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Detecting viral contaminants in biologic-based medicines-and identifying their source-requires a holistic testing approach.
Viruses replicate or grow within living organisms, from man to microbes. Therefore, any biological system used to generate biological or biopharmaceutical products could be contaminated with viruses. Viruses can cause serious or fatal illness. Even viruses not known to be highly pathogenic, or to cause significant disease in humans, could potentially do so if introduced into an immune-compromised host and/or by a non-natural route of infection (e.g., intravenous or intramuscular inoculation rather than respiratory or oral spread). Some viruses can result in chronic infections, or persist silently within cells for long periods. For example, retroviruses, a family of viruses known to cause serious disease in humans and animals, integrate into the host cell genome as part of their lifecycle, and can result in life-long infection or cellular change.
There are several, well-documented historical instances of virus transmission to humans through contaminated vaccines. Some early batches of yellow fever vaccine, for example, produced in hens’ eggs were found to be contaminated with avian leucosis virus. In the late 1950s and early 1960s, batches of poliovirus and adenovirus vaccines were contaminated with Simian vacuolating virus 40 (SV40) from rhesus monkey kidney cells. Viruses have also been detected in cell culture-derived products without transmission to humans or animals. Examples are shown in Table I.
Cell culture-derived source
Bovine diarrhea virus
Live veterinary vaccines
Epizootic haemorrhagic disease virus
Live veterinary vaccine
Murine minute virus
BHK-derived veterinary vaccine
BHK cell product
Virus contamination could arise due to the presence of virus in the original cell line or biological system used, or to the inadvertent introduction of virus into cell cultures during a manufacturing process. Raw materials of animal origin, a manufacturing process operator, or even materials contaminated by contact with animals or animal-derived material are possible contamination sources. Contamination of Chinese hamster ovary (CHO) cell banks with murine minute virus (MMV), a virus primarily found in the urine of rats and mice, could potentially have resulted from this latter route.
Although there is significant effort to move away from the use of animal-derived materials such as bovine serum or porcine trypsin for cell culture, many cell lines have historically been exposed to these materials and could potentially be infected with a range of bovine and porcine viral contaminants. For example, the most likely source of contamination of live rotavirus vaccine with porcine circovirus, identified initially in 2010 via next generation sequencing, appears to have been non-irradiated porcine trypsin used in the 1990s to manufacture the Vero cell banks used for vaccine production (1-3).
Three principal and complementary approaches are generally applied for controlling viral contaminants in biologics: the selection and control of starting materials; the testing of source materials and the products; and viral clearance. This article primarily focuses on testing of source materials and products.
Regulatory authorities require testing to be carried out at every stage of the manufacturing process of the product, including cell substrate banks, viral seed banks, raw materials of animal origin, bulk harvests, and batches of the final manufactured clinical product. The samples must be tested to recognized international guidelines, such as the International Council for Harmonization (ICH) guidelines, FDA Vaccine 2010 guidelines (4), and/or European Pharmacopeia guidelines, as appropriate.
There are many factors to be taken into consideration when planning a viral testing strategy, and these will include assessment of the risk of contamination from endogenous and adventitious viruses due to the starting materials and reagents used, as well as the nature and intended use of the final product. All potential contaminants may not be predicted. Novel infectious agents are being increasingly reported (e.g., novel coronaviruses, such as severe acute respiratory syndrome [SARS] and novel influenza virus variants), and no single assay method or approach will detect all viruses. A combination of approaches for virus detection, including general and specific assays, is therefore required. Potential test methods typically include adventitious agent tests, usually non-specific tests capable of detecting a broad range of viruses; species-specific assays, designed to detect the presence of identified potential contaminants (e.g., specific bovine or porcine viruses in serum or trypsin, respectively, or murine viruses in mouse cells); and tests for retroviruses.
Viruses can be detected by using assays that look for evidence of infectivity in a living system (in-vitro or in-vivo assays), or for viral markers including the presence of the virus genome (e.g., polymerase chain reaction [PCR]-based methods), viral proteins (e.g., enzyme assays for reverse transcriptase in retroviruses), or virus particles (e.g., by electron microscopy).
