In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy

Authors

Krishnakumar Chullipalliyalil, Centre for Advanced Photonics & Process Analysis, Munster Technological University Cork, T12P928, IrelandFollow
Khaled ElKassas, SSPC Centre for Pharmaceutical Research, School of Pharmacy, University College Cork, T12 YT20, Ireland
Michael McAuliffe, Centre for Advanced Photonics & Process Analysis, Munster Technological University Cork, T12P928, IrelandFollow
Sonja Vucen, SSPC Centre for Pharmaceutical Research, School of Pharmacy, University College Cork, T12 YT20, Ireland
Abina Crean, SSPC Centre for Pharmaceutical Research, School of Pharmacy, University College Cork, Cork T12 YT20, Ireland

ORCID

https://orcid.org/0000-0002-8803-8200

Document Type

Article

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Disciplines

Analytical Chemistry | Chemistry | Physical Chemistry

Publication Details

Published in Analytical Chemistry on 25th January 2023.

Abstract

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Recommended Citation

(1) Chullipalliyalil, K.; Elkassas, K.; McAuliffe, M. A. P.; Vucen, S.; Crean, A. In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy. Analytical Chemistry 2023, 95 (5), 2774–2782. https://doi.org/10.1021/acs.analchem.2c03912. ‌ ‌