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Blood, 15 December 2001, Vol. 98, No. 13, pp. 3626-3634
HEMATOPOIESIS
Sugar profiling proves that human serum erythropoietin differs
from recombinant human erythropoietin
Venke Skibeli,
Gro Nissen-Lie, and
Peter Torjesen
From the Section for Doping Analysis, Hormone
Laboratory, Aker University Hospital, Oslo, Norway.
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.
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