Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches

MAbs. 2013 Jul-Aug;5(4):565-75. doi: 10.4161/mabs.24814. Epub 2013 Apr 25.


In developing biosimilar or biobetter products, comparability to the reference product is required to claim similar integrity or intended purpose. In this work, an anti-CD20 monoclonal antibody developed using RNA interference to decrease core fucosylation (RNAi-mediated) was comprehensively characterized by LC-MS and compared with the commercially-available anti-CD20 rituximab (MabThera (®) ). As anticipated, < 30% core fucose was found within the RNAi-produced molecule (compared with > 90% in rituximab), and the reduction in fucose resulting in a significant improvement in FcγRΙΙΙa binding and antibody-dependent cell-mediated cytotoxicity. Two mutations, S258Y (fully mutated) and F174I/L (partially mutated), however, were detected in the production of the RNAi-mediated molecule. An alternative LC-MS approach using dimethyl labeling (i.e., 2CH 2 for rituximab and 2CD 2 for the RNAi-mediated molecule) was developed to additionally compare the two mAbs and confirm the full sequence with the two mutation sites. Furthermore, disulfide linkages were found to be the same for the two antibodies, with a small portion of unpaired cysteines in both products. Disulfides were correctly linked if the samples were prepared at low pH (i.e., enzymatic digestion by pepsin at pH 2); however, trace amounts of scrambling were found by trypsin digestion at pH 6.8, and this scrambling increased significantly at pH 8. Typical modifications, such as pyro-Glu formation at the N-terminus, K clipping at the C-terminus, oxidation at Met, and deamidation at Asn, were also detected with no significant differences between the two products. Using the LC-MS approaches for the comparability study, product integrity with critical structure information was revealed for confirmation of intended purpose (core fucosylation), identification of critical parameters (e.g., sample pH), and correction as needed (amino acid mutation).

Keywords: LC-MS; biosimilar; disulfide scrambling; free cysteine; sequence mutation; structure characterization.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Monoclonal, Murine-Derived / biosynthesis
  • Antibodies, Monoclonal, Murine-Derived / chemistry*
  • Antibodies, Monoclonal, Murine-Derived / genetics
  • Antibodies, Monoclonal, Murine-Derived / immunology
  • Fucose / chemistry*
  • Fucose / genetics
  • Fucose / immunology
  • Glycosylation
  • Humans
  • Mass Spectrometry*
  • Point Mutation
  • Protein Engineering / methods
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • RNA Interference*
  • Rituximab


  • Antibodies, Monoclonal, Murine-Derived
  • Fucose
  • Rituximab