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.