SmartEnzymes™ Simplify Characterization of Charge Variants by Native Mass Spectrometry

November 19, 2019 | References |

Monoclonal antibodies (mAbs) are complex macromolecules that undergo a wide range of post-translational modifications that can result in charge heterogeneity. Ion exchange (IEX) chromatography is considered the gold standard for characterization of charge variants. This approach separates charge variants and allows for the collection of isoforms for protein identification by mass spectrometry (MS). However, the collection of multiple fractions often needs to be combined with top-down and bottom-up characterization methods, which is both resource and time consuming.

 

In a recent study by LeBlanc et al., (2019) from LFB Biotechnologies, a newly developed cation exchange column technology was evaluated for its ability to separate charge variants under native conditions with direct coupling to MS. In total, three different mAbs with high basicisoelectric points (pI) and a monoclonal antibody reference material from NIST were analyzed in both their native and proteolyzed forms. The fragments were obtained using the IgG-specific proteases FabRICATOR®, which digests below the hinge, and FabALACTICA®, which digests above the hinge.

 

The column was shown to separate mAbs with high basicpI, demonstrating that mAbs having a strong retention on cation exchange media could be separated. A good repeatability was achieved in terms of resolution, recovery and retention times. Overall, the results demonstrate how SmartEnzymes have made mAb charge variant characterization, which was formerly challenging, more user-friendly with an easy-to-replicate protocol.

 

Read more about FabRICATOR and FabALACTICA.

 

Leblanc, Y. et al., 2019. A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry, Journal of Chromatography B. doi: https://doi.org/10.1016/j.jchromb.2019.121814