Problematic HCP
The leading cause of particle formation following enzymatic degradation
pathway is from a group of host cell proteins, which exhibit polysorbate
degradation activity. Lipoprotein lipase (LPL) hydrolyze ester bonds
within triglycerides to form alcohols and fatty acid molecules. Knockout
mutants of LPL reduce polysorbate degradation by 50% (Chiu et al.,
2017). In the development of mAb1, we observed elevated sub visible and
visible particles after long term storage at 2-8oC and
also showed high PS20 degradation over 4 weeks storage at ambient
temperature. LPL was detected in the bulk drug substance in parts per
billion (ppb) levels (data not shown). In our proteomic studies, LPL,
was found in all three culture processes (FB, SS, NSS). After the ProA
purification, LPL was only detected in the fed-batch process, suggesting
fed-batch culture process may be harboring higher LPL levels (Table 2).
The DDA method was not adequately sensitive to detect the lipases in
purified samples, such as ProA pool. Instead, the PRM method was used to
further detect low ppm levels of lipases in mAb1 process sample. The
data-acquisition method for PRM contains a list of peptides, which are
exclusively present only in proteins that are of interest to the
researcher, and their elution time window. After the data is acquired
and analyzed, the information on the abundance of peptides observed by
PRM is then used to estimate the abundance level of their corresponding
proteins. PS degradation was also reported during the long-term storage
of CHO-derived purified mAbs with detectable quantities of endogenous
lysosomal phospholipase A2 isomer X1 (LPLA2) (Hall et al., 2016) and
PLBL2 (Dixit et al., 2016). We used PRM method for targeted quantitative
analysis of peptide pairs to identify LPL, LPLA2 and PLBL2 (Table 2).
Compared from the FB process, both perfusion processes shows less
overall lipases and later removed or greatly reduced the quantity after
the ProA purification.
Peroxiredoxin 6 (Prdx6) is the uniquely 1-Cys member of the
peroxiredoxin family with calcium-independent phospholipase A2
activities (Shanshan et al., 2017). Phospholipases such as LPLA2, Prdx6
and phospholipase A-2 activating protein (PLAA) were detected in
fed-batch and NSS perfusion processes but not in SS perfusion process.
LPLA2, Prdx6 and PLAA were also detected in fed-batch intermediate
purification pool after ProA purification but not in the both perfusion
processes.
For long-term storage of mAbs formulated with PS, difficult to remove
lipases that were identified need to be further cleared during the
downstream purification. In order to evaluate the mAb1 product
stability, PS was analyzed using two different methods after 4 weeks of
storage at 25oC. Fluorescence micelle assay (FMA) was
utilized for PS quantification and 4-methylumbelliferyl decanoate
(4-Mud) assay for lipase activity. Table 2 summarized the PS
quantification, lipase activity and its relative abundance in different
processes samples. In HCCF, PS degraded from 400 ppm at day 0 to
approximately 20 ppm after 4 weeks of incubation. This result was
expected due to the high HCP concentration in the cell culture fluid.
After ProA purification, PS content was measured to above 200 ppm for
both perfusion processes but less than 100 ppm for the fed-batch
process. Lipase activity also shows great differences between perfusion
and fed-batch processes after the ProA purification. Combining all the
data collected in this study lead to the fact that fed-batch samples
contained more lipases involved in polysorbate degradation and particle
formation compared to both perfusion processes. These lipases were not
removed by chromatography methods.