Meston Daniel, Maloney Todd D, Stoll Dwight R
Department of Chemistry, Gustavus Adolphus College, Saint Peter, MN 56082, United States.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, United States.
J Chromatogr A. 2025 Feb 8;1742:465643. doi: 10.1016/j.chroma.2024.465643. Epub 2024 Dec 27.
Determination of quality attributes of antisense oligonucleotides (ASOs) such as purity, potency, and sequence is challenging due to their relatively large size, polyanionic nature, and large number of synthetic modifications. Chromatography technologies are evolving rapidly to meet these challenges, and one area of particularly rapid change at this time is the use of hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide (ON) separations. Relatively little has been published on the factors that dictate the kinetics of these separations. This knowledge gap consequently makes it difficult to know what gains might be made during method development by changing flow rate or particle size, for example. In this work we have taken initial steps to address this gap by examining the dependence of plate height and resolution on flow rate for separations of 23-mer ASOs under HILIC conditions. Such work is complicated by the fact that the retention of these molecules decreases dramatically with increasing pressure. After adjusting mobile phase composition to hold retention factor nominally constant for each flow rate used, we find that plate height increases strongly with increasing flow rate such that the plate height increases about ten-fold over the range of flow rate of 0.1 to 4.0 mL/min. when using a 4.6 mm i.d. column. However, the minimum reduced plate height observed at the lowest flow rate is quite impressive at around 2. Finally, we find that this dependence of plate height on flow rate translates, as expected, to an improvement in resolution as flow rate is decreased, both in conventional one-dimensional separations, and in the second dimension of a two-dimensional separation. We expect to use this work as a foundation to build on as we deepen our understanding of the kinetics of ON separations.
由于反义寡核苷酸（ASO）相对较大的尺寸、多阴离子性质以及大量的合成修饰，测定其质量属性（如纯度、效价和序列）具有挑战性。色谱技术正在迅速发展以应对这些挑战，目前变化特别迅速的一个领域是使用亲水相互作用液相色谱（HILIC）进行寡核苷酸（ON）分离。关于决定这些分离动力学的因素，相对较少有文献发表。因此，这一知识空白使得例如在方法开发过程中难以知道通过改变流速或粒径可能会有哪些收获。在这项工作中，我们通过研究在HILIC条件下23聚体ASO分离时塔板高度和分辨率对流速的依赖性，迈出了填补这一空白的初步步骤。由于这些分子的保留率会随着压力增加而急剧下降，这项工作变得复杂。在调整流动相组成以使每个使用的流速下保留因子名义上保持恒定时，我们发现塔板高度随着流速增加而强烈增加，以至于在使用内径为4.6 mm的色谱柱时，在0.1至4.0 mL/min的流速范围内，塔板高度增加了约十倍。然而，在最低流速下观察到的最小折合塔板高度约为2，相当可观。最后，我们发现塔板高度对流速的这种依赖性正如预期的那样，在传统的一维分离以及二维分离的第二维中，随着流速降低分辨率都会提高。我们期望将这项工作作为基础，随着我们对ON分离动力学的理解不断深入，在此基础上进一步拓展。
