Automated high-throughput capillary circular dichroism and intrinsic fluorescence spectroscopy for rapid determination of protein structure

Charles Moore-Kelly, John Welsh, Alison Rodger, Tim R. Dafforn, Owen R.T. Thomas*

*Corresponding author for this work

Research output: Contribution to journalArticle

2 Citations (Scopus)
2 Downloads (Pure)

Abstract

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Original languageEnglish
Pages (from-to)13794-13802
Number of pages9
JournalAnalytical Chemistry
Volume91
Issue number21
DOIs
Publication statusPublished - 5 Nov 2019

Bibliographical note

Copyright the Publisher 2019. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

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