Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP–SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-A...

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Bibliographic Details
Published in:Analytical biochemistry 2008-07, Vol.378 (2), p.115-122
Main Authors: Hawe, Andrea, Friess, Wolfgang, Sutter, Marc, Jiskoot, Wim
Format: Article
Language:English
Subjects:
Aggregation
Asymmetrical flow field flow fractionation
Chromatography, Gel - methods
Circular Dichroism
Fluorescent dyes
Fluorescent Dyes - analysis
Fluorescent Dyes - chemistry
Fractionation, Field Flow - methods
High-pressure size exclusion chromatography
Hot Temperature
Humans
Immunoglobulin G - analysis
Immunoglobulin G - chemistry
Light
Monoclonal antibody
Online Systems
Protein characterization
Protein Structure, Quaternary
Scattering, Radiation
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
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Summary:The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP–SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP–SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP–SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2008.03.050