New Dual Electrode µ-PrepCell™ for Efficient Reduction of Disulfide Bonds in Proteins/Peptides ASMS 2018 San Diego, CA, USA

Outline Cell configuration and workwise Cell evaluation - Influence of flow rate and %FA - Long-term stability - Cell to cell repro Reduction of Fab fragment Conclusion The lecture will explore on-line EC/MS as a powerful technique to investigate various oxidation and reduction processes in life sciences.

Cell Configuration Inversed applied potential Ti inlet block Pt counter electrode

Instrumental Setup FIA-EC-MS 1 3 2 5 4 1) Sample infusion pump, 2) injection valve, 3) HPLC loading pump, 4) ROXY Potentiostat equipped with dual electrode µ-PrepCell, 5) MS

Pulse Settings Pulse settings: E1 = 1.5V, t1 = 1s, E2 = 0V, t2 = 0.1s, ts = 40 ms  

Structure of Intact and Reduced Insulin SH HS SH Chain A Chain B Intact insulin: 2 inter disulfide bonds between chain A and B 1 intra disulfide bond in chain A between Cys 6 - Cys 11 Reduced insulin: Formation of chain A and B and reduced disulfide bond between Cys 6 - Cys 11

MS Spectra of Intact and Reduced Insulin Cell OFF: intact Cell ON: reduced [M + 6H+]6+ [M + 5H+]5+ Insulin Chain B [M + 4H+]4+ [M + 5H+]5+ Chain A [M + 3H+]3+ Pulse settings: E1=1.5V, E2=0V, t1=1.0s, t2=0.10s

MS Voltammogram Insulin Reduction vs. Voltage

Reduction Efficiency - Insulin 99% Insulin 10 µg/mL, diluted in 1%FA 50% Acetonitrile/H2O, 50 µL/min, for clarity only Chain B recorded

Flow Rate vs. Reduction Efficiency optimal flow rate range 20 – 150 µL/min Insulin 10 µg/mL, diluted in 1%FA 50% Acetonitrile/H2O

Acidity vs. Reduction Efficiency 98% 92% 67% 56%

Reproducibility (short-term) 5 consecutive injections of insulin 

Reproducibility (long-term) 100 injections of insulin 

Cell to Cell Reproducibility Cell #1 Cell #2 Cell #3 3 Different cells, randomly assembled

Cell Contamination/Fouling Pt counter electrode Ti inlet block No visible contamination or fouling of cell (inlet block & counter electrode), after several days of operation

Reduction of Avastin® Fab Fragment 50 kDa Cell off Cell on E= 1.5V Lc 75 kDa 25 kDa 100 kDa Left: Spectrum of Avastin Fab digest consisting of intact 100 KDa fragment and different digest forms of 75, 50 and 25 kDa. Separated by on-line Capillary LC-EC-MS. Cell off Right Spectrum after reduction, E = 1.5 V, clearly visible light chain (Lc) with mass of 23437.44 Da.  

Spectrum of Light Chain (Lc) Annotation of some charge states, A16 - A25

TP237, 10:30 am - 2:30 pm

Conclusions EC Reduction The new dual electrode µ-PrepCell for disulfide bond reduction in proteins provides the following features: High reduction efficiency Robust and stable reduction w/o cell fouling Good cell to cell reproducibility Ideal for on-line reduction of proteins, mAbs, etc., in top-down, bottom-up and HDX-MS Proteomics   19

Acknowledgement Dr. Theo M. Luider et al., Erasmus MC, Rotterdam. The Netherlands For the data on Avastin Fab reduction   20