Electron microscopy (EM) is used widely in the industry as a generalized, non-specific screening method. It allows direct visualization of cells or samples, and can allow for the detection of a broad range of viruses as well as other micro-organisms, included bacteria, fungi, and mycoplasma. Although a useful screen, the sensitivity of the method is low. For example, virus present in a very small proportion of a cell bank (e.g., 1 in 1000 cells) may not be detected in a standard assay where 200-300 cell profiles may be examined. EM can also be a useful diagnostic and investigative tool if the presence of virus is suspected in cell cultures.
Almost all cells contain retrovirus sequences. Some cell lines commonly used in the industry, such as murine hybridomas and CHO cells, are known to release retrovirus-like particles. Although the retrovirus particles released from CHO cells have not been shown to be infectious, murine cells can often release infectious virus. Regulations require the use of EM to look for the presence of retrovirus in cells and to quantify the load of retrovirus-like particles in bulk harvests when rodent cells such as CHO are used.
Retroviruses express an enzyme known as reverse transcriptase (RT), which converts virus RNA to DNA and facilitates integration of the virus DNA into the host cell genome. It is virtually unique to retroviruses, so RT activity is an indication of potential retrovirus contamination. Regulations call for the use of PCR-based RT (PBRT) or product-enhanced RT (PERT) assays to detect retroviral RT enzyme activity (4-10). Although PERT assays are highly sensitive, they can sometimes give false positives from high background cellular DNA polymerase activity, so unexpected PERT positive results need to be confirmed by retroviral infectivity testing, using a relevant detector cell line(s) (11-14).
Quantitative PCR-based assays (QPCR), such as TaqMan (Thermo Fisher) technology, or other nucleic acid tests (NAT), are widely used to look for species-specific contaminants by amplification of viral DNA or RNA. These methods are considered specific, and therefore it is only possible to detect what is looked for. The appropriate contaminants to analyze for are selected based on a risk assessment of what the material is, and what it is ultimately going to be used for, as well as relevant regulatory guidance.
Not all viruses grow or are detectable in cell culture or in-vivo systems, and PCR-based assays are therefore useful for detection of such contaminants. However, a positive PCR result only indicates the presence of virus DNA or RNA, rather than reflecting the presence of infectious virus. Bovine polyoma virus, for example, is a well-known contaminant of serum, but a positive PCR result from serum cannot determine whether there is replicating virus present or merely DNA fragments. Therefore, positive results from PCR assays may require further investigation, and use of infectivity assays, if available, to determine if infectious virus is present.
There are also practical considerations with respect to the use of these assays. For example, the PCR target must be highly conserved and the assay highly sensitive (<100-1000 copies per 200,000 non-infected cells for cell banks or <2000 copies per mL for vaccine seeds). Due to the high sensitivity, PCR is highly prone to contamination, so rigorous controls must be put in place to segregate the PCR reagents and test materials in different cleanrooms and to ensure that the workflow moves only in one direction.
Industry interest in next generation sequencing (NGS), also known as massive parallel sequencing, deep sequencing, or metagenomics is strong. Using this method, a population of sequences in the sample can be surveyed, allowing the detection of unknown viruses, integrated retrovirus provirus, transcripts and endogenous sequences.
Assays that detect virus markers, including virus DNA or RNA, protein or particles, are an important component of a virus testing strategy. Interpretation of results can be difficult, however, as these assays may not distinguish between infectious and non-infectious virus. Many of these assays are also highly specific (e.g., PCR, enzyme assays). Therefore, additional approaches, including the use of assays capable of detecting a broad range of infectious viruses, are also required.
Infectivity assays are routinely used to screen for the presence of infectious virus contaminants. These may be non-specific, such as in-vitro and in-vivo assays for the detection of adventitious virus contaminants, or specific bovine or porcine virus assays or mouse antibody production tests (MAPs)/hamster antibody production tests (HAPs) for the detection of specific bovine, porcine, murine, or hamster viruses.
Cell culture-based (in-vitro) assays for the detection of adventitious virus contaminants involve inoculating selected indicator cells in culture with the test sample, maintaining the cells for a period of time to allow virus growth and spread, and looking for non-specific or general signs of infection common to many viruses, such as cell death or cytopathology, or hemadsorption (the ability of infected cells to bind red blood cells). This process is illustrated in Figure 1.
Figure 1. In-vitro assays: Cell-culture assays for detection of adventitious virus contaminants. CHO is Chinese hamster ovary. BHK is baby hamster kidney cells.
