Emerging manufacturing modalities
Newer manufacturing technologies (e.g. continuous manufacturing, gene
therapy vectors) require special consideration when designing processes
and scale-down viral clearance studies for viral filtration.
For very small gene therapy vectors, like AAV, it is conceivable to use
large virus retentive filters in downstream manufacturing as a risk
mitigation barrier for larger contaminating viruses. AAV is predicted to
pass through these filters, while larger viruses like RVLPs are
entrapped. While there are some studies assessing the use of viral
filters in this field (Adams, Bak et al.
2020, Barone, Wiebe et al. 2020), this
concept is being adopted on a case-by-case basis in this therapy class.
Inherent to continuous manufacturing is the seamless transition from one
unit operation to the next, which presents a challenge for the viral
filtration unit operation. Besides the unique spiking strategies, which
are discussed below, integrated unit operations also have to consider
the impacts of longer processing times, continuous but potentially low
flow rates, and the higher level of impurities and product titer that
will be experienced in an end-to-end processing unit operation linkage.
One potential strategy to avoid the potential issues of clogging and
filter overload is to implement a parallel switch-in and switch-out
filtration scheme before the filters reach a validated total volumetric
throughput. Monitoring of cumulative flow volume, in addition to
traditional filtration pressure monitoring, would allow for control of
switching from the first filter to the second once the established
filter capacity is reached. Several groups have conducted
proof-of-concept studies for such studies and showed equitable clearance
values to traditional batch viral filtration
(Patt, Dong et al. 2015,
Swalec, Feng et al. 2015,
Johnson, Brown et al. 2017).
The concept of “filter trains” for continuous viral filtration have
also been posited as a potential strategy to allow for increased
impurity clearance and improve overall viral filter lifetimes in the
extended processing times (Arnold, Lee et
al. 2019). Several filters are placed in series with no line or
pressure breaks for clarification by depth filtration, TFF for product
concentration, a charged membrane filter as a low pressure option for
further impurity removal, and finally viral filtration for primary viral
clearance. The train allows for reduced facility footprint as well as
potentially faster processing times, but discussions on how to validate
orthogonal viral clearance steps within these trains is required.
As discussed above, the development of the scale down models for
integrated continuous viral filtration may pose a few challenges
compared to batch viral filtration. These challenges include 1)
performing virus filtration under constant flow, 2) extended volumetric
throughputs and extended processing times, and 3) the potential for a
dynamic product fluid stream potentially due to significant fluctuations
in protein and buffer concentrations
(Lute, Kozaili et al. 2020). While the
use of constant flow may not be a difficult challenge, as this mode of
operation can also occur in batch mode, proper modeling of the pump
behavior may be required to avoid pulsations at small scale. A bigger
challenge is how to perform virus spiking for the extended processing
volumes and processing times. The traditional approach to virus spiking
(bolus spike) may be prohibitive because the increased throughputs would
require a higher virus spike concentration or a larger volume of virus.
This could also lead to overloading of the filter with virus, which was
previously discussed as a known concern for some virus filters
(Lute, Riordan et al. 2008). Conversely,
a low titer virus spike may be implemented to avoid overloading;
however, this may lower the clearance values that can be achieved due to
assay sensitivity and lowered linear range. Another challenge for virus
spiking is maintaining the infectivity of the virus over the extended
processing time. A bolus spike may experience a significant loss of
infectivity over the course of the filtration study
(David, Maiser et al. 2019,
Lute, Kozaili et al. 2020). Alternative
spiking strategies, have been proposed
(Johnson and Roush 2018,
Lute, Kozaili et al. 2020). Proposed
bracketed spiking strategies involve spiking a high virus load in a
small volume at the beginning and end of the filtration study, with
either no virus or low titer virus spike for the majority of the process
volume. This approach was able to achieve similar total virus loads and
clearance values, for up to 2300 L/m2, while avoiding
potential virus overloading and loss of infectivity by only spiking
virus at the beginning and end of the filtration study
(Johnson and Roush 2018).
Another approach to avoiding a loss of infectivity is to have a fresh
daily spike of virus throughout the filtration study. In this approach,
a fresh spike can be applied to the filter every 24 hours for the
duration of the experiment (Lute, Kozaili
et al. 2020). To avoid overloading of the filter with virus, the total
virus spike should be determined prior to the study and back calculated
for a reasonable load titer per day. Care should be taken to seamlessly
integrate the fresh load and avoid the introduction of air bubbles or
pressure fluctuations that may negatively impact filtration study. This
approach was able to demonstrate an LRV of >6 log10 for up to 4 days and 2900 L/m2(Lute, Kozaili et al. 2020).
One final spiking approach mixed the virus stock with the feed solution
to desired viral titer, then prefiltered the final feed solution through
a sterile 0.1µm filter to mimic the practice of prefilter usage for
aggregates. The feed was then pumped at a continuous rate through the
viral filter(s) with a constant flux of 0.3L/m2/hour
for up to 72 hours. Load samples were taken twice a day
(log10 Day) and overnight without fresh spike being
added to the feed solution. To account for potential loss of viral
titer, the study calculated LRVs as equal to the log10of the daily load titer minus the log10 daily pool
sample titer (David, Maiser et al. 2019).
These studies have provided frameworks for spiking strategies and
provide strong supportive evidence that valid models for continuous
viral filtration exist. Though the extended processing times in these
scale-down models do not appear to affect viral reduction, other aspects
like filter leachables need consideration.