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Electrochemical Reduction of Biopharmaceuticals for Superior HDX/MS


The controlled electrochemical reduction of the S-S bonds has been successfully applied for the unfolding of larger proteins such as antibodies, replacing the harsh chemical reduction that often jeopardized the H/D exchange. Outer regions of the protein undergo easy exchange of Hydrogen (H) by Deuterium (D). However inner regions particularly in larger proteins are not easily accessible to the deutereation and need to be opened (unfolded) by reducing the S-S bonds. Using classical reducing agents such as DTT or TCEP are often ineffective under quenching conditions (i.e., low pH and temperature). Hence, the use of ROXY EC with its proprietary Titanium based electrodes for the gentle and controlled cleavage of the S-S bond opens new opportunities in HDX/MS workflows, resulting in significant higher sequence coverage.

Below the sequence coverage for Nerve Growth Factor-β with chemical and electrochemical (EC) reduction is shown. The integration of the electrochemical flow cell into the HDX/MS workflow resulted in a much higher sequence coverage than chemical reduction (99% vs. 46%) allowing a much more comprehensive characterization of NGF. Adapted from Trajberg E. et al., Anal. Chem.87 (2015) 8880.


Chemical (TCEP) Reduction

Electrochemical Reduction

Application Notes

  • 210_009_05 - Electrochemical Reduction of Biopharmaceuticals in HDXMS

Application Notebook

26249042 - Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow.
Trabjerg E, Jakobsen RU, Mysling S, Christensen S, J›rgensen TJ, Rand KD;

24251601 - Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry.
Mysling S, Salbo R, Ploug M, J›rgensen TJ.; Anal Chem. 2013 Nov 19. [Epub ahead of print]

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