3. Results
3.1. Column chromatography
and filtration for mAb with aggregate spike
The effect of column chromatography and nylon prefilter treatments on
mAb filterability is shown in Figure 1A and 1B. Protein aggregate
characterization by SEC analysis is shown in Figure 1C and a comparison
of dimer and trimer or larger aggregate and HCP content are shown in
Figure 1B. From the figures, we see that the purified mAb (reference
solution) with 175 ppm HCP and 1.3% dimer content has 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 trimer or 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 trimer or
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 shows
markedly higher filterability, surprisingly higher than the reference.
These chromatography processes effectively reduced HCP and aggregate,
particularly reducing trimer or 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 the trimer or 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 are
shown in Figure 1C for the reference, control, mixed-mode AEX1 output
and nylon prefilter output. The aggregate-spiked mAb (control) has
increased dimer and especially more trimer or larger aggregate content,
which is believed to have a great impact on filterability in the virus
filtration step. For mixed-mode AEX1 output, which shows improved
filterability at the virus filtration step, the molecular weight
distribution profile clearly shows that this processing almost
completely removed trimer or larger aggregates and reduced the dimer
content. Filtering the solution with a nylon prefilter moderately
improved filterability and it decreased the proportion of trimer or
larger aggregates, but its effect on improving filterability is very
small compared to mixed-mode AEX1.
3.2. Column chromatography
and filtration for plasma IgG with aggregate spike
Figure 2 shows the effect of column chromatography on the filterability
of polyclonal plasma IgG isolated from plasma derivatives with 0.5%
aggregate spike. Aggregate content by SEC analysis is shown in Figure
2A. Plasma IgG spiked with 0.5% IgG aggregate (control) had increased
trimer or larger aggregate content (from 0.3% for the reference
solution to 0.5%) and 7.8% dimer content as shown in Figure 2A. The
figure clearly shows that the 0.5% IgG aggregate spike causes a marked
decrease in filterability as evidenced by achieving flux of 100 LMH at
nearly 80 L/m2 for the reference, while the flux of
the control had become nearly zero and the run was ended by 12
L/m2. Normal AEX processing of aggregate-spiked plasma
IgG produced no aggregate removal and there was no improvement in
filterability.
Mixed-mode AEX1 shows more than double the throughput of the control and
high aggregate removal with dimer content decreasing from 7.8% to 5.8%
and trimer or larger aggregate content decreasing from 0.5% to below
detectable level. On the other hand, while trimer or larger aggregate
content was also reduced to below detectable level by modified CEX1 and
modified CEX2 processing, dimer was not reduced markedly from the
control and remained at 7.3% and 7.7%, respectively. Interestingly,
modified CEX output shows significantly higher filterability than the
mixed-mode AEX1 output and even exceeds the flux of the reference in the
early phase of filtration (Figure 2B). Modified CEX1 and modified CEX2
both show high filterability, but modified CEX2 output shows a greater
flux decay than does modified CEX1 output.
3.3. Determination of
clogging factor k for model analysis
For clogging model analysis, clogging factor k is determined by applying
the obtained filtration data (filtration volume and flow rate) to
Equations 2, 4, 6 and 8 for the cake filtration model, intermediate
blocking model, standard blocking model and complete blocking model,
respectively. Graphical results of applying the control (aggregate
spike) filtration results of both mAb and plasma IgG to each clogging
model and finding the line of best fit using the least squares method
are shown in Figure 3. Based on the overlap between experimental and
calculated data, the mAb is best fit to the standard blocking model
(Figure 3C) and plasma IgG is best fit to the complete blocking model
(Figure 3D).
3.4. Clogging model
analysis for aggregate-spiked mAb
Evaluation of filtration behavior for the reference (no spike) and
control (aggregate spiked) mAb solutions in Figure 1B with each of the
models is shown in Figure 4A and 4B, respectively. For the reference (no
aggregate spike), values calculated based on each of the models overlap
with experimental values, indicating that pronounced clogging was not
observed for this purified protein (Figure 4A). In contrast, the control
(aggregate-spiked mAb) had distinct plots for each clogging model, and
the standard blocking model plot overlapped the most with experimental
values (Figure 4B).
