The use of confocal laser scanning microscopy to study the transport of biomacromolecules in a macroporous support

Abstract

Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 _m in diameter and have 3.5% solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites hroughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.

Characterization and application of a sterol esterase immobilized on polyacrylate epoxy-activated carriers (Dilbeads™)

“…This technique has been employed to visualize the distribution of biomolecules throughout the bead (esp. in hydrogels) as well as to evaluate restrictions to diffusion within the support [29][30][31][32]. A typical confocal image for FITC-labeled enzyme immobilized in Dilbeads TM TA is shown in Fig.…”

Comparison of chitosan nanoparticles and chitosan hydrogels for vaccine delivery

“…The release of active ingredients from gels is generally diffusion controlled, with diffusion being determined by the porosity of the gel and volume of the water present (Mi et al 1999;Kofuji et al 2004;Subramanian & Hommerding 2005). The shape, size and distribution of pores are also important parameters for the release of the antigen.…”

Ion exchange chromatography of antibody fragments

“…Most of these studies have been carried out as batch or column experiments in which the adsorbent is considered as a bulk phase. Recently, confocal scanning laser microscopy has been introduced as an alternative and complementary method for studying protein adsorption at the matrix level (Dziennik et al, 2003;Hubbuch et al, 2003a,b;Linden et al, 1999Linden et al, , 2002Ljunglöf and Hjorth, 1996;Ljunglöf and Thömmes, 1998;Ljunglöf et al, 1999;Subramanian and Hommerding, 2005;Zhou et al, 2006). With this technique it is possible to study intra-particle protein and DNA concentrations, and also ligand distributions, directly within individual adsorbent particles (Larsson et al, 2002(Larsson et al, , 2003Ljunglöf et al, 2000).…”