To be detected, a virus must be able to infect and grow within the indicator cell line(s) under the conditions used, and induce cytopathology or an alternative detectable effect (Figure 2). By using a range of cell lines capable of supporting the growth of different viruses, and appropriate end-points, in-vitro assays may allow the detection of a broad range of adventitious virus contaminants.
Figure 2. Adenovirus cytopathic effect.
The precise assay conditions selected will depend, for example, on the origin of the cell bank, the type of virus that may be present, the intended use of the product, and the requirements of the regulating authority. For example, ICH Q5A guidelines (6) advise that human and/or non-human primate cells susceptible to human viruses be used as indicator cell lines and end points for detection of cytopathic and hemadsorbing viruses. Likewise, the FDA Points to Consider (PTC) (1993, 1997) (8,9) recommend the use of at least three cell types (cells of the same species and tissue type as the production line, where possible; human diploid cells; and monkey kidney cells).
Vero cells are a commonly used monkey kidney cell line and can detect human adenoviruses, herpesviruses (HSV), arboviruses, picornaviruses (polio, enterovirus), SV40, vesicular stomatitis virus (VSV), paramyxoviruses (including mumps, measles, parainfluenza), and orthomyxoviruses. MRC-5 cells (a human dipoid cell line) can detect human herpesviruses, picornaviruses (polio, echo, rhino, coxsackie), VSV Indiana, paramyxoviruses (parainfluenza), and orthomyxoviruses. The use of MRC-5 and Vero cells, therefore, provides two indicator cell lines susceptible to a range of human viruses.
Cell culture-based assays can also be specific, for example, by -including a specific end-point such as immunofluorescence with a specific antibody. This approach is used to detect specific viruses that may go undetected by other endpoint methods (e.g., non-cytopathic viruses) and this method is used routinely as part of 9 Code of Federal Regulations 113 for the detection of bovine and porcine viruses in fetal bovine serum, cell banks, and viral seeds.
Cell-culture assays are frequently performed for the detection of infectious retroviruses, either by infection of relevant cells in culture with a test sample or by co-cultivation with live test cells. Retrovirus infection is often non-cytopathic, and therefore relevant endpoints are typically included, such as the use of a PERT assay for detection of RT activity in culture supernatants, or a sarcoma-positive, leukemia-negative (S+L-) cell line, which results in transformed foci in response to infection with a relevant retrovirus. These assays include the XC, S+L- and Mus dunni assays used for rodent cell lines and their harvests, and co-cultivation assays using human cell lines for detection of human-tropic retrovirus.
No amount of testing can ever definitively prove the absence of an infectious agent. Clearly, it is not possible to test all of a product, so if a contaminant is present at low level then there is a statistical chance that it will not be detected. Furthermore, not all viruses or all individual virus isolates will necessarily be detected in the assay systems used.
Virus safety testing therefore requires a risk-based approach involving a combination of methods, dependent on factors such as the production parameters, the viral risk assessment of raw materials, and the clinical application of the product.
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2. WHO, Questions and Answers Relating to Finding of Porcine Circoviruses in Rotavirus Vaccines (June 3, 2010), accessed Sept. 19, 2017.
3. WHO, “Porcine Circoviruses and Rotavirus Vaccines,” Extract from report of GACVS meeting of 16-17 June 2010 (July 23, 2010) accessed Sept. 19, 2017.
4. FDA, Guidance for industry. Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (Rockville, MD, February 2010).
5. EurPh, General Chapter 5.2.3 Cell Substrates for the Production of Vaccines for Human Use (EDQM, Strasbourg, France).
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8. FDA, Point to Consider in the Manufacturing and Testing of Monoclonal Antibody Products for Human Use, Report (1997).
9. FDA, Points to Consider (PTC) in the Characterization of Cell Lines Used to Produce Biologics, Report (1993).
10. WHO, Recommendations for the Evaluation of Animal Cell Cultures as Substrates for the Manufacture of Biological Medicinal Products and for the Characterization of Cell Banks, Technical Report Series No. 978, Annex 3 (2013).
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14. K. Brorson, et al., Biotechnology Progress, 17 (1) 188-196 (2001).
Volume 30, Number 10
When referring to this article, please cite it as R. Adair, “Control Viral Contaminants with Effective Testing," BioPharm International 30 (10) 2017.