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.