Impact of Supercharging on Top-Down Characterization of Monoclonal Antibodies by Collision Induced Dissociation.

Native top-down characterization of monoclonal antibodies (mAbs) is an attractive approach due to the narrow charge state distribution of the intact mAb and the spectral decongestion afforded by relatively low charge product ions being spread over a broad m/z range. However, low precursor charge and extensive disulfide bonding together greatly limit characterization by collision induced dissociation (CID). This study evaluates solution phase supercharging in ammonium acetate electrospray solutions to produce charge state distributions with intermediate charge, i.e., somewhere between native and fully denatured. Sequence coverage and interchain disulfide bond cleavage were evaluated as a function of precursor charge and collision energy for denatured, native, and supercharged "native" mAb precursor ions. Native ESI produced low charge precursors (z~22-23) that required high collision energies (~200-225 eV/q) for interchain disulfide cleavages (i.e., light chain (LC) release) and yielded modest sequence coverage. Formic acid, 50:50 acetonitrile/water with formic acid, dimethyl sulfoxide, propylene carbonate, and sulfolane were compared as additives; many increased precursors charge states but produced broader envelopes and degraded spectral quality. In contrast 3% sulfolane in 100 mM ammonium acetate increased average charges to ~39 for Infliximab and ~42 for SigmaMAb and reduced the dissociation threshold to 125 eV/q as expected for higher charge precursor ions. Optimized conditions produced abundant [LC-S]/[LC]/[LC+S] triplets and extended b/y ions into CDR3 regions. Overall, adding 3% sulfolane in ammonium acetate provides a practical way to overcome key CID limitations for intact mAbs and enables more extensive sequence characterization.