In this manuscript, the application of high-resolution sampling (HRS) two-dimensional liquid chromatography (2D-LC) in the detailed analysis of key linker drug intermediate is presented. Using HRS, selected regions of the primary column eluent were transferred to a secondary column with fidelity enabling qualitative and quantitative analysis of linker drugs. The primary column purity of linker drug intermediate ranged from 88.9% to 94.5% and the secondary column purity ranged from 99.6% to 99.9%, showing lot-to-lot variability, significant differences between the three lots, and substantiating the synthetic and analytical challenges of ADCs. Over 15 impurities co-eluting with the linker drug intermediate in the primary dimension were resolved in the secondary dimension. The concentrations of most of these impurities were over three orders of magnitude lower than the linker drug. Effective peak focusing and high-speed secondary column analysis resulted in sharp peaks in the secondary dimension, improving the signal-to-noise ratios. The sensitivity of 2D-LC separation was over five fold better than conventional HPLC separation. The limit of quantitation (LOQ) was less than 0.01%. Many peaks originating from primary dimension were resolved into multiple components in the complementary secondary dimension, demonstrating the complexity of these samples. The 2D-LC was highly reproducible, showing good precision between runs with%RSD of peak areas less than 0.1 for the main component. The absolute difference in the peak areas of impurities less than 0.1% were within ±0.01% and for impurities in the range of 0.1%–0.3%, the absolute difference were ±0.02%, which are comparable to 1D-LC. The overall purity of the linker drug intermediate was determined from the product of primary and secondary column purity (HPLC Purity = %peak area of main component in the primary dimension × %peak area of main component in the secondary dimension). Additionally, the 2D-LC separation enables the determination of potential impurities that could impact the downstream process, like ADCs stability, efficacy and patient safety. Peak capacity of this magnitude, sensitivity and reproducibility of 2D-LC for resolving structurally similar impurities co-eluting with the main component has not been demonstrated to date. This application clearly demonstrates the power of 2D-LC in detailed analysis of structurally similar, co-eluting impurities from key linker drug intermediate used in ADCs that is impossible to achieve by conventional 1D-LC.

本文介绍了高分辨率采样（HRS）二维液相色谱（2D-LC）在关键接头药物中间体的详细分析中的应用。使用HRS，可以将第一色谱柱洗脱液的选定区域保真地转移至第二色谱柱，从而可以对接头药物进行定性和定量分析。接头药物中间体的一级色谱柱纯度在88.9％至94.5％之间，二级色谱柱纯度在99.6％至99.9％之间，显示批间差异，三批之间的显着差异以及证实了合成和分析难题ADC。在次要维度中解决了超过15种与接头药物中间体共同洗脱的杂质。这些杂质中大多数的浓度比接头药物低三个数量级。有效的峰聚焦和高速二级色谱柱分析在二级维数上产生了尖锐的峰，从而改善了信噪比。2D-LC分离的灵敏度比常规HPLC分离高五倍以上。定量限（LOQ）小于0.01％。许多来自初级维的峰都被分解为互补次级维的多个分量，这说明了这些样品的复杂性。2D-LC具有很高的重现性，在两次运行之间显示出良好的精度，主成分的峰面积的％RSD小于0.1。小于0.1％的杂质峰面积的绝对差在±0.01％以内，对于0.1％–0.3％的杂质，绝对差为±0.02％，与1D-LC相当。根据一级和二级色谱柱纯度的乘积（HPLC纯度=一级维主要成分的峰面积×二级维主要成分的峰面积），确定接头药物中间体的总体纯度。此外，二维液相色谱分离能够确定可能影响下游工艺的潜在杂质，例如ADC的稳定性，功效和患者安全性。迄今为止，尚未证明具有如此大小的峰容量，2D-LC的灵敏度和重现性，以解决与主要组分共同洗脱的结构相似的杂质。此应用程序清楚地展示了2D-LC在结构相似的详细分析中的强大功能，