Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples

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 (D-2). 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 (D-1) separation coupled with different D-2 separation, such as ProA-IEX and ProA-HIC.