A native multi-dimensional monitoring workflow for at-line characterization of mAb titer, size, charge, and glycoform heterogeneities in cell culture supernatant.

With growing maturity of the biopharmaceutical industry, new modalities entering the therapeutic design space and increasing complexity of formulations such as combination therapy, the demands and requirements on analytical workflows have also increased. A recent evolution in newer analytical workflows is that of multi-attribute monitoring workflows designed on chromatography-mass spectrometry (LC-MS) platform. In comparison to traditional one attribute per workflow paradigm, multi-attribute workflows are designed to monitor multiple critical quality attributes through a single workflow, thus reducing the overall time to information and increasing efficiency and throughput. While the 1 generation multi-attribute workflows focused on bottom-up characterization following peptide digestion, the more recent workflows have been focussing on characterization of intact biologics, preferably in native state. So far intact multi-attribute monitoring workflows suitable for comparability, utilizing single dimension chromatography coupled with MS have been published. In this study, we describe a native multi-dimensional multi-attribute monitoring workflow for at-line characterization of monoclonal antibody (mAb) titer, size, charge, and glycoform heterogeneities directly in cell culture supernatant. This has been achieved through coupling ProA in series with size exclusion chromatography in 1 dimension followed by cation exchange chromatography in the 2 dimension. Intact paired glycoform characterization has been achieved through coupling 2D-LC with q-ToF-MS. The workflow with a single heart cut can be completed in 25 mins and utilizes 2D-liquid chromatography (2D-LC) to maximize separation and monitoring of titer, size as well as charge variants.