Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography

Klausen, Niels Kristian; Kornfelt, Troels

doi:10.1016/0021-9673(95)00656-7

Cited by 42 publications

(23 citation statements)

References 33 publications

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“…The use of 1,5-diaminopentane (cadaverine) exhibited the same glycoform pattern. Analysis times, however, are relatively long (up to 100 min) [497]. Ovalbumin glycoforms have been analyzed using a 100 m~ sodium borate buffer, pH 8.5 containing 3 m~ 1,4-diaminobutane [498].…”

Section: Cations As Modifiersmentioning

confidence: 99%

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications

1998

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This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electroosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.

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Section: Cations As Modifiersmentioning

confidence: 99%

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications

1998

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“…Landers et al [16], Che et al [24], and Estrella and Pedrosa [21] showed that the use of PUT and SPD, respectively, as buffer additives resulted in high-resolution for the components of OVA-glycoform in a bare capillary tube. Their effectiveness has been verified by applying the approach to different glycoproteins [15,17,20,22,23]. The efficiency of polyamines was thus recognized.…”

Section: Discussionmentioning

confidence: 99%

“…Such unfavorable adsorption can be interrupted by modification of the surface by coating with neutral polymers [1][2][3][4][5][6][7][8][9][10][11][12] or neutralization of the surface charge by ionic additives of countersign [13,14]. The most successful examples resulting in high-performance separation of proteins with microheterogeneity are polyacrylamide coating [8-10, 25, 26] for the former and addition of polyamine [15][16][17][18][19][20][21][22][23][24] to the electrolyte buffer solution for the latter. In each case, high resolution is achieved by the reduction of protein-wall interaction.…”

Section: Introductionmentioning

confidence: 99%

Assessment of the capillary zone electrophoretic behavior of proteins in the presence of electroosmotic modifiers: Protein-polyamine interaction studied using a polyacrylamide-coated capillary

Kubo

Hattori

2001

Electrophoresis

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The use of polyamines as electroosmotic modifiers has been shown to be effective in enhancing resolution of protein glycoforms in capillary zone electrophoresis (CZE) using a bare capillary tube. In this study, effectiveness was evaluated by using a polyacrylamide-coated capillary tube instead of a bare capillary tube. Electropherograms obtained in the presence of polyamines were inferior to those obtained in their absence with respect to resolution. Electrophoretic mobility of the proteins decreased and their peaks were broadened by polyamines bound to them. This unfavorable effect was dependent on both the species of polyamines and the pH values of the electrolyte buffer. The reduction of resolution caused by polyamines was in the following order: spermidine (SPD) approximately spermidine-tri-hydrochloride (SPD-HCI) > putrescine (PUT) > hexamethonium chloride (HMC). The observed effect can be ascribed to the formation of complexes between the proteins and the polyamines. In addition, for the bare capillary tube the complexes showed interaction with the inner surface, resulting in local suppression of electroosmosis and poor resolution. The high resolution obtained in the coated capillary tube was reduced in the presence of the polyamines. Thus, the use of the polyamines has a negative effect on the analysis of protein microheterogeneity as a result of protein-polyamine interaction.

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“…The total capillary length was reduced to 67 cm (60 cm effective length), and PBS present in the VEGF 165 sample was removed using centrifugal filter devices. To avoid the unspecific binding of the protein to the capillary wall putrescine, in the range of 1-4 mM, was employed as a dynamic coating in the following development [32][33][34][35][36]. Best results were obtained with 2 mM putrescine in the BGE.…”

Section: Assignment Of Vegf 165 Peaks: Computer Program and Migrationmentioning

confidence: 98%

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

et al. 2009

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The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF165 arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF165 are developed. Using a computer program previously developed in‐house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF165 expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF165, mean a starting point that permits the study of the role of VEGF165 as a potential biomarker of different diseases and physiological processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF165 have been developed.

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Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography

Cited by 42 publications

References 33 publications

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications

Assessment of the capillary zone electrophoretic behavior of proteins in the presence of electroosmotic modifiers: Protein-polyamine interaction studied using a polyacrylamide-coated capillary

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

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