Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation-exchange HPLC and capillary isoelectric focusing

Anal Biochem. 1999 Nov 1;275(1):98-108. doi: 10.1006/abio.1999.4275.


Cation-exchange liquid chromatography (CIEX) and capillary isoelectric focusing (cIEF) methods have been developed for the routine analysis of a recombinant, human anti-tumor necrosis factor monoclonal antibody D2E7. Both of these methods can separate heavy-chain C-terminal variants of this antibody. Various enzymatic digestion methods have also been developed for the identification of the antibody C-termini lysine (Lys) variants. A comparison of conventional CIEX-HPLC and cIEF methods has been made for the analysis of antibodies. cIEF can also be used to determine the isoelectric points (pI) of antibody variants based on the use of internal pI standards. Different C-termini Lys variants have been separated and collected from the CIEX column and subsequently analyzed by cIEF and mass spectrometry.

MeSH terms

  • Antibodies, Monoclonal / analysis*
  • Antibodies, Monoclonal / immunology
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Isoelectric Focusing / methods*
  • Mass Spectrometry
  • Recombinant Proteins / analysis
  • Reproducibility of Results
  • Tumor Necrosis Factor-alpha / immunology*


  • Antibodies, Monoclonal
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha