UV-C versus small, non-enveloped viruses

By Dr. Ray Nims

Small, non-enveloped viruses (especially the circoviruses, parvoviruses, picornaviruses, caliciviruses, and polyomaviruses) and bacteriophage with similar characteristics represent a special challenge to the biologics industry.

In fact, contamination events have occurred with each of these virus families; in some cases more than once. Within the Circoviridae, the primary concern has been the porcine circovirus discovered in rotavirus vaccine. At least four contamination events involving the parvovirus, mouse minute virus, have been reported. The calicivirus vesivirus 2117 has contaminated biologics manufacturing processes on at least two occasions. The picornaviruses are a threat which has yet to be realized in the manufacturing context; but a viral safety test conducted for a biologics cell bank was the scene of a contamination with equine rhinitis A virus. The polyomavirus SV40 was found to have survived the formaldehyde inactivation used in the preparation of polio vaccines and therefore made its way into the doses of vaccine delivered to millions of individuals during the decade between 1955 and 1965 (being born in 1952, it is possible that the author received such tainted vaccine!).

The contamination events described above are, at least in part, a reflection of the ability of these viruses to withstand conditions that would lead to inactivation of other types of viruses. Inactivation strategies that are typically employed for viral safety include chemical (low pH, high pH, disinfectants, solvents, detergents, etc.) and physical (heat, irradiation, pressure, etc.) means. In a previous posting, the efficacy of gamma irradiation for inactivating various types of viruses within frozen animal serum was discussed. The use of UV-A in combination with riboflavin has also been discussed previously.

It is perhaps fortunate that the efficacies of different inactivation approaches are in many cases complementary. For instance, it appears that the efficacy of gamma irradiation for inactivation of viruses decreases as viral particle size decreases (although this relationship is not strictly linear). The outcome of this is that gamma irradiation does not appear to be particularly effective for inactivating small, non enveloped viruses from the circovirus, parvovirus, and polyomavirus families. On the other hand, ultraviolet radiation in the C range (254 nm is the most commonly employed wavelength) appears to be more effective for the inactivation of smaller viruses than for larger viruses (or bacteria).

The table below is a compilation of the UV-C inactivation constants (K, defined as the log₁₀ reduction in titer per mJ/cm² fluency) for various families of small, non-enveloped viruses. These K values represent inactivation of the viruses in a variety of matrices, ranging from water to protein-containing matrices such as albumin or complete culture medium). These results suggest that UV-C irradiation may be a viable approach for inactivating many of these problem viruses in raw materials or process intermediates used in biologics manufacture.

references for K values: * Lytle and Sagripanti 2005, ¶ Kowalski et al. 2009; † Maier 2007. The other values are the mean K values assembled by the author from the inactivation literature.

The one exception appears to be the polyomaviruses, which appear to be relatively resistant to the inactivating effects both of gamma irradiation and UV-C irradiation. This family of viruses may best be inactivated using high-temperature short-time treatment (HTST), though the efficacy of this approach for the polyomaviruses has yet to be demonstrated empirically.

The inactivation literature for circoviruses

by Dr. Ray Nims

The Circoviridae family of viruses represent an extreme case for small, non-enveloped viruses. We have posted previously that the latter group constitutes a high risk for manufacturers of biologicals due to the difficulty of eradicating the viruses from raw materials or from a contaminated facility.  At 17-25 nm particle size, the circoviruses are among the smallest of the animal viruses. These viruses represent more of an economic threat than a threat to human health. The porcine circoviruses (PCV-1 and PCV-2) and the chicken anemia virus are well known examples of the circoviruses and these have been studied extensively due to their impact on the swine and poultry industries. So why care about the circoviruses in the biologics industry?

We begin being concerned about porcine circoviruses in the context of xenotransplantation of porcine tissues (e.g., islet cells) into humans. The worry was that a porcine circovirus might be transmitted to a patient via the porcine donor tissue. More recently, PCV genomic sequences were discovered in rotavirus vaccines manufactured by GlaxoSmithKline and Merck. The presence of the genomic material was attributed to the use of porcine trypsin during the culture of the cell substrates in which the vaccines were manufactured (see previous post).

