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>

Porcine circoviruses, vaccines, and trypsin

By Dr. Ray Nims

It has now been more than a year since the announcements by GlaxoSmithKline (GSK) and Merck of the presence of porcine circovirus (PCV) genomic material in their rotavirus vaccines.

The presence of the PCV viral sequences was, in both cases, provisionally attributed to the use of porcine trypsin during the culture of the cell substrates used in the manufacture of the vaccines. It has been reported that the genomic sequences were associated with low levels of infectious PCV in the GSK vaccine.     

As mentioned in a previous posting, an expected outcome of these disclosures was heightened regulatory expectations, going forward, for PCV screening of porcine raw materials and of Master and Working cell banks which were exposed to porcine ingredients (e.g., trypsin) at some point in their development. In January of 2011, the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use was revised to include the following instruction: “Trypsin used for the preparation of cell cultures is examined by suitable methods and shown to be sterile and free from mycoplasmas and viruses, notably pestiviruses, <circoviruses> and parvoviruses.” The addition of circoviruses to the list of viruses of concern (previously, mainly bovine viral diarrhea virus and porcine parvovirus) in Ph. Eur. 7.2 was not unexpected, based on the rotavirus vaccine experience.

A more broad expectation going forward may also be that vaccine and biologics production cell banks be proactively screened for unexpected, perhaps previously undetectable, viruses using detection techniques such as the deep sequencing used initially to detect the PCV in the GSK rotavirus vaccine. A related technique referred to as massively parallel sequencing (Massively Parallel Sequencing (MP-Seq), a New Tool For Adventitious Agent Detection and Virus Discovery) has been adopted for detection of viral contaminants in cells and viral seed stocks and for evaluating vaccine cell substrates by the contract testing organization BioReliance.

The more important sequella of the porcine circovirus disclosures may therefore be the proactive use of these new and powerful virus detection techniques for ensuring the viral safety of production cell banks, going forward.

Ridding serum of viruses with gamma irradiation: part 1

by Dr. Ray Nims

Blood serum, while at times required as a medium component for cell growth in vitro, is an animal-derived material that can introduce contaminating viruses such as Cache Valley virus, REO virus, vesivirus, and epizootic hemorrhagic disease virusinto a biological product. If animal serum must be used in upstream manufacturing processes, the risk of introducing a virus may be mitigated by gamma irradiation of the frozen serum prior to use. How effective is this treatment, and against which viruses?

To answer this question, I have surveyed the literature from the past two decades. A number of investigations have been conducted and the results are in the public domain. The most useful of these studies have examined the dose-response relationships for viral inactivation (rendering of the virus as non-infectious) by gamma irradiation.

In the table below, I have assembled the results obtained for 7 viruses, including four that might be expected to be found in bovine serum: (bovine viral diarrhea virus [BVDV], infectious bovine rhinotracheitis virus [IBR], respiratory-enteric orphan virus [REO virus], and parainfluenza type 3 virus [PI3]). The other three (canine adenovirus, porcine parvovirus [PPV], and mouse minute virus [MMV]), while perhaps not expected to be found in bovine serum, have been studied as model viruses for the adenovirus and parvovirus families (click on table to enlarge).

The efficacy of gamma irradiation for viral inactivation is reported as log10 reduction in titer per kGy, rather than the more commonly employed D10 (Mrad dose required to reduce the titer by 1 log10), as I find the former value to be more useful. To estimate the effectiveness of a given dose of gamma radiation for inactivation of a virus, just multiply the dose in kGy by the log10 reduction in titer per kGy value from the table. The result is the number of logs of inactivation estimated to be achieved for that virus at that radiation dose.

These data tell us that the mid- to large-sized viruses BVDV, IBR, PI3, REO, and CAV should be readily inactivated at the gamma radiation doses normally applied to frozen serum for risk mitigation (25-45 kGy). On the other hand, the two parvoviruses, PPV and MMV, are more difficult to inactivate, presumably due to their small size. Parvoviruses are often used to challenge viral removal and inactivation processes due to their size and lack of an envelope. Higher kGy dose levels may increase the effectiveness of inactivation for these viruses, although at such levels the performance of the animal serum being irradiated may be adversely impacted.

