Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze-thaw stresses revealed by hydrogen exchange

Pharm Res. 2012 Jan;29(1):236-50. doi: 10.1007/s11095-011-0538-y. Epub 2011 Jul 30.


Purpose: Aggregation of monoclonal antibodies (mAbs) is a common yet poorly understood issue in therapeutic development. There remains a need for high-resolution structural information about conformational changes and intermolecular contacts during antibody aggregation.

Methods: We used hydrogen exchange mass spectrometry (HX-MS) to compare the aggregation mechanism and resultant aggregate structures of the pharmaceutical antibody Bevacizumab under freeze-thaw (F/T) and thermal stresses.

Results: Bevacizumab aggregation increased with number of F/T cycles and decreased with protein concentration. HX-MS showed native-like aggregates. Conversely, thermal stress triggered non-native aggregation at temperatures below melting point of the least stable CH2 domain. Under these conditions, HX was significantly enhanced in much of the Fab fragment while being decreased relative to native HX in CDRs. Analysis of intrinsic fluorescence Trp and extrinsic ANS dye binding supported structural differences between two antibody aggregates formed by F/T vs. thermal stresses.

Conclusions: Reduced hydrogen exchange in three CDRs suggests these residues may form strong intermolecular contacts in the antibody aggregates; regions of enhanced HX indicate unfolding. Residue level modeling methods with varying levels of atomistic detail were unable to identify aggregation patterns predictively.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal, Humanized / chemistry*
  • Bevacizumab
  • Chromatography, Gel
  • Complementarity Determining Regions / chemistry*
  • Deuterium Exchange Measurement / methods*
  • Freezing
  • Humans
  • Immunoglobulin Fab Fragments / chemistry*
  • Mass Spectrometry
  • Molecular Sequence Data
  • Protein Multimerization
  • Protein Stability
  • Temperature


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Humanized
  • Complementarity Determining Regions
  • Immunoglobulin Fab Fragments
  • Bevacizumab