Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

Tantipolphan, Ruedeeporn; Romeijn, Stefan; Engelsman, John den; Torosantucci, Riccardo; Rasmussen, Tue Kruse; Jiskoot, Wim

doi:10.1016/j.jpba.2010.01.014

Cited by 40 publications

(31 citation statements)

References 36 publications

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“…23,24 In particular, reduction of the 273-nm CD band is observed when insulin is dissociated. 25,26 Plain insulin presented a higher negative molar ellipticity band when compared with PEG-insulin, which can be explained by the association state of plain insulin differing from that of PEG-insulin, because in the free protein, hexamer prevails 27 (Fig. 5f and Table 3), whereas the conjugated protein appeared to be in the dimeric state, as reported by Hinds et al 10 Intrinsic tyrosine fluorescence spectroscopy was used to further analyze the tertiary structure of the proteins.…”

Section: Characterization Of Insulin and Peg-insulinmentioning

confidence: 55%

“…The signal in this region is due to the absorbance of insulin's tyrosine, phenylalanine, and disulfides, and is substantially affected by its self‐association state 23,24. In particular, reduction of the 273‐nm CD band is observed when insulin is dissociated 25,26. Plain insulin presented a higher negative molar ellipticity band when compared with PEG–insulin, which can be explained by the association state of plain insulin differing from that of PEG–insulin, because in the free protein, hexamer prevails27 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Plain and Mono-pegylated Recombinant Human Insulin Exhibit Similar Stress-Induced Aggregation Profiles

Torosantucci

Kükrer

Mero

et al. 2011

Journal of Pharmaceutical Sciences

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Section: Characterization Of Insulin and Peg-insulinmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Plain and Mono-pegylated Recombinant Human Insulin Exhibit Similar Stress-Induced Aggregation Profiles

Torosantucci

Kükrer

Mero

et al. 2011

Journal of Pharmaceutical Sciences

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“…This technique was employed for several purposes: to detect and quantify the percentage of monomer loss, and the formation of covalent dimers and high-molecular-weight oligomers (HMWO) during MCO, by using UV detection at 276 nm (note that the composition of the mobile phase does not allow us to estimate the amount of non-covalent aggregates since these will be dissociated because of the organic solvent and the low pH of the eluent (31)); to monitor Tyr fluorescence in oxidized insulin, by using an excitation wavelength of 275 nm with emission set at 302 nm, and to detect the development of DOPA and DOCH through derivatization with ABS. Furthermore, we used SEC to monitor the presence of fragments after prolonged oxidation.…”

Section: Resultsmentioning

confidence: 99%

Chemical Modifications in Aggregates of Recombinant Human Insulin Induced by Metal-Catalyzed Oxidation: Covalent Cross-Linking via Michael Addition to Tyrosine Oxidation Products

et al. 2012

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PurposeTo elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).MethodsInsulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.ResultsThe results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.ConclusionMCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.

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“…[9,63–65,98–100] These ionic interactions can result in adsorption of the protein, [74] shifts in retention time, [5] peak tailing or peak asymmetry, [6] or to changes in the three dimensional conformation of the protein. [63,101] As previously mentioned, chromatographic stationary phases and mobile phases have been used to mitigate nonideal interactions.…”

Section: Methods Of Optimizationmentioning

confidence: 99%

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

Hong

Koza

Bouvier

2012

Journal of Liquid Chromatography & Related Technologies

445

291

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In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.

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Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

Cited by 40 publications

References 36 publications

Plain and Mono-pegylated Recombinant Human Insulin Exhibit Similar Stress-Induced Aggregation Profiles

Plain and Mono-pegylated Recombinant Human Insulin Exhibit Similar Stress-Induced Aggregation Profiles

Chemical Modifications in Aggregates of Recombinant Human Insulin Induced by Metal-Catalyzed Oxidation: Covalent Cross-Linking via Michael Addition to Tyrosine Oxidation Products

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

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