Virus Removal by Filtration: Points to Consider
Virus safety of biotech- and plasma-derived therapeutics is ensured through complementary manufacturing and quality control measures that include the control and monitoring of raw materials, the validation and implementation of effective virus clearance technology, and the monitoring of final filled product for the presence of virus. Virus filtration, which is considered a robust and effective virus clearance technology, is a common unit operation in the manufacture of biologicals. In this article, we review the points that need to be considered when selecting a virus retentive filter. The areas covered include regulatory considerations; selecting, optimizing, and validating a virus filtration step; and process scale implementation—areas that are of critical importance to users of virus filters.
Some biological therapeutic products are produced using mammalian cell lines or human plasma. The risk of contamination with either known or unknown viruses in these products has been demonstrated; hence, regulatory agencies have mandated that manufacturers evaluate the risks of virus contamination and take necessary measures to mitigate these risks. In addition to ensuring the purity of source materials, manufacturers are encouraged to institute steps in the purification process that will clear endogenous and adventitious viruses. Filtration has been successfully used in numerous processes as a robust step for virus clearance.
To implement virus retentive filtration successfully within a process, several points should be considered. These can be broadly
categorized as follows:
This article reviews each of these categories and suggests factors to consider when selecting and implementing a virus filter.
To ensure the virological safety of biological therapeutics, regulatory guidance advocates virus control at various stages of the drug manufacturing process. Specifically, manufacturers should (a) select and test source materials for the absence of viruses; (b) test the capacity of the production process to remove or inactivate viruses; and (c) test the product at appropriate stages of production for freedom from detectable viruses.1
Regulators require that an overall safety margin, such as <1 virus particle per 106 doses, be used to demonstrate the virus safety of the manufacturing process. The drug manufacturer is required to quantify the virus "load" in the process. For biotech products derived from murine cell lines such as Chinese hamster ovaries (CHO) and nonsense oligonucleotide (NSO), this typically translates to ~12–18 log10 clearance for endogenous retroviruses and ~6 log10 removal for adventitious viruses. The following is a composite summary of regulatory guidance on virus clearance, with an emphasis on how the guidance relates to virus filtration. 2–5
Depending on regulatory requirements, one needs to consider if the process requires retrovirus clearance, or retrovirus and
parvovirus removal. Virus clearance filters are broadly classified into two categories:
After regulatory requirements have been addressed, it is time to look at the protein purification process, not only at the current scale but also at the potential manufacturing scale, to ensure that virus filtration is implemented at the best location in the process.
Parvoviruses have a diameter of ~18–26 nm, but a typical monoclonal IgG antibody has a hydrodynamic diameter of ~8–12 nm. To achieve >4 log10 retention of the viruses and >99% recovery of the protein, parvovirus filters are required to have a very narrow pore size distribution. They are, therefore, generally sensitive to the presence of impurities in the feed solution. Thus, optimizing a virus filtration process involves evaluating the effect of a variety of process parameters to arrive at conditions that will ensure a robust, consistent, economical, and scalable operation.6–9
Impact of Location in the Downstream Process Train
Impact of Feed Concentration
Impact of Prefiltration
Prefiltration of the feed solution can have a dramatic impact on filter performance. Prefiltration is targeted to remove various impurities or contaminants, such as protein aggregates, DNA, and other trace materials. Although larger impurities can be removed by prefiltering using 0.2 μm or 0.1 μm microfilters, smaller impurities, such as protein aggregates that may be only marginally larger than the protein product, are not easily removed using size-based removal methods. Prefiltration through adsorptive depth filtration has been observed to provide significant protection for certain virus removal filters.7 The impact of prefiltration can be dramatic, with up to a 10-fold reduction in required filter area.
Impact of Hold Times and Freeze-Thaw Cycles
Some proteins exhibit time-dependent aggregate formation or will form low concentrations of aggregates when subjected to a freeze–thaw cycle. If a hold step or a freeze-thaw cycle is expected to be a part of the process, it is important to evaluate the effects of these during filter optimization. Furthermore, although the actual purification process may not have a freeze–thaw step, feed samples required for virus retention testing are often conveniently submitted in a frozen form due to material stability considerations.
