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Antibody Engineering by Electrochemistry


This novel electrochemical approach provides a fast and efficient method for the selective reduction, conjugation and re-oxidation of antibodies without the need of reducing chemicals. The reduction of antibodies into its Light Chains (Lc) and Heavy Chains (Hc) is performed by the electrochemical method previously published based on square-wave potential pulses applied to Antec’s proprietary Ti electrode. Subsequently, the free cysteine generated by the electrochemical reduction of the antibody can be conjugated. Finally, the free cysteine on both Lc and Hc can be very rapidly and selectively re-oxidized to reform the antibody in high yields. Electrochemical treatment did not affect the binding affinity of antibodies, while eliminating the need for harsh chemicals and additional tedious cleaning steps.

Such an approach is likely to prove itself as a major technological advancement not only in the analysis of antibodies but also in the design of novel antibody-based therapeutics, such as bispecific antibodies or antibody-drug conjugates.

High Resolution Mass Spectra of a Monoclonal Antibody.


Infusing an acidified antibody solution in the mass spectrometer with the μ-PrepCell turned OFF (A) resulted in a charge envelope spreading between m/z 2200 and 4000, with the intact antibody carrying approximately 40 to 60 charges. When the cell was turned ON (B), a shift of the antibody charge envelope toward lower m/z (~1000-2500) was observed. The spectrum (C) results from the deconvolution of the MS signal (A) obtained with the cell OFF and confirms that the antibody is intact. D was obtained with the cell turned ON, and mostly shows species with molecular weights of approximately 25 and 50 kDa corresponding to the masses of Lc and Hc. The very low intensity of the MS signal for species with molecular weights greater than 50 kDa further confirms the selective reduction of the antibody inter-chain disulfide bonds in high yields. 

Evaluation of the ligand binding affinity by ELISA. 

The curves obtained in ELISA for the three different antibodies - in native form (green), electrochemically reduced and re-oxidized (red and blue) - show excellent agreement, illustrating that electrochemical treatment of antibodies does not affect their binding activity nor their biological activity. Additionally, it can be concluded that keeping the reduced antibody for several hours in acidic medium did not affect its binding activity.

Application Notes

Application Notebook

26369777 - In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry
Switzar L, Nicolardi S, Rutten JW, Oberstein SA, Aartsma-Rus A, van der Burgt YE;

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;

ac500383c - Structural Analysis of an Intact Monoclonal Antibody by Online Electrochemical Reduction of Disulfide Bonds and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Simone Nicolardi, Andre M. Deelder , Magnus Palmblad , and Yuri E. M. van der Burgt; Anal. Chem., 2014, 86 (11), pp 5376-5382

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]

24077854 - A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.
Kraj A, Brouwer HJ, Reinhoud N, Chervet JP.; Anal Bioanal Chem. 2013 Sep 29. [Epub ahead of print]

24018861 - On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin.
Nicolardi S, Giera M, Kooijman P, Kraj A, Chervet JP, Deelder AM, van der Burgt YE.; J Am Soc Mass Spectrom. 2013 Sep 10. [Epub ahead of print]

22448817 - Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
Zhang Y, Cui W, Zhang H, Dewald HD, Chen H.; Anal Chem. 2012 Apr 17;84(8):3838-42. doi: 10.1021/ac300106y. Epub 2012 Apr 4.

21197958 - Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds.
Zhang Y, Dewald HD, Chen H.; J Proteome Res. 2011 Mar 4;10(3):1293-304. doi: 10.1021/pr101053q. Epub 2011 Feb 14.

24300002 - Online Electrochemical Reduction of the Disulfide Bond(s) in Oxytocin and Hepcidin Results in Different CID and ETD Fragmentation Spectra
Martin A. Giera; Simone Nicolardi; Pieter Kooijman; Agnieszka Kraj; Jean-Pierre Chervet; Andre M. Deelder; Yuri E.M. van der Burgt; ASMS 2013 poster

2011-06 - Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds
Y. Zhang, Hao Chen et al., J. Proteome Res., 2011, 10(3), 1293-1304;

2012-01 - Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins
Y. Zhang, Hao Chen et al., Anal. Chem, 2012, 84(8), 3838-3842;