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 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.
Luykx
Dion M A M
DM
Centre for Biological Medicines and Medical Technology, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands.
Goerdayal
S S
SS
Dingemanse
P J
PJ
Jiskoot
W
W
Jongen
Peter M J M
PM
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002942:Circular Dichroism; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000302:isolation & purification; D004364:Pharmaceutical Preparations / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins