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European Biopharmaceutical Review

Biomanufacturing: Cell Lines

The realm of viruses is vast, incorporating all the major animal kingdoms as well as the plant, aquatic and insect worlds, and therefore risk management plays a key role in designing and implementing an effective virus safety strategy. No two products are identical and will present different historical exposure risks. The risks for each product need to be clearly defi ned and evaluated, ideally through a formalised risk assessment procedure aimed at measuring and identifying the most appropriate measures for reducing risk at the level of sourcing; implementing the correct testing strategy based on the defi ned risks; and proactively designing the manufacturing process to reduce risk where possible. The risk evaluation procedure needs to be applied to both the product itself as well as any excipients, media components or APIs used in the manufacturing process.

Examples of Contamination

Table 1 lists some of the better known examples of virus contamination observed in well-characterised cell lines. The use of qualifi ed cell banks provides the opportunity to detect and identify (and thereby exclude) possible contaminating viruses prior to their use in manufacture. Recombinant cell-based systems have not been without virus contamination events, and therefore the characterisation of in vitro systems must be carefully implemented to ensure that contaminants are indeed excluded. Figure 1 summarises the key aspects for controlling virus risk in a master cell bank.

The main challenges for virus risk in recombinant cell-based systems originate from:

  • Cell line derived contaminants such as endogenous retroviruses, for example latent herpesviruses
  • Animal-derived components used either in the past or during manufacture; contamination events have been observed with porcine parvovirus (PPV), porcine Cirovirus (PCV), bovine diarrhea virus (BVDV), bovine polyomavirus (BPyV), Cache Valley virus (CVV), bovine herpesvirus (BHV) and others (see Table 1)
  • Other virus risks with as yet non clearly defi ned aetiology such as mouse minute virus (MMV) and V2117 (probably animal derived)

Why Was the Contamination Missed?

The largest single root cause of virus contamination in well-characterised systems has been the use of animal-derived components. Media formulations, even if they do not use bovine serum, may still use purifi ed animal-derived factors which can present a risk.

Important lessons can be learned through a root cause analysis focusing on why contamination events with viruses from animal components occured. Where the root cause could be clearly identified, the main reasons were:

  • The virus was not permissive for the cell lines used for testing; cell culture or in vivo tests cannot detect all known potential contaminants
  • Detection of virus was masked by the presence of neutralising antibodies (such as BVDV). Matrix-induced inhibition of the assay may mask a potential contaminating virus and it is difficult to include and test interference controls for every possible virus contaminant. Current guidelines require the inclusion of controls for such neutralising antibodies (2)
  • The virus was below the limit of detection. The limit of sensitivity for current cell culture and PCR-based tests can never assure viral sterility, and the volumes tested are significantly lower than the volumes used in manufacturing
  • No steps were present for the effective removal of virus. History has demonstrated that tested products in the absence of robust virus inactivation or removal steps can still result in virus transmission (3). Even with extensive testing, the possibility exists for the introduction of virus contaminants from other sources (such as facility rodents as has been demonstrated with mouse minute virus (MMV)(4) and V2117)

Factors Affecting Virus Risk from Animal-Derived Source Materials

As the large majority of virus contamination events have resulted from the use of animal-derived components, there is a strong argument for using alternatives. However, even where such components are not used for routine manufacture, the cell line or media ingredients could have been exposed (perhaps indirectly), and therefore it is important to understand the risk factors impacting on the likelihood of contamination (see Figure 1). The following factors need to be formally assessed for all animal-derived components to which the cell line could have been exposed:

  • Location of the donor population (5)
  • Management and collection practices, including donor selection, donor monitoring, physical barriers to infection (such as SPF or closed herds), and preventive measures such as collection to prevent cross contamination
  • Age of animals, for example for control of transmissible spongiform encephalopathies
  • QC testing: should include generic tests as well as tests for specific viruses identified as risks based on epidemiological data

PCV Contamination and the Importance of Epidemiology

PCV1 was identified as a contaminant of the Rotarix vaccine through massively parallel sequencing (6). Investigation by the manufacturer identified contamination to have originated during preparation of the MCB back in 1983, and all subsequent materials produced from this MCB were also positive (the most likely source of the contamination was porcine trypsin used during the subculture of the cells).

