Viral clearance studies …. are they needed for proteins produced using bacterial or yeast fermentation processes?

Dr. Ray Nims

In E. coli or Pichia pastoris-based bioproduction of recombinant proteins, there are no suitable host cells for amplification of viruses that are infectious for humans. Bacteria such as E. coli and yeast such as P. pastoris can only support the growth of certain bacteriophage or yeast viruses, respectively. These types of viruses are not infectious for humans or animals.

The potential for viral infection of a bacterial cell process or a yeast cell process used for production of a recombinant protein is therefore a business risk, not a patient safety risk. This is the reason why case studies of bacteriophage infection of bacterial fermentation processes or of viral contamination of yeast cell processes have not appeared in the literature (or in the news). Unlike virus contamination of animal cell-based production processes, the events involving bacterial or yeast do not have patient safety implications. When a bacteriophage or yeast virus contamination occurs, manufacturers quietly go about the business of restarting the fermentation. Remediation and prevention of future occurrences is driven primarily by business concerns, as opposed to regulatory concerns.

Manufacturers of recombinant proteins produced using bacterial or yeast cell substrates are not required to conduct cell line viral testing (though phage induction studies may be performed as part of bacterial cell line characterization – to mitigate business risk!), nor are they required to conduct lot-by-lot viral testing of bulk harvest samples. Such requirements are mandated for production processes using animal or human cells. As the title of the document “Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin” indicates, the ICH Q5A (R1) guidance mandating such testing is clearly directed toward manufacturing processes utilizing the types of cells which may support growth of viruses infectious for human or animal cells. This ICH document also provides guidance on the conduct of viral clearance studies.

Viral clearance studies are intended to provide evidence of the ability of downstream purification steps of a manufacturing process to remove or inactivate viruses that potentially may contaminate the process, make their way into final product, and represent a health risk to patients. As such, the evaluation typically involves both relevant viruses (i.e., viruses that are known to contaminate these kinds of processes) and model viruses (i.e., viruses representative of the types of viruses that could contaminate these processes). In the case of bacteria- and yeast-based manufacturing processes, the relevant viruses (bacteriophage and yeast viruses, respectively) are not infectious for humans or animals. In addition, there are no model viruses that are capable of infecting these kinds of cells that are of concern for humans or animals. Therefore, viral clearance studies are typically not required or conducted.

Are there any circumstances where a bacterial or yeast fermentation process could be expected to harbor a virus capable of infecting humans? The only that I can imagine is a process utilizing primary or secondary animal-derived raw materials in rather large quantities. The worry would not be that viral amplification might occur, but that some carryover of surviving animal viruses to the final product might be possible. The use of such animal-derived materials by a manufacturer of a therapeutic protein must be justified based on risk analysis. If substantial risk is introduced by the use of such raw materials, then perhaps a raw material treatment approach would need to be validated.

In the absence of the use of animal derived materials or plant materials not subject to processing steps that would inactivate contaminating animal viruses, incorporation and validation of viral clearance steps into protein production processes using bacteria or yeast cell substrates is not expected, nor would this be of practical value in assuring patient safety.

A much improved Ph. Eur. Chapter 5.3.2

By Dr. Ray Nims

Vaccine manufacturers intending to market in the EU should be aware of a recent change in the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use. This chapter addresses the characterization of vaccine cell substrates. The section on Test Methods for Cell Cultures within the chapter includes an instruction to perform a co-cultivation study. The language previously was as follows: “Co-cultivation. Co-cultivate intact and disrupted cells separately with other cell systems including human cells and simian cells. Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses. The cells comply with the test if no evidence of any extraneous agent is found.”

This section has been changed, as of Ph. Eur. version 7.2 effective in January of 2011, to the following: “Co-cultivation. For mammalian and avian cell lines, co-cultivate intact and/or disrupted cells separately with other cell systems including human cells and simian cells. For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21). Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses, or on cells to detect haemadsorbing viruses. The test for haemagglutinating viruses does not apply for arboviruses to be detected in insect cells. The cells comply with the test if no evidence of any extraneous agent is found.”

So what is the big deal? Co-cultivation is a commonly employed technique for detecting infectious retrovirus in a cell bank. It is effective for this purpose because the chances for spread of infectious virus from test cell to indicator (host) cell are optimized by the cultivation of live cells of each kind in close proximity. The endpoint of the retrovirus assay, be it reverse transcriptase enzyme induction or rescue of an S+L- virus, is not interfered with by the presence of two cell types in one culture. The same is not always true for a co-cultivation of a test cell with an indicator (host) cell for detection of infectious virus when morphological changes (viral cytopathic effects) are one of the assay endpoints. The reason is that the diploid human cells (e.g., MRC-5 or WI-38) used as one of the indicator cells in such assays are rapidly displaced during co-cultivation with intact continuous cell lines used to produce vaccines, such as the simian cell Vero. The result of this is that within a short period of time in co-cultivation, the test culture is no longer predominated by the diploid cell but rather by the test cells and observation of the culture for cytopathic effects becomes problematic. Changing the language of this section to read “…co-cultivate intact and/or disrupted cells separately with other cell systems…” allows the user to eliminate the inoculation of intact test cells onto a diploid indicator cell.

