Richard Adair, Virology Manager, SGS Life Sciences, Glasgow, UK11.13.18
Proving the absence of something is far more complicated that detecting its presence, so tests to rule out the presence of viral contaminants in biopharmaceutical manufacturing will never yield a definitive result. Additionally, since it is obviously not possible to test the entire product, there is a statistical chance that low-level contaminants may be missed during analysis. These are just two of the challenges that the industry faces as biologic-based medicines continue to grow in importance.
In theory, one single infectious virus particle may be sufficient to initiate an infection that could have potentially dangerous, or even fatal, consequences in humans, and this is especially true in vulnerable patients, whose immune system could already be compromised. Even viruses that are not known to cause significant disease in humans could, potentially, do so if introduced by a non-natural route of infection, such as by injection rather than respiratory or oral spread. Contamination with retroviruses is a particular concern, because they integrate into the host cell genome as part of their lifecycle, and can result in life-long infection or cellular change.
Since viruses replicate within living organisms, any system used to generate biological or biopharmaceutical products could be at risk of viral contamination. There have been a number of well-documented instances of contaminated vaccines transmitting viruses to humans, such as batches of poliovirus and adenovirus vaccines that were found to be contaminated with Simian vacuolating virus 40 (SV40) from rhesus monkey kidney cells in the late 1950s and early 1960s; and some early batches of yellow fever vaccine produced in hens’ eggs were found to be contaminated with avian leucosis virus.
More recently, there has been transmission of hepatitis A, B and C viruses, HIV and B19 via human blood products. However, viral contamination does not necessarily result in infection, and there are also examples of viruses being detected in cell culture-derived products without transmission to humans or animals (see Table 1).
Virus contamination could arise from the presence of virus in the original cell line or biological system used, or from the inadvertent introduction of virus into cell cultures during a manufacturing process. Potential contamination sources could include raw materials of animal origin, the human operator, or materials contaminated by contact with animals or animal-derived material. For example, contamination of Chinese hamster ovary (CHO) cell banks with murine minute virus (MMV)—a virus primarily transmitted in the urine of rats and mice—could, potentially, have resulted from this latter route. Although there is significant pressure in the industry to move to serum-free conditions for cells to reduce the risk of contaminants, if cell banks and cell lines have been exposed historically to bovine serum or porcine trypsin, then they could potentially be affected by a range of bovine and/or porcine viral contaminants.
Three principal and complementary approaches are generally applied for controlling viral contaminants in biologics: first, the selection and control of starting materials; second, the testing of source materials and the products; and finally, viral clearance.
Regulatory authorities require testing to be carried out at every stage of the product’s manufacturing process. These include cell substrate banks, viral seed banks, raw materials of animal origin, bulk harvests, and the batches of the final manufactured clinical product. The samples must be tested to recognized international guidelines, such as the International Conference on Harmonization (ICH) guidelines, FDA Vaccine 2010 guidelines, and/or European Pharmacopeia guidelines, as appropriate.
As there is no single method that will be able to detect all potential viral contaminants, a holistic approach is required involving a combination of methodologies with both general and specific assays. These will 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.
In vitro and in vivo assays can be used to look for evidence of infectivity in a living system, while electron microscopy (EM), polymerase chain reaction (PCR)-based methods, and immunoassays look for viral markers, including the presence of virus particles, the virus genome, viral proteins or enzyme activity (see Figure 1).
Infectivity assays are routinely used to screen for the presence of infectious virus contaminants, whether these are non-specific for the detection of adventitious virus contaminants, or specific for the detection of specific bovine, porcine, murine or hamster viruses. If a virus is to be detectable, it 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. By using a range of cell lines capable of supporting the growth of different viruses, and appropriate end-points, in vitro assays may allow a broad range of adventitious virus contaminants to be detected.
Electron microscopy is used widely as a generalized non-specific screening method. It allows direct visualization inside cells or samples, and can enable the detection of a broad range of viruses, as well as other micro-organisms including bacteria, fungi and mycoplasma. It can also be a useful diagnostic and investigative tool in cell cultures if the presence of virus is suspected.
Drawbacks to using EM are that it is a labor-intensive method and has low sensitivity, so a 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.
Quantitative PCR (qPCR) and nucleic acid tests (NAT) are also widely used in the industry. Virus qPCR assays, such as TaqMan, are used to look for species-specific contaminant by nucleic acid amplification of the viral genome. Testing companies have hundreds of validated PCR reactions for a wide range of different potential viral contaminants of cell banks and cell lines, and these are selected based on a risk assessment of what the material is, and what it is ultimately going to be used for.