As a result of the heightened awareness of the contamination threat posed by the porcine circoviruses, infectivity assays for these viruses are now being offered at contract testing organizations (e.g., BioReliance, MICROBIOTEST, and WuXi Apptec), for raw material testing, cleaning efficacy testing, and for evaluating the clearance of the circoviruses by purification processes. As might be expected based on our experience with other small, non-enveloped viruses, inactivation approaches that are effective against many larger non-enveloped  or enveloped viruses have little efficacy for the circoviruses.

So how much do we actually know about the inactivation of  circoviruses? The literature on the subject is fairly extensive for PCV-2 and for chicken anemia virus, if one is willing to spend time digging deeply. I have done the digging, and have assembled the literature into the following categories of inactivants: heat, irradiation, and disinfectants/chemicals. Keep in mind that the inactivation potential of the physical or chemical agents depends greatly upon the matrix in which the virus is present as well as the temperature and contact time with inactivant. The matrices evaluated varied for the studies described below, and the reader is directed to the individual papers for this critical detail. In addition, some variability in results may be expected because of the relative difficulty in assaying infectivity of the circoviruses.

A number of studies on the thermal stability of the circoviruses have been published. In general, it appears that 15 or more minutes of exposure to wet heat (heating of viruses spiked into solutions) at temperatures ≥80 ◦C should provide extensive inactivation (3-5 log₁₀) of circoviruses. Dry heat (heating of freeze-dried virus and, by implication, viruses on the surfaces of coupon materials) appears to be much less effective, resulting in <1.5 log₁₀ inactivation even at temperatures as high as 120 ◦C . The results of at least one investigator suggest that a temperature of 95 ◦C will be sufficient for high temperature short time (HTST) treatment for mitigating the risk of introducing a circovirus in a process solution.

A number of studies on the inactivation of the circoviruses by disinfectants and other chemicals have appeared in the literature, reflecting the relatively great economic threat of the circoviruses to the swine, poultry, and exotic bird industries. While many of the chemicals/disinfectants had little efficacy for inactivation of the circoviruses (as might be expected for a non-enveloped virus), certain treatments appear to have been highly effective. These included the following: a) glutaraldehyde at 1% or 2% and 10 or more minutes contact time; b) sodium hypochlorite at 6% and 10 or more minutes contact time; c) sodium hydroxide  at 0.1 N or greater; d) Roccal® D Plus at 0.5% and 10 minutes contact time; e) Virkon® S at 1% and 10 minutes contact time; f) β-propiolactone at 0.4% and 24 hours contact time; and g) formaldehyde at 10% and 2 hours contact time. Variable results were obtained for the iodine-containing disinfectants. These ranged from <1.0 log₁₀ inactivation for iodine (10%; 30 minutes contact time; 20 ◦C) to ≥5.5 log₁₀ for Cleanap® (1%; 2 hours contact time; 37 ◦C). A third study involving an iodine-containing disinfectant, FAM®30 (Biocide30) used at 1% or 2%; 30 minutes contact time, and 10 ◦C temperature demonstrated ≥3.5 log₁₀ inactivation.

The literature on inactivation of circoviruses by irradiation is scant, to say the least. Plavsic and Bolin showed that gamma irradiation of PCV-2-spiked fetal bovine serum at the radiation doses normally employed for serum treatment (30 and 45 kGy) resulted in ≤1.0 log₁₀ reduction in virus titer. Gamma irradiation (at the doses normally used) appears to be relatively ineffective for inactivating the very smallest of the viruses (parvoviruses and circoviruses) in serum so this result is perhaps not surprising. One approach that offers hope for inactivation of circoviruses is ultraviolet radiation (specifically UV-C) treatment, as this technology appears to be effective for smaller non-enveloped viruses such as the parvoviruses and caliciviruses. I expect that studies to demonstrate efficacy of UV-C for inactivating circoviruses will be performed in the near future.

In summary, there is ample evidence in the literature that effective inactivation approaches exist for the circoviruses. Careful selection of inactivation technologies that is based on the body of evidence accumulated by workers in the swine and poultry industries should enable appropriate risk mitigation and facility cleaning strategies to be adopted in the biologics industry.

<This material was excerpted in part from Nims and Plavsic, Bioprocessing J, 2012; 11:4-10>

What's up with USP Chapter 1050?

by Dr. Ray Nims

United States Pharmacopeia (USP) general chapter <1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin originally appeared in supplement 10 to USP23-NF18 in May 1999 and was at that time essentially a verbatim adoption of the International Conference of Harmonization (ICH) Guideline Q5A (R1) having the same title.

The chapter describes the methods of evaluating the viral safety of biotechnology pharmaceutical products that are manufactured using cell lines of human or animal origin.

In 2006, an ad hoc advisory panel was assembled by the USP and tasked with revision of this chapter. The goals were to update the chapter and, more specifically, to add greater detail in the viral clearance validation section. The hope was that a user following the recommendations set forth in the general chapter would have greater confidence that viral clearance validation data generated would prove acceptable to the regulatory agencies.

The organization of the revised chapter <1050> was not changed. It comprised the following main sections: 1) Introduction; 2)Potential sources of viral contamination; 3) Cell line qualification: testing for viruses; 4) Testing for viruses in unprocessed bulk; 5) Rationale and action plan for viral clearance studies and viruses tests on purified bulk; and 6) Evaluation and characterization of viral clearance procedures. The changes proposed for the initial 5 sections were minor and primarily reflected attempts to update the chapter and to align the chapter more closely with FDA guidance documents. The most extensive changes were to section 6, in keeping with the goals described above.

The revised chapter was published for public comment in Pharmacopeial Forum 36(3) in the fall of 2010. Comments received as a result of the public review apparently suggested that a more extensive update of the chapter was warranted. At any rate, the revised chapter was not made effective during the USP’s 2005-2010 revision cycle. A new ad hoc advisory panel now being assembled as part of USP's 2010-2015 revision cycle will take over the responsibility for moving the revision of this chapter forward.

Remember....bacteriophage are viruses too

by Dr. Ray Nims

Are you using bacterial cells to produce a biologic? Do not make the mistake of thinking that your upstream process is safe from infection by adventitious viruses. True, you are not required to test for the usual viruses of concern using a lot release adventitious virus assay. But bacterial production systems are susceptible to introduction of viruses just as mammalian cell processes are. In this case, the viruses just happen to be referred to as bacteriophage. Other than this, the putative contaminants have the same nasty property exhibited by viruses that can contaminate mammalian cell processes, that is … their small size (24-200 nm) allows them to readily pass through the filters used to “sterilize” process solutions. So media, buffers, induction agents, vitamin mixes, trace metal mixes, etc. that are fed into the fermenter without proper treatment can introduce a bacteriophage. Especially worrisome in this regard are raw materials that are generated through bacterial fermentation (such as amino acids, antibiotics). A fermenter infected with a lytic phage exhibits a clear signal that the bacterial substrate is unhappy. The trick then is to discover where the phage originated and to mitigate the risk of experiencing it again.

How can you mitigate the risk of experiencing a bacteriophage infection? Many of the same strategies used to protect mammalian cell processes may be applicable to the bacterial fermentation world. Raw materials and/or process solutions may be subjected to gamma-irradiation, to ultraviolet light in the C range, to prolonged heating or to high temperature short time treatment, to viral filtration, etc. In addition, mitigation of risk of bacteriophage contamination may require filtration of incoming gasses using appropriate filters. 

A sampling of the data available on inactivation of bacteriophage by various methods is shown in the table below. The literature is extensive, and as with viral inactivation, the inactivation of phage by certain of the methods (e.g., UVC, gamma-irradiation) may be dependent both upon the matrix in which the phage is suspended as well as the physical properties of the phage (e.g., genome or particle size, strandness, etc.). For fairly dilute aqueous solutions, gamma-irradiation, UVC treatment, or parvovirus filtration should represent effective inactivation/removal methods. HTST at temperatures effective for parvoviruses (102°C, 10 seconds) should be effective for most bacteriophage, although this is an area that needs further exploration.

Mitigating the risk of experiencing a bacteriophage contamination of a bacterial fermentation process is possible if one remembers that bacteriophage are similar to mammalian viruses. Strategies that are effective for small-non-enveloped mammalian viruses (i.e., the worst case for mammalian viruses) should also be effective for most bacteriophage.

A possible exception to this is prophage. In analogy with the presence of endogenous retroviruses in certain mammalian cells (i.e., rodent, human, monkey), there is a possibility of encountering integrated bacteriophage (prophage) in certain bacterial cell lines. Like endogenous retroviruses, prophage may result in the production of infectious particles under certain conditions. This phenomenon deserves some discussion, but this will have to be deferred to a future blog.

References: Purtel et al., 2006; Ward, 1979; Sommer et al., 2001.

Is Clarence calculating clearance correctly?

by Dr. Ray Nims

As pointed out by Dr. Rudge in a recent posting “Do we have clearance, Clarence?”, spiking studies conducted for the purpose of validating impurity clearance are often done at only one spiking level (indeed often at the highest possible impurity load attainable). This is especially true for validation of adventitious agent (virus and mycoplasma) clearance in downstream processes. The studies are done in this way in order to determine the upper limit of agent clearance (in terms of log10 reduction) by the process. Such log10 reduction factors from individual process steps are then summed in order to determine the overall capability of the downstream processes to clear adventitious agents. The regulatory agencies have fairly clear expectations around such clearance capabilities which must generally be met by biologics manufacturers.

The limiting factor in such clearance studies is typically the amount or titer of the agent that is able to be spiked into the process solution, which is determined by: 1) the titer of the stock used for spiking, and 2) the maximum dilution of the process solution allowed during spiking (typically 10%). Under these circumstances, as Scott points out, there is a possibility that the determined clearance efficiency (i.e., the percentage of the load which is cleared during the step) is an underestimate of the actual clearance that might be obtained at lower impurity loading levels.

Adventitious agent clearance is comprised of two possible modalities, removal and inactivation. Removal refers to physical processes designed to eliminate the agent from the process solution, usually through filtration or chromatography. Removal efficiency through filtration would not be expected to display variability based on impurity loading. On the other hand, chromatographic separation of agents (by, for example, ion-exchange columns) may display saturation at the highest loadings, and therefore use of the highest possible loading levels may result in underestimates of removal efficiency at lower (i.e., more typical) impurity levels.

Inactivation refers to physical or chemical means of rendering the agent non-infectious. Agent inactivation is not always a simple, first-order reaction. It may be more complex, with a fast phase 1 stage of inactivation followed by a slow phase 2 stage of inactivation. An inactivation study is planned in such a way that samples are taken at different times so that an inactivation time curve can be constructed. As with removal studies, the highest possible impurity levels are typically used to determine inactivation kinetics.

Source: Omar et al. Transfusion 36:866-872, 1996

While the information obtained through clearance studies of this type may be incomplete from the point of view of understanding the relationships between impurity loading levels and clearance efficiency, the results obtained are consistent with the regulatory expectation that the clearance modalities be evaluated under worst-case conditions. Therefore, at least in the case of adventitious agent clearance validation, I would say that Clarence is calculating clearance correctly!

Riboflavin plus UVA irradiation: another inactivation approach to consider

by Dr. Ray Nims

Short-wavelength ultraviolet irradiation (UVC) has been used for years to disinfect air, surfaces, and thin liquid films because it is effective in inactivating a variety of bacteria, protozoa, phage, and viruses. More recently, UVC (100-280 nm) irradiation has been shown to be useful for viral risk mitigation in biologics manufacturing. UVC-treatment of culture media and other liquid reagents has been demonstrated to inactivate potential adventitious viral contaminants, including those which are resistant to inactivation by other physical means (e.g., murine minute virus; calicivirus; and porcine parvovirus and SV-40 [Wang et al., Vox Sanguinis 86: 230-238, 2004]).