The other useful modification to the language of this section is the following addition: “For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21).” Testing of insect cells for extraneous virus is only marginally effective when it is conducted per the usual method of inoculating another insect cell. Why? The insect cells that are available are most commonly suspension cultures, making observation for cytopathic effect problematic. The extraneous viruses that are of most concern for an insect production cell are the arboviruses (viruses transmitted via insect vectors). It has been known for some time that the Syrian hamster cell line BHK-1 is an excellent host cell for detecting arboviruses. The new language in this section of Ph. Eur. chapter 5.2.3 now clears the way for the use of the monolayer BHK-1 cell line to be used for the testing of insect cells for extraneous virus. In this regard the Ph. Eur. chapter is now more closely aligned with the World Health Organization’s 2009 Evaluation of cell substrates for the production of biologicals: revision of WHO recommendations. The latter has the following passage: "For instance, in the case of insect cell substrates, certain insect cell lines may be used for detection of insect viruses, and BHK cells may serve for the detection of arboviruses."

   

Taken together, the recent changes to Ph. Eur. Chapter 5.2.3 greatly improve the chapter and the viral safety testing of vaccine production cell banks specifically proscribed within it.

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.

Cell Culturists….Are your human cells authenticated?

by Dr. Ray Nims

Until fairly recently, it has been common practice to authenticate human cell cultures using phenotypic status (e.g., receptor or protein expression) and animal species of origin testing. This level of authentication is better than none, but it is not sufficient to unambiguously identify a human cell culture. The result has been that we are still hearing about cases of misidentified human cells being used for biomedical research.

There are now methods available that are capable of rapidly and unambiguously identifying human cell lines, tissues, and cell preparations to the individual level. The recent demonstration of the potential utility of molecular technologies such as short tandem repeat (STR) and single nucleotide polymorphism (SNP) profiling for cell authentication has provided the impetus for development of a new standardized method for human cell authentication.

To this end, an ATCC Standards Development Organization workgroup with international representation has spent the past two years developing a consensus standard for the Authentication of Human Cell Lines through STR Profiling. The forthcoming Standard will provide guidance on the use of STR profiling for authenticating human cells, tissue, and cell lines. It will contain methodological detail on the preparation and extraction of the DNA, guidance on the appropriate numbers and types of loci to be evaluated and on interpretation and quality control of the results. Associated with the standard itself will be the establishment of a public STR profile database which will be administered and maintained by the National Center for Biotechnology Information (NCBI). The database primarily will contain STR profiles of commonly used cell lines.

                                            STR Profiling of Hela Cells

 An announcement that the Standard is now available for public 45-day review, comment, and vote was published in the October 22, 2010 issue of the ANSI newsletter Standards Action.

The benefits of the Standard will depend on the degree to which it is adopted and followed in the biomedical research and development and biopharmaceutical  communities. Taking a broader view, it is hoped that funding agencies and journals will begin to use such authentication standards as important considerations for funding or publishing research employing human cells. The quality and validity of funded and published research should benefit greatly as a result of the reduction in frequency of use of misidentified human cells.

The deadline for comments is December 6, 2010. There is still time to review the draft Standard and to voice your opinions and concerns.

Manufacturing Biologics with CHO Cells? What’s the Risk for Viral Contamination?

by Dr. Ray Nims

Chinese hamster ovary (CHO) cells are frequently used in the biopharmaceutical industry for the manufacture of biologics such as recombinant proteins, antibodies, peptibodies, and receptor ligands. One of the reasons that CHO cells are often used is that these cells have an extensive safety track record for biologics production. This is considered to be a well-characterized cell line, and as a result the safety testing required may be less rigorous in some respects (e.g., retroviral safety) than that required for other cell types. But how susceptible is the cell line to viral contamination?

There are a couple of ways of answering this question. One way is to examine, in an empirical fashion, the susceptibility of the cell type to productive infection by model exogenous viruses. This type of study has been conducted at least three times over the past decades by different investigators. Wiebe and coworkers (In: Advances in Animal Cell Biology and Technology for Bioprocesses. Great Britain, 1989; 68-71) examined over 45 viruses from 9 virus families for ability to infect CHO-K1 cells, using immunostaining and cytopathic effect to detect infection. Only 7 of the viruses (Table 1) were capable of infecting the cells. Poiley and coworkers (In Vitro Toxicol. 4: 1-12, 1991) followed with a similar study in which 9 viruses from 6 families were evaluated for ability to infect CHO-K1 cells as detected by cytopathic effect, hemadsorption, and hemagglutination. This study did not add any new viruses to the short list (Table 1). The most recent study was conducted by Berting et al. This study involved 14 viruses from 12 families. The viruses included a few known to have contaminated CHO cell-derived biologics in the past two decades, and therefore did add some new entities to the list in Table 1. Still, the list of viruses that are known to replicate in CHO cells is relatively short.

