Biomedical Engineering Reference
In-Depth Information
weight above the clearance limit of the kidneys may be
relevant.
The extraordinarily long half-life of albumin makes it an
attractive fusion partner to improve the pharmacokinetics of
therapeutic proteins. Given that albumin is a naturally
occurring protein, that is widely distributed and has no
enzymatic activity, it is unlikely to be toxic or immunogenic
[9,10]. The structure of albumin has been well characterized
and thus helps to predict how it may interact with other
protein moieties [10]. These known properties have allowed
recombinant albumin to be fused successfully to both small
peptides and large, complex molecules such as cytokines [2]
and coagulation factors [11,12]. In addition, recombinant
albumin fusion proteins are technically easier and more
efficient to produce than other fusion proteins involving
the Fc fragment of IgG. Many recombinant albumin fusion
proteins can be manufactured efficiently in yeast expression
systems on a commercial scale and at typically lower costs
than other methods of generating long-acting biopharma-
ceuticals [13]. Finally, because only monomeric fusions are
possible, dosing is simplified [13].
Recombinant albumin fusion proteins can be produced
with single-step expression techniques, making production
and purification relatively straightforward and economical
[14]. This avoids the need for complicated chemical modi-
fications during the manufacturing process [14]. In most
cases, fusion protein technology yields a homogenous prod-
uct, and fusion to naturally occurring and abundant proteins
such as albumin can improve the solubility and stability of a
therapeutic protein while reducing renal clearance [2] and
intracellular degradation. Finally, recombinant fusion pro-
tein technology allows genes to be fused in any order,
making it possible to construct multiple variants of the
same fusion protein; this design flexibility can help optimize
the biological activity of the product [2].
Theoretically, fusion to recombinant albumin could allow
for less frequent administration of a therapeutic protein
while maintaining the same efficacy. Fusion may also
lead to more consistent and sustained protein concentrations,
thereby improving tolerance and safety by maintaining
blood levels within the therapeutic window. The improved
pharmacokinetics of the therapeutic protein often means that
fewer administrations can be used to achieve the same effect.
These features can make therapy more convenient for both
clinicians and patients, leading to improvements in treat-
ment compliance. In addition, the reduced dosing frequency
and improved safety profile may enable prophylaxis in areas
where this was previously unfeasible.
part on the specific nature of the therapeutic protein in
question. In general, cDNA sequences encoding recombi-
nant human albumin (rHA) and the therapeutic protein are
isolated using standard laboratory techniques and fused in
a plasmid vector. The recombinant albumin fusion tech-
nology permits the creation of various orientations that
provide design simplicity and flexibility. The albumin
molecule has two-preferred sites, particularly the C- and
N-termini, where proteins can be attached. Additionally, it
is possible to fuse two different proteins together with
recombinant albumin to give two different functions
(bivalent) at the same time. rHA can be fused with biologi-
cally active compounds of varying sizes, from small
peptides to large cytokines. Moreover, the two genes
can be directly joined together or separated by a linker
to maximize the interaction between the therapeutic pro-
tein and its target molecule(s). Some linker sequences
are cleavable and can therefore separate the two protein
moieties under specific conditions.
The cDNA construct is created in a prokaryotic system
and then used to transfect eukaryotic cells such as Pichia
pastoris [2,15]. Although rHA can be expressed in both
prokaryotic and eukaryotic systems [6], eukaryotic sys-
tems are more efficient in producing the final albumin
structure [15]. P. pastoris is often used because, compared
to other eukaryotic expression systems, it is economical
and readily scaled-up for mass production [16]. For pro-
teins with complex posttranslational modifications, how-
ever, the use of animal or human cell lines is mandatory.
During fermentation, the fusion gene is transcribed and
translated as a single polypeptide, that is secreted into the
fermentation broth. The broth can then be separated
and clarified by centrifugation or filtration, and the protein
can be purified using standard separation techniques
[15,17-20].
10.3 TYPICAL APPLICATIONS AND
INDICATIONS
Recombinant albumin fusion technology has been success-
fully applied to various proteins and peptides with clinical
applications in a broad range of diseases (Table 10.1).
Three recombinant albumin fusion proteins are in late-
stage clinical development, demonstrating that albumin
fusion technology is feasible to improve the pharmaco-
kinetics of therapeutic proteins while retaining their bio-
logical activity. Numerous other recombinant albumin
fusion proteins are in preclinical or early-stage clinical
development; the variety of protein partners fused to
recombinant albumin and the many different diseases
for which these proteins are being developed are indicative
of the broad spectrum of potential applications of recom-
binant albumin fusion proteins.
10.2 TECHNOLOGICAL ASPECTS
Several methods have been used to generate recombinant
albumin fusion proteins, and the specific methods depend in
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