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

Publication Type:

Journal Article

Source:

Analytical Biochemistry, Volume 378, Number 2, pp. 115-122 (2008)

ISBN:

0003-2697

DOI Name (links to online publication)

10.1016/J.Ab.2008.03.050

Keywords:

protein characterization; aggregation; fluorescent dyes; high-pressure size exclusion; chromatography; asymmetrical flow field flow fractionation; monoclonal antibody; intramolecular charge-transfer; monoclonal-antibody; protein aggregation; light-scatter

Abstract:

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. (C) 2008 Elsevier Inc. All rights reserved.

28/10/2009