PCV was first identified in 1982 as a contaminant in a porcine cell line, and is known as a contaminant in pigs with a high sero-prevalence and high titre viraemia (7,8). A formal risk analysis of potential viruses of concern, focusing on those viruses at high sero-prevalence and high titre, would have identified PCV as a potential virus of concern, and highlights the importance of epidemiology in identifying potential risks. The human plasma industry has long recognised the power of virus epidemiology and its role in minimising the risk for virus transmission (9). As PCV is a virus which would not be detected using standard cell culture tests for porcine viruses, other testing methodologies (such as PCR) could be employed to test for potential contamination.

Similar issues have been noted for BPyV contamination of serum. Seventy per cent of bovine serum lots are positive for BPyV by PCR and manufacturers are encouraged to apply infectivity tests to evaluate the potential threat or concern (2). However, infectivity tests for BPyV generally have a low level of sensitivity and a rather long incubation time. Up to now it was not possible to detect infectious BPyV in serum samples using the infectivity assay currently available.

Understanding the Impact of the Limit of Detection

Irrespective of the type of test used, all virus assays have a defined limit of detection (LOD) dependent on the type of cell line used, the virus in question and the volume of sample tested. A negative result therefore does not necessarily mean that the sample is negative. Furthermore, many animal and human sourced materials are pooled prior to testing for virus contamination, and the impact of such pooling on the probability of detecting virus requires careful attention. Factors having an impact on the likelihood of detecting a particular contamination event include the peak virus titre (higher titres during viraemia will result in a higher risk) and the pooling strategy. The larger the pool, the higher the likelihood of a contaminated donation entering manufacture – but a large pool could also dilute the virus to a level that it is no longer detectable in the QC assay. Thus, low titres in a pool may result in a failure to detect the virus even where a risk may be present.

A paradigm relying on sourcing and testing alone can result in significant levels of residual risk. Testing has its limitations, in that it cannot assure viral sterility for a product no matter how much it is tested. Furthermore, we know that in vitro or in vivo based tests cannot detect all known potential contaminants.

Significant experience with human plasma-derived products has shown that where the plasma is QC tested only and no robust dedicated virus inactivation or removal steps are built into the manufacturing process, virus transmissions still occurred (10). The reason why such transmission events have continued is rooted in the limits of detection of the tests and the volumes of product used per dose. It can be calculated that sourcing policies alone can reduce risk for a given virus by around 100-fold and that subsequent testing by qPCR can reduce this risk an additional 100-1,000-fold (11). The overall risk reduction in comparison to the general population is reduced around 10,000 to 100,000-fold, but the primary factor for determining residual risk is still the LOD of the qPCR assay. For a product requiring one litre of plasma to manufacture one dose, and with a limit of detection for the qPCR assay of 100 copies/ml, a total of 100,000 units of virus could still be entering the manufacturing process per patient dose.

With a potential load of 100,000 virus particles, the only risk control measure able to reduce the risk sufficiently to assure safety is robust, dedicated virus inactivation or removal steps. In comparison to sourcing and testing, the level of risk reduction through virus clearance is significantly greater. Controlling the input load is still important and testing should be based on appropriate epidemiological evaluation of the donor population, but combined with robust inactivation procedures (for example, where complete inactivation or removal to below the limit of detection is consistently observed). It was only the introduction of highly robust inactivation (that is, more than six or seven logs through S/D treatment) that prevented transmission of HCV through plasma products.

A similar issue of scale and the volume of the components used in manufacture exists for well characterised cells. In a standard QC testing of bovine serum, a volume of up to 10ml may be tested, equivalent to a theoretical LOD of 0.3IU/ml using the Poisson equation. The calculation of the potential level of virus which might be present in a 1,000L bioreactor where bovine serum is present at five per cent is given in Table 2 (first calculation column). It is clear that a residual virus load of 15,000IU entering the bioreactor is an unacceptable level of risk, and that a different paradigm to testing alone is required to bring such a risk under control. For comparison, Table 2 provides the same residual risk calculation for bovine serum where more than five logs virus inactivation via gamma-irradiation of the fetal bovine serum (FBS) is included as a risk control measure. A residual risk of 0.15 IU represents a significantly more acceptable risk level.

Examples of Contamination with Unclear Aetiology- MMV and V2117

One aspect that has received increasing scrutiny has been the control of suppliers of components (for example, cell culture medium and APIs). Examples of virus contamination with no clear aetiology (such as MMV and V2117), and the conclusions that such contaminants might have been introduced through a component such as the cell culture medium, raise the question of supplier control. The example of MMV highlights the difficulty of controlling such risks.

