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Detection and characterization of protein aggregates by fluorescence microscopy.

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 degrees 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.

journal_name

Int J Pharm

journal_title

International journal of pharmaceutics

authors

Demeule B,Gurny R,Arvinte T

doi

10.1016/j.ijpharm.2006.08.024

subject

Has Abstract

pub_date

2007-02-01 00:00:00

pages

37-45

issue

1-2

eissn

0378-5173

issn

1873-3476

pii

S0378-5173(06)00684-3

journal_volume

329

pub_type

杂志文章

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