Figure Legends
Figure 1. (I) Chemical structure of major expected POE esters in polysorbates, the polysorbates are mainly composed of fatty acid esters sharing common sorbitan or isosorbide head group. Lauric acid is the main fatty acid for PS20 and oleic acid is the main fatty acid for PS80. (II) CAD chromatography of PS20 standard (A) and PS20 in mAb formulation (B) separated and detected by 2D-LC-CAD, (III) Representative total ion current (TIC) profile of PS20, with major peaks labeled as POE sorbitan monolaurate (1), POE isosorbide monolaurate (2), POE sorbitan monomyristate (3), POE isosorbide monomyristate (4), POE isosorbide monopalmitate (5), POE isosorbide monosterate (6), POE sorbitan mixed diesters (7-9), POE sorbitan trilaurate and POE sorbitan tetralaurate (10). (IV). CAD chromatography of PS80 standard (A) and PS80 in mAb formulation (B) separated and detected by 2D-LC-CAD (V). Representative total ion current (TIC) profile of PS80, with major peaks labeled as POE sorbitan monolinoleate (1), POE sorbitan monooleate (2), POE isosorbide monooleate and POE monooleate (3), POE sorbitan di-oleate (4), POE isosorbide di-oleate and POE di-oleate (5), POE sorbitan mixed trioleate and tetraoleate (6).
Figure 2. Western blot of PLBD2, Lane 2 is 40 ug mAb-1 containing PLBD2, lane 4 is 10ng PLBD2 purchased from Origene, lane 5 is 10ng CHO PLBD2 tagged with mmHis tag (made in-house).
Figure 3. (I) Chromatogram of 0.1% PS20 in 200 µg/mL commercial PLBD2 spiked in 150 mg/mL mAb-3 incubated @ 45 ºC in 10mM Histidine, pH6 for for 0 day (A, T0), and 5 days (B, T5). (II) Chromatogram of 0.1% PS20 in 200 µg/mL CHO PLBD2 spiked in 150 mg/mL mAb-3 incubated @ 45 ºC in 10mM Histidine, pH6 for for 0 day (A, T0), and 5 days (B, T5). (III) Chromatogram of 0.1% PS80 in 200 µg/mL commercial PLBD2 spiked in 150 mg/mL mAb-3 incubated @ 45 ºC in 10mM Histidine, pH6 for for 0 day (C, T0), and 5 days (D, T5). (IV) Chromatogram of 0.1% PS80 in 200 µg/mL CHO PLBD2 spiked in 150 mg/mL mAb-3 incubated @ 45 ºC in 10mM Histidine, pH6 for for 0 day (C, T0), and 5 days (D, T5).
Figure 4. (I) Upper panel: Chromatogram of 0.1% PS20 in 75 mg/mL mAb-2 (generated by PLBD2 knockout cell line) incubated @ 45 ºC in 10mM Histidine, pH6 for 0 day (A, T0), and 5 days (B, T5). The percentage of PS20 degradation is calculated using equation\(\%PS20=1-\frac{\int_{27.5}^{33}{I\left(t\right)\text{dt}}@T5}{\int_{27.5}^{33}{I\left(t\right)\text{dt}}@T0}\), I(t) is the intensity of the peak at any time point. Lower panel: Chromatogram of 0.1%PS80 in 100 mg/mL mAb-2 incubated @ 45 ºC in 10mM Histidine, pH6 for 0 day (C, T0), and 5 days (D, T5). The percentage of PS20 degradation is calculated using equation\(\%PS80=1-\frac{\int_{30}^{35}{I\left(t\right)\text{dt}}@T5}{\int_{30}^{35}{I\left(t\right)\text{dt}}@T0}\), I(t) is the intensity of the peak at any time point. (II). PLBD2 knockout showed similar or higher lipase activity for PS20 and PS80 as control cell line. Upper panel: comparison of 0.1% PS20 degradation incubated with 75 mg/mL mAb-1 generated from control cell line at pH 6.0 (A) and 75 mg/mL mAb-2 generated from PLBD2 knockout cell line at pH 6.0 (B); Bottom panel: comparison of 0.1% PS80 degradation incubated with 100 mg/mL mAb-1 generated from control cell line at pH 6.0 (C) and 100 mg/mL mAb-2 generated from PLBD2 knockout cell line at pH 6.0 (D); (III). Western blot of PLBD2 in mAb-1 and mAb-2 PLBD2 knockout cell line. Lane 2 is 40ug mAb-1 and lane 3 is 40ug mAb-2 generated by PLBD2 knockout cell line.
Figure 5. Schematic diagram of the PLBD2 depletion experiment. Dynabeads magnetic beads were covalently coupled with Anti-PLBD2 monoclonal antibody and used for immunoprecipitating (IP). The original mAb (A) and flow through (B) were incubated with 0.1% PS at 45 ºC for 5 days and subject to PS degradation measurement. A non-relevant antibody was served as the negative control by replacing anti-PLBD2 monoclonal antibody (C).
Figure 6. (I) Western blot of PLBD2, Lane 1 is Mw standard, Lane 2 is 40 µg mAb-1 alone, lane 3 is 40 µg mAb-1 with PLBD2 being depleted completely, lane 4 is 40 µg mAb-1 with PLBD2 being partially depleted and lane 5 is 40 µg mAb-3 containing no PLBD2. (II) The percentage of PS20 remaining in original mAb-1, PLBD2-completely depleted mAb-1 and PLBD2 partially depleted mAb-1 are plotted against incubation time. The original mAb, PLBD2 completely-depleted mAb and PLBD2 partially depleted mAb are indicated by filled circle with solid line, filled diamond with dashed line and filled triangle with dotted line. (III) The percentage of PS80 remaining in original mAb-1, PLBD2-completely depleted mAb-1 and PLBD2 partially depleted mAb-1 are plotted against incubation time. The original mAb, PLBD2 completely-depleted mAb and PLBD2 partially depleted mAb are indicated by filled circle with solid line, filled diamond with dashed line and filled triangle with dotted line.
Figure 7. Left panel: calibration curve of two selected peptides YQLQFR (filled square) and SVLLDAASGQLR (filled circle) from recombinant CHO PLBD2 with mAb-3 as matrix. Right panel: correlation curve between remaining PS20 percentage and PLBD2 concentration. PLBD2 concentration were quantitated by MRM-MS using the calibration curve (SVLLDAASGQLR) in the left panel. The percentage of PS20 remaining was determined by using LC-CAD after 0.1% PS20 was incubated with various mAbs (filled circles, 75 mg/mL) at 45 ºC for 5 days.