Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography–mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.

中文翻译：

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使用4D HPLC / MS快速，自动表征抗体变异体

鉴定未知单克隆抗体（mAb）变体的特征很重要，以便确定其对安全性，效力和稳定性的潜在影响。在常规质量控制（QC）中，离子交换色谱（IEC）与UV检测结合经常用于分离和定量mAb变体。但是，表征由IEC分离产生的色谱峰是非常耗时的过程，涉及许多繁琐的步骤。这里介绍的是一种在线四维高效液相色谱-质谱（4D HPLC / MS）方法，旨在克服这些局限性。为此，通过引入其他模块扩展了2D HPLC系统，从而实现了单克隆抗体的全自动生物分离，峰分离，还原，胰蛋白酶消化，反相分离（RP）分离所得肽，然后进行MS检测。未知峰的整个分离和分析过程都在不到1.5小时的时间内完成，从而大大节省了时间，并具有相当的序列覆盖率。为了显示与传统脱机过程的可比性，本文介绍了使用先前表征的mAb1进行的概念验证研究。