The control, output from normal AEX and output from nylon prefilter (all
three solutions having relatively high proportions of trimer or larger
aggregate content) had markedly higher k than column output from
mixed-mode AEX (low proportion of aggregate), and the higher the
aggregate content, the larger the k (Figure 4C). Further, for all three
solutions with trimer or larger aggregates, the k decreased in the order
of cake filtration, intermediate blocking, standard blocking and
complete blocking models. The differences between experimental and
calculated values for each solution with all four clogging models in
Figure 4D show that for solutions with high aggregate content and high
k, the standard blocking model is the best fit for mAb.
3.5. Clogging model
analysis for aggregate-spiked plasma IgG
Evaluation of the filtration behavior for the reference and control for
plasma IgG solutions in Figure 2B with each of the four clogging models
is shown in Figure 5A and 5B. While the calculated results for all four
clogging models overlap with experimental results (Figure 5A),
indicating no significant clogging for the reference (no aggregate
spike), control (spiked with aggregate) had distinct plots for each
clogging model, and the complete blocking model showed the smallest
differences between experimental and calculated values (Figure 5B).
Plots of k and mean differences between experimental and calculated
values for filtration behavior (Figure 5C and 5D) show that plasma IgG
solutions with relatively high proportions of trimer or larger aggregate
content (control and normal AEX output) showed the best fit with the
complete blocking model, which is a different from that for mAb
(standard blocking model). A deeply interesting point is that the output
for mixed-mode AEX1, which removed the trimer or larger aggregates
through column chromatography and had lower dimer content than the
outputs for modified CEX1 and modified CEX2 (5.8% vs. 7.3% and 7.7%,
respectively), had a markedly higher k than the output for both modified
CEX resins and the reference, which had 0.3% trimer or larger aggregate
content as shown in Figure 5C. Furthermore, the differences between
experimental and calculated values for each solution in Figure 5D show
that mixed-mode AEX1 output has the best fit with complete blocking
model. These results suggest that there are components besides
aggregates detected by SEC that impede the filterability of plasma IgG
on the virus filter, and the components formed in the process of
producing aggregates are reduced by processing with modified CEX but not
by mixed-mode AEX1.
4. Discussion
Plots of filterability profiles for mAb (Figure 1) and plasma IgG
(Figure 2) with aggregate spike and after processing with various
chromatography columns clearly show that chromatography processing
significantly affects filterability for both solutions, but with
different results for various chromatography resins. It should be noted
that all runs were conducted with the same solution conditions for
control and simplicity of the experimental design, but optimizing
solution conditions for each different resin could potentially result in
different outcomes. Differences in
initial flux, even for constant pressure filtration at the same
pressure, may be due to differences in viscosity resulting from the
chromatography processes.
Based on analysis of filtration behavior and the addition of column
chromatography on filterability, users can consider choosing
chromatography resins that will 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 a primary amine and butyl
base and mixed-mode AEX2 has a tertiary amine and phenyl group, and as
such, these mixed-mode AEX resins 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 dextran sulfate
being more effective for flow-through processing. However, while both
mixed-mode AEX and modified CEX column chromatography removed trimer or
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 based
on filtration behavior, we see that mAb with aggregate was best fit to
the standard blocking model (Figure 4) and plasma IgG with aggregate was
best fit to the complete blocking model (Figure 5). Appropriate
selection of the best-fit model for each molecule was shown as the
lowest k for both solutions with aggregate spike (control) and solutions
with moderate reduction of aggregate following chromatography
processing. Based on selection of the standard blocking model for mAb,
the pores of the filter are likely narrowed by molecules adhering to the
walls of the pores. In contrast, plasma IgG likely obstructs the pores
based on the selection of the complete blocking model.
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 clogging models to filtration behavior
could be an insightful way to characterize filtration processes. Our
findings indicate that, by selecting chromatography processes that are
compatible with virus filtration and that improve the filterability of
the feed stream, the capacity of production processes can be increased.
These processes can be conducted at large scales of at least 1000
L/m2, and even larger throughput can be expected, for
example, as has already been put into practice (Lute et al.,
2020).[12]
Optimizing filterability through consideration of aggregate removal is
of great interest for downstream process development.
As
future work, correlation of clogging model results and analytical
results of solution characteristics including aggregate content along
with the use of visualization techniques will deepen our understanding
of filtration mechanisms. Applying filtration data from higher
throughput runs (more than 500 L/m2) to clogging model
analysis will provide guidance for scaling up.