title: High-throughput assessment of antigen conformational stability by ultraviolet absorption spectroscopy and its application to excipient screening.
abstract: In high-throughput screening (HTS) assays, the use of ultraviolet absorption spectroscopy (UA) is commonly limited to concentration and turbidity measurements. Our aim was to evaluate microplate-based UA and its second-derivative [(2d)UA] for measuring the conformational stability of two recombinant antigenic proteins in the presence of 44 excipients. Protein conformational stability was assessed by (2d)UA upon titration with guanidine hydrochloride. (2d)UA was compared with tryptophan fluorescence spectroscopy (TF) and differential scanning fluorimetry (DSF), both commonly used techniques for measuring protein conformational stability. The HTS data were corrected for plate, row and column effects by applying a median polish procedure. Irrespective of the unfolding method applied, similar stabilizing excipients were identified by all analytical methods for a given antigen. The native forms of both antigens were destabilized by arginine, hydroxypropyl-β-cyclodextrin, and sodium docusate, and were protected by polyols. The median polish correction improved the quality of the prediction models and the screening resolution. The higher sensitivities of TF and DSF compared with (2d)UA allowed the identification of a larger number of stabilizing excipients. However, similar screening resolutions (z'-factor > 0.8) were observed for 2dUA, TF, and DSF in a HTS of excipients applied to one of the antigens. Therefore, (2d)UA deserves more attention in HTS studies focused on protein conformational stability.
authors.affiliation: Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Université catholique de Louvain, Brussels, Belgium.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dasnoy
authors.forename: Vivien
authors.initials: V
authors.lastname: Le Bras
authors.forename: Véronique
authors.initials: V
authors.lastname: Préat
authors.forename: Dominique
authors.initials: D
authors.lastname: Lemoine
mesh_terms: D000941:Antigens / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry* / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D008956:Models, Chemical; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014364:Tryptophan
chemical_list: D000941:Antigens; D005079:Excipients; D011994:Recombinant Proteins; D014364:Tryptophan