Gamma-irradiation can effectively mitigate the risk of introducing other potential contaminants of bovine serum, including Cache Valley virus, blue tongue virus, and epizootic hemorrhagic disease virus. Like the parvoviruses, however, other relatively small non-enveloped viruses of the calicivirus, picornavirus, polyomavirus, and circovirus families may represent cases where gamma irradiation is less effective at the doses normally applied. Other means of mitigating the risk associated with these viruses may need to be considered.

< This information was excerpted in part from Nims, et al. Biologicals (2011) >


Information sources: Daley et al., FOCUS 20(3):86-88, 1998; Wyatt et al. BioPharm 1993: 6(4):34-40; Purtel et al., 2006; Hanson and Foster, Art to Science 16:1-7, 1997; Hyclone Labs Art to Science 12(2): 1-6, 1993; Gauvin and Nims 2010; Plavsic et al. BioPharm 2001: 14(4):32-36.

Small, non-enveloped viruses: number 1 threat to biologics manufacture

by Dr. Ray Nims

Perhaps surprisingly, few types of viruses have infected biologics manufacture since the 1980s when the first recombinant proteins began to be produced in mammalian cells. While the list of contaminating viruses has included some relatively large enveloped and non-enveloped viruses (Reovirus type 2, epizootic hemorrhagic disease virus, Cache Valley virus, human adenovirus), by far the most problematic contaminants have been the small non-enveloped viruses. Why? For the most part, the contaminations involving the larger viruses have been attributed to the use of non-gamma irradiated bovine serum or to operators conducting open vessel manipulations. Remediating the manufacturing processes to include gamma irradiation of the serum (or elimination of the use of serum altogether), and eliminating wherever possible open vessel operations should mitigate the risk of experiencing these viruses.

Now we come to the small non-enveloped viruses, the real problem. Foremost among these has been murine minute virus (MMV). This 20-25 nm non-enveloped parvovirus has infected biologics manufacturing processes using Chinese hamster cell substrates on at least four occasions, affecting at least three different manufacturers (Genentech, Amgen, and Merrimack). In each case, the source of the contamination has been unclear, making remediation of the processes difficult. Due to the ability of these viruses to survive on surfaces and their resistance to inactivation by detergents and solvents, eliminating the agent from contaminated facilities may require drastic measures such as fumigation with vaporous hydrogen peroxide .

A second problem virus is the 27-40 nm non-enveloped calicivirus, vesivirus 2117. This is the virus that was found to have infected the Genzyme Allston manufacturing facility in 2009. The same virus had appeared already once in the past, at a manufacturing facility in Germany. Both of the infected processes involved Chinese hamster production cells and both involved the use of bovine serum at some point in the manufacturing process. Whether or not the animal-derived material was the actual source of the infection was not proven in either case. Unfortunately, if the source was the bovine serum, gamma irradiation probably would not mitigate the risk, as gamma irradiation is less effective for inactivating the smaller non-enveloped viruses. This is another virus that may be able to survive on facility surfaces. As in the case with MMV, ridding a manufacturing facility of vesivirus may require entire facility fumigation with vaporous hydrogen peroxide, as was done at Genzyme.

Another problem virus is the 17-20 nm porcine circovirus that was found to contaminate a rotavirus vaccine in 2010. This virus was thought to have originated in contaminated porcine trypsin used in the manufacturing process. Wouldn’t this contaminant have shown up in the raw material testing done for the trypsin, or in the extensive cell bank testing required for vaccine production substrates? The answer is no. The circovirus would not have been detected using the 9CFR-based detection methods used for trypsin at this time (and at present). And the required testing for cell banks used to produce vaccines would not have detected this particular virus. To make matters worse, gamma irradiation of the trypsin would not be expected to inactivate this virus. How can we mitigate the risk of this virus going forward? As described in a previous posting, manufacturers may need to apply specific nucleic acid tests for the circovirus as part of the raw material release process for trypsin.

These and other small non-enveloped viruses represent the greatest risk for biologics manufacturing because they are more difficult to inactivate in raw materials, and more difficult to eradicate from the facility once infected, and because the source of the infection is not always clear. There must be analogous small-non-enveloped bacteriophage lurking out there that represent, for the same reasons, special threats to the fermentation industry.

FDA to viral vaccine makers: it's time to update viral testing methods

By Dr. Ray Nims

If you have been following the recent (2010) unfolding of the discovery of porcine circovirus DNA contamination in rotavirus vaccines from GSK and Merck, you may not be surprised to hear that the FDA has asked viral vaccine manufacturers to outline, by October, their plans to update their testing methodologies to prevent future revelations of this type.
 
