Abstract
Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.
Highlights
Monoclonal antibodies and their variants make up an increasingly large field within biotherapeutics
Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents
Complete electrochemical reduction of an antibody can be carried out inline with liquid chromatography-mass spectrometry (LC-MS) analysis without the need for reducing agents and denaturing agents, save for common organic solvents
Summary
Monoclonal antibodies (mAb) and their variants make up an increasingly large field within biotherapeutics. Subunit production is often carried out by non-biological chemical reaction and/or enzyme digestion.[10,11] Consistent subunit production is essential in middle-up and middledown mass spectrometry workflows requiring effective reduction steps Both inter- and intra-chain disulfide bonds are common and important features of antibodies. Electrochemical reduction of disulfide bonds in proteins using a flow-through reactor cell prior to mass spectrometry analysis has been successfully applied as an alternative method previously. With the switching valve initially held in the 1 : 6 position for intact mAb analysis 5 μg of mAb sample was injected through the electrochemical cell at a flow rate of 50 μL min−1 fed from pump 1 and trapped onto a MAbPacTM RP column, 4 μm, 2.1 mm × 50 mm. The mobile phase was increased to 32% B over 6 minutes and up to 80% for a further 10 minutes, held at 80% B for 3 minutes before dropping the mobile phase down to 20% B for 2 minutes for a total length of 30 minutes
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