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High-throughput assessment of antigen conformational stability by ultraviolet absorption spectroscopy and its application to excipient screening

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Dasnoy, Sébastien [UCL] Le Bras, Vivien Préat, Véronique [UCL] Lemoine, Dominique [UCL]

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.

Document type Article de périodique (Journal article) – Article de recherche
Access type Accès restreint
Publication date 2012
Language Anglais
Journal information "Biotechnology and Bioengineering" - Vol. 109, no.2, p. 502-516 (2012)
Peer reviewed yes
Publisher JohnWiley & Sons, Inc. ((United States) Hoboken)
issn 0006-3592
e-issn 1097-0290
Publication status Publié
Affiliation UCL - SSS/LDRI - Louvain Drug Research Institute
MESH Subject Antigens ; Reproducibility of Results ; Spectrophotometry, Ultraviolet ; Tryptophan ; Chemistry, Pharmaceutical ; Drug Stability ; Excipients ; High-Throughput Screening Assays ; Models, Chemical ; Protein Conformation ; Protein Stability ; Recombinant Proteins
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Bibliographic reference Dasnoy, Sébastien ; Le Bras, Vivien ; Préat, Véronique ; Lemoine, Dominique. High-throughput assessment of antigen conformational stability by ultraviolet absorption spectroscopy and its application to excipient screening. In: Biotechnology and Bioengineering, Vol. 109, no.2, p. 502-516 (2012)
Permanent URL http://hdl.handle.net/2078.1/120122