Depletion of PLBD2 Does Not Decreases Level of PS20
Degradation
To further examine whether PS degradation is caused by the presence of
PLBD2 in the formulated mAbs, a PLBD2 depletion experiment was
performed. If PS20 degradation is caused by PLBD2, its depletion should
result in diminished PS20 degradation. The relative extent of
degradation should also depend on how much PLBD2 has been removed. While
the knockout experiment is complicated by the possibility of additional
lipase being upregulated in the PLBD2 knockout cells, the depletion
experiment may provide a more direct answer as to the potential role of
PLBD2 in PS degradation. Figure 5 shows the schematic workflow of the
depletion experiment. An antibody against CHO PLBD2 was covalently
coupled to Dynabeads for depletion of PLBD2. Specificity of the the
anti-PLBD2 antibody used in this experiment was evaluated by Western
blot, demonstrating that all three forms of PLBD2 present in mAb-1
including proenzyme at 64 kDa, mature protein at 40 k Da and prodomain
at 28 kDa were recognized by the anti-PLBD2 antibody (Figure 6-I lane
2). PLBD2 in mAb-1 sample can be partially (Figure 6-I lane 4) or
completely depleted (Figure 6-I lane 3) by adjusting the ratio of
anti-PLBD2 to mAb-1 sample during depletion. Complete depletion of PLBD2
in mAb-1 was performed by incubating 10 mg mAb-1 with 50 µg anti-PLBD2
conjugated magnetic beads while partial depletion of PLBD2 in mAb-1 was
achieved by incubating 10 mg mAb-1 with 10 µg anti-PLBD2 conjugated
magnetic beads. The percentage of PLBD2 depletion from mAb-1 was
estimated by Western blot. Antibody mAb-3 which is PLBD2 free (Figure
6-I Lane 5) served as the negative control.
Prior to PLBD2-depletion, mAb-1 sample exhibited approximately 28.0% of
PS20 degradation after 5 day incubation at 45°C. After PLBD2-depletion,
the PS degradation was 23.2% for partially and 27.7% for the
completely depleted sample, respectively (Figure 6-II). Similar results
were observed for PS80 degradation performed under the same conditions
(Figure 6-III), with 18.9% PS80 degradation observed prior to
depletion, 19.32% and 20.8% PS80 degradation observed in partially and
completely depleted PLBD2 samples, respectively, after 5-day incubation
at 45°C. The lack of correlation between PLBD2 level in the samples and
the extent of PS degradation in this depletion study suggested PLBD2 was
unlikely the enzyme responsible for polysorbate degradation.