Another approach that has been used recently, especially in the ophthalmologic and blood products communities, is UVA (315-400 nm) in the presence of the photosensitizer riboflavin. Riboflavin interacts with nucleic acid and photosensitizes to damage by UVA leading to direct electron transfer, production of singlet oxygen, and generation of hydrogen peroxide. The treatment results in oxidation and ring-opening of purines and in DNA strand breakage. The advantage of riboflavin over other photosensitizers (e.g., methylene blue, psoralens, etc.) is that riboflavin (vitamin B2) is an endogenous physiological substrate.

 

The photosensitizer interacts with nucleic acids.

Upon irradiation, the results may include cross-linking, mutation, or strand breakage.
Source: Bryant and Klein

Riboflavin/UVA treatment has been explored in ophthalmology applications such as infectious keratitis and keratomycosis. The typical treatment paradigm involves application of a solution of 0.1% riboflavin (as riboflavin-5-phosphate) followed by irradiation using 365 nm light (5 to 10 J/ml). The approach has shown effectiveness against a variety of pathogenic bacteria, including drug-resistant strains. Effectiveness against fungal pathogens requires combination therapy with amphotericin B.

In the blood products community, photosensitizer/UV treatment is being explored for pathogen reduction. For instance, riboflavin/UV treatment is being evaluated for platelet and plasma pathogen reduction, for prevention of graft versus host reactions, and for pathogen reduction in whole blood products. In the proprietary application (Mirasol PRT®), blood product pools are combined with riboflavin (final concentration 50 µM) and the solutions are irradiated with 6.24 J/ml broadband (265-370 nm) UV light. The technology has been shown to be effective for a variety of pathogenic bacteria and viruses (see Table 3 in the review by Bryant and Klein).

Will this approach be useful in the biopharma industry? It appears so. Recently, irradiation with UVA (365 nm) light in the presence of 50 µM riboflavin has been evaluated for controlled inactivation of gene transfer (adenovirus, adeno-associated virus, lentivirus) virus preparations. Complete inactivation was obtained in each case within 90 minutes.

Can Cache Valley Virus Trash Your Manufacturing?

By Dr. Ray Nims

Cache Valley virus is a single-stranded RNA virus of family bunyavirus, genus Bunyavirus. It is enveloped and nominally 80-120 nm in diameter. Cache Valley virus was first isolated in Utah in 1956 and is carried by mosquitoes. It has since been found to be a widespread virus, having been isolated in Texas, Michigan, North Carolina, Indiana, Virginia and Maryland, for example.

source: Nims et al., BioPharm. Int. 21: 89-94, 2008

Basis of Concern. Cache Valley virus is known to infect livestock, causing birth defects. There have been two reports of encephalitic disease in humans attributed to Cache Valley virus. This virus has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions from 2000 to 2004 (Nims et al., BioPharm. Int. 21: 89-94, 2008). The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated bovine serum is considered to be the most likely source. Thus far the virus has only been known to infect manufacturing processes employing bovine serum.

Regulatory Expectations. Cache Valley virus is not mentioned specifically in any regulatory guidance, as the detection of this virus in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).

Mitigating Risk. Although many Contract Testing laboratories offer rapid nucleic acid-based detection assays for Cache Valley virus, raw material screening for this virus using such assays does not appear to be a viable means of eliminating risk. The industry experience thus far indicates that Cache Valley virus may be a low level, non-homogeneous contaminant of bovine serum. Viral screening performed on one bottle out of a large lot of serum therefore is no guarantee that this virus will not be encountered. Elimination of animal-derived materials (esp. bovine serum) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the serum or serum-containing media should be considered. Gamma-irradiation has been demonstrated to be effective in inactivating this virus in bovine serum (Gauvin, 2009). UVC treatment of media containing bovine serum also appears to be quite effective at inactivating Cache Valley virus (Weaver, 2009).

Conclusions. Cache Valley virus infects livestock and has been found to contaminate biologics manufacturing processes employing bovine serum. It is a virus of concern for biologics manufacturers employing bovine serum which has not been gamma-irradiated. Risk of infection of biological products with Cache Valley virus through use of bovine serum may be mitigated through implementation of gamma-irradiation of the serum, or UVC- or high-temperature short-time (HTST) treatment of media containing the serum and of viral purification processes capable of removing and inactivating enveloped viruses.