HALF-LIFE EXTENSION THROUGH O-GLYCOSYLATION - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 200

on the surface of erythroid progenitors resulting in replica-
tion and maturation to functional erythrocytes by an incom-
pletely understood mechanism. Recombinant human (rhu)
EPO has been used successfully in a variety of clinical
situations to increase production of red blood cells. Cur-
rently, this agent is licensed for use in the treatment of the
anemia of renal failure, the anemia associated with HIV
infection in zidovudine (AZT) treated patients, and anomie
associated with cancer chemotherapy. Administration of
rhuEPO has become routine in the treatment of anemia
secondary to renal insufficiency, where doses of 50-
75 U/kg given three times per week are used to gradually
restore hematocrit and eliminate transfusion dependency.
One major issue regarding the clinical use of EPO is its
relatively short half-life in vivo due to its rapid clearance
(

week as 5 m g/kg per injection. These results indicated the
importance of sustained blood levels, rather than total dose
of EPO. These findings are consistent with the hypothesis
that the ability of a single injection of EPO-CTP to increase
hematocrit, results from its increased stability in the circu-
lation. The increased biopotency of the chimera may reflect
a change in their metabolic clearance in vivo. Detecting the
half-lives of EPO analogs in mice indicated that a higher
level of the chimera is still detectable in serum after 24 h.
The half-life of EPO-CTP is increased twofold to threefold
comparing to EPO-WT. These data suggest that the mecha-
nism of EPO metabolic clearance is affected by the presence
of CTP [24].
Other variant of long-acting EPO was design by intro-
duction of additional N-linked glycosylation into the protein
backbone. Using site-directed mutagenesis, the DNA
sequence was changed to design hyperglycosylation recom-
binant EPO analog, (novel erythropoisis-stimulating protein,
NESP) which contain five N-linked oligosaccharide chains
(two more than EPO-WT) [25,26]. It was found that NESP
distinct from rHuEPO having an increased molecular weight
and greater negative charge. Compared with rHuEPO, it has
an approximate threefold longer serum half-life, greater
in vivo potency, and can be administered less frequently
to obtain the same biological response. On the other hand,
removal of the sialic acid from either native EPO or rHuEPO
resulted in molecules having an increased activity in vitro,
but very low activity in vivo, presumably due to removal
from circulation by the asialoglycoprotein receptor in the
liver. Similarly, it was shown that EPO molecules, which
have been deglycosylated to remove COH (or produced in
Escherichia coli to allow expression of only the EPO
polypeptide), are active in vitro, but have very low
in vivo activity [27]. RHuEPO and NESP were tested for
EPO receptor-binding activity in the radioreceptor assay.
Consistent with the results obtained in this assay for the
isolated isoforms and the hypothesis, the relative EPO
receptor-binding affinity was inversely correlated with the
COH content. The relative affinity of NESP for the EPO
receptor was 4.3-fold lower than that of rHuEPO [25].

5 h) from the circulation when it is injected intravenously.
Thus, the therapeutic protocol used in the treatment of
patients, required frequent injections of EPO. The recom-
mended therapy with rhuEPO is two to -three times per
week by subcutaneous or intravenous injections. Therefore,
it was anticipated that enhancing the in vivo half-life of EPO
will reduce the number of injections per week.
The CTP was ligated to the coding sequence at the
C-terminal end of EPO. The in vitro biological activity of
EPO analogs was demonstrated by measuring their ability to
stimulate proliferation of erythroid burst forming colonies
(BFU-E) from human peripheral blood. BFU-E colonies
were grown from blood of healthy donors using a microwell
modification of the methylcellulose technique. The optimal
formation of BFU-E colonies achieved by EPO-CTP was
similar to that achieved by EPO-WT and rhuEPO using
1 U/mL of the protein. Receptor-binding assay indicated that
ligation of the CTP to the carboxyl-terminal of EPO has no
significant effect on the affinity of the hormone to the
receptor.
For further pharmacological evaluation of EPO-CTP,
comparative pharmacodynamic studies of EPO-CTP and
commercial rhuEPO were performed in male C57BL

mice (n 7/group) using different frequencies and wide

dose range. The in vivo efficacy was obtained by measuring
the mean values of hematocrit percentage in the blood. The
results indicated that EPO-CTP is significantly (P

¼

0.05)
more efficient than EPO-WT when administered IV once a
week with a dose of 5 m g/kg. EPO-CTP can successfully
increase the hematocrit when administered once a week with
a dose of 15 m g/kg. Once weekly dosing with the same
concentration of commercial rhuEPO or EPO-WT was
significantly (P

13.3.3.4 Human Growth Hormone (GH). Growth
hormone is secreted by the somatotrophs of the anterior
pituitary gland and regulates a wide variety of physiological
processes including growth and differentiation of muscle,
bone, and cartilage cells.
The use of hGH for the treatment of children with
impaired linear growth has been accepted as an important
therapeutic modality for many years. In addition, hGH
substitution in adults increases muscle mass by 5-10%,
but part of the effect is attributed to rehydration rather
than protein accretion [28-31]. One major issue regarding
the clinical use of hGH is its short half-life due to its rapid
clearance (

<

0.001) less efficient than once weekly
dosing of EPO-CTP.
An interesting observation from the present study was the
ability of a single injection once a week of EPO-CTP
(15 m g/kg) to increase the levels of hematocrit, whereas
the same effect was achieved by administration of the
same total dose of rhuEPO administered three times a

<

12 min) from the circulation. Previous studies