Chinese hamster cells possess an endogenous retrovirus which expresses its presence in the form of retroviral particles, however these particles have been consistently found to be non-infectious for cells from other animals, including human cells. This endogenous retrovirus therefore does not present a safety threat (Dinowitz et al. Dev. Biol. Stand. 76:210–207, 1992).

A second way of looking at the question of viral susceptibility of CHO cells is to examine the incidence and types of reported viral contaminations of manufacturing processes employing CHO cell substrates. This subject has been reviewed a number of times, most recently by Berting et al. The types of viral contaminants fill a fairly short list (Table 2). In most cases, the contaminations have been attributed to the use of a contaminated animal-derived raw material, such as bovine serum.

Sources: Rabenau et al.1993; Garnick 1996; Oehmig et al., 2003; Nims Dev. Biol. 123:153-164, 2006; Nims et al., 2008; Genzyme 2009..

Considering the frequency with which CHO cell substrates have been used in biologics production, this history of viral contamination is remarkably sparse. This is further testament to the overall safety of this particular cell substrate.

The nuts and bolts of retrovirus safety testing

by Dr. Ray Nims


Retroviruses may integrate into the genome of host animals. For this reason they are often referred to as endogenous viruses. Viral particles may or may not be expressed in the host cell. Expressed viruses may be infectious or non-infectious, and infectious virus may have tropism for (ability to infect) the same or different animal species relative to the host cell of origin. Infection results from a process of reverse transcription of the viral RNA leading to proviral DNA. To accomplish this, retroviruses have a specialized enzyme known as reverse transcriptase. Through this process (see figure below), the infected cell may be enlisted to produce viral progeny. Certain of the retroviruses are known to be oncogenic (e.g., human T-lymphotropic virus 1, feline leukemia virus, Raus sarcoma virus, etc.). Other retroviruses are of concern as a result of disease syndromes caused in humans (e.g., human immunodeficiency virus 1 in acquired immunodeficiency syndrome, and the possible role of xenotropic murine leukemia virus-related virus in chronic fatigue syndrome). From a biosafety standpoint, there is a worry that under some conditions, integrated viruses in cell substrates employed to produce biopharmaceuticals which do not normally express their presence may be induced to produce infectious particles.

Source: AccessExcellence

Retrovirology safety testing for biologics manufacture can be confusing to those not familiar with the subject. Here is a brief overview.

Demonstrating retroviral safety typically involves a combination of the following three components:
• detecting infectious retrovirus through cell culture assays (XC plaque, cocultivation with mink lung or Mus dunni cells, etc.).
• measuring reverse transcriptase enzyme activities either through tritiated thymidine incorporation into templates, or through product amplification (PCR) techniques (PERT, etc.). This is not required if infectious retrovirus is detected.
• Visualizing and enumerating retroviral particles in supernatants or in fixed cells using transmission electron microscopy.

The various assays are applied during cell bank characterization (including end of production cell testing), during evaluation and validation of purification processes, and in some instances, as bulk harvest lot-release assays (results from 3 lots at pilot or commercial scale are submitted with the marketing application). For processes using well-characterized rodent cells known to contain endogenous retrovirus (CHO, C127, BHK, murine hybridoma), retroviral infectivity testing of the processed bulk is not required provided that adequate downstream clearance of the particles has been demonstrated.

Infectivity testing can be particularly confusing, due to the variety of cell-based assays employed. These include both direct and indirect assays. An example of a direct assay is the XC-plaque assay for ecotropic (a term meaning the virus is infectious for mouse cells) murine retroviruses. By definition, therefore, this would only be used to assay production cells of mouse origin.

Indirect assays are those in which a second endpoint is required to assess positive or negative outcome. Indirect assays include the various co-cultivation assays in which the test cells are co-cultivated with host cells such as mink lung, Mus dunni, and any of a number of human cells (see Table 4 within USP <1237> Virology Tests for a list of commonly used host cells). The indirect assays are performed to detect xenotropic retroviruses (retroviruses which are capable of infecting only animals other than the species of origin). The secondary endpoints used to assess outcome include reverse transcriptase activity, sarcoma virus rescue (S+L- focus formation assays), or enzyme immunoassay. The indirect assays are used in the retrovirus testing of mouse, hamster, monkey, and human production cell substrates. The selection of the host cell for the cocultivation assay is dependent upon the species of origin of the production cell, recognizing that cocultivation host cells from a species other than that of the production cells must be used. For production processes using rodent or other non-human cells, one or more human host cells are typically used for the cocultivation assay, as xenotropic retroviruses infectious for human cells are of obvious concern.

