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

Digging deeper in every direction: developing a next-generation platform for high-throughput multi-faceted protein characterization (#854)

AARON O BAILEY 1 , JIMMY Y ZENG 1 , GUANGHUI HAN 1 , JOSHUA SILVEIRA 2 , KRISTINA SRZENTIC 3 , CHRISTOPHER MULLEN 2 , JOHN E.P. SYKA 2 , ROMAIN HUGUET 2
  1. BGI Americas, San Jose, California, United States
  2. Thermofisher scientific, San Jose, California, United States
  3. Thermo Fisher scientific, Cambridge, MASSACHUSETTS, United States

So-called “comprehensive” LC-MS-based characterization of therapeutic proteins requires attaining a detailed molecular knowledge of every residue and sidechain.We have designed a cutting-edge platform for addressing the versatility and throughput requirements of performing comprehensive characterization.We have developed advanced versions of multiple key characterization workflows using state-of-the-art LC-MS methods performed on a single Orbitrap mass spectrometer. We used the Fc-fusion protein etanercept to optimize intact mass and peptide mapping strategies for characterizing highly glycosylated therapeutic proteins.Specifically, we show that native SEC-MS intact mass strategy is significantly improved with the combined use of proton transfer charge reduction (PTCR) to provide additional separation of the broad isoform profiles at sequential charge state in the complex intact mass spectra.For peptide mapping, etanercept was digested using trypsin and AspN. Intact and IdeS-digested Fc and TNFR subunits were deglycosylated with combinations of PNGase F, O-glycosidase, and sialidase and analyzed by native SEC-MS followed by PTCR. LC-MS was accomplished using a Vanquish UHPLC connected to a Thermo Scientific™ Orbitrap Eclipse Tribrid mass spectrometer equipped with PTCR and extended mass range detection and isolation. Our studies showed highly complex isoform distributions consistent with previously published reports. We performed peptide mapping using data dependent acquisition (DDA) to fragment selected peptide ions by HCD and ETD. This effort resulted in 100% coverage of the etanercept amino acid sequence, including 3 N-glycan sites and 13 O-glycosylation sites. We analyzed etanercept in intact and subunit form after combined deglycosylation treatments and determined the main sources of glycoform heterogeneity for each preparation. For highly complex preparations we show that high mass isolation and PTCR are powerful tools to further separate sequential charge states.This form of charge reduction intact mass analysis enhanced our ability to accurately assess glycoform identity directly from a highly complex isoform background and can be implemented in a high throughput fashion.