at a constant mobile phase composition. After taking these factors into account, we find that resolution of the oligonucleotides does increase with the square root of column length, as predicted by the well-established theory for small molecules. However, we also find that this relationship is only found when the gradient slope is held constant while varying the column length, and that if this is not done it is actually possible to observe that resolution decreases with increasing column length. Thus, the design of experiments used to evaluate the role of column length in separation performance is critical. In addition to the importance of these findings to development of LC methods for ON separations in general, they will be especially impactful in two-dimensional (2D) separations of ONs where there is more or less freedom to choose parameters from a wide range of possibilities depending on the mode of 2D separation that is used.
随着治疗性寡核苷酸（ONs）在制药行业的重要性持续增长，用于表征它们的高性能分析方法的重要性也在增加。这些分子的特性（例如，带高电荷的磷酸骨架，以及甲基化和氟化等微小但重要的修饰）使得使用传统液相色谱（LC）条件对其进行全面分析变得困难。最近，其他研究小组一直在强调超短（<< 50 mm）色谱柱对蛋白质和其他大型生物分子的实用性，并指出长色谱柱只会增加不必要的峰展宽，而不会比短色谱柱提供更高的分离度。这些说法自然会让人质疑长期以来关于小分子LC分离的理论，该理论认为通过在最高可用操作压力下工作，然后在范德姆特最佳流速下工作时选择尽可能长的色谱柱，可以使分离性能最大化。这种明显的矛盾反过来又引发了一个问题——对于哪些类型的大型生物分子，已确立的色谱理论不再适用？在本研究中，我们进行了实验和计算，旨在回答这个问题，即针对质量约为6 kDa的治疗性ONs的离子对反相分离。这包括在建立保留、流动相组成和流速之间的经验关系后，测量这些分子的等度塔板高度，因为ONs的保留对压力极其敏感（每巴压力降k增加20%），因此在恒定流动相组成下，保留随流速变化。考虑到这些因素后，我们发现，正如小分子的既定理论所预测的那样，寡核苷酸的分离度确实随色谱柱长度的平方根增加。然而，我们还发现，只有在改变色谱柱长度时保持梯度斜率不变，才会发现这种关系，如果不这样做，实际上可能会观察到分离度随色谱柱长度增加而降低。因此，用于评估色谱柱长度在分离性能中作用的实验设计至关重要。除了这些发现对一般ONs分离的LC方法开发的重要性之外，它们在ONs的二维（2D）分离中将特别有影响力，在二维分离中，根据所使用的二维分离模式，从广泛的可能性中或多或少可以自由选择参数。
