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Simultaneous detection and analysis of protein aggregation and protein unfolding by size exclusion chromatography with post column addition of the fluorescent dye BisANS.

Abstract：

:For development and optimization of protein formulation sensitive analytical tools are required to follow both aggregation and changes in protein structure. The latter can be seen as the beginning of physical instability leading to aggregation. The focus of this work laid on the development of a novel analysis simultaneously detecting changes in protein conformation and the formation of oligomers. By adding the extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) after size exclusion chromatography (SEC) and UV detection, it was possible to separate protein monomer and oligomers by size, analyze the amount of formed oligomers quantitatively using UV detection, and observe changes in protein structure of different protein species by fluorescence detection. This enabled us to distinguish between native-like and denatured oligomers and monomers formed under different stress conditions. Correspondingly, increased fluorescence reflecting partial unfolding was assigned specifically to monomer, oligomer, or both. The unfolding of monomer is not traceable by commonly used detection methods, but its monitoring may provide important information about activity and long-term stability. By adding the dye after SEC and UV detection, interferences with prior detectors are precluded, excipients are separated avoiding interferences with the protein-dye interaction and, in addition, the dye-protein interaction cannot impact the aggregation formation, as added after the separation of monomer and aggregates.

journal_name

J Pharm Sci

journal_title

Journal of pharmaceutical sciences

authors

Printz M,Friess W

doi

10.1002/jps.22808

subject

Has Abstract

pub_date

2012-02-01 00:00:00

pages

826-37

issue

2

eissn

0022-3549

issn

1520-6017

pii

S0022-3549(15)31735-4

journal_volume

101

pub_type

杂志文章

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