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

Linden, Thomas; Ljunglöf, Anders; Hagel, Lars; Kula, Maria‐Regina; Thömmes, Jörg

doi:10.1081/ss-120000319

Cited by 66 publications

(50 citation statements)

References 12 publications

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“…The final result is that all protein molecules are evenly distributed throughout the whole particle. This result is in accordance with Linden et al (2002) who reported that changes in the mobile phase conditions (pH and ionic strength) lead to a switch in the protein uptake pattern for a monoclonal IgG2A. From a situation that appeared to be consistent with the physical picture of the shrinking core model, the protein uptake changed to a pattern where the protein molecules were transported further towards the particle core subsequent to binding to outer regions of the adsorbent.…”

Section: Multi-color Analysissupporting

confidence: 81%

“…Multi-dye analysis was performed according to the method developed by Linden et al (2002). The method is based on the consecutive incubation of adsorbent particles with the same protein but with different fluorophores.…”

Section: Multi-dye Analysis Of Protein Adsorptionmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

An exclusion mechanism in ion exchange chromatography

Harinarayan

Mueller

Ljunglöf³

et al. 2006

Biotech & Bioengineering

136

107

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Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes. ß

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Section: Multi-color Analysissupporting

confidence: 81%

Section: Multi-dye Analysis Of Protein Adsorptionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An exclusion mechanism in ion exchange chromatography

Harinarayan

Mueller

Ljunglöf³

et al. 2006

Biotech & Bioengineering

136

107

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“…These profiles also indicate that uptake reaches completion much more quickly in this limit than at low ionic strength. A similar transition from sharp fronts to diffuse profiles has been reported in confocal uptake studies of proteins in SP Sepharose FF after increases in the ionic strength and pH (12).…”

Section: Figmentioning

confidence: 49%

“…A second promising route is the use of scanning confocal microscopy to image protein uptake into individual chromatography particles. Results reported thus far have been limited to studies of large proteins with relatively slow transport rates because contacting and imaging were performed sequentially rather than simultaneously, introducing a time lag (9)(10)(11)(12). We report here results using confocal microscopy to perform imaging of protein uptake into single ion exchange particles packed in a flow cell.…”

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confidence: 99%

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Dziennik

Belcher²,

Barker³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

126

145

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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...

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High Throughput Screening Techniques in Protein Purification

Lacki¹,

Brekkan²

2011

Methods of Biochemical Analysis

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

Cited by 66 publications

References 12 publications

An exclusion mechanism in ion exchange chromatography

An exclusion mechanism in ion exchange chromatography

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

High Throughput Screening Techniques in Protein Purification

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