Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC

The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coeffic...

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Bibliographic Details
Published in:European biophysics journal 2018-10, Vol.47 (7), p.761-768
Main Authors: Hoffmann, Andreas, Grassl, Kerstin, Gommert, Janine, Schlesak, Christian, Bepperling, Alexander
Format: Article
Language:English
Subjects:
Amino acid sequence
Amino acids
Biochemistry
Biological and Medical Physics
Biomedical and Life Sciences
Biopharmaceuticals
Biophysics
Cell Biology
Coefficients
Data points
Fc receptors
Fusion protein
Glycosylation
Life Sciences
Membrane Biology
Monoclonal antibodies
Nanotechnology
Neurobiology
Optics
Original Article
Photometry
Proteins
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Summary:The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).
ISSN:0175-7571
1432-1017
DOI:10.1007/s00249-018-1299-x