Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

MAbs. 2013 Nov-Dec;5(6):925-35. doi: 10.4161/mabs.26192. Epub 2013 Aug 19.


Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as "shoulder," indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1-3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.

Keywords: antibody size variant; design of experiments; hydrophobic interaction chromatography; monoclonal antibody; size exclusion chromatography; third light chain.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • CHO Cells
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Immunoglobulin Light Chains / chemistry*
  • Molecular Weight


  • Antibodies, Monoclonal
  • Immunoglobulin Light Chains