Biomedical Engineering Reference
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authors were able to produce Fab fragments of increased
molecular mass which were stable in serum (indicating that
the sialic acid chains would not be cleaved in vivo) and had
only a moderate (approximately fourfold) decrease in affin-
ity for antigen when compared to the unmodified Fab. Effect
of polysialylation on pharmacokinetics was determined in
mice and the circulating half-life of H17E2-derived Fab
shown to be increased by up to threefold following poly-
sialylation. Although chemical methods were used to couple
sialic acid chains in this study, the authors are also investi-
gating the use of engineered glycosylation motifs to allow
polysialylation of recombinant proteins during expression,
overcoming the requirement for further downstream proc-
essing [41].
Other investigators are also studying the use of geneti-
cally fused hydrophilic polymers as an alternative to PEGy-
lation. XL-protein has developed PASylation technology,
which involves genetic fusion of recombinant proteins to
polypeptide sequences containing the amino acids Pro, Ala,
and Ser (PAS), leading to an increase in molecular size
without affecting the biological activity of the therapeutic
protein (Skerra, unpublished work). PAS sequences adopt a
conformationally disordered structure under physiological
buffer conditions whose spatial dimensions are adjustable by
the length of the PAS repeat unit, thereby increasing the
apparent molecular size of the therapeutic protein above the
pore size of the renal glomeruli and reducing renal clearance
rates of PASylated proteins; in fact, these investigators show
that this can lead to a reduction of in vivo clearance rates of
between 10- and 100-fold compared to the unmodified
protein.
A similar half-life extension technology has also been
developed by Stemmer and colleagues, in which nonrep-
etitive amino acid sequences containing the residues Ala,
Glu, Gly, Pro, Ser, and Thr are genetically fused to proteins
and peptides, increasing their hydrodynamic radius and
delaying clearance in much the same way as PASylation
and PEGylation technologies. Fusion of an 864 amino acid
sequence, referred to as EXTEN, to the GLP-1 peptide
analog Exenatide increased the circulating half-life in rat
from 2.5 to 29 h. The Exenatide-EXTEN fusion protein also
displayed significantly improved biological activity in mice
compared to the free peptide, consistent with its improved
pharmacokinetic profile [42].
Although half-life extension technologies based on albu-
min-binding molecules other than single domain antibodies
are currently being developed, these technologies also have
potential limitations in addition to their advantageous prop-
erties. Albumin-binding peptides, which can be fused genet-
ically to therapeutic proteins and are much smaller than
immunoglobulin variable domains, are not based on natu-
rally occurring epitopes and it therefore remains to be seen
whether addition of such synthetic sequences could result in
immunogenic responses upon repeat dosing or when using
extended half-life formats. Peptide sequences used in this
manner may also be susceptible to proteolytic cleavage
in vivo.
Albutag technology, which is based on addition of chem-
ical groups with affinity for albumin, is unlikely to cause
problems with immunogenic responses but the chemical
coupling methods used to attach these albumin-binding
groups may be subject to the same downstream processing
issues associated with the production of PEGylated mole-
cules, in particular, heterogeneity of product or modifica-
tions in the amino acid sequence of the protein required to
develop site-specific coupling chemistries.
Novel half-life extension technologies, which are not
based on albumin-binding functionality, are also currently
being developed. The major advantage to such technologies
is the reduced requirement for downstream processing com-
pared to PEGylation, for example, glycosylation and PASy-
lation result in addition of hydrophilic polymers during
expression via recombinant means rather than chemical
coupling of products to PEG following expression and
purification. It remains to be seen how the resulting improve-
ments in half-life using these technologies compare with
those obtained using serum albumin binding strategies,
however.
11.6 CONCLUSIONS
Although PEGylation technologies have been the most
widely used method of improving the pharmacokinetics
of clinically relevant recombinant proteins over the last
30 years, the limitations associated with this technology,
in particular, the associated cost of downstream processing,
have led to the development of alternative half-life extension
technologies based around the use of serum albumin fusion
proteins. Although a number of recombinant HSA fusion
proteins are currently in late stage clinical development,
HSA fusion technology is also associated with a number of
process issues due to the large and complex nature of the
serum albumin molecule. This in turn has served as a driver
for the development of further half-life extension technol-
ogies based on the use of serum albumin binding molecules.
We have developed human immunoglobulin variable
domains that specifically bind to serum albumin (AlbudAbs)
as a means of extending the half-life of therapeutic mole-
cules to which they are fused. AlbudAbs with desirable
biophysical properties have been isolated, and using IFN as
an example of a therapeutic molecule with an inherently
poor half-life in man, we have used AlbudAb technology to
develop a molecule with therapeutic potential, which has an
extended half-life, improved in vitro potency and greater
in vivo efficacy in comparison to HSA-fused IFN. AlbudAbs
can also be expressed in a variety of bacterial, yeast, and
mammalian expression hosts, offering alternatives to the
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