Designing Virus Retention Qualification Studies
Spiking studies should be designed to reflect the virus clearance capability of the process-scale unit operation.2 Therefore, the level of purification of the scaled-down version should represent the production process as closely as possible,
by reproducing the key operating parameters that have an effect on purification and on virus clearance. The critical operating
parameters will vary depending on clearance technologies (due to the different mechanisms of action). For filtration, scale-down
will focus on such parameters as:
Typically, the study sponsor may rely on the filter manufacturer to supply data required to support the claim of scale-down validity.
Although not specified in regulatory guidance, some manufacturers choose to use worst-case process-variable settings in the design of the spiking study.
Appropriate guidance on how to plan and carry out virus retention qualification studies is available from regulatory agencies.3 Documents such as the Parenteral Drug Association Technical Report 41 provide guidance on how to determine the quantity of virus spike needed to achieve necessary log reduction values (LRV).10
VIRUS FILTER–RELATED CONSIDERATIONS
After the virus clearance step has been optimized and virus retention studies completed, an implementation strategy is required for robust process operation. After determining the filter capacity (L/m2) required for a process during process simulation, process scale-up, and virus retention studies, the filter area required for processing a given batch volume can be calculated. Various filter configurations are made available by manufacturers to facilitate large-scale implementation. When multiple filter modules are required to process a given batch volume, the modules may be installed in parallel within a multi-round housing.
A typical sequence of operations in a virus filtration process includes the following steps. (Some of these will be discussed
later in more detail.)
Sanitization and Sterilization
In a typical downstream purification process, virus clearance filters are used downstream of a chromatography column and upstream of an ultrafiltration/diafiltration step, neither of which is considered an aseptic operation. However, there appears to be an industry trend to sanitize or sterilize the virus filter to reduce the bioburden. Some virus filters are available pre-sterilized and, therefore, will eliminate the sanitization step. It is important to ensure that the filters are compatible with a sanitization or sterilization method that is likely to be implemented at manufacturing scale. Furthermore, it is important to ensure that process steps used during large-scale processing are also carried out during the scaled-down virus retention studies.
To ensure that virus clearance is consistent with results obtained during virus retention studies, it is recommended that
filter integrity be checked both pre- and post-use.14,15 To facilitate this, filter manufacturers have developed a variety of destructive and non-destructive physical integrity tests
that are related to virus retention. Ultimately, the objectives of properly designed physical integrity testing are threefold:
To satisfy these requirements, a series of tests is needed to confirm filter integrity. Some of these tests, typically carried out by the end-user, are better suited for confirming proper installation and for confirming the absence of gross defects. Other tests, generally performed by the filter manufacturer, may be better suited for detecting subtle changes in filter pore size distribution. A more detailed summary of the various tests can be found in Parenteral Drug Association Technical Report 41.
It is important to ensure that conditions used during processing are within the parameters used during the scaled-down virus retention assessment studies. Because most parvovirus retentive filters exhibit a decline in virus retention ability with flow decay, it is important to ensure that flow decay during processing is comparable to that observed during retention studies. Other parameters to be monitored during processing include solution conditions, volumetric throughput, and post-production buffer flush.
Virus filtration is a critical component in the manufacture of biological therapeutics. Implementation of a virus retentive filter is one of many steps a manufacturer will take to ensure product safety. The choice of a virus filter is driven mainly by robust virus retention. Nevertheless, robust retention should be achieved as economically as possible. This brief overview of regulatory-, process-, and filter-related considerations should aid filter users in selecting the right virus filter and in initiating filter optimization studies.
Gerd Kern is technology manager, virus management solutions, for Millipore SAS, 39, route industrielle de la Hardt, 67120 Molsheim,France, +33.(0).126.96.36.199.00, fax: +33.(0).188.8.131.52.93, email@example.com
Mani Krishnan is program manager, virus and biomolecular clearance, for Millipore Corporation, 80 Ashby Road, Bedford, MA 01730, 800.645.5476, fax: 800.645.5439, firstname.lastname@example.org
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