Column chromatography and filtration for mAb with aggregate
spike
From the filtration results shown in Figure 1 and protein
characterization data shown in Table 1, we see that the purified mAb
(reference solution) with 175 ppm HCP and 1.3% dimer content showed a
stable flow rate on Planova BioEX. The control, which is
aggregate-spiked mAb (spiked at 1.0%), has markedly lower throughput
from shortly after the start of filtration, showing that an increase in
larger aggregate (trimer or larger species) content from 0.2% to 1.2%
is the likely cause for the marked decrease in filterability. Further,
aggregate-spiked mAb processed in flow-through mode with normal AEX has
similarly high larger aggregate content and filtration behavior that is
almost the same as the control, showing that normal AEX column
chromatography has no impact on improving filterability. The moderate
improvement by the nylon prefilter on filterability of the
aggregate-spiked mAb can be attributed to the reduction of larger
aggregates to 0.5%, which is less than half of the level in the
control. The output from mixed-mode AEX1 and mixed-mode AEX2 showed
markedly higher filterability, surprisingly higher than the reference.
These chromatography processes effectively reduced HCP and aggregate,
particularly reducing larger aggregates to below detectable levels. HCP
was also decreased for both outputs, but the similar improvement in
filterability increase observed for both despite mixed-mode AEX2 output
having twice the HCP as mixed-mode AEX1 output suggests that the cause
of decreased filterability for aggregate-spiked mAb is larger aggregates
more so than HCP. These results suggest that filterability was not
greatly impacted by the HCP or mAb dimer content at these
concentrations.
The molecular weight distribution profiles based on SEC analysis shown
in Figure 2 for the reference, control, mixed-mode AEX1 output and nylon
prefilter output shows the changes in aggregate content and changes in
the distribution for these various solutions. The aggregate-spiked mAb
(control) has increased dimer and larger aggregate content, which is
believed to have a great impact on filterability in the virus filtration
step. Further, filterability at the virus filtration step is markedly
increased by mixed-mode AEX1 processing, and the molecular weight
distribution profile clearly shows that this processing almost
completely removed larger aggregates and reduced the dimer content.
Filtering the solution with a nylon prefilter moderately improved
filterability and it decreased the proportion of larger aggregates, but
its effect on improving filterability is very small compared to
mixed-mode AEX1.