title: Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze-thaw stresses revealed by hydrogen exchange.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904-4741, USA.
authors.forename: Aming
authors.initials: A
authors.lastname: Zhang
authors.forename: Satish K
authors.initials: SK
authors.lastname: Singh
authors.forename: Michael R
authors.initials: MR
authors.lastname: Shirts
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D000068258:Bevacizumab; D002850:Chromatography, Gel; D022801:Complementarity Determining Regions / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D005615:Freezing; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D055550:Protein Stability; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D000068258:Bevacizumab