I had predicted earlier that biologics manufacturers would be asked to provide evidence, going forward, that their porcine raw materials (trypsin being the most common) are free of porcine circovirus. This testing has not been manditory in the past, but adding this to the porcine raw material virus screening battery moving forward is a prudent action in light of the recent rotavirus vaccine experience.

The FDA has appropriately gone a little farther in it's request to the viral vaccine manufacturers. The regulators would like to assure that the future will not bring additional discoveries of viral contaminants in licensed vaccines, and the best way to accomplish this at the moment appears to be to request implementation of updated viral screening methodologies. Does this mean that viral vaccine makers will need to employ deep sequencing on a lot-by-lot basis? Most likely not. It appears that reliance on the in vivo and in vitro virus screening methods which have been the gold standards since the 1980s will, however, no longer be sufficient. So what does this leave us with? What FDA appears to be asking for is a relatively sensitive universal viral screening method.

The in vivo and in vitro methods were, until now, the best option for this purpose. These methods detect infectious virus only and depend upon the ability of the virus to cause an endpoint response in the system (cytopathic effect, hemagglutination, hemadsorption, or pathology in the laboratory animal species used). So viral genomic material would not be detected, and the methods have had to be supplemented with specific nucleic acid-based tests for viruses which could not otherwise be detected (e.g., HIV, hepatitis B, human parvovirus B9, porcine circovirus).

Some options for sensitive and universal viral screening methods which might fit the requirements include DNA microarrays and universal sequencing methods performed on cell and viral stocks. The latter technology may be preferable, as microarrays are constructed to detect known viruses, while the desire is that the technology be universal in the sense that it detect both known and unknown viruses. Such a test will provide additional assurance that the virus and cell banks used to manufacture viral vaccines do not harbor a viral contaminant.

Other universal viral screening methods which are less labor intensive than the sequencing technologies may be developed in the near future and addition of one of these to the release testing battery for viral vaccine lots may need to be considered in satisfying the FDA's goals.

Using porcine trypsin in biologics manufacture?

by Dr. Ray Nims

On March 22, 2010, a press release from GlaxoSmithKline (GSK) announced that porcine circovirus 1 (PCV 1) DNA had been detected in their rotavirus vaccine. On May 6, Merck disclosed that it had found DNA fragments of both PCV types 1 and 2 in its rotavirus vaccine. The PCV 2 findings in Merck's vaccine may be of greater concern, due to the fact that this virus causes disease in pigs, while PCV 1 apparently does not. However, the relative amounts of PCV DNA found in the GSK vaccine appear to be much greater (the lab discovering the PCV DNA in the GSK vaccine did not detect any in the Merck vaccine), and the worry in this case is that some of the genomic material may be associated with infectious PCV 1 virus. In both cases, the presence of the PCV genomic material has been attributed to the use of porcine trypsin at some point in the vaccine manufacturing process.


The FDA convened an advisory committee meeting on May 7th to discuss the findings of PCV DNA in the two licensed rotavirus vaccines. What was the result of the advisory committee meeting? The advisory committee felt that the benefits of the rotavirus vaccines clearly outweigh the risks. This, added to the fact that there appears to be little human health hazard associated with these viruses, led to the FDA clearing the two vaccines for continued use on May 14th. The product labels will be updated to reflect the presence of the PCV DNA in these products. In the longer term, these products may need to be "reengineered" to remove the PCV DNA. This may involve the preparation of new Master and Working cell banks and thus will take some time.

Another likely outcome of the advisory committee’s meeting may be heightened expectations, going forward, for PCV screening of porcine raw materials and of Master and Working cell banks which were exposed to porcine ingredients (e.g., trypsin) at some point in their development. Porcine-derived raw materials which are used in the production of biologics are to be tested per 9 CFR 113.53 Requirements for ingredients of animal origin used for production of biologics for a variety of viruses of concern. In the case of ingredients of porcine origin, those viruses of concern are listed in 9 CFR 113.47 Detection of extraneous viruses by the fluorescent antibody technique. These include rabies, bovine viral diarrhea virus, REO virus, porcine adenovirus, porcine parvovirus, transmissible gastroenteritis virus, and porcine hemagglutinating encephalitis virus. While porcine circovirus may not be specifically mentioned in the 9 CFR requirements, it will be prudent to add a nucleic acid-based assay for detection of this virus to the porcine raw material testing battery going forward. Similarly, Master and Working cell banks exposed to porcine raw materials (e.g., trypsin) during their developmental history should be assayed for PCV prior to use.

