RECOMBINANT ALBUMIN FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 165

and diastolic blood pressure and provided a sustainable
reduction in arterial pressure for more than 2 days. When
given at a dose of 6 nmol/kg, albumin-BNP increased cGMP
levels 5.6-fold compared to baseline. Notably, the elimina-
tion half-life was 3min for wild-type BNP and much longer
at 12-19 h for albumin-BNP. Other studies have shown that
the renal-enhancing properties of albumin-BNP are pro-
longed in animal models [17]. Compared to wild-type
BNP, the fusion protein provided sustained improvements
in markers of BNP activity, natriuresis, diuresis, renal blood
flow, and glomerular filtration rate. It also reduced cardiac
filling pressure compared to BNP. The development of a
long-acting BNP could have important implications in the
management of CHF, since it would allow for long-term use
in earlier stages of CHF, the postacute follow-up phase, and
following myocardial infarction [28].

and was markedly higher than that of BARH 6 (0.15 days).
Thus, albumin fusion appeared to slow the clearance of
barbourin without interfering with its platelet-aggregating
effects [33].

10.3.2.8 Hirudin Hirudin is a small protein (65 amino
acids) derived from leeches and is a potent and direct
inhibitor of thrombin [69]. Recombinant forms of hirudin,
such as desirudin and lepirudin, are used to inhibit thrombus
formation. Hirudin increases the risk of hemorrhagic com-
plications, however, and this narrow therapeutic window has
limited its clinical use. The small size and compact structure
of hirudin leads to rapid clearance from the circulation, and
these pharmacokinetic properties present further clinical
challenges [70].
Initial attempts to create albumin-hirudin fusion proteins
indicated that the biological activity of the fusion protein
depended on the way the fusion protein was constructed
[40]. Fusion of albumin to the C-terminus of hirudin with a
linker separating the two moieties (termed HLA for hirudin
linked to albumin) produced a fusion protein that inhibited
thrombin, whereas the reverse order, in which albumin was
attached to the N-terminus of hirudin (termed ALH for
albumin linked to hirudin), did not inhibit thrombin, indi-
cating that a free N-terminus is necessary for hirudin
activity. In rabbits, HLA prolonged the half-life by more
than twofold compared to hirudin and retained potent anti-
coagulant and antithrombotic effects [40]. As with its prede-
cessor, however, the HLA fusion protein also increased the
risk of bleeding, and the use of lower doses in combination
with antiplatelet

10.3.2.6 Erythropoietin Recombinant human erythro-
poietin (rhEpo) is used to treat anemia and has a half-life
of 4-13 h when given intravenously. This short half-life
necessitates 2-3 doses weekly and produces excessive
peak/trough concentrations [38]. Subcutaneous administra-
tion is associated with slightly better maintenance of plasma
concentrations, but still necessitates dosing at equal frequen-
cies each week. Joung et al. [38] fused recombinant albumin
to erythropoietin using a flexible linker (GGSGG) 4 to protect
against cross-interaction between the two proteins. The
fusion protein, rHA-EPO, was expressed in mammalian
cells (CHO) and purified from culture supernatant using
chromatography. The fusion protein had a similar half-life to
that of darbepoetin, a glycosylation variant with increased
half-life, when given intravenously in rodents. A cleavage
site was found in the linker sequence, and the formulation
was modified to include a stabilizer, which prevented 80% of
cleavage after 1 month. In terms of biological activity, rHA-
EPO was nearly eightfold more potent than was rhEpo in
increasing hematocrit in normal mice.

therapy only moderately reduced this

effect [71].
The stable but inactive ALH fusion protein was then
reconfigured to include a linker sequence that contained a
plasmin cleavage site [69]. With this configuration, hirudin
is liberated from albumin in the presence of plasmin, whose
activity is tightly restricted to the site of thrombus and/or
hemostatic plug formation. When tested in vivo, this fusion
protein was found to have an elimination profile similar to
that of albumin within the 96 h observation period and no
hirudin activity until it was liberated by plasmin, after which
it inhibited thrombin in solution and bound to fibrin clots.
Notably, the fusion protein did not promote bleeding in
mice, indicating that this approach may overcome one of the
major disadvantages of recombinant hirudin. Compared to
other measures to inactivate hirudin by altering its N-termi-
nus, the use of albumin appears to shield hirudin more
effectively, thereby reducing bleeding risk [71].

10.3.2.7 Barbourin Barbourin is a 73-amino acid pro-
tein that inhibits platelet aggregation by binding to the
platelet integrin a IIb b 3 [33]. It is found in the venom of
the southwestern US pigmy rattlesnake Sistrurus miliarius
barbouri and inspired the rational design of the peptide-
based antiplatelet drug eptifibatide. Because barbourin is
rapidly cleared from the circulation, a fusion protein of
rabbit serum albumin (RSA) and recombinant His 6 -tagged
barbourin (BARH 6 ) was developed (BLAH 6 ) [33]. The
P. pastoris expression system was used to produce
BARH 6 , RSAH 6 , and the fusion protein BLAH 6 . The fusion
protein BLAH 6 had inhibitory effects on platelet aggrega-
tion in vitro comparable to that of BARH 6 using both human
and rabbit platelets, and both proteins formed a complex
with a IIb b 3 . In rabbits, the terminal half-life of BLAH 6
approached that of RSAH 6 (3.4 and 4.0 days, respectively)

10.3.2.9 Factor VII Hemophilia A is a hereditary bleed-
ing disorder characterized by a lack of coagulation factor
VIII (FVIII). Replacement therapy with FVIII products helps
to reduce bleeding complications, improve quality of life,
and allow hemophilia patients to undergo major surgery [10].