Ion-Exchange Chromatography of Proteins

Yamamoto, S.; Nakanishi, Kazahiro; Matsuno, Ryuichi

doi:10.1201/b15751

Cited by 173 publications

(135 citation statements)

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“…Using activated PEG molecules of different molecular weights, lysozyme was randomly PEGylated and BSA mono-PEGylated. Linear gradient elution (LGE) experiments were carried out, and the number of binding sites was determined [13][14][15] to examine how PEGylation affects the binding mechanism. For lysozyme, enzyme activity was measured using two different substrates (suspended or soluble) to determine how the PEG chain affects the enzyme activity.…”

Section: Research Articlementioning

confidence: 99%

“…4 and 5). The number of binding sites, B values and the parameter A (including the equilibrium coefficient, the binding site and the ion-exchange capacity) were determined from the GH-I R curves [13][14][15] (Tables 1 and 2). This method is very suitable for this type of difficult separation as a number of peaks must be separated, which is not easy by a standard isocratic elution method.…”

Section: Binding Sitementioning

confidence: 99%

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Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abe

Akbarzaderaleh

Hamachi

et al. 2010

Biotechnology Journal

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The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

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Section: Research Articlementioning

confidence: 99%

Section: Binding Sitementioning

confidence: 99%

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abe

Akbarzaderaleh

Hamachi

et al. 2010

Biotechnology Journal

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“…This correlation suggests that a simple set of isocratic retention measurements can be used as the basis for predicting conditions for the change in uptake mechanism and for narrowing down conditions for optimizing DBC as a function of flow rate. Indeed, the k' vs. ionic strength curve can be estimated with reasonable accuracy from a small set of gradient elution experiments coupled with a parametric equation relating k' to ionic strength [59,60].…”

Section: Origins Of Change In Uptake Mechanism and Maximum In Dynamicmentioning

confidence: 99%

Chromatography of proteins on charge-variant ion exchangers and implications for optimizing protein uptake rates

Langford

Yan

et al. 2007

Journal of Chromatography A

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Intraparticle transport of proteins usually represents the principal resistance controlling their uptake in preparative separations. In ion-exchange chromatography two limiting models are commonly used to describe such uptake: pore diffusion, in which only free protein in the pore lumen contributes to transport, and homogeneous diffusion, in which the transport flux is determined by the gradient in the total protein concentration, free or adsorbed. Several studies have noted a transition from pore to homogeneous diffusion with increasing ionic strength in some systems, and here we investigate the mechanistic basis for this transition. The studies were performed on a set of custom-synthesized methacrylate-based strong cation exchangers differing in ligand density into which uptake of two proteins was examined using confocal microscopy and frontal loading experiments. We find that the transition in uptake mechanism occurs in all cases studied, and generally coincides with an optimum in the dynamic binding capacity at moderately high flow rates. The transition appears to occur when protein-surface attraction is weakened sufficiently, and this is correlated with the isocratic retention factor k' for the system of interest: the transition occurs in the vicinity of k' ~ 3000. This result, which may indicate that adsorption is sufficiently weak to allow the protein to diffuse along or near the surface, provides a predictive basis for optimizing preparative separations using only isocratic retention data.

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“…In addition, the diffusion rate of the acylase-polyA C H T U N G T R E N N U N G (Lys) complex from the outer stream to the central stream is lower than the cross-linker from the central to the outer stream. (Roughly calculating based on the literature, [26] the diffusion coefficients are 910 mm 2 sec À1 for the cross-linker and 70 mm 2 sec À1 for acylase.) These factors would lead to the enzyme-polymer being promoted more preferentially in the vicinity of the inner wall of CEM-tube than at the central region.…”

Section: Discussionmentioning

confidence: 99%

Facile Preparation of an Enzyme‐Immobilized Microreactor using a Cross‐Linking Enzyme Membrane on a Microchannel Surface

et al. 2006

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Abstract:The enzyme microreactor has considerable potential for use in biotechnological syntheses and analytical studies. Simplifying the procedure of enzyme immobilization in a microreactor is attractive, and it is achievable by utilizing enzyme immobilization techniques and taking advantage of the characteristics of microfluidics. We previously developed a facile and inexpensive preparation method for an enzyme-immobilized microreactor. The immobilization of enzymes can be achieved by the formation of an enzyme-polymeric membrane on the inner wall of the microchannel through cross-linking polymerization in a laminar flow. However, this method is unsuitable for use in conjunction with electronegative enzymes. Therefore, a novel preparation method using poly-l-lysine [polyA C H T U N G T R E N N U N G (Lys)] as a booster and an adjunct for the effective polymerization of electronegative enzymes was developed in this study. Using aminoacylase as a model for an electronegative enzyme, the reaction conditions for the enzymecross-linked aggregation were optimized. On the basis of the determined conditions, an acylase-immobilized tubing microreactor was successfully prepared by cross-linking polymerization in a concentric laminar flow. The resulting microreactor showed a higher stability against heat and organic solvents compared to those of the free enzyme. The developed method using polyA C H T U N G T R E N N U N G (Lys) was applicable to various enzymes with low isoelectric points, suggesting that this microreactor preparation utilizing a cross-linked enzyme in a laminar flow could be expanded to microreactors in which a broad range of functional proteins are employed.

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Ion-Exchange Chromatography of Proteins

Cited by 173 publications

References 0 publications

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Chromatography of proteins on charge-variant ion exchangers and implications for optimizing protein uptake rates

Facile Preparation of an Enzyme‐Immobilized Microreactor using a Cross‐Linking Enzyme Membrane on a Microchannel Surface

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