Method Development and Case Studies of Large Molecule Separations: SEC, HIC, Mixed-Mode and CE-SDS

摘要

Protein therapeutics are diverse molecules requiring a wide array of tools to assess purity including aggregates, fragments, glycosylation and various other PTMs that may occur during manufacture and stability. Size exclusion chromatography (SEC) is the primary method used for monitoring aggregation because it is traditionally performed under native conditions. A significant drawback to SEC is the undesired protein-column interactions that occur during analysis. These interactions should be thoroughly investigated during method development. Protein-column interaction can be exploited to separate species of interest in a mixed-mode separation (SEC and HIC). Complementary to SEC, Capillary Electrophoresis-SDS (CE-SDS) is the major workhorse for monitoring small fragments and clippings due to its superior resolution. This talk will focus on SEC method development for mAbs and protein therapeutics, including the use of Multi Angle Light Scattering (MALS) to confirm and identify species under native conditions. We will focus on the recovery and throughput as well as ways to assess and extend column lifetime. In addition, we will discuss case studies on the use of mixed-mode and CE-SDS separations of large molecules.