Contamination events with MMV started to be noted in the 1990s in CHO fermenters and resulted in multiple contamination events (4). In all instances, the root cause of the contamination could not be traced, but it was assumed to have originated from facility rodents. What is not clear is if this was facility rodents present in the GMP manufacturing facility (such risks should be controlled through appropriate pest control procedures), or from rodents present in the suppliers for excipients or media components?

MMV is an endemic virus in mice with seroprevalence of up to 70 per cent (12). The virus is present in high titres in multiple tissues (titres up to 107 per ml) and the virus is excreted in the urine of infected animals (the most likely source of contamination where exposure to mice is not controlled). Furthermore, the virus is highly resistant to inactivation procedures. Parvoviruses are among the most resistant viruses used in virus validation studies and so will also survive for extended periods in the environment (13).

Potential options for controlling MMV risk could include:

  • Controlling exposure to mice for areas of risk which should include effective pest control procedures both at the GMP manufacturing plant but also at the suppliers of media components or APIs. The question of how far back in the supply chain this risk should be controlled is a question each manufacturer should evaluate carefully through systematic risk evaluation procedures
  • Incoming materials could also be tested for MMV, but as a risk control measure this is unlikely to prevent entry into GMP manufactured product (due to the limitations of testing discussed above). Many manufacturers implement tests for MMV at the bulk harvest level, but by this time it is too late to prevent a GMP contamination and the eventual clean down, which for MMV is not easy due to its high resistance
  • The risk from incoming materials could be reduced through the implementation of an effective virus inactivation or removal step. The success of gamma-irradiation in reducing the residual risks from bovine serum (such as the risk remaining following appropriate sourcing and testing) to a level where contamination by bovine viruses is effectively controlled highlights the power of this approach

Given the impact that virus contamination events cause, both in terms of company image as well as the resources required to investigate and clean contaminated facilities, consideration of control measures becomes more attractive.

V2117 was first identified as a contaminant of CHO bioreactors at the end of the 1990s where companies observed 40nm virus particles in CHO cultures exhibiting cytopathic changes (14). The nucleotide sequence of isolate 2117 is most similar to that of canine caliciviruses, but also showed significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In 2009, Genzyme again reported a number of contamination incidents with V2117.

In all incidents, statements have been made that the virus is most likely of bovine origin, but direct data in support of this is lacking. Sequence comparisons with nucleotide databases continue to show highest levels of homology with Canine Calicivirus, and in fact homology with bovine caliciviruses does not score high in such searches. The question of the origin of V2117 therefore appears to be still open and the assertation that this virus is of bovine origin has not yet been proven. The MMV precedent has shown us that manufacturers should be prepared to expect unusual sources of potential contamination.

Other Potential Sources of Contamination

As always, manufacturers should be proactive in identifying from where potential risks from viruses could arise. Potential sources of risk could include:

  • Arboviral insect vectors: a large number of viruses are transmitted by insects and dead insects present in, for example, powdered media could theoretically be a source of contamination. Most Arboviruses are enveloped viruses, and so might not survive for long periods in the environment, although there are exceptions to this rule
  • Infected workers at the GMP plant: many viruses are asymptomatic and so would not necessarily be apparent in workers in a GMP facility. Furthermore, viruses could be carried by fomites from infected pets
  • Controlling the supply chain: a major question is how far back in the supply chain do you go? Some viruses have very high resistance to inactivation and could potentially survive for extended periods in the environment and the extent of supplier auditing required should be given careful consideration

Prevention is always better than cure, and so it is important to understand where the risks might originate from and then to implement the most appropriate steps to reduce that risk, either through sourcing, QC testing or dedicated robust virus inactivation/removal steps.

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Andy Bailey is a virologist who has been actively involved in the pathogen safety of biopharmaceuticals for over 16 years. Originally a biochemist, Andy served for nine years at the MRC Virology Unit in Glasgow, Scotland. In 1995, he moved as Director of Virus Validation services to Q-One Biotech Ltd, and later to the Pathogen Safety group of Baxter Healthcare in Vienna, Austria. Since 2005, Andy has been a founder and CEO of ViruSure in Vienna, Austria, a company specialising in virus and prion safety testing of biopharmaceutical products. Andy has presented at numerous regulatory agencies either in support of products or as an invited speaker at expert workshops, including the UK MHRA, German PEI, French AFFSAPS, US FDA, EMEA and JMHLW (Japan).
Andy Bailey of ViruSure GmbH
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