Biomedical Engineering Reference
In-Depth Information
within the Fc portion of the molecule, while all the anti-Gal-
-1,3-Gal IgE binding to
cetuximab was shown to be directed to the highly exposed sugars in the Fab variable
region [12]; (3) typical N-linked oligosaccharides at Asn-297 of IgG antibodies, such as
in palivizumab and motavizumab, are sequestered within the Fc domain polypeptide
structure and are sterically hindered from interactions with other macromolecules [13];
(4) the presence of Gal-
a
-1,3-Gal glycoforms on the two Fab arms of the cetuximab
molecule could provide a means by which cross-linking of IgE on the surface of mast
cells could occur, resulting in a strong stimulus for the inflammatory and anaphylactic
response [12]. The low levels of Gal-
a
-1,3-Gal glycoforms within the Fc domains of
palivizumab and motavizumab would not be conducive to such cross-linking. (5) The
intravenous route of administration of cetuximab may be responsible for the rapid
hypersensitivity response; palivizumab andmotavizumab are delivered by intramuscular
injection.
Motavizumab and palivizumab have a small proportion of high mannose-type
structures (Man-5 and Man-6) and negligible proportions of the hybrid-type oligosac-
charides (Fig. 4.6). It is recognized that higher mannose forms (in glycoprotein
populations) may have a faster rate of clearance because of binding to the mannose
receptor [13]. However, the likelihood that the variations in these glycoforms in
motavizumab or palivizumab could affect PK is low, because the levels of Man-5 and
Man-6 are consistently low in manufactured lots.
In addition to MS, some peaks in the oligosaccharide profile were also identified by
spiking with 2-AB-labeled G0, G2, and Man-5 standards. Two positional isomers of
2-AB-labeled G1 were identified through sequential exoglycosidase digestion steps by
using
a
a
-N-acetylhexosaminidase and
a
-1-2,3 mannosidase [14], and the Gal-
a
-1,3-Gal
linkages were confirmed by
-galactosidase digestion [15].
A number of glycoforms were generated by enzymatic remodeling. Treatment with
a
a
-galactosidase was used to remove terminal galactose sugars from the G2 and G1 forms
and to generate an enriched G0 glycoform (74.4%). Treatment with
-galactosyltrans-
ferase and UDP galactose was used to extend both arms of the oligosaccharide chain and
to generate an enriched G2 glycoform (69.7%). The oligosaccharide side chain
was removed after treatment with PNGase F (deglycosylated motavizumab), and Fab
and F(ab
0
)
2
were generated after treatment with pepsin and papain, respectively [16, 17]
(Fig. 4.7).
Motavizumab variants were evaluated for F protein binding activity and RSV
microneutralization (Table 4.1). The results demonstrated that the N-linked oligosac-
charide side chain has no significant effect on binding activity to RSV F protein by
ELISA and surface plasmon resonance (SPR) or microneutralization, and, furthermore,
the Fc region is not required for RSV neutralizing activity.
These results are similar to those observed with palivizumab binding to F protein, as
shown in Table 4.2. All the palivizumab variants had comparable F protein binding
activity (by ELISA and SPR) as well as neutralizing activity, indicating that the
oligosaccharides side chains do not affect the in vitro functional activity.
Similar studies carried out byWu and coworkers [18] showed that both palivizumab
and a palivizumab F(ab
0
)
2
fragment could inhibit RSV replication in vitro with inhib-
itory concentration (IC
50
) values of 3.6 and 1.4 nM, respectively. This work further
a
