ALBUMIN-BINDING FUSION PROTEINS IN THE DEVELOPMENT OF NOVEL LONG-ACTING THERAPEUTICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 175

being developed, for example, other albumin-binding moie-
ties, including camelid antibody domains (nanobodies),
small molecules and peptides, and addition of other poly-
mers besides PEG, for example, sialic acid (polysialylation),
Pro-Ala-Ser containing sequences (PASylation), and non-
repetitive sequences containing residues Ala, Glu, Gly, Pro,
Ser, and Thr (EXTEN technology).
In this chapter, the relative merits and challenges associ-
ated with the use of AlbudAb technology are discussed and
compared with clinically validated half-life extension plat-
forms such as PEGylation and serum albumin fusion. The
basic concepts behind other half-life extension technologies
currently in development are also discussed and their rela-
tive advantages and challenges compared with AlbudAb
technology highlighted.

group of surface exposed lysine residues. This approach
requires no modification of the recombinant protein prior to
PEGylation, and the availability of surface exposed lysine
residues means recombinant proteins can be PEGylated with
high efficiency using this method. However, because of the
presence of multiple residues available for PEGylation, a
high degree of heterogeneity is often observed in reaction
products following the use of this type of chemistry, which
can present further downstream processing challenges. In
certain circumstances, addition of PEG chains to randomly
distributed lysine residues can also impact negatively on the
biological activity of the molecule. Site-specific chemistries,
in which thiol-reactive PEG molecules such as maleimide-
PEG are attached to reactive cysteine residues, provide an
alternative approach and result in much more homogenous
reaction products. However, this approach requires the
presence of a free cysteine, which is not found in a large
number of recombinant proteins. Site-directed mutagenesis
can be used to engineer the addition of a reactive cysteine in
recombinant proteins [6] although changing the amino acid
sequence of the therapeutic protein in this manner may
present additional risk in terms of adverse effects on activity
and introduction of immunogenicity, which can in turn
impact development costs and timelines.
In summary, the applications of PEGylation technologies
such as those described earlier have resulted in the develop-
ment of therapeutics with improved pharmacokinetics for
use in the treatment of a variety of diseases; however, the
process challenges associated with production of PEGylated
proteins have led to the development of alternative half-life
extension technologies, which is discussed here.

11.2 CLINICALLY VALIDATED HALF-LIFE
EXTENSION TECHNOLOGIES—AN OVERVIEW

11.2.1 PEGylation

Polyethylene glycol (PEG) is a polyether compound used in
a variety of industrial applications. Owing to its high solu-
bility and low toxicity, it can be attached covalently to
therapeutic proteins using a range of chemistries. This
has the effect of increasing the molecular size of the
PEGylated protein, thereby delaying the rate of renal clear-
ance significantly [1]. PEGylated proteins have been used as
therapeutic agents in the treatment of a variety of human
diseases over the last 30 years, for example, PEGylated
interferon- a (IFN- a ) (Pegasys 1 and Pegintron 1 ) in the
treatment of chronic hepatitis C virus (HCV) infection
[2,3], PEGylated granulocyte colony stimulating factor
(Neulasta 1 ) used as an immunostimulator in patients under-
going chemotherapy [4], and PEGylated asparaginase
(Oncaspar 1 ) used in the treatment of acute lymphocytic
leukemia and non-Hodgkin's lymphoma [5]. PEGylation of
human IFN- a 2b (Pegintron, Scherring Plough) by attach-
ment of 12 kDa linear PEG moieties results in a preparation
consisting of predominantly mono-PEGylated IFN- a 2b
with multiple positional isomers present. Biological activity
is comparable to the non-PEGylated Intron-A 1 protein, and
PEGylation in this manner increases the serum half-life in
humans to

11.2.2 Serum Albumin Fusion

Human serum albumin (HSA) is the most abundant protein
in the serum, functions in the regulation of colloid osmotic
pressure in the circulation, and acts as a carrier protein in the
transport of fatty acids, hormones, bilirubin, and cations [7].
Synthesized in liver, the mature 585 amino acid polypeptide
of 67 kDa has a circulating half-life of greater than 20 days

in man. The exceptionally long half-life of serum albumin is
due in part to its size (it is above the 60 kDa threshold for

renal clearance) but also due to protection from intracellular
degradation in the lysosome. Serum albumin is pinocytosed
nonspecifically by the cell and accumulates in the early
endosomal compartment. Owing to the low pH environment
found within the early endosome, serum albumin is able to
bind to the MHC-related receptor for IgG (FcRn), which
facilitates recycling back to the cell surface, releasing
albumin into the extracellular space [8]. Serum albumin
is thus able to avoid the alternative fate of lysosomal
degradation undergone by proteins, which traffic from the
endosome to the lysosome in the absence of FcRn binding.
This FcRn-mediated “recycling” receptor is also involved in

40 h (compared with 7-9 h for non-PEGylated
IFN- a 2b) and enables once-weekly dosing schedules of
PEGylated IFN to replace the once-daily or three times
weekly dosing required with the non-PEGylated form.
Owing to their improved pharmacokinetics, which translates
directly into improved clinical efficacy, PEGylated IFN
molecules in combination with the nucleoside analog Riba-
virin now form the current standard of care in the treatment
of chronic HCV infections. As in the example provided
earlier, PEG is most commonly attached to the -amino

e