Genentech Research and Early Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Genentech Research and Early Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
J Chromatogr A. 2020 Jun 7;1620:460987. doi: 10.1016/j.chroma.2020.460987. Epub 2020 Feb 20.
It is critical to determine the chiral impurity profile of pharmaceutical compounds. The rising trend of drug candidates bearing multiple chiral centers has aggravated the analytical challenges. The traditional chiral HPLC methods can take gruelingly long time to develop yet may not offer sufficient resolution for all stereoisomers. A fast analytical strategy with a high success rate is in urgent demand for compounds with multiple chiral centers. In this study, we have developed an effective and fast multiple heart-cutting (MHC) multicolumn two-dimensional liquid chromatography (LC-mLC) platform approach. The m in the name of LC-mLC highlights the employment of multiple chiral columns with different chiral selectors and mobile phases in the second dimension (D) within the same run. A short achiral HPLC method in the first dimension (D) allows the separation of diastereomers and other achiral impurities, followed by D analysis enabling different chiral columns and different mobile phases on each coeluted D peak for maximum resolution. This LC-mLC strategy breaks down the complex multiple-chiral-center separation problems into simple individual one-chiral-center separation, which dramatically reduces chiral method development time and sample analysis turnaround. Its versatile nature and fast turnaround approach have made it a highly efficient strategy to enable quick stereoselective synthetic route development. This platform LC-mLC strategy has been successfully demonstrated in separating eight stereoisomers for a pharmaceutical compound with 3 chiral centers, within total method development time of less than 2 hours and a final analysis time of less than 24 min, including column equilibration time. It was also proved highly efficient in separating multiple chiral and achiral components in an in-process sample containing structurally similar starting materials, intermediates, side products and multiple stereoisomers of the product with 3 chiral centers, with minimal method development time.
确定药物化合物的手性杂质概况至关重要。具有多个手性中心的候选药物呈上升趋势，这增加了分析挑战。传统的手性 HPLC 方法可能需要很长时间来开发，而且对于所有立体异构体可能无法提供足够的分辨率。对于具有多个手性中心的化合物，迫切需要一种具有高成功率的快速分析策略。在本研究中，我们开发了一种有效的快速多中心切割（MHC）多维液相色谱（LC-mLC）平台方法。LC-mLC 中的 m 突出了在同一运行中，在第二维（D）中使用具有不同手性选择器和流动相的多个手性柱。在第一维（D）中使用短的非手性 HPLC 方法允许分离非对映异构体和其他非手性杂质，然后进行 D 分析，允许每个共洗脱 D 峰上的不同手性柱和不同流动相以获得最大分辨率。这种 LC-mLC 策略将复杂的多手性中心分离问题分解为简单的单个单手性中心分离，这大大缩短了手性方法开发时间和样品分析周转时间。其多功能性和快速周转方法使其成为一种高效的策略，可快速实现立体选择性合成路线的开发。该平台 LC-mLC 策略已成功用于分离具有 3 个手性中心的药物化合物的 8 个立体异构体，总方法开发时间不到 2 小时，最终分析时间不到 24 分钟，包括柱平衡时间。它还被证明在手性和非手性成分的分离方面非常高效，包括含有结构相似起始原料、中间体、副产物和具有 3 个手性中心的产物的多个立体异构体的过程样品，方法开发时间最短。
