Modern size exclusion chromatography (SEC) can be defined by the use of relatively small columns (e.g., 150 × 4.6 mm) packed with sub-3 μm particles, allowing a 3- to 5-fold increase in method throughput compared to that of conventional SEC. The quick success of the first sub-2 μm SEC column introduced in 2010 led to the development of numerous ultra-high performance (UHP)-SEC columns for the analysis of therapeutic monoclonal antibody (mAb)-based products. Aggregates also known as high-molecular-weight species (HMWS) are indeed one of the most important critical quality attributes (CQAs) of mAbs, as HMWS may decrease the product efficacy or cause immunogenicity effects. Therefore, the confident characterization of mAbs requires strong knowledge of not only modern SEC performance (i.e., selectivity and efficiency) but also the inherent limitations caused by non-specific interactions more likely to occur with complex antibody drug conjugates (ADCs) and some commercial mAb products. This review discusses the importance of liquid chromatographic (LC) instrumentation in order to exploit the full potential of modern SEC columns and current trends to hyphenate SEC to mass spectrometry (MS). Recent applications for antibody-based products (i.e., mAbs, ADCs, Fc-Fusion proteins and bispecific antibodies) are presented. Finally, tips and tricks are provided to further optimize SEC separations and maintaining their performance over time with better understanding of unexpected SEC results.

现代尺寸排阻色谱法（SEC）可以通过使用相对较小的色谱柱（例如150×4.6 mm）填充以小于3μm的颗粒来定义，与传统方法相比，方法通量可提高3至5倍SEC。2010年推出的首个亚2μmSEC色谱柱迅速获得成功，导致了众多用于分析治疗性单克隆抗体（mAb）产品的超高性能（UHP）-SEC色谱柱的开发。聚集体也称为高分子量物种（HMWS）实际上是mAb的最重要的关键质量属性（CQA）之一，因为HMWS可能会降低产品功效或引起免疫原性。因此，要对mAb进行可靠的表征，不仅需要现代SEC性能的丰富知识（即，选择性和效率）以及由于非特异性相互作用引起的固有局限性，更容易发生在复杂抗体药物偶联物（ADC）和某些市售mAb产品上。这篇综述讨论了液相色谱（LC）仪器的重要性，以便充分利用现代SEC色谱柱的潜力以及当前将SEC联用到质谱（MS）的趋势。提出了基于抗体的产品（即mAb，ADC，Fc融合蛋白和双特异性抗体）的最新应用。最后，提供了提示和技巧，以进一步优化SEC分离并随着时间的推移保持其性能，从而更好地了解意外的SEC结果。这篇综述讨论了液相色谱（LC）仪器的重要性，以便充分利用现代SEC色谱柱的潜力以及当前将SEC联用到质谱（MS）的趋势。提出了基于抗体的产品（即mAb，ADC，Fc融合蛋白和双特异性抗体）的最新应用。最后，提供了提示和技巧，以进一步优化SEC分离并随着时间的推移保持其性能，从而更好地了解意外的SEC结果。这篇综述讨论了液相色谱（LC）仪器的重要性，以便充分利用现代SEC色谱柱的潜力以及当前将SEC联用到质谱（MS）的趋势。提出了基于抗体的产品（即mAb，ADC，Fc融合蛋白和双特异性抗体）的最新应用。最后，提供了提示和技巧，以进一步优化SEC分离并随着时间的推移保持其性能，从而更好地了解意外的SEC结果。