title: High concentration formulation studies of an IgG2 antibody using small angle X-ray scattering.
abstract: OBJECTIVE
  Concentrated protein formulations are strongly influenced by protein-protein interactions. These can be probed at low protein concentration by e.g. virial coefficients. It was recently suggested that interactions are attractive at short distances and repulsive at longer distances. Measurements at low concentrations mainly sample longer distances, hence may not predict high concentration behavior. Here we demonstrate that small angle X-ray scattering (SAXS) measurements simultaneously collect information on interactions at short and long distances.
  
  
  METHODS
  IgG2 antibody samples at concentrations up to 122 mg/ml are analyzed using SAXS and compared to Circular Dichroism (CD), Fluorescence, Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) analysis.
  
  
  RESULTS
  DLS and SEC analyses reveal attraction between antibodies at high concentrations. SAXS data analysis provides an elaborate understanding and shows both attractive and repulsive forces. The protein-protein interactions are strongly affected by excipients. No change in the solution state of IgG2 is observed at pH 4-8, while samples at pH 3 exhibit heavy oligomerization. The solution conformation of the examined IgG2 derived from SAXS data is a T-shape.
  
  
  CONCLUSIONS
  SAXS analysis resolves simultaneous attractive and repulsive interactions, and details the effect of excipients on the interactions, while providing three-dimensional structural information from low-concentration samples.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Charlotte Rode
authors.initials: CR
authors.lastname: Mosbæk
authors.forename: Petr V
authors.initials: PV
authors.lastname: Konarev
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Christian
authors.initials: C
authors.lastname: Rischel
authors.forename: Bente
authors.initials: B
authors.lastname: Vestergaard
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D066246:ErbB Receptors / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D000077544:Panitumumab; D011487:Protein Conformation; D053838:Scattering, Small Angle; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D012996:Solutions; D000077544:Panitumumab; C512478:EGFR protein, human; D066246:ErbB Receptors