The success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical quality attributes (CQAs) during production. The current workflow to measure CQAs of antibodies involves multiple one-dimensional liquid chromatography methods, including Protein-A (ProA), ion-exchange (IEX), reversed-phase, size exclusion (SEC), hydrophilic interaction, and hydrophobic interaction (HIC). Recent advances in commercial two-dimensional liquid chromatography (2D-LC) affords an opportunity to perform two separations at once to measure multiple CQAs in a single assay. Here, we describe the development of a 2D ProA-SEC method using entirely commercially available instrumentation. Each individual separation and the transfer of material between dimensions were optimized to develop a method that measures titer and aggregation of a target antibody from harvested cell culture fluid in under 5 min. We determined the effects of each parameter of the method on mAb recovery and stability, as well as speed, robustness, resolution, and accuracy of the aggregate amount detected in the second dimension (2D). While there are still sources of error caused by hardware limitations, our rapid ProA-SEC method is an effective screening tool with a significant throughput advantage over previously described methods. Additionally, this work serves as a basis for developing other 2D-LC methods with ProA as the first dimension (1D) separation coupled with different 2D separation, such as ProA-IEX and ProA-HIC.

中文翻译：

---

单克隆抗体的快速二维Protein-A尺寸排阻色谱法，用于从收获的细胞培养液样品中进行滴度和聚集测量。

单克隆抗体（mAb）治疗剂的成功增加了mAb生产中的制药投资，这导致对有效表征这些生物治疗剂的技术有更大的需求。mAb的大尺寸和异质性要求在生产过程中测量多个关键质量属性（CQA）。当前测量抗体CQA的工作流程涉及多种一维液相色谱方法，包括Protein-A（ProA），离子交换（IEX），反相，尺寸排阻（SEC），亲水相互作用和疏水相互作用（HIC） ）。商业二维液相色谱（2D-LC）的最新进展提供了一次执行两次分离以在单个测定中测量多个CQA的机会。这里，我们描述了使用完全可商购的仪器开发2D ProA-SEC方法的过程。优化每个个体的分离和尺寸之间材料的转移，以开发一种方法，可在5分钟内测量收获的细胞培养液中目标抗体的效价和聚集。我们确定了该方法的每个参数对mAb回收率和稳定性以及在第二维（2D）中检测到的总量的速度，鲁棒性，分辨率和准确性的影响。尽管仍然存在由硬件限制引起的错误源，但我们的快速ProA-SEC方法是一种有效的筛选工具，与先前描述的方法相比，具有显着的吞吐量优势。此外，