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European Biophysics Journal

, Volume 47, Issue 7, pp 761–768 | Cite as

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

  • Andreas Hoffmann
  • Kerstin Grassl
  • Janine Gommert
  • Christian Schlesak
  • Alexander Bepperling
Original Article

Abstract

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).

Keywords

Analytical ultracentrifugation Extinction coefficient Refractive index increment Biopharmaceutical Multi-signal sedimentation velocity Monoclonal antibody 

Notes

Compliance with ethical standards

Conflict of interest

All authors are or have been employees of HEXAL AG. None of the authors owns stock of HEXAL AG (Novartis).

Supplementary material

249_2018_1299_MOESM1_ESM.pdf (1 mb)
Supplementary material 1 (PDF 1061 kb)

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Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  1. 1.Hexal AGOberhachingGermany
  2. 2.Roche Diagnostics GmbHPenzbergGermany

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