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.