In vitro stoichiometry of complexes between the soluble RANK ligand and the monoclonal antibody denosumab

Biochemistry. 2012 Jan 24;51(3):795-806. doi: 10.1021/bi2007806. Epub 2012 Jan 12.


The in vitro binding stoichiometry of denosumab, an IgG2 fully human monoclonal therapeutic antibody, to RANK ligand was determined by multiple complementary size separation techniques with mass measuring detectors, including two solution-based techniques (size-exclusion chromatography with static light scattering detection and sedimentation velocity analytical ultracentrifugation) and a gas-phase analysis by electrospray ionization time-of-flight mass spectrometry from aqueous nondenaturing solutions. The stoichiometry was determined under defined conditions ranging from small excess RANK ligand to large excess denosumab (up to 40:1). High concentrations of denosumab relative to RANK ligand were studied because of their physiological relevance; a large excess of denosumab is anticipated in circulation for extended periods relative to much lower concentrations of free soluble RANKL. The studies revealed that an assembly including 3 denosumab antibody molecules bound to 2 RANKL trimers (3D2R) is the most stable complex in DPBS at 37 °C. This differs from the 1:1 binding stoichiometry reported for RANKL and osteoprotegerin (OPG), a soluble homodimeric decoy receptor which binds RANKL with high affinity. Denosumab and RANKL also formed smaller assemblies including 1 denosumab and 2 RANKL trimer molecules (1D2R) under conditions of excess RANKL, 3 denosumab molecules and 1 RANKL trimer (3D1R) under conditions of excess denosumab, and larger assemblies, but these intermediate species were only present at lower temperatures (4 °C), shortly after mixing denosumab and RANKL, and converted over time to the more stable 3D2R assembly.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal, Humanized
  • Buffers
  • CHO Cells
  • Cricetinae
  • Denosumab
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Glycosylation
  • Humans
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / genetics
  • Protein Interaction Mapping*
  • Protein Stability
  • RANK Ligand / antagonists & inhibitors*
  • RANK Ligand / blood
  • RANK Ligand / chemistry*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Solubility


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Buffers
  • Immunoglobulin G
  • RANK Ligand
  • Rank-Fc
  • Recombinant Fusion Proteins
  • TNFSF11 protein, human
  • Denosumab