Anders Ljunglöf

Visualising intraparticle protein transport in porous adsorbents by confocal microscopy

Confocal scanning microscopy was used to study protein uptake to porous adsorbents during batch experiments in a finite bath. By coupling of a fluorescent dye to the protein molecules the penetration of single adsorbent particles at different times during batch uptake could be observed visually. Intensity profiles of the protein distribution within a single particle were obtained by horizontal scanning. After integration of the profiles the overall fluorescence within a bead could be calculated. Relating the overall fluorescence at different incubation times to the value at equilibrium allowed the construction of the fractional approach to equilibrium versus time. These data were compared to uptake curves, which had been obtained by measurements of the protein concentration in the supernatant and an excellent agreement of the curves was detected. The procedure was performed for two different proteins (lysozyme and human IgG) on two different media for protein adsorption (SP Sepharose Fast Flow and SP Sepharose XL; Pharmacia Biotech) and in all cases it could be shown, that the results from the direct measurements by confocal microscopy correspond very well to the data obtained from the indirect measurements in the fluid phase.

Confocal microscopy as a tool for studying protein adsorption to chromatographic matrices

Confocal scanning laser microscopy was used for studying protein adsorption to affinity chromatography matrices. The adsorption of Protein A to IgG Sepharose 6 Fast Flow was studied by batch incubation with varying amounts of fluorescently labeled Protein A. At low sample amounts, Protein A had been adsorbed to a thin outer layer. By increasing the sample-IgG Sepharose ratio, the adsorption layer also increased. Likewise, the adsorption depth was dependent on the incubation time. Finally, a stack of confocal images separated in space was used for three-dimensional reconstruction of the adsorption pattern in a particle.

Direct visualisation of plasmid DNA in individual chromatography adsorbent particles by confocal scanning laser microscopy.

Confocal microscopy was used for the measurement of plasmid DNA adsorbed to individual adsorbent particles intended for anion-exchange and triple helix affinity chromatography. Plasmid DNA was visualized with the fluorescent dye YOYO-1, that forms a highly fluorescent complex with double stranded DNA. Confocal images were translated into fluorescence intensity profiles and the distribution of plasmid DNA in the particles was measured. The results that adsorption of plasmid DNA mainly takes place in an outer layer of the particles. The described procedure can also be advantageously used to demonstrate triple helix formation between plasmid DNA and immobilized oligonucleotides.

Visualizing patterns of protein uptake to porous media using confocal scanning laser microscopy

Confocal scanning laser microscopy has been used to visualize the uptake of fluorescence-labeled proteins to porous stationary phases in finite bath adsorption experiments. Reference proteins were labeled with three different fluorescent dyes and a porous cation exchanger was sequentially incubated with solutions of these protein–dye conjugates. This sequential incubation experiment was used to investigate the pattern of protein uptake during adsorption. The confocal images obtained during the experiments clearly visualized that under certain conditions the adsorbent particles are gradually saturated from the rim to the core, a pattern consistent with the shrinking core model. Changes in the mobile phase conditions (ionic strength, pH) can lead to significant shifts in the uptake pattern, i.e., the further transport of initially bound molecules to the core, thus making binding sites at the rim available for adsorption of new molecules. At low pH and ionic strength BSA showed an uptake pattern, which was completely in accordance with the shrinking core model, but a clear deviation from this profile was observed at increased ionic strength. During adsorption of a monoclonal antibody a change in pH was sufficient to change the uptake pattern in finite bath adsorption completely. An attempt was made to correlate this behavior to the shape of the equilibrium-binding isotherm. The technique presented here allows unique insights into the details of protein adsorption to porous media and will provide a valuable extension of the existing experimental methods for studies of mechanistic aspects of protein chromatography.

Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy

The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.

Measurement of ligand distribution in individual adsorbent particles using confocal scanning laser microscopy and confocal micro-Raman spectroscopy.

Two methods, confocal scanning laser microscopy and confocal micro-Raman spectroscopy were used to analyse the distribution of IgG antibodies immobilized on CNBr-activated agarose beads. In the first method the internal distribution profile of fluorescent labelled Protein A was used as an indirect measure of the distribution of IgG, while the second method detects vibrations originating from aromatic amino acids present in the immobilized antibodies. Both these methods indicate an homogeneous ligand distribution within IgG Sepharose 4 Fast Flow and IgG Sepharose 6 Fast Flow.

Ligand Distributions in Agarose Particles as Determined by Confocal Raman Spectroscopy and Confocal Scanning Laser Microscopy

Confocal Raman spectroscopy and confocal scanning laser microscopy have been used to analyze ligand distributions within individual chromatographic adsorbent particles. Three different types of particles have been investigated. The first type was synthesized to have a uniform distribution of allyl groups, whereas the two others were designed to have a surface layer of sulphopropyl groups and cores containing allyl groups and dextran, respectively. With confocal Raman spectroscopy it was possible to follow the distribution of both the surface layer and the interior. The distribution of sulphopropyl groups was evaluated with both confocal scanning laser microscopy and confocal Raman spectroscopy, whereas the distributions of allyl groups and dextran were evaluated only with the latter method. The results from the confocal measurements showed the expected result with a uniform distribution of allyl groups in the first type of particle and surface layers of sulphopropyl groups and cores with dextran or allyl groups for the two others.