Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin.
Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)-specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports.
Skibeli
V
V
Section for Doping Analysis, Hormone Laboratory, Aker University Hospital, Oslo, Norway. venke.skibeli@ioks.uio.no
Nissen-Lie
G
G
Torjesen
P
P
D000581:Amidohydrolases / Q000378:metabolism; D000740:Anemia / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008280:Magnetics; D008863:Microspheres; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000097:blood*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins
D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D009439:Neuraminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase