Virus removal from factor IX by filtration: Validation of the integrity test and effect of manufacturing process conditions

Virus removal from a high purity factor IX, Replenine^®-VF, by filtration using a Planova 15N filter has been investigated. A wide range of relevant and model enveloped and non-enveloped viruses, of various sizes, were effectively removed by this procedure. Virus removal was confirmed to be effective when different batches of filter were challenged with poliovirus-1. It was confirmed that intentionally modified filters that failed the leakage test had completely lost the ability to remove virus, thus confirming that this test demonstrates gross filter failure. In the case of the more sensitive integrity test based on gold particle removal, it was found that a pre-wash step was not essential. Planova filters that had been modified by sodium hydroxide treatment to make them more permeable, and filters manufactured with varying pore-sizes over the range of 15–35 nm, were tested. The integrity test value that resulted in the removal of >4 log₁₀ of poliovirus-1 from the product correlated with that recommended by the filter manufacturer. Virus removal from the product was not influenced by filter load mass, flow-rate or pressure. These studies confirm the robustness of this filtration procedure and allow suitable process limits to be set for this manufacturing step.     

Scrapie removal using Planova virus removal filters.

As a possible method for reducing the risk of transmissible spongiform encephalopathy (TSE) infection, Planova virus removal filters were tested for their ability to remove scrapie agent ME7. Albumin solution was spiked with high-titre ME7 and filtered through three different pore sizes of Planova filters. Infectivity of the pre- and post-filtration samples was assayed in log dilutions by intracerebral inoculation into C57B1/6 mice. Filtration of albumin solution in the absence or presence of a detergent (Sarkosyl) with Planova 35N (35+/-2 nm mean pore size) removed the contaminating scrapie agent with reduction factors of 4.93 log10 and 1.61 log10, respectively. Filtration, both in the absence and presence of detergent with Planova 15N (15+/-2 nm mean pore size), and in the presence of detergent with Planova 10N (9+/-2 nm mean pore size), showed high levels of scrapie reduction of >5.87 log10, >4.21 log10, and >3.80 log10, respectively, with no residual infectively detected in any of the filtrate samples. The effectiveness of Planova 35N filtration for the removal of infectivity of this TSE agent is greatly reduced in the presence of a strong detergent, but Planova filters with 15 nm or smaller pore size membranes can remove such infectivity at high reduction rates.     

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (～20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (～100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co‐spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in‐house viral validation studies and the results from the co‐spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log₁₀ reduction factors (LRF) to support clinical trial applications in both USA and Europe. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:79–85, 2014     

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles

Virus filtration with nanometer size exclusion membranes (“nanofiltration”) is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18–26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova? BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0–49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294–1302, 2017     

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.     

A preliminary study on viral decontamination of chicken serum using gamma-irradiation

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral ‘decontamination’ of chicken serum.     

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Improvement of virus safety of an antihemophilc factor IX by virus filtration process

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized productionscale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Nonenveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were (i)v.12 for HAV, (i)t.28 for PPV, (i)u.33 for EMCV, (i)u.51 for HIV, (i)u.17 for BVDV, and (i)u.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.     

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.     

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.     

Achieving high mass‐throughput of therapeutic proteins through parvovirus retentive filters

Parvovirus retentive filters that assure removal of viruses and virus‐like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size‐based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass‐throughput values of 7.3, 26.4, and 76.2 kg/m² were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass‐throughput values of 73, 137, and 192 kg/m² were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large‐scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small‐scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large‐scale filter capacity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The removal of viruses during the purification of equine antisera using filtration aids Hyflo Super-Cel and Fulmon Super A.

The manufacturing process in Australia for equine antisera against various venoms/toxins is based primarily on ammonium sulphate precipitation of pepsin-digested IgG, whereby Fc and F(ab')(2)fragments are separated. The capacity of the process to remove non-enveloped and enveloped model viruses was assessed using a scaled-down process. Each virus was added to mid-process samples from equine plasma before the material was applied to Hyflo Super-Celtrade mark filtration followed by Fulmonttrade mark Super A filtration. Samples were analysed pre- and post-filtration and the log clearance of the viruses calculated. The mean clearance factors for viral load of canine adenovirus type II (CAV(2)), poliovirus type 1 (PV1), infectious bovine rhinotracheitis virus (IBR) and canine distemper virus (CDV) were 5.3 logs, 4.2 logs, 5.7 logs and 4. 0 logs respectively. Clearance results as virus is adsorbed to the filtration aids which are removed from the process, thereby demonstrating improved viral safety of equine antisera produced by CSL.     

