Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Abstract

Scanning confocal fluorescence microscopy and multiphoton fluorescence microscopy were used to image the uptake of the protein lysozyme into individual ion exchange chromatography particles in a packed bed in real time. Self-sharpening concentration fronts penetrating into the particles were observed at low salt concentrations in all of the adsorbents studied, but persisted to 100 mM ionic strength only in some materials. In other adsorbents, diffuse profiles were seen at these higher salt concentrations, with the transition region exhibiting a pronounced fluorescence peak at the front at intermediate salt concentrations. These patterns in the uptake profiles are accompanied by significant increases in protein uptake rates that are also seen macroscopically in batch uptake experiments. The fluorescence peak appears to be a concentration overshoot that may develop, in part, from an electrokinetic contribution to transport that also enhances the uptake rate. Further evidence for an electrokinetic origin is that the effect is correlated with high adsorbent surface charge densities. Predictions of a mathematical model incorporating the electrokinetic effect are in qualitative agreement with the observations. These findings indicate that mechanisms other than diffusion contribute to protein transport in oppositely charged porous materials and may be exploited to achieve rapid uptake in process chromatography.T he dramatic growth of the biotechnology industry in recent years has depended critically on preparative separation techniques for protein products. Ion exchange chromatography, introduced for protein separations in the 1950s, remains one of the most widely used preparative-scale purification and separation methods (1). Ion exchange adsorbents have been designed to offer a range of mechanical and chemical properties for chromatographic separation and purification (2), but a fundamental understanding of transport mechanisms in these materials remains elusive. For most materials, simple diffusive mechanisms are thought to control intraparticle transport, but studies using batch or column techniques (3-7) yield model-dependent transport coefficients, and they cannot definitively determine the physical transport mechanism. With the projected acceleration in the discovery and development of protein therapeutics as a result of the human genome project, the demand for improved chromatographic materials and methods for optimizing protein separation and purification will increase. Therefore, a more fundamental understanding of the transport mechanisms in these materials is required to develop better heuristics for process development as well as methodologies for the design of new adsorbents.A key complication in the interpretation of batch or column studies is that the transport is coupled to adsorption, making additional assumptions regarding the adsorption behavior necessary to characterize the transport. Microscopic techniques have a greater potential to resolve the resulting ambiguities regarding transport mechanisms...

An exclusion mechanism in ion exchange chromatography

“…Such a hypothesis may provide a path to reconciling present findings with a wider range of media and proteins as well as the work of earlier scientists (e.g., Dziennik et al, 2003;Pujar and Zydney, 1998) and the obvious complexity of protein transport and binding in charged hydrogels (Carlsson et al, 2001;Lewus and Carta, 2001;Liapis et al, 2001).…”

“…Several models describe the complex adsorption mechanisms in IEX (Brooks and Cramer, 1992;Chang and Lenhoff, 1998;Gallant, 2004;Hunter and Carta, 2000;Lewus and Carta, 2001;Liapis et al, 2001;Nash and Chase, 1998;Zhang et al, 2004a). Confocal microscopy has recently been used to further elucidate the complex nature of IEX adsorption and transport mechanisms (Dziennik et al, 2003;Hubbuch et al, 2002Hubbuch et al, , 2003Kasche et al, 2003;Laca et al, 1999;Linden et al, 1999Linden et al, , 2002Ljunglöf and Hjorth, 1996;Ljunglöf and Thömmes, 1998).…”

Bioseparations

“…New adsorbents are being developed (Hjorth, 2003;Palmgren et al, 2003;Sarkar et al, 2003) and used (Hahn et al, 2002;Jungbauer et al, 2003;Phillips, 2003;Pieracci et al, 2003;Teeters et al, 2002). Even such widely accepted transport mechanisms as intraparticle diffusion have been found to be more complex than previously believed (Dziennik et al, 2003). Computational chemistry (DePhillips and Lenhoff, 2001;Koehler et al, 2000) and atomic probes (Tibbs and Fernandez, 2003) are increasingly helpful in choosing extractants, and protein databases (e.g., http://www.rcsb.org/ pdb/index.html) are simplifying the hunt for effective separating agents.…”

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

“…In a previous study, a microtome was used to section fluorescently labeled beads to evaluate the effects of antibody density on a support's efficiency (6). Several groups of researchers have used confocal laser scanning microscopy (CLSM) to measure the uptake of dyes and proteins on individual particles under varying binding conditions (7)(8)(9)(10)(11). Using CLSM, it is possible to obtain three-dimensional images of individual chromatographic support particles that directly illustrate the amount of protein uptake.…”