PS20 and PS80 Degradation Pattern with human PLBD2 and CHO
PLBD2
Polysorbates degradation by recombinant human PLBD2 or CHO PLBD2 was
monitored as outlined in the Material and Methods section. Recombinant
human PLBD2 (Figure 3-I, III) and the CHO PLBD2 (Figure 3-II, IV) with
concentration at 200 µg/mL were incubated with 0.1% of PS20 (upper
panel) and PS80 (lower panel) for 5 days. Significant PS20 degradation
was observed for both human PLBD2 and CHO PLBD2 but in different
patterns. By incubating PS20 with OriGene human PLBD2, a decrease in
signal intensity occurred on POE-ester peaks eluting at between 27.5 and
38 min. The peaks eluted before 34 min were POE monoester containing
short fatty acid chain, i.e., POE sorbitan monolaurate, POE isosorbide
monolaurate, POE sorbitan monomyristate and POE isosorbide
monomyristate. The POE esters with longer chains, eluted between 34-38
min, including POE isosorbide monopalmitate, POE isosorbide monosterate
and POE sorbitan diester also showed notable reduction in intensity.
Higher order triesters and tetraesters, which eluted after 38 min, did
not show noticeable changes during incubation (Figure 3-I). For CHO
PLBD2, similar degradation was observed in most PS20 species except the
first peak representing POE sorbitan monolaurate (Figure 3-II). As for
PS80 degradation, incubation with both human PLBD2 and CHO PLBD2 showed
significant degradation on peaks eluting between 30 and 35 min,
representing POE sorbitan monolinoleate, POE sorbitan monooleate and POE
isosorbide monooleate and POE monooleate (Figure 3-III and Figure 3-IV).
The CHO PLBD2 exhibited a higher degree of degradation compared to the
human PLBD2. The CHO PLBD2 also showed a noticeable intensity reduction
between elution time of 39 to 41 min, corresponding to degradation POE
sorbitan dioleate. POE sorbitan dioleate. The distinct degradation
pattern induced by these two types of PLBD2 (human vs Chinese hamster)
could be due to inherent differences between human and CHO PLBD2, or due
to differences in impurities.