PCR is highly prone to contamination, so rigorous testing 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 to avoid cross-contamination. Other key controls are that nucleic acid recovery in each test item extraction is qualified by use of an internal spike control, and pathogen nucleic acid is also spiked into a test sample to ensure there is no interference from the presence of the test material.
PCR can detect both infectious and non-infectious viruses, which is important because some contaminant viruses will not grow in culture systems. The method could, therefore, give an indication of a possible contaminant that may not be detected by an infectivity assay.
However, a positive PCR result only indicates the presence of virus DNA or RNA, rather than reflecting whether the virus is infectious. 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 confirmatory tests to determine if infectious virus is present, such as infectivity assays.
The specificity of PCR is both an advantage and a drawback, because the technique cannot find contaminants that are not being looked for. Furthermore, because PCR is specific, the target has to be highly conserved and the PCR reaction highly sensitive (<100-1000 copies per 200,000 non-infected cells for cell banks or <2000 copies per ml for vaccine seeds). For this reason, there is now a lot of interest in the industry in next generation sequencing (NGS), also known as massive parallel sequencing, deep sequencing or metagenomics. It is, essentially, a tool to screen a population of sequences in the sample and can be used to detect unknown viruses, integrated retrovirus provirus, transcripts and endogenous sequences.
As stated above, retroviruses are of particular concern, and because they do not always exhibit a cytopathic effect, they cannot be detected by conventional culture methods. However, they express an enzyme called reverse transcriptase (RT), which is largely found in retroviruses, so RT activity is an indication of potential retrovirus contamination (see Figure 2).
Retroviral RT enzyme activity can be detected by the use of PCR-based RT (PBRT) or product-enhanced RT (PERT), but although PERT assays are highly sensitive, they can sometimes give false positives from high background cellular DNA polymerase activity. Therefore, unexpected PERT positive results need to be confirmed by retroviral infectivity testing, using a relevant detector cell line(s).
The third stage of controlling viral contaminants in biopharmaceuticals is viral clearance—downstream processing of the product by putting it through a filtration process or subjecting it to high pH conditions or high temperatures to further reduce the risk of the presence of infectious virus contaminants. But while this is suitable for some biologics, it cannot be used in the manufacture of live viral vaccines or viral vectors because it would render the product ineffective. For this reason, the focus on the testing of the materials and the products is crucial in viral vectors and vaccines manufacture.
All of these methodologies have limitations, in terms of both their respective limits of detection and levels of specificity. The absence of an infectious agent can never be entirely proven—it is only possible to achieve negative test results. The only realistic approach to take is to assess the risk based on the materials being used, the production methods and the eventual clinical application, and then test accordingly.
In theory, one single infectious virus particle may be sufficient to initiate an infection that could have potentially dangerous, or even fatal, consequences in humans, and this is especially true in vulnerable patients, whose immune system could already be compromised. Even viruses that are not known to cause significant disease in humans could, potentially, do so if introduced by a non-natural route of infection, such as by injection rather than respiratory or oral spread. Contamination with retroviruses is a particular concern, because they integrate into the host cell genome as part of their lifecycle, and can result in life-long infection or cellular change.
Since viruses replicate within living organisms, any system used to generate biological or biopharmaceutical products could be at risk of viral contamination. There have been a number of well-documented instances of contaminated vaccines transmitting viruses to humans, such as batches of poliovirus and adenovirus vaccines that were found to be contaminated with Simian vacuolating virus 40 (SV40) from rhesus monkey kidney cells in the late 1950s and early 1960s; and some early batches of yellow fever vaccine produced in hens’ eggs were found to be contaminated with avian leucosis virus.
More recently, there has been transmission of hepatitis A, B and C viruses, HIV and B19 via human blood products. However, viral contamination does not necessarily result in infection, and there are also examples of viruses being detected in cell culture-derived products without transmission to humans or animals (see Table 1).
Virus contamination could arise from the presence of virus in the original cell line or biological system used, or from the inadvertent introduction of virus into cell cultures during a manufacturing process. Potential contamination sources could include raw materials of animal origin, the human operator, or materials contaminated by contact with animals or animal-derived material. For example, contamination of Chinese hamster ovary (CHO) cell banks with murine minute virus (MMV)—a virus primarily transmitted in the urine of rats and mice—could, potentially, have resulted from this latter route. Although there is significant pressure in the industry to move to serum-free conditions for cells to reduce the risk of contaminants, if cell banks and cell lines have been exposed historically to bovine serum or porcine trypsin, then they could potentially be affected by a range of bovine and/or porcine viral contaminants.
Three principal and complementary approaches are generally applied for controlling viral contaminants in biologics: first, the selection and control of starting materials; second, the testing of source materials and the products; and finally, viral clearance.
