title: High-throughput screening of excipients intended to prevent antigen aggregation at air-liquid interface.
abstract: OBJECTIVE
  The aim was to develop a high-throughput screening method compatible with low protein concentrations, as present in vaccines, in order to evaluate the performance of various excipients in preventing the aggregation at air-liquid interface of an experimental recombinant antigen called Antigen 18A.
  
  
  METHODS
  Aggregation of Antigen 18A was triggered by shaking in a half-filled vial or by air bubbling in a microplate. Size-exclusion chromatography, turbidimetry, Nile Red fluorescence spectroscopy, and attenuated total reflection Fourier-transform infrared spectroscopy were used to assess Antigen 18A aggregation. A high-throughput method, based on tryptophan fluorescence spectroscopy, was set up to screen excipients for their capability to prevent Antigen 18A aggregation at air-liquid interface.
  
  
  RESULTS
  While a similar aggregation profile was obtained with both stress tests when using size-exclusion chromatography, spectroscopic and turbidimetric methods showed an influence of the stress protocol on the nature of the aggregates. The high-throughput screening revealed that 7 out of 44 excipients significantly prevented Antigen 18A from aggregating. We confirmed the performance of hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin, as well as poloxamers 188 and 407, in half-filled shaken vials.
  
  
  CONCLUSIONS
  A high-throughput screening approach can be followed for evaluating the performance of excipients against aggregation of a protein antigen at air-liquid interface.
authors.affiliation: Louvain Drug Research Institute, Université catholique de Louvain, Unité de Pharmacie Galénique, Avenue E. Mounier 73, UCL 7320, 1200, Brussels, Belgium.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dasnoy
authors.forename: Nancy
authors.initials: N
authors.lastname: Dezutter
authors.forename: Dominique
authors.initials: D
authors.lastname: Lemoine
authors.forename: Vivien
authors.initials: V
authors.lastname: Le Bras
authors.forename: Véronique
authors.initials: V
authors.lastname: Préat
mesh_terms: D000388:Air; D000818:Animals; D000941:Antigens / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005079:Excipients / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D014867:Water / Q000737:chemistry
chemical_list: D000941:Antigens; D005079:Excipients; D006023:Glycoproteins; D011994:Recombinant Proteins; D014867:Water