Biomedical Engineering Reference
In-Depth Information
identified the best disulfide bond in terms of retention of
activity. After introducing the cysteine pair in the peptide
region to form the disulfide bridge that constrained the
peptide, we were able to eliminate protease cleavage, and
consequently, the fusion protein proved to be stable both in
vitro and in vivo while retaining the majority of its potency
(data not shown).
susceptibility. In this example, a substantial portion of the
protein-A purified MIMETIBODY protein was aggregated
as demonstrated by analytical High Performance Liquid Chro-
matography (HPLC) (Figure 8.3A). A series of ladder-like high
molecular weight bands was observed on an SDS-PAGE gel
under nonreducing conditions (Figure 8.3B, left lane), and
these bands collapsed to a single band under reducing condi-
tions (Figure 8.3B, right lane), strongly suggesting that the
aggregation is disulfide bond mediated. After incubating the
aggregates at
8.6.3 Improving Pharmacokinetic Profile
100
m
g/mL in a phosphate buffer with the
presence of thio-redox reagents (50 mM phosphate buffer,
pH 7.2, 1mM reduced glutathione, 0.1 mM oxidized glutathi-
one) at 4
C for 36 h, more than 50%of aggregatewas converted
to the monomer (data not shown), further indicating that
theaggregationiscausedbydisulfidebonds.Webelieved
that the misformed intermolecular disulfide bridges cross-
linked the proteins together to form oligomers as illustrated
in Figure 8.3, which migrated as the ladder-like bands observed
on SDS-PAGE (Figure 8.3B). These oligomers can further
associate to form larger aggregates as shown in HPLC analysis
(Figure 8.3A). We hypothesized that adding carbohydrates to
the flanking region of the peptide could prevent the formation
of interchain disulfide bonds due to steric hindrance as illus-
trated in Figure 8.4. To test this hypothesis, several MIMETI-
BODY constructs were made, in which N-glycosylation
sites were introduced near the peptide region. As expected,
the aggregation level of some of these constructs was
significantly reduced on the basis of size exclusion HPLC
analysis (Figure 8.3C) while retaining most of their activity
(data not shown).
As intended, peptides fused to Fc demonstrate significantly
improved PK profiles. For example, we have shown that
CNTO 530 has extended clearance kinetics in mice with a
terminal t
1/2
of
40 h compared to only several minutes for
the naked EMP1 peptide [92]. The clearance of CNTO 530
was comparable to the clearance of a humanized IgG4ala-
ala in mice [96], suggesting that the mechanism of clear-
anceforCNTO530maybesimilartothatofanantibody
and that CNTO 530 may also have extended pharmaco-
kinetics in humans. It suggested that protease cleavage
may not play a significant role in the clearance of CNTO
530. The Phase I clinical trial of CNTO 528 showed that
the mean circulating half-life reached 7.6 days in humans
at high doses (0.9mg/kg) [92]. Despite the fact that
peptide-Fc fusions have a prolonged serum half-life, care-
ful protein engineering, especially at the peptide and Fc
junction region, can affect the protein PK profile. This
junction region is flexible and probably accessible to prote-
ases. For example, we were able to eliminate the protease
cleavage sites by reengineering the junction region between
EMP1 and Fc in the CNTO 528 construct. A rat PK study
showed improvement of the mean terminal serum half-life of
the new construct from 1.54 days to 3.03 days [95]. The
improvement of in vitro activity and serum half-life results in
significantly higher in vivo potency in rat [92].
8.6.5 Reducing Heterogeneity
Protein heterogeneity is another potential challenge for drug
development simply because it puts a huge burden on the
protein purification process. Many factors, such as protease
cleavage, posttranslational modification, and oligomeriza-
tion, can cause protein heterogeneity. For example, one of
our MIMETIBODY proteins migrated on the SDS-PAGE as
multiple but very close bands under reducing conditions.
Mass spectrum analysis revealed that these bands have an
8.6.4 Improving Solubility and Reducing Aggregation
It
is highly desirable to formulate protein therapeutics at
100mg/mL to have a small enough volume for subcutaneous
administration. However, some peptides have a tendency to
form oligomers and induce aggregation, especially at high
concentration, which can cause decreased activity, decreased
bioavailability, and increased immunogenicity in vivo [97].
Although hydrophobic interactions were shown to be the
predominant reason for protein aggregation, there are
many other possiblemechanisms. Aggregates may be classified
in different ways, including soluble/insoluble, covalent/
noncovalent, reversible/irreversible, and native/denatured
[98]. Understanding the causes of aggregation and then engi-
neering around it is the approachwe regularly use. For example,
one of our MIMETIBODY constructs contained a metaboli-
cally labile peptide, in which a pair of cysteines was introduced
to constrain the peptide by a disulfide bond to reduce protease
>
900 Da difference in molecular weight, which we hypoth-
esized to be the O-linked carbohydrates populating to
varying extents the flexible hinge region. Using site-directed
mutagenesis, we eliminated the potential O-linked glyco-
sylation sites, and the multiple bands are reduced to a single
band as shown in Figure 8.5.
In another interesting example, we found that sometimes
the signal peptidase can potentially act at alternative cleav-
age sites to generate heterogeneity. Mass spectrometry and
amino acid sequencing analysis revealed that a small portion
of one of our MIMETIBODY proteins missed three amino
acids at the N-terminus of one chain but not the other chain.
Interestingly, after a series of experiments, we found that the
Search WWH ::
Custom Search