Biology Reference
In-Depth Information
•
Does treatment with rHuEPO induce non-neutralizing anti-EPO antibodies?
•
What are the most efficient assays to detect antibodies and when should a
treating physician require such assays?
•
What are the physician's options for patients found to have neutralizing
anti-EPO antibodies?
•
What could be the reasons for the sudden appearance of pure red cell apla-
sia due to anti-EPO antibodies?
Structure of EPO and rHuEPO
Endogenous EPO is a glycoprotein of 165 amino acids. The full molecular
mass is approximately 30 Kd,with the average molecular mass of the protein
moiety approximately 18 Kd and the remaining mass attributed to the carbo-
hydrates. The carbohydrates are linked to three asparagine residues (Asn24,
Asn38, Asn83) and one serine residue (Ser126). Although sugars are not
involved in EPO receptor (EPOR) binding or activation and are not necessary
for
in vitro
biologic activity [4, 5], the deglycosylated molecule is biological-
ly inactive
in vivo
because of its rapid clearance. Moreover, the deglycosylat-
ed molecule exhibits a rather high tendency to aggregate. Solubility of ungly-
cosylated EPO can be increased by changing the asparagine residues bearing
carbohydrates to charged amino acids, such as lysine. Such a modified form of
EPO has been co-crystallized with the extracellular domain of EPOR [6]. The
EPO molecule stacks in four alpha helices connected by less structured loops.
(See Chapter 3 for more information.) Two disulfide bridges stabilize this ter-
nary structure. One disulfide bridge (Cys7-Cys161) connects the
N
- and
N C
-ter-
minal regions of the molecule and the other (Cys29-Cys33) forms a small
bridge inside the loop connecting the two first helices. Both these disulfide
bonds are absolutely required for biologic activity. Careful analysis of circu-
lating endogenous EPO and of rHuEPO has shown that the protein part of the
molecules was identical, but that the glycosylation pattern could be different.
Indeed,all the currently commercially available forms of rHuEPO (epoetin
alpha, epoetin beta, epoetin omega) have more branched oligosaccharides like
tetra-antennary structures that seem to be lacking in endogenous EPO. Most of
these structures are fully sialylated, and rHuEPO exhibits more acidic forms
than endogenous EPO. A high proportion of these tetra-antennary structures is
present on the epoetin beta molecule and to a less extent on the epoetin alpha
and epoetin omega molecules. Since both epoetin alpha and epoetin beta are
produced in Chinese hamster ovary (CHO) cells, the difference in carbohy-
drate composition between these two molecules is most likely due to differ-
ences in the purification process. Epoetin omega, which is produced in baby
hamster kidney (BHK) cells, exhibits roughly the same glycosylation pattern
as epoetin alpha [7].
