Determinants of protein retention characteristics on cation-exchange adsorbents

DePhillips, Peter; Lenhoff, Abraham M.

doi:10.1016/s0021-9673(01)01275-4

Cited by 104 publications

(73 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The difference in the strength of action of the sulfates and carboxylates exemplifies the importance of the type of charged group. Similar effects of tighter electrostatic binding of protein to sulfates compared to carboxylates have been observed previously in protein adsorption on chromatographic media with different surface groups 21 and have been explained on the basis of hydration effects. 22 The data also show, however, that the hydrophobic protein-surfactant interactions are also of primary importance in mediating the protein-protein interactions.…”

Section: Discussionsupporting

confidence: 77%

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization

Velev¹,

Pan²,

Kaler³

et al. 2004

Crystal Growth & Design

View full text Add to dashboard Cite

Surfactants are used as additives in some protein separations and crystallization procedures, but the mechanism of their action is still poorly understood. By measuring the osmotic second virial coefficients of lysozyme solutions by static light scattering, we show that small amounts of anionic surfactants of varying molecular weight always make the protein-protein interactions in solution more attractive and that both the charge of the headgroup and the length of the hydrophobic tail mediate the interactions. The same surfactants also modify lysozyme crystallization and promote formation of twinned phases with gross morphologies different from those seen in the absence of surfactant, some of which display remarkably structured patterns on a micrometer scale. The surfactant effects are, however, often only kinetic, as the phases obtained initially recrystallize slowly into large stable crystals. These crystals are of excellent X-ray diffraction quality and resolution (up to 1.4 Å). Their symmetry, unit cell dimensions, crystal contacts, and protein backbone conformation are the same as those commonly observed for lysozyme, but sometimes occur at atypical pH values. The data suggest new techniques for modification of protein crystallization.

show abstract

Section: Discussionsupporting

confidence: 77%

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization

Velev¹,

Pan²,

Kaler³

et al. 2004

Crystal Growth & Design

View full text Add to dashboard Cite

show abstract

“…At low ligand density, capacities ascend with increasing density until the ligand spacing is comparable to the diameter of the protein that is adsorbed. 30 At ligand densities well above this critical value additional binding sites can have a negative influence on the capacity because proteins will only pack into the pore space to a certain density as electrostatic effects repel adjacent protein molecules. Once that packing density has been achieved, the repulsive forces between the proteins dominate and additional ligands are not beneficial.…”

Section: Cation-exchange Chromatography Of Monoclonal Antibodiesmentioning

confidence: 99%

Cation-exchange chromatography of monoclonal antibodies: Characterisation of a novel stationary phase designed for production-scale purification

Urmann¹,

Graalfs²,

Joehnck³

et al. 2010

mAbs

View full text Add to dashboard Cite

“…7A). Therefore, accounting for finite These values were determined from manufacturers' ion-exchange capacity information and surface area estimates based on pore size distributions (20). adsorption kinetics leads to an overshoot in pore concentration when electrophoresis is included in the description; the lack of an overshoot for the materials with lower surface charge densities would then be caused by weaker potential gradients along the pore.…”

Section: Fig 4 (A-e)mentioning

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.

127

145

View full text Add to dashboard Cite

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...

show abstract

Determinants of protein retention characteristics on cation-exchange adsorbents

Cited by 104 publications

References 48 publications

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization

Cation-exchange chromatography of monoclonal antibodies: Characterisation of a novel stationary phase designed for production-scale purification

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Contact Info

Product

Resources

About