High-performance anion-exchange chromatography combined with intrinsic fluorescence detection to determine erythropoietin in pharmaceutical products.
A high-performance anion-exchange chromatography (HPAEC) method was developed for determination of recombinant human erythropoietin (EPO) in pharmaceutical products. A fluorescence detector was added to the HPLC system as intrinsic fluorescence detection compared favourably to UV detection regarding sensitivity and selectivity. The HPLC method has been successfully applied to analyse erythropoietin products even in the presence of albumin as excipient. The intrinsic fluorescence chromatograms of both proteins revealed various peaks attributed to either micro-heterogeneous erythropoietin or albumin variants. The intrinsic fluorescence signal was linear over the range 10-200 microg/ml erythropoietin corresponding to pharmaceutically relevant concentrations. The HPLC method appeared to be a suitable method for differentation between recombinant human erythropoietin epoetin-alpha and -beta as they revealed different intrinsic fluorescence elution profiles. In conclusion, this study contributes to the development of a straightforward physicochemical method for specific quantification of recombinant human erythropoietin in pharmaceutical preparations.
Luykx
Dion M A M
DM
Centre for Biological Medicines and Medical Technology, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
Dingemanse
Pieter J
PJ
Goerdayal
Soenita S
SS
Jongen
Peter M J M
PM
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000032:analysis*; D006801:Humans; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods
D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D012709:Serum Albumin; C103998:epoetin beta; D004921:Erythropoietin; D000068817:Epoetin Alfa