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

Structural characterizations of intact monoclonal antibodies by native MS (#951)

Angela Criscuolo 1 2 3 , Tabiwang N. Arrey 1 , Eugen Damoc 1 , Thomas Möhring 1 , Catharina Crone 1 , Martin Zeller 1 , Markus Kellmann 1
  1. Thermo Fisher Scientific (Bremen) GmbH, Bremen, HB, Germany
  2. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany
  3. Center for Biotechnology and Biomedicine, University of Leipzig, Leipzig, Germany

In the production of therapeutic monoclonal antibodies (mAbs) intact mass spectrometry analysis has become a key step. In particular, the characterization of PTMs is a fundamental regulatory requirement as these modifications may affect quality, efficacy and safety of these biopharmaceuticals. These MS analyses can be performed under denaturing or native conditions. The main advantage of native MS is to avoid acid and organic solvents. The protein, which then can maintain a three dimensional structure, will accept less charges during the ionization process with an improvement in terms of spectral resolution. Most commonly, these analyses are performed by size exclusion chromatography (SEC) coupled with MS. Alternatively, charge variant analysis by ion exchange chromatography (IEC) has shown is potential to reveal mAb heterogeneity.

Preliminary experiments were performed on Trastuzumab by size exclusion chromatography coupled to a modified quadrupole Orbitrap mass spectrometer. The protein was eluting in a sharp peak, about 20 sec at the baseline. The average spectrum over the elution time shows a nicely distributed envelope with charge state between 21 and 30. Zooming inside a single charge state revealed five major glycoforms baseline separated. Then, the datasets were analyzed using ReSpect algorithm in Thermo Scientific™ BioPharmaFinder™ 3.0 software. The deconvoluted observed masses matched very well to the average theoretical masses for the known amino acid sequence with various combinations of glycoforms (delta ppm < 7). 

SEC experiments were compared with IEC, particularly cation exchange. Experiments conducted by Bailey et al. [1], Trappe et al. [2] and Fuessl et al. [3] report this new tool as a promising technique to complete structural elucidation of therapeutic monoclonal antibodies. This technique adds another dimension of separation, the retention time which is influenced by the chemical nature of the PTM. Consequently mAbs variants characterized by minimal mass difference may be now identified.