Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection

Dou, Peng; Liu, Zhen; He, Jiangang; Xu, Jing‐Juan; Chen, Hong‐Yuan

doi:10.1016/j.chroma.2008.03.001

Cited by 47 publications

(30 citation statements)

References 39 publications

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“…After separation, proteins were immobilized to the capillary wall and detected with primary and HRP-conjugated secondary antibodies. Both gH/gL and pentameric complex appeared as broad peaks, suggesting that both were heterogeneous in charge, a common phenomenon for CHO expressed heavily glycosylated macromolecules (24,25). As expected, gH/gL migrated between pH 6.0 -6.5 consistent with its theoretical pI of 6.2.…”

Section: Soluble Gh/gl and Gh/gl/pul128 -131 Pentameric Complex Retaisupporting

confidence: 54%

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Loughney

Rustandi

Wang

et al. 2015

Journal of Biological Chemistry

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Background: HCMV gH/gL/pUL128 -131 pentameric complex is a main target for potent neutralizing antibody responses. Results: Soluble gH/gL/pUL128 -131 inhibits viral entry and presents neutralizing epitopes. Conclusion:The biological activities of gH/gL/pUL128 -131 confirm the critical role of the native complex as an immunogen. Significance: The functional differences between gH/gL and gH/gL/pUL128 -131 must be considered when designing vaccines and assessing immune responses.

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Section: Soluble Gh/gl and Gh/gl/pul128 -131 Pentameric Complex Retaisupporting

confidence: 54%

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Loughney

Rustandi

Wang

et al. 2015

Journal of Biological Chemistry

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“…The resolution is similar to the one reported by Hunt et al [33]. The resolution ability and rapidness of the imaged CIEF will be a powerful tool for routine analysis as indicated for glycoform analysis of erythropoietin [34].…”

Section: Evaluation Of Modification Of Igg Molecules By Capillary Isosupporting

confidence: 53%

Evaluation of glycosylation for quality assurance of antibody pharmaceuticals by capillary electrophoresis

Kamoda¹,

Kakehi

2008

Electrophoresis

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The development of monoclonal antibody (mAb) pharmaceuticals has rapidly generated in this decade, and there are currently 23 FDA-approved mAb pharmaceuticals. Carbohydrate chains in mAb pharmaceuticals play important roles for the expression of their biological activities such as antibody-dependent cellular cytotoxicity, whereas the oligosaccharide profile is easily changed depending on the manufacturing conditions. Therefore, the evaluation of carbohydrate chains in detail is quite important for quality control in the development of mAb from early phases, but oligosaccharides in mAb have high heterogeneity and full characterization of oligosaccharide moiety in mAb has been a challenging target. Capillary electrophoresis (CE) is a powerful tool for the evaluation of glycan occupancy and oligosaccharide profile. This review focuses mainly on the application of CE to the analytical studies on glycosylation in mAb pharmaceuticals developed in our laboratory. The related techniques including the full-image microchip isoelectric focusing and the newly developed mass spectrometry technologies are also shown. The combination of the techniques described in this review will be a good guide for the evaluation of glycosylation for quality assurance of antibody pharmaceuticals by CE from the early stage of the development to the marketing stage.

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“…Previous works [41,42] indicated that the presence of certain buffer additives such as putrescine and urea is critical for the resolution of EPO glycoforms. These additives improve the stability and solubility of EPO in the BGE, decrease the adsorption of the protein on the capillary wall, and also reduce the intermolecular interactions of EPO isoforms.…”

Section: Effect Of Additive In Sample Matrixmentioning

confidence: 99%

Combination of large volume sample stacking and reversed pH junction in capillary electrophoresis for online preconcentration of glycoforms of recombinant human erythropoietin

Liu

Liu²,

Kang

et al. 2009

J of Separation Science

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Resolution of glycoforms of a glycoprotein is usually a challenge for an analytical chemist, which is often associated with the difficulty of detecting a large number of glycoforms with sufficient sensitivity. CE is the main workhorse for the analysis of glycoproteins; however, the current methods are only applicable to high concentration samples. Therefore, the development of online preconcentration approaches is important for the CE analysis of glycoproteins. In this study, we present a combined strategy for online preconcentrating glycoforms of glycoproteins, which couples two individual online preconcentration techniques, i.e., large volume sample stacking (LVSS) and reversed pH junction (RPHJ). LVSS allows for compressing a large injected sample volume into a narrow sample zone, contributing to the main sensitivity improvement, while RPHJ coordinates the preconcentration process to improve the resolution. This strategy was verified with recombinant human erythropoietin (rhEPO), a typical glycoprotein, as the test analyte, and the effects of experimental conditions were investigated. It was found that there is a compromise between sensitivity enhancement and resolution for online preconcentration of glycoprotein glycoforms. By using this strategy, the detection sensitivity can be improved by 50-100 times for rhEPO.

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Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection

Cited by 47 publications

References 39 publications

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Evaluation of glycosylation for quality assurance of antibody pharmaceuticals by capillary electrophoresis

Combination of large volume sample stacking and reversed pH junction in capillary electrophoresis for online preconcentration of glycoforms of recombinant human erythropoietin

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