Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Improved middle-down characterization of antibodies using multiple ion activation techniques and Proton Transfer Charge Reduction on an Orbitrap Eclipse mass spectrometer  (#431)

Romain Huguet 1 , Steve Binos 2 , Kristina Srzentic 1 , John E.P. Syka 1 , Christopher Mullen 1 , Joshua Silveira 1 , Jennifer Sutton 1 , Luca Fornelli 3
  1. Thermo Fisher Scientific, San Jose, CA, United States
  2. Thermo Fisher Scientific, Parkville, VIC, Australia
  3. University of Oklahoma, Normal, OK, United States

Relative to peptide-based mass spectrometry approaches, middle-down (MD) strategies offer the advantage of higher molecular sequence integrity in the characterization of biotherapeutics. However, reaching the protein sequence coverage required to confidently verify the complete mAb primary sequence or map post-translational modifications in MD experiments can be challenging. We apply advanced ion activation techniques, including ultraviolet photodissociation (UVPD) and electron transfer dissociation (ETD) in combination with Proton Transfer Charge Reduction (PTCR) reactions to improve IgG1 mAb characterization via liquid chromatography (LC)-MS. We have demonstrated that rapid (8 min long) gradient middle-down LC-MS/MS analyses of digested NIST mAb collectively yielded sequence coverage in excess of 80%. 90% sequence coverage was observed for Fc/2 and LC and around 80% for Fd mAb subunits. ETD and UVPD spectra of polypeptide precursors over 15 kDa are extremely complex and therefore are challenging to process with current m/z to mass spectral conversion software. The high spectral density of these product ion peaks is such that isotopic peak clusters often overlap and are frequently not sufficiently resolved to be differentiated from noise, nor to accurately assign a charge state and monisotopic peak, and thus not properly converted to neutral masses. To achieve more complete sequence coverage, product ion peak clusters associated with fragmentation in the middle region of the mAb subunits must be observed and properly mass deconvoluted. We extend our LC-MS/MS analyses of mAb subunits by utilizing PTCR subsequent to ETD and UVPD to enhance sequence coverage. In a single LC run, we obtained for Fc/2 and Fd over 50% and around 60% for LC mAb subunits. All the Complementarity Determining Regions (CDRs) were partially to fully sequenced. In case of Fc/2, this strategy unraveled an extensive series of large z-ions, unambiguously confirming the glycosylation site of the most abundant glycan variant.