Biomedical Engineering Reference
In-Depth Information
10-fold higher activity in the presence of PSA-producing
prostate cancer cells [70].
Other mutagenesis approaches are directed to the presence
of disulfide bridges. Cysteins can be added, removed, or
repositioned. In many cases, the introduction of disulfide
bonds improves stability. Heterodimers between V
H
and
V
L
of antibody fragments are relatively unstable but can be
stabilized by adding a cystein in each of the fragments to
generate a disulfide bridge. Although there are two possible
positions, only one locus retained the full binding activity
[71]. Even the rearrangement of existing disulfide bridges is
possible as demonstrated with erythropoietin (EPO) where a
cystein was moved fromposition 33 to 88. This modified EPO
in fusion to an Fc part exhibited superior dimerization capa-
bilities, better glycosylation stability and improved phar-
macokinetic properties [72]. Stabilization by introducing
disulfide bridges can also suppress immunogenicity as shown
with an immunotoxin that was mutagenized in the domain III
of Pseudomonas exotoxin A [73]. However in some cases the
presence of an unpaired cystein can cause aggregation prob-
lems. A fusion protein between IFN-
a
2b and HSA (IFN-
a
2b-
HSA) aggregated and caused immunogenicity issues. Only
replacing the free cystein by serine abolished the effect,
leading to a more stable and less immunogenic protein
[74]. Sometimes disulfide-induced dimerization has multiple
effects. The Fab-PE38 dimer with PE38 fused to the light
chain and linked through a disulfide bridge in the hinge region
had a 16-fold higher refolding yield and 2.5-fold better
activity than the initial monomer [75].
Not only free cysteins can cause aggregation, much more
frequent is the exposure of hydrophobic residues at protein
surfaces that leads to aggregation. In a systematic study,
these critical positions and their respective ideal aa of scFvs
were identified. Two positions on the V
H
(82, 85) and also on
the V
L
(36, 60) were found, whose replacement with more
ideal aa resulted in a significant improvement of stability and
yield [76]. A similar approach with another scFv revealed
three other heavy chain and one light chain residue that
contained not conserved aa. Triple V
H
mutants resulted in
eightfold higher yields that could be increased to 20-fold,
when the orientation was reversed. Interestingly, the muta-
tions did not have a negative effect on binding affinity, but
improved plasma stability [77]. Aggregation can also occur
by the lack of glycosylation when proteins are expressed in
E. coli. In the case of erythropoietin (EPO) this was coun-
teracted by replacing asparagine residues with lysine. Due to
the increased isoelectrical point (pI) the protein became
positively charged under physiological conditions thus elim-
inating aggregation [78].
Mutagenesis can also have an effect on the activity profile
of proteins. For instance, the replacement of the plasmino-
gen activator inhibitor-1 binding site in Tenecteplase
1
by
exchanging four aa resulted in a significantly longer activity,
because the inhibitor could no longer bind [79].
1.5 MANUFACTURING
The first recombinant protein product, insulin, was initially
produced as two separate chains that were conjugated
chemically. But soon thereafter the commercial process
utilized expression in E. coli and subsequent enzymatic
maturation to generate insulin from proinsulin [80]. Since
that time more than 120 therapeutic proteins, including a
number of fusion proteins, have been manufactured for
human use in bacteria, yeasts, or animal cells [81]. From
a manufacturing perspective in most cases it is not necessary
to differentiate between fusion proteins or regular singular
therapeutic proteins. The production typically covers three
complex steps: upstream processing (molecular biology and
fermentation), downstream processing (capture and purifi-
cation), and finally formulation (transforming the protein
into a storable and administrable form). One of the many
advantages of fusion proteins is the uninterrupted manufac-
turing process of a single protein molecule with several
functions.
1.5.1 Upstream Process
The choice of the expression system depends heavily on the
properties of the desired protein product such as glycosyla-
tion, disulfide bridges or other post-translational modifica-
tions that can only be obtained from eukaryotic cells [82].
Also protein size plays a role; usually, proteins larger than
100 kDa are by default produced in eukaryotic cells, whereas
proteins below 30 kDa are expressed in bacteria. A recent
analysis revealed that 39% of recombinant proteins were
expressed in E. coli, 35% by Chinese hamster ovary (CHO)
cells, 15% by yeasts, and 10% by other mammalian cells, but
only 1% by other systems [83]. Interestingly, 17 of the 58
approved therapeutic proteins between 2006 and 2010 were
manufactured in E. coli [84]. The following paragraph
focuses on the two major host organisms, E. coli and
CHO cells, while Pichia pastoris has been primarily used
for albumin and transferrin fusion proteins [85]. Secreted
Fc-fusion proteins in P. pastoris require individual optimi-
zation of upstream conditions [86].
The modern upstream process consists of three elements:
the expression construct, the host cell, and the cultivation
conditions. The expression construct in microbial expression
is mostly a plasmid vector coding for a resistance gene as
selection marker, the gene for the protein to be expressed,
both accompanied by the respective promoter sequences,
and a replication origin to maintain the plasmid during cell
division [87]. It is generally recommended to optimize the
codon usage according to the selection of the host organism.
Usually, microbial cells are transfected and kept as clonal
glycerol stock to serve as master cell bank. Alternatively, the
expression cassette can also be integrated into the host
genome to create a stable transfected cell. This is the
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