Amount of PLBD2 in Formulated mAb cannot be positively
correlated to PS20 loss over time
To further prove that PLBD2 does not contribute to polysorbate
degradation, we determined levels of PLBD2 in a number of formulated
mAbs. Two PLBD2 peptides (SVLLDAASGQLR and YQLQFR) were chosen to
quantitate PLBD2 in formulated mAbs using multiple reaction monitoring
mass spectrometry (MRM-MS) technology. CHO PLBD2 spiked-in mAb-3 was
used to create the calibration curve. Standard curves (10-500 ppm) with
coefficients 0.9965 and 0.9943 were generated for each of the peptide
(Figure 7, left panel), concentration of PLBD2 in each sample was then
obtained by extrapolating it peak area onto the curve. In total, six
mAbs were subjected to PLBD2 quantitation. Quantitative examination of
peak areas of these 6 mAbs determined the concentration of PLBD2 in the
formulated mAb to be between 0 to 230 ng/mg mAb.
PS20 degradation was then measured for the same 6 mAbs after each sample
was concentrated and buffer exchanged to 10 mM Histidine buffer, pH 6.
The percentage of the intact PS20 was plotted against PLBD2
concentration and correlation coefficient R2 was
calculated to evaluate the linear dependence of the two variables
(Figure 7 right panel). A slight downhill linear relationship with
calculated Pearson correlation coefficient of 0.0042 indicates no
correlation between these two variables, suggesting PLBD2 concentration
in drug substances is not correlated to the PS20 loss during incubation.
Among the samples tested, mAb-4 showed strong lipase activity with no
detectable level of PLBD2, indicating another lipase/esterase was
responsible for PS20 degradation in that drug substance. In a contrast,
mAb-6 had high concentration of PLBD2 but showed no lipase activity,
suggesting PLBD2 is unlikely the root cause of PS20 degradation.