TY - JOUR
T1 - Automated high-throughput capillary circular dichroism and intrinsic fluorescence spectroscopy for rapid determination of protein structure
AU - Moore-Kelly, Charles
AU - Welsh, John
AU - Rodger, Alison
AU - Dafforn, Tim R.
AU - Thomas, Owen R.T.
N1 - 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.
PY - 2019/11/5
Y1 - 2019/11/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85073753186&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.9b03259
DO - 10.1021/acs.analchem.9b03259
M3 - Article
C2 - 31584804
AN - SCOPUS:85073753186
SN - 0003-2700
VL - 91
SP - 13794
EP - 13802
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 21
ER -