In vivo deamidation characterization of monoclonal antibody by LC/MS/MS

Anal Chem. 2005 Mar 1;77(5):1432-9. doi: 10.1021/ac0494174.


The spontaneous nonenzymatic deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo deamidation mechanisms of protein drug candidates. Several methods are available to characterize the deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several "hot spots" for spontaneous deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidation at this site greatly decreases the binding activity. MAb was administered to cynomolgus monkeys by intravenous and subcutaneous routes. At various times after dosing, monkey serum was prepared and MAb captured by the immobilized antigen or a goat anti-human IgG Fcgamma antibody. The captured MAb was treated with trypsin followed by endoproteinase Glu-C. The digests were separated on RP-HPLC and analyzed by MS/MS on Q-Tof Global mass spectrometer. Using this method, we were able to determine the deamidation half-life of amino acid residue Asn55 in vivo and the ratio of the deamidated derivatives, i.e., isoAsp55 and Asp55. The method is rapid and sensitive with low-nanogram quantities of protein detected in the biological matrix.

MeSH terms

  • Amides / chemistry
  • Amides / metabolism*
  • Animals
  • Antibodies, Monoclonal / blood
  • Antibodies, Monoclonal / metabolism*
  • Antibodies, Monoclonal / pharmacokinetics
  • Asparagine / metabolism
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Chromatography, Liquid / methods
  • Glutamine / metabolism
  • Humans
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / immunology
  • Immunoglobulin G / metabolism
  • Immunosorbent Techniques
  • Kinetics
  • Macaca fascicularis
  • Tandem Mass Spectrometry / methods*
  • Temperature


  • Amides
  • Antibodies, Monoclonal
  • Immunoglobulin G
  • Glutamine
  • Asparagine