Large molecule biologics, encompassing cell and gene therapies, monoclonal antibodies, and antibody-drug conjugates, continue to be a major focus in the pharmaceutical market. These biopharmaceutical drugs require rigorous testing and monitoring throughout the production lifecycle beginning with the manufacturing processes and ending with lifetime monitoring for stability and efficacy. At each step within the lifecycle, high-performance liquid chromatography (HPLC) is a key analytical tool used to garner information about the purity, stability, and safety of the drug product. With the advent of biopharmaceuticals occurring nearly half a century ago, there is a need for the transfer of established methods to contemporary analytical platforms designed for biologics.

          This study focuses on the transfer of two chromatographic techniques – Size Exclusion Chromatography (SEC) and Ion-Exchange Chromatography (IEX) – from a legacy HPLC system to a modern HPLC system designed for biologics analyses. SEC employs a buffered mobile phase system designed to prevent analyte interaction with the column packing and chromatographic hardware, allowing separation based on the hydrodynamic radius of the molecules. IEX, on the other hand, employs a polar stationary phase to bind charge variants, with a salt or pH gradient facilitating the release of adsorbed compounds based on their charge characteristics. While these techniques yield distinct information, they share a common challenge: their high-salt method conditions can exert significant stress on a chromatographic system.

          The modern HPLC platform used in this study enhances ease-of-use for the analyst while leveraging surface modification technology to mitigate off-target interaction of analytes within the chromatographic flow path. Several critical quality attributes such as aggregation, fragmentation, and lysine charge variants of a monoclonal antibody drug are monitored and compared between systems. Additionally, chromatographic performance characteristics, including peak tailing, area reproducibility, and retention time stability are systematically compared. Ultimately, the transfer of these methods to a modern HPLC platform demonstrates improved analytical efficiency and robustness