Still confused? Don’t worry. An individual with virology testing expertise can assist in designing the appropriate retrovirus testing battery for your biologic.

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.

What cell line is this anyway?

By Dr. Ray Nims

For about as long as scientists have been using cell cultures in biomedical research, there have been cases of cell line misidentification. This has been especially true for continuous cell lines, with the increased probability over time of mislabeling or cross-contamination. The primary cross-contaminant historically has been HeLa, a human cervical carcinoma cell which, given the opportunity, could outgrow most other cells in culture. More recently, the use of feeder cells for the propagation of human stem cells, and the use of xenografting for the propagation of human tumor cells, has provided additional opportunities for cell line cross-contamination and misidentification.


In the past, confirmation of cell line species of origin has been the main approach for authenticating cell lines. This was done initially by karyotyping or by immunological techniques, but more recently it has been done through the technique of isoenzyme analysis. An example of an isoenzyme analysis is shown below for Peptidase B and Aspartate Aminotransferase.  These agarose gels show a positive control, a negative control (this is the band that does not line up with the others), the test article and a standard extract.  These gels confirmed the identity of the test article as mouse derived, as expected. 

Isoenzyme analysis has the advantage that it is rapid, not very technically demanding, and may be used not only to confirm species of origin but also to detect the presence of a cross-contaminating cell if the latter is present in the culture at 10% or greater (Nims et al., Sensitivity of Isoenzyme Analysis for the Detection of Interspecies Cell Line Cross-Contamination. In Vitro Cell. Dev. Biol.-Animal 34:35-39, 1998). In fact, isoenzyme analysis is currently the primary method employed within the biopharmaceutical industry for cell line authentication in satisfaction of 1993 Points to Consider and ICH Q5D guidance.

Recent advances in molecular diagnostic techniques have made possible the authentication of human cell lines to the individual level. DNA fingerprinting technologies have matured to the point that some of them, especially single nucleotide polymorphism (SNP) typing and single tandem repeat (STR) profiling, are now considered to be viable options for standardizing human cell authentication (see ATCC SDO newsletter article, page 5. For both human and animal cells, DNA fingerprinting provides a means of determining authenticity to the individual level. However, the primary drawback is that the fingerprinting techniques as routinely performed will be less or not at all useful for detecting interspecies cocultivations or cross-contaminations. For this purpose, it may be necessary to retain isoenzyme analysis as part of the authentication armament even when the molecular technologies become the definitive authentication practices for human and animal cell lines.

Should we care about…Vesiviruses?

By Ray Nims

Vesiviruses are single-stranded RNA viruses of family calicivirus, genus Vesivirus. They are non-enveloped and 30-40 nm in diameter, and the genus includes feline calicivirus, vesicular exanthema of swine virus, rabbit vesivirus, and San Miguel sea lion virus, as well as vesivirus isolate 2117.

source: Stewart McNulty, Queens University, Belfast, UK

Basis of Concern. Vesivirus 2117 has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions, the first being reported in 2003 (Oehmig et al., J. Gen. Virol. 84, 2837-2845, 2003), and additional occurrences being reported in 2008 and 2009.
The susceptibility of relevant manufacturing cell lines of different animal species to infection by this virus appears to be limited to the Chinese hamster. When infected, these cells undergo a relatively rapid lytic infection. The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated animal-derived materials, such as bovine sera, is considered to be the most likely source.

Regulatory Expectations. Vesivirus is not mentioned specifically in any regulatory guidance, as the detection of the 2117 isolate 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. At least three Contract Testing laboratories have announced rapid nucleic acid-based detection assays for vesivirus isolate 2117 within the past year. These assays are available for raw material screening and for in-process testing of biologics bulk harvest samples. Elimination of animal-derived materials (esp. bovine sera) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the sera or sera-containing media should be considered. Studies on the inactivation of caliciviruses indicate that UV treatment may be effective (Duizer et al., Appl. Env. Microbiol. 70, 4538-4543, 2004; de Roda Husman et al., Appl. Env. Microbiol. 70, 50989-5093, 2004). Gamma-irradiation at the dosages normally used does not appear to be effective, as might be expected for a virus of this relatively small size. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for vesiviruses in general.

Conclusions. Vesivirus isolate 2117 preferentially infects Chinese hamster cells and has been found to contaminate biologics manufacturing processes employing this cell substrate. It is now a virus of concern for the biopharmaceutical industry. Risk of infection of biological products with vesiviruses through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UV or HTST treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.