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Two‐dimensional electrophoresis of recombinant human erythropoietin: A future method for the European Pharmacopoeia?
Author(s) -
Schlags Wolfgang,
Lachmann Bodo,
Walther Michael,
Kratzel Martin,
Noe Christian R.
Publication year - 2002
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200206)2:6<679::aid-prot679>3.0.co;2-f
Subject(s) - isoelectric focusing , glycosylation , recombinant dna , chemistry , chromatography , gel electrophoresis , isoelectric point , biochemistry , capillary electrophoresis , sialic acid , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , enzyme , proteomics , gene
Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post‐translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon® (rh‐EPO, Roche) for our studies to demonstrate the efficiency of two‐dimensional electrophoresis (2‐DE) to analyse post‐translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (p I ) 3.5–4.5 and 32–45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2‐DE for the analysis of rh‐EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh‐EPO glycoforms. Sequential deglycosylation of sialic acids, N ‐glycosides and the O ‐glycoside lead to significant shifts both in apparent relative molecular mass and p I . Comparing the 2‐DE patterns of rh‐EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2‐DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2‐DE has improved significantly over the last decade, we see 2‐DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.