Detection and characterization of protein aggregates by fluorescence microscopy - Analytical Methods & Tools for Lyophilization

Detection and characterization of protein aggregates by fluorescence microscopy
Received 23 February 2006; revised 11 August 2006; accepted 15 August 2006. Available online 26 August 2006.
Barth?lemy Demeulea, Robert Gurnya and Tudor Arvinte, a,
International Journal of Pharmaceutics
Volume 329, Issues 1-2 , 1 February 2007
ScienceDirect
Copyright ? 2006 Elsevier B.V. All rights reserved.
aDepartment of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
Abstract
Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed in the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 90? light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation.
Keywords: Immunoglobulin; Calcitonin; Aggregation; Nile Red; Microscopy; Physical stability

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