Selecting the Optimal Column for Native SEC-MS of Monoclonal Antibodies

Characterization of monoclonal antibodies (mAbs) is essential to product safety and efficacy. Determining the purity and characterizing the impurities such as dimers or fragments are two critical quality parameters. Size exclusion chromatography (SEC) coupled with mass spectrometry (MS) is increasingly used to identify the accurate molecular mass of mAbs. However, traditional SEC typically generates high particle shedding, which decreases ionization efficiency over time, even when operating in high molecular weight (HMW) m/z ranges. To avoid shedding for MS and multi-angle light scattering (MALS) applications, Tosoh Bioscience developed TSKgel® UP-SW3000-LS U/HPLC size-exclusion columns.

In this application note, a TSKgel UP-SW3000-LS column was coupled with an MS instrument for the analysis of a mAb standard. Data demonstrates that the TSKgel UP- SW3000-LS column surpasses competitive UHPLC columns and a dedicated low shedding column for SEC of proteins in terms of particle shedding observed by MS. Moreover, this column helps maintain ionization efficiency in the electrospray ionization (ESI) source >90% compared to the initial injection over >50 injections.

Columns:

• TSKgel UP-SW3000-LS, 4.6 mm ID x 15 cm, 2 μm (P/N 0023547)
  UHPLC column for SEC of proteins, 4.6 mm ID x 15 cm, 1.7 μm
  Dedicated light scattering column, 4.6 mm ID x 15 cm, 3 μm