Extrapolating process capacity using clogging models
From Figure 7b, reference plasma IgG (no aggregate spike) showed good agreement between the experimental and calculated values for all four clogging models, but this is based on experimental results from filtration throughput up to 100 L/m2. It is of great interest and thought to be of importance to know whether this experimental data could be used to predict filtration behavior for throughput beyond this range. In order to calculate and compare the maximum practical throughput for the filtration of plasma IgG solution without aggregate spiking shown in Figure 6a, the theoretical filtration behavior plots calculated using each clogging factor and clogging model for much larger filtration volumes is shown in Figure 8. From this extrapolation, for larger filtration volumes, the different clogging models have varying maximum filtration volumes: about 250 L/m2 for complete blocking model, about 350 L/m2 for standard blocking model, about 1000 L/m2 for intermediate blocking model and 2000 L/m2 or more for the cake filtration model.
Discussion
Based on analysis of filtration behavior and the impacts of the addition of column chromatography on filterability, users can consider choosing chromatography resins to improve the overall performance of their virus filtration process. For aggregate-spiked mAb processing, the output from mixed-mode AEX1 and mixed-mode AEX2 showed improved filterability while normal AEX did not. Based on manufacturer information on the resins, mixed-mode AEX1 has primary amine and butyl base, and mixed-mode AEX2 has tertiary amine and phenyl group, and as such, these mixed-mode AEX media do not rely on the strength and weakness of an anion exchange group and hydrophobic group. Similarly, for aggregate-spiked plasma IgG processing, filterability was improved over reference by two resins with a sulfate ligand, modified CEX1 with dextran sulfate and modified CEX2 with cellulose sulfate, indicating that plasma IgG filterability improvement is due to the more effective dextran sulfate flow-through processing. However, while both mixed-mode AEX and modified CEX column chromatography removed larger aggregates from plasma IgG, there were differences in removal of dimers by these two methods. Despite greater removal by mixed-mode AEX, modified CEX showed markedly better improvement in filterability with flux at the start of the filtration exceeding that for the reference. This observation suggests that the decrease in filterability of plasma IgG, which is polyclonal, is not dependent solely on the aggregate content determined by SEC, unlike the pattern observed for mAb solutions.
From clogging model results, we see that the clogging model that best matches the filtration behavior for each protein filtration under constant pressure filtration with Planova BioEX filters clearly differed with the properties of the solution being filtered. Solutions showing extreme clogging, such as plasma IgG aggregate spike, follow the complete blocking model, and solutions showing minor clogging, such as aggregate-spiked mAb, follow the standard clogging model. The results from all four clogging models are practically indistinguishable for solutions that do not include any spiked substances that cause clogging, with the cake filtration model having a slightly better fit than other clogging models.
Although the clogging models assume simplified uniform cylindrical pores, which may not be exactly representative of virus filters, based on the studies and analyses presented here, applying the appropriate clogging models to filtration behavior is an insightful way to characterize filtration processes.
By selecting chromatography processes that are compatible with virus filtration and that improve the filterability of the feed stream, users can increase the capacity of their production processes. For mAb, for example, these processes can be conducted at large scales of at least 1000 L/m2, and even larger throughput can be expected as has already been put into practice (Lute et al., 2020). By evaluating process filterability, determining the best fit clogging model and utilizing it, total virus filtration process throughput can be estimated and used for planning purposes.
As future work, application of visualization techniques will add valuable information and contribute to better understanding of clogging mechanisms.