HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this...

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Bibliographic Details
Published in:Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2005-07, Vol.821 (1), p.45-52
Main Authors: LUYKX, Dion M. A. M, GOERDAYAL, S. S, DINGEMANSE, P. J, JISKOOT, W, JONGEN, Peter M. J. M
Format: Article
Language:English
Subjects:
Analysis
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromatography, Gel
Chromatography, High Pressure Liquid - methods
Circular Dichroism
Fundamental and applied biological sciences. Psychology
General pharmacology
Humans
Interferon-alpha - chemistry
Interferon-alpha - isolation & purification
Medical sciences
Pharmaceutical Preparations - chemistry
Pharmacology. Drug treatments
Protein Conformation
Recombinant Proteins
Reproducibility of Results
Sensitivity and Specificity
Spectrometry, Fluorescence - methods
Spectrophotometry, Ultraviolet
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Summary:High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities.
ISSN:1570-0232
1873-376X
DOI:10.1016/j.jchromb.2005.04.005