Regulatory authorities require testing to be carried out at every stage of the product’s manufacturing process. These include cell substrate banks, viral seed banks, raw materials of animal origin, bulk harvests, and the batches of the final manufactured clinical product. The samples must be tested to recognized international guidelines, such as the International Conference on Harmonization (ICH) guidelines, FDA Vaccine 2010 guidelines, and/or European Pharmacopeia guidelines, as appropriate.
As there is no single method that will be able to detect all potential viral contaminants, a holistic approach is required involving a combination of methodologies with both general and specific assays. These will 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.
In vitro and in vivo assays can be used to look for evidence of infectivity in a living system, while electron microscopy (EM), polymerase chain reaction (PCR)-based methods, and immunoassays look for viral markers, including the presence of virus particles, the virus genome, viral proteins or enzyme activity (see Figure 1).
Infectivity assays are routinely used to screen for the presence of infectious virus contaminants, whether these are non-specific for the detection of adventitious virus contaminants, or specific for the detection of specific bovine, porcine, murine or hamster viruses. If a virus is to be detectable, it 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. By using a range of cell lines capable of supporting the growth of different viruses, and appropriate end-points, in vitro assays may allow a broad range of adventitious virus contaminants to be detected.
Electron microscopy is used widely as a generalized non-specific screening method. It allows direct visualization inside cells or samples, and can enable the detection of a broad range of viruses, as well as other micro-organisms including bacteria, fungi and mycoplasma. It can also be a useful diagnostic and investigative tool in cell cultures if the presence of virus is suspected.
Drawbacks to using EM are that it is a labor-intensive method and has low sensitivity, so a 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.
Quantitative PCR (qPCR) and nucleic acid tests (NAT) are also widely used in the industry. Virus qPCR assays, such as TaqMan, are used to look for species-specific contaminant by nucleic acid amplification of the viral genome. Testing companies have hundreds of validated PCR reactions for a wide range of different potential viral contaminants of cell banks and cell lines, and these are selected based on a risk assessment of what the material is, and what it is ultimately going to be used for.
PCR is highly prone to contamination, so rigorous testing 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 to avoid cross-contamination. Other key controls are that nucleic acid recovery in each test item extraction is qualified by use of an internal spike control, and pathogen nucleic acid is also spiked into a test sample to ensure there is no interference from the presence of the test material.
PCR can detect both infectious and non-infectious viruses, which is important because some contaminant viruses will not grow in culture systems. The method could, therefore, give an indication of a possible contaminant that may not be detected by an infectivity assay.
However, a positive PCR result only indicates the presence of virus DNA or RNA, rather than reflecting whether the virus is infectious. 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 confirmatory tests to determine if infectious virus is present, such as infectivity assays.
The specificity of PCR is both an advantage and a drawback, because the technique cannot find contaminants that are not being looked for. Furthermore, because PCR is specific, the target has to be highly conserved and the PCR reaction highly sensitive (<100-1000 copies per 200,000 non-infected cells for cell banks or <2000 copies per ml for vaccine seeds). For this reason, there is now a lot of interest in the industry in next generation sequencing (NGS), also known as massive parallel sequencing, deep sequencing or metagenomics. It is, essentially, a tool to screen a population of sequences in the sample and can be used to detect unknown viruses, integrated retrovirus provirus, transcripts and endogenous sequences.
As stated above, retroviruses are of particular concern, and because they do not always exhibit a cytopathic effect, they cannot be detected by conventional culture methods. However, they express an enzyme called reverse transcriptase (RT), which is largely found in retroviruses, so RT activity is an indication of potential retrovirus contamination (see Figure 2).
Retroviral RT enzyme activity can be detected by the use of PCR-based RT (PBRT) or product-enhanced RT (PERT), but although PERT assays are highly sensitive, they can sometimes give false positives from high background cellular DNA polymerase activity. Therefore, unexpected PERT positive results need to be confirmed by retroviral infectivity testing, using a relevant detector cell line(s).
The third stage of controlling viral contaminants in biopharmaceuticals is viral clearance—downstream processing of the product by putting it through a filtration process or subjecting it to high pH conditions or high temperatures to further reduce the risk of the presence of infectious virus contaminants. But while this is suitable for some biologics, it cannot be used in the manufacture of live viral vaccines or viral vectors because it would render the product ineffective. For this reason, the focus on the testing of the materials and the products is crucial in viral vectors and vaccines manufacture.
All of these methodologies have limitations, in terms of both their respective limits of detection and levels of specificity. The absence of an infectious agent can never be entirely proven—it is only possible to achieve negative test results. The only realistic approach to take is to assess the risk based on the materials being used, the production methods and the eventual clinical application, and then test accordingly.
