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.