Biomedical Engineering Reference
In-Depth Information
plasma after incubation at 37
C. The results confirmed that the acidic isoforms were due
to deamidation and identified the primary site of deamidation as Asn-387, which is
located on the H37 peptide of the heavy chain in the Fc region (Table 4.5). The samples
were also characterized by densitometry of the native IEF gels and by analysis of binding
and biological activity. Native IEF was performed on the same samples and the ratios of
band 1 and band 2 ODs were determined by densitometry. The “OD ratios” from native
IEF gels represent the level of deamidation as the ratio of optical density values
determined by densitometry for the major product band (represented by band 1) and
acidic isoform bands (represented by band 2). Lower OD ratio values indicate increased
proportions of the acidic isoforms and, therefore, increased deamidation levels and vice
versa. Total deamidation of motavizumab ranged from 25.4% at the initial time point to
77.2% after 5 weeks of exposure to human plasma at 37
C.
Deamidated motavizumab (up to 77.2% deamidation) exhibited F protein binding
activity, as measured by ELISA and SPR, and was able to neutralize RSV. For these
assays, the variability was greater than typically observed in lot release, characterization,
and stability testing because the antibody isolated from the human serumwas at very low
concentrations. For SPR, the k
off
was evaluated because it was the best indicator of a
change in the ability to bind to either F protein because the SPR off-rate is independent of
protein concentration. All k
off
values were comparable to each other and to the initial
value (Table 4.5). To overcome the limitations of the low protein concentration in the
plasma incubation study, a control study was conducted by incubating higher protein
concentration motavizumab samples at 37
C in pH 8.5 buffer for a period of up to 5
weeks (Table 4.6). Deamidation sites were confirmed to be the same as described earlier
for the human plasma incubation site. In this study, binding activity was observed for
deamidation levels of up to 79.0%. Hence, the buffer-incubated deamidated preparation
was subsequently used in the nonclinical PK and bioactivity studies described later.
In the plasma incubation study, OD ratios as low as 0.25 (with a pI of greater than
8.65) resulted in some bioactivity. In the buffer incubation study, a similar level of
deamidation (as determined by peptide mapping) resulted in an OD ratio of 0.50 (with a
pI of greater than 8.65) and no reduction in binding to F protein. Because there is
variability in the OD ratio at high deamidation levels, an OD ratio of 0.50 is presently
designated as the lower OD ratio limit of the prior product knowledge for an acceptable
level of deamidation.
The results of the incubation studies support the conclusion that the deamidation
occurs naturally in human plasma. For OD ratios greater than 0.5 and pI values greater
than 8.65, deamidation does not impact motavizumab binding or biological activity.
N
ONCLINICAL AND
C
LINICAL
S
TUDIES
—M
OTAVIZUMAB
. The level of deamidation in
motavizumab produced under typical manufacturing conditions is approximately 20%,
and it increases upon incubation in plasma. Motavizumab deamidated by incubation in
buffer at pH 8.5was evaluated in a cotton rat PK study. Deamidatedmotavizumab (1- and
5-week incubation), as well as unmodified motavizumab, was administered to groups of
rats. Motavizumab levels were measured in serum (Fig. 4.22), BAL (Fig. 4.23), and lung
homogenates (Fig. 4.24). The results showed no differences in the levels of motavizumab
compared to deamidated motavizumab in serum, BAL, and lung.