Removal of minute virus of mice-mock virus particles by nanofiltration of culture growth medium supplemented with 10% human platelet lysate

Background aimsPlatelet concentrates (PCs) are pooled to prepare human platelet lysate (HPL) supplements of growth media to expand primary human cells for transplantation; this increases the risk of contamination by known, emerging, and unknown viruses. This possibility should be of concern because viral contamination of cell cultures is difficult to detect and may have detrimental consequences for recipients of cell therapies. Viral reduction treatments of chemically defined growth media have been proposed, but they are not applicable when media contain protein supplements currently needed to expand primary cell cultures. Recently, we successfully developed a Planova 35NPlanova 20N nanofiltration sequence of growth media supplemented with two types of HPL. The nanofiltered medium was found to be suitable for mesenchymal Stromal cell (MSC) expansion.MethodsHerein, we report viral clearance achieved by this nanofiltration process used for assessing a new experimental model using non-infectious minute virus of mice-mock virus particle (MVM-MVP) and its quantification by an immunoqPCR. Then, high doses of MVM-MVP (1012 MVPs/mL) were spiked to obtain a final concentration of 1010 MVPs/mL in Planova 35N-nanofiltered growth medium supplemented with both types of HPLs [serum converted platelet lysate SCPL) and intercept human platelet lysate (I-HPL)] at 10% (v/v) and then filtering through Planova 20N.ResultsNo substantial interference of growth medium matrices by the immune-qPCR assay was first verified. Log reduction values (LRVs) were ≥ 5.43 and ≥ 5.36 respectively, SCPL and I-HPL media. MVM-MVPs were also undetectable by dynamic light scattering and transmission electron microscopy.ConclusionsThe nanofiltration of growth media supplemented with 10% HPL provides robust removal of small nonenveloped viruses, and is an option to improve the safety of therapeutic cells expanded using HPL supplements.     

New insights into the performance characteristics of the Planova-series hollow-fiber parvovirus filters using confocal and electron microscopy

Virus filtration remains a critical step in the downstream process for the production of monoclonal antibodies and other mammalian cell-derived biotherapeutics. Recent studies have shown large differences in virus capture behavior of different virus filters, although the origin of these differences is still unclear. The objective of this study was to use confocal and scanning electron microscopy to directly evaluate the capture of virus-size nanoparticles in Planova 20N and BioEX hollow-fiber virus filters. Confocal images of fluorescent nanoparticles were quantified using ImageJ image processing software based on the measured fluorescence intensity of the labeled nanoparticles. Nanoparticle capture by the Planova BioEX was independent of transmembrane pressure from 10 to 45 psi. In contrast, the Planova 20N showed significant differences in nanoparticle capture profile at low pressure, consistent with literature data showing virus breakthrough under these conditions. Images obtained after a process interruption show significant migration of previously captured nanoparticles in the Planova 20N filters but not in the BioEX. These results provide important insights into the nature of virus capture in different virus filters and its dependence on the underlying structure of the virus filtration membranes.     

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

Inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation

Human tissue allografts are widely used in a variety of clinical applications with over 1.5 million implants annually in the US alone. Since the 1990s, most clinically available allografts have been disinfected to minimize risk of disease transmission. Additional safety assurance can be provided by terminal sterilization using low dose gamma irradiation. The impact of such irradiation processing at low temperatures on viruses was the subject of this study. In particular, both human tendon and cortical bone samples were seeded with a designed array of viruses and the ability of gamma irradiation to inactivate those viruses was tested. The irradiation exposures for the samples packed in dry ice were 11.6-12.9 kGy for tendon and 11.6-12.3 kGy for bone, respectively. The viruses, virus types, and log reductions on seeded tendon and bone tissue, respectively, were as follows: Human Immunodeficiency Virus (RNA, enveloped), >2.90 and >3.20; Porcine Parvovirus (DNA, non-enveloped), 1.90 and 1.58; Pseudorabies Virus (DNA, enveloped), 3.80 and 3.79; Bovine Viral Diarrhea Virus (RNA, enveloped), 2.57 and 4.56; and Hepatitis A Virus (RNA, non-enveloped), 2.54 and 2.49, respectively. While proper donor screening, aseptic technique, and current disinfection practices all help reduce the risk of viral transmission from human allograft tissues, data presented here indicate that terminal sterilization using a low temperature, low dose gamma irradiation process inactivates both enveloped and non-enveloped viruses containing either DNA or RNA, thus providing additional assurance of safety from viral transmission.