Context in overall viral safety program
In modern biotechnology manufacturing (or bioprocessing), there are
typically three or four unit operations in the overall purification
train that are capable of removing or inactivating viruses. These
include certain chromatography steps (e.g. protein A or anion exchange),
incubations with low pH or detergents, and virus retentive filters. Not
all of these steps will be robust or even effective in removal or
inactivation for all viruses. For example, low pH incubations are
generally ineffective for non-enveloped virus inactivation
(Miesegaes, Lute et al. 2010)) although
quite robust for enveloped viruses
(Brorson, Krejci et al. 2003). Under
certain operating conditions, anion exchange columns may not bind and
remove neutral isoelectric point viruses from product streams
(Riordan, Brorson et al. 2009), but are
effective for acidic viruses (Strauss,
Lute et al. 2009). It is the combination of the three to four
independent and orthogonal unit operations that together assure viral
safety for biotechnology products.
It is commonly considered that a robust, effective and reliable process
step will be able to remove or inactivate a substantial amount of virus
(typically defined as 4 log10 or greater, where the log
reduction value or LRV is calculated as the log10 of the
total input divided by the total output virus). However, the LRV cannot
be used as the single absolute measure of the effectiveness of a step. A
robust, effective and reliable step should be easy to model, be
relatively insensitive to changes in process conditions, and be
effective for a range of viruses (WHO
2004). Virus filtration is generally agreed to be one such robust and
effective process step and is a key component in an overall strategy to
minimize the risks of adventitious and endogenous viral particles during
the manufacturing of biotechnology products.
Viral filters are typically understood to function through a robust,
size-based retention mechanism. Based on this robust mechanism of
action, virus filters are more likely to provide predictable viral
retention for a range of viruses than the chromatography steps. This is
because filters are less likely to be influenced by differences in the
physicochemical properties of different viruses, and the virus-resin
interactions modulated by operating conditions. Therefore, a viral
filtration step is commonly utilized in a well-designed recombinant
therapeutic protein purification
process(EMEA 1996) and also has proven to
provide robust performance in the plasma processing industry
(Roth, Dichtelmüller et al. ,
Junter and Lebrun 2017).
Introduction of viral filters with a historical contextVirus filters are polymeric membranes with complex pore structures
designed to provide high retention of 20—140 nm viral particles while
allowing the smaller product molecules to pass freely. Due to the high
selectivity required to distinguish closely sized viruses and molecules
and the desire to perform at high flux and high throughput conditions,
viral filter manufacture requires more stringent quality control
relative to sterilizing grade filters and thus virus filtration can
comprise one of the most expensive unit operations
(Phillips, Bolton et al. 2007).
There are two types of viral filters typically used in bioprocessing. A
recent ANSI accredited PDA standard classified filters into two
categories, large virus retentive filters, which are designed to retain
viruses larger than about 60 nm, and small virus retentive filters
designed to retain viruses larger than 20 nm
(Lute, Riordan et al. 2008,
PDA 2021). In the last decade or so, new
filters have been predominantly designed for parvovirus retention but
can function as a retrovirus filter. The filters vary in their formats
and materials of construction. The filters comprise either two or three
layers of flat sheets, or consist of hollow fibers. The filters are made
using one to three layers of the following hydrophilic polymers:
polyvinylidene fluoride (PVDF), hydrophilic polyethersulfone (PES), or
cuprammonium regenerated cellulose. The membranes may be symmetric or
asymmetric in structure (Gefroh, Dehghani
et al. 2014). Table 1 summarizes commercial viral filters available
currently, although it is likely that additional filters may be
developed in the future.
The operation of virus retentive filters is independent of the
size-based retention mechanisms. Earlier filters were designed to be run
in tangential flow filtration (TFF) mode to reduce fouling
(DiLeo, Allegrezza et al. 1992). Though
many hollow fiber virus filters are capable of running in TFF mode,
filters are now typically run in normal flow filtration (NFF) mode which
is also referred to as direct flow filtration (DFF). Operation in NFF
mode provides consistent process performance and eliminates the
complexity of controlling feed and permeate flow rates required for TFF
mode operation. The development of new parvovirus filters capable of NFF
mode operation, which are robust, effective and reliable in the
clearance of parvovirus, led the industry to widely migrate to the use
of parvovirus retention filters and reduce usage of retrovirus-specific
filters.
To improve virus filter throughput and economics, prefilters are often
used in-line with the virus filter. These prefilters remove trace
impurities that could otherwise foul virus filters, thereby increasing
throughput and decreasing area requirements
(Bolton, Spector et al. 2006,
Brown, Bechtel et al. 2010). Initially
prefilter options were limited to microfiltration membranes (e.g. 0.2 µm
filters) and diatomaceous earth-based depth filters
(Bolton, Spector et al. 2006).
Diatomaceous earth-based depth filters have been shown to be effective,
though they pose an increased leachables risk compared to other filter
types. They are also known to release beta-glucans, which can interfere
with endotoxin assays (Gefroh, Hewig et
al. 2013). These risks can be mitigated with a water flush, buffer
flush, or carbonate buffer flush. The carbonate flush has been shown to
reduce beta-glucan levels in filter effluents
(Holstein, Jang et al. 2021). Absorptive
membranes utilizing ion exchange functionality were also developed to
mitigate some of these challenges and more recently synthetic depth
filters have been developed. Many virus filter manufacturers currently
offer specialized prefilters to increase the capacity and throughput of
their respective virus filters. Table 1 also includes some common
prefilter options.