title: Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations.
abstract: The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.
authors.affiliation: Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Julich, Germany. j.hubbuch@fz-juelich.de
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
authors.forename: Thomas
authors.initials: T
authors.lastname: Linden
authors.forename: Esther
authors.initials: E
authors.lastname: Knieps
authors.forename: Jörg
authors.initials: J
authors.lastname: Thömmes
authors.forename: Maria-Regina
authors.initials: MR
authors.lastname: Kula
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D021381:Protein Transport