Routine nucleic acid-based testing for PCV should detect the genomic sequences for this virus should intact infectious or non-infectious PCV be present in the test materials. Now that this virus is one of concern to the FDA and to the public, performing the appropriate raw material and cell bank testing for it will most likely become an expectation for vaccine and biologics manufacturers.

Oops, adventitious viral DNA fragments in a vaccine

by Dr. Ray Nims

On March 22, 2010, a press release from GlaxoSmithKline (GSK) announced that DNA from porcine circovirus had been detected in their rotavirus vaccine. According to GSK, the DNA ”was first detected following work done by a research team in the US using a novel technique for looking for viruses and then confirmed by additional tests conducted by GlaxoSmithKline”. As a result of this finding, the FDA “is recommending that US clinicians and public health professionals temporarily suspend the use of Rotarix as a precautionary measure. The FDA have also stated that they intend to convene an advisory committee, within approximately four to six weeks, to review the available data and make recommendations on rotavirus vaccines licensed in the USA. The FDA will also seek input on the use of new techniques for identifying viruses in vaccines.” The EMEA, on the other hand, does not appear to consider this finding to be a safety concern, citing the fact that porcine circovirus is not infectious for human cells and does not cause disease in humans.

Porcine circovirus

Source: Meat and Livestock Commission, UK

What is porcine circovirus? Porcine circovirus (PCV) is a member of the family Circoviridae, among the smallest of the known animal DNA viruses. It is approximately 17 nm in diameter, non-enveloped, with icosahedral symmetry. The virus was originally identified as a noncytopathic contaminant of the PK-15 porcine kidney cell line (Tischer et al., Zentralblatt Bakt Hyg A 226:153-167, 1974). Like many very small, non-enveloped viruses, PCV represents a challenge for removal and inactivation.

Why is this finding coming to light now, or stated another way, why wasn’t the PCV DNA detected when the vaccine was initially tested and approved for human use? The press release wasn’t specific as to method used. It has subsequently been revealed, however,  that these fragments were detected using sequence-independent amplification (deep sequencing; or as Eric Delwart calls it, metagenomics). The resulting library of amplified sequences is characterized by BLAST searching using identification algorithms. Confirmation in this case was obtained using microarray and PCR analyses. The sequencing techniques have been available for some time, and have proven useful for identification of viruses in an academic setting, though they have not been applied to safety testing of biopharmaceuticals until fairly recently due to the relatively high costs associated with the analyses.

The finding of viral DNA should not be equated with detecting the infectious virus in the product. The sequence-independent amplification, microarray, and virus-specific PCR assays used can detect viral nucleic acid, but as normally performed do not indicate whether infectious virus is present. Generally, with the possible exception of transforming viruses, it is the infectious virus that is of concern, not its DNA. Efforts to assess the presence of intact viral genomic material and of infectious porcine circovirus in this vaccine are most likely underway at this time.

The presence of the PCV viral sequences has provisionally been attributed to the use of porcine trypsin during the culture of the Vero cell substrate in which the vaccine is manufactured. The trypsin used had been gamma-irradiated to inactivate adventitious viruses prior to use. While it would be comforting to believe that the PCV DNA may simply have reflected carryover of non-infectious, lethally-irradiated PCV1 from the trypsin, the fact is that gamma-irradiation is not very effective at inactivating this small, non-enveloped virus (Plavsic and Bolin. Resistence of porcine circovirus to gamma irradiation. Biopharm Int. 14:32-36, 2001). In the case of porcine circovirus, there is little evidence to indicate that the virus is infectious or pathogenic for humans. So regardless of the outcome of the various ongoing studies, it is likely that the use of the GSK rotavirus vaccine will be re-instated after the FDA convenes and discusses the implications of this finding.

I predict that there will be more and more of this kind of revelation in the future as the sequencing techniques display stretches of viral or other contaminant DNA within samples of biopharmaceuticals. I would hate to see revelations like this impede the use of the sequencing technologies going forward, as these technologies are going to be very useful to the industry as rapid detection methods for contaminant identification.