General methods to render macroporous stationary phases nonporous and deformable, exemplified with agarose and silica beads and their use in high-performance ion-exchange and hydrophobic-interaction chromatography of proteins

Mohammad, Jamil; Eriksson, Kjell-Ove; Liao, Jiali

doi:10.1007/bf02290503

Cited by 27 publications

(9 citation statements)

References 25 publications

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“…Bare silica beads used in chromatographic columns have the great drawback to adsorb proteins strongly. Therefore, we developed a coating method to make also all surfaces of these beads hydrophilic, non‐charged and pH stable 30. This approach is now used by most companies selling packed, chromatographic columns or columns in the monolithic version.…”

Section: Resultsmentioning

confidence: 99%

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Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

2010

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Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii-vii) and how to overcome them, which also may help to overcome the difficulty (ii), which in turn minimizes difficulty (i).

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Section: Resultsmentioning

confidence: 99%

“…Silica gels should be employed only if they are deactivated, as described in Ref. 30. In many of the commercial silica columns this or a modified deactivating technique is used.…”

Section: Resultsmentioning

confidence: 99%

Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

2010

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“…105 Although these materials compress under flow, this deformation can be turned into an advantage, as controlled compression of a packed bed results in improved column efficiency due to reduced interparticle diffusion paths. 106 An inherent and problematic property of biological macromolecules as chromatographic supports is their vulnerability to break-down by biological activity. Although some polysaccharides are subject to break-down by enzymatic action, it is particularly important to protect the supports from becoming consumed or contaminated by microbial activity.…”

Section: Agarosementioning

confidence: 99%

“…However, in recent years a number of rigid phases have become available 252,253 based on silica, 160,252 crosslinked agarose, 106 and synthetic polymers, 254,255 with a variety of functional groups including short-chain alkyl chains, phenyl groups, 254 oligoethyleneglycol, 252 and intermediately polar groups such as poly(alkylaspartamides). 160 The polar -apolar characteristics of nine different RPHPLC and HIC columns were investigated by Rippel et al 256 using homologous series as probes.…”

Section: Hydrophobic Interaction Chromatographymentioning

confidence: 99%

High‐Performance Liquid Chromatography of Biological Macromolecules

Irgum¹

2000

Encyclopedia of Analytical Chemistry

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“…HIC is often employed to remove dimer and/or multimer species from the monomeric forms of protein therapeutics by exploiting differences in the hydrophobicities of the respective species. Despite being commonly used as an effective separation tool, the mechanism for HIC adsorption is quite complex, and depends on a number of process parameters, including the temperature, pH, salt concentration, adsorbent ligand density, and adsorbent ligand hydrophobicity [4][5][6][7][8][9][10][11][12]. Several theories have been proposed to explain the HIC adsorption process, including the Solvophobic [13,14] and preferential interaction theories [15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

McCue

Engel

et al. 2008

Bioprocess Biosyst Eng

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Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.

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General methods to render macroporous stationary phases nonporous and deformable, exemplified with agarose and silica beads and their use in high-performance ion-exchange and hydrophobic-interaction chromatography of proteins

Cited by 27 publications

References 25 publications

Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

High‐Performance Liquid Chromatography of Biological Macromolecules

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

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