Mapping Antibody Impurities Coupling Size-based Electrophoresis and Capillary Zone Electrophoresis Mass Spectrometry (CZE-MS)

The characterization of mAb impurities is essential to assess their safety, quality and efficacy, and usually needs high-resolution separations and mass spectrometric identification. A plethora of size-based electrophoretic methods i.e., SDS-PAGE, cSDS or GXII are used as stand- alone methods to profile impurities. However, the identification of unknown features from these electrophoretic methods are challenging and often requires LC-MS to assist in identification of unknown features.

We have used LC-MS to identify HHL species and quantify HHL species based on ion signals, A good correlation is observed for HHL fragment between the GXII fluorescence intensities (FI) and the mass spec ion current. However, the LOD of mass spec signals are much lower than FI. Thus, it is much critical to couple high-resolution CE-MS to improve the mapping of impurities detected by sized-based electrophoretic methods performed routinely in biopharma for deep characterization of impurities in a single workstream. We use a “impurities standard” to benchmark size-based electrophoresis methods. In addition to CE-SDS peak identification, our CZE-MS approach also has the potential of providing improved quantitative analysis of impurities. This workflow will be applied to other commercial mAb standards that are prone to degradation.

Presenter: Jim Lau, PhD (HRAMS LC/MS Application Engineer, Agilent Technologies, Inc.)

Jim Lau received his Ph.D. From the University of Colorado. There he worked in the laboratories of Peter Albersheim and Robert Shapiro. This work involved the structure elucidation of plant natural products and, in particular, elucidating the structures of oligosaccharides derived from plant cell wall polysaccharides. This work was done in the early days of LCMS and made use of DLI-LCMS and (at the time) recently invented Thermospray-LCMS.

After the completion of his Ph.D., Jim joined Hewlett-Packard to work on LCMS ionization techniques with Paul Goodley during the early days of Electrospray-LCMS. He then moved to work on field applications and collaborations with Hewlett-Packard’s LCMS customers. More than 30 years later, Jim continues to work with Agilent’s customers in High Resolution Accurate Mass applications of LCMS. While involved in many areas of HRAMS LCMS, his recent work has focused on the area of Screening/Qual&Quant of small molecules and on the high throughput confirmation of oligonucleotidesynthesis.