Need for New Technologies for Detection of Adventitious Agents in Vaccines and Other Biological Products

Routine Testing Strategy for Viral Adventitious Agents

Overall, the routine testing strategy for viral adventitious agents is based on three main complementary approaches: (i) Testing of raw materials from animal origins (e.g., serum, trypsin) using 9 CFR tests (3) on indicator cells with cytopathic effects (CPEs), hemadsorption (HAd), and immunofluorescence readouts. (ii) Employing a broad, overlapping viral testing package on cell banks using non-specific tests (to detect known/unknown agents) such as in vitro tests using indicator cells (with CPE & HAd readouts); in vivo tests including adult and suckling mice, embryonated eggs, and sometimes guinea pigs or rabbits; retrovirus detection/quantitation by transmission electron microscopy, reverse transcriptase detection (PERT)/retrovirus infectivity, as well as specific tests (to detect known agents) by polymerase chain reaction (PCR); or by in vivo antibody detection assays. (iii) Testing of seed lots (for vaccines), crude harvests/unprocessed bulks using in vitro and in vivo tests (for vaccine seed lots), in vitro tests using indicator cells, PCR, and so forth, as well as conducting some additional tests on control cells or control eggs (for vaccine production).

Adventitious Agent Testing Key Challenges

The current testing approaches for viral adventitious agents present four key challenges: the lack of global harmonization of testing requirements, the lack of publicly available validation data, the technical issues encountered with compendial tests, and the limitations of these compendial tests.

The challenge befalling the global harmonization of testing requirements is illustrated in Table I where, although excellent monographs and guidelines are available and overall requirements are well framed for the testing for adventitious agents in biological products, subtle differences exist.

Despite all the regulations and guidances provided for adventitious agents testing, there are still key challenges from an industrial perspective due to the fact that subtle differences exist across the monographs and guidelines. Global harmonization of testing requirements would be beneficial especially when developing products for worldwide use and registration. The ICH has succeeded in establishing harmonized best practices for some approaches; however, specific differences in the requirements from one region to another in terms of testing procedure, stage of testing, and nature of test article still remain.

One example is the in vitro adventitious agent test using indicator cells. This broad spectrum screening assay is generally carried out at the bulk harvest stage, prior to the purification step, or on cell substrate using cell lysates or live cells. Table II illustrates the highlighted differences in the required test article, type and number of indicator cells, incubation time, and method employed (for example, the requirement for subculturing). The observation of cytopathic effects on the indicator cells (following 14 or 28 days), or hemadsorption and hemagglutination of erythrocytes endpoints, are recorded.

Another example of non-harmonized testing procedures is the in vivo test for adventitious agents using adult mice and embryonated eggs (allantoic route) as described in Tables III and IV. Slight differences in the testing procedures make it difficult to comply with all regulations if registration of a global product is being sought. The differences in the number of animals used, sub-passaging requirements, and the duration of the observation period make it difficult to rationalize what specific testing method should be carried out while also respecting the ethical use of animals.

The second major challenge with conventional tests for adventitious agents is the lack of full validation by today's standards (9). The tests were developed for clinical diagnostics in the mid-20th century for detection of non-specific adventitious agents. The breadth of viruses able to be detected and their detection limit have not been systematically assessed and published. In vitro test validation data are available with some model viruses at the level of manufacturers and some contract research organizations. However, validation data for in vivo tests are not publicly available. In addition, the specificity for detecting relevant field virus isolates is unknown. Overall, this lack of data makes it difficult to introduce alternative testing methods. However, preliminary results from a study conducted by the NIH (10) using a panel of 16 viruses tested using both in vitro and in vivo tests highlighted the poor performance of in vivo tests to detect five of 10 viruses (not detected at even the highest concentration tested) with the exception of influenza in eggs and Vesicular Stomatitis Virus in eggs and mice where the detection in vivo was better than in vitro. In contrast, 16 of 16 viruses tested were detected in cell culture (although special conditions for detection or for sensitive detection were required for two viruses). This study constitutes the first effort to evaluate in parallel the performance of in vivo and in vitro adventitious agent tests using a significant panel of viruses. These results highlight the limitations of the current compendial adventitious virus testing strategies, in particular the breadth of specificity and the sensitivity of the in vivo tests, which were published during the preparation of this paper (11). Using these viruses and the study results as the basis for the evaluation of new broad alternative molecular methods is an ideal starting point for their implementation in the near future.

The third major challenge with the use of the conventional methods for adventitious agents testing is the technical issues encountered with the procedure and test article or matrix. For example, in vitro and in vivo tests generally require neutralization when testing vaccine viral seeds or viral harvests. The neutralization step requires that antiserum be produced by using antigen produced in cell species different from that used for production of the vaccine. In addition, due to the potential for cross-neutralization of adventitious human viruses, neutralizing antibodies should generally not be prepared from human and primate sera (4). Historically some viruses, such as poxviruses, are very difficult to neutralize and a cocktail of polyclonal antisera, monoclonal antibody, and inhibitory drugs may be needed to get successful neutralization. Moreover, producing large quantities of quality antiserum requires extensive efforts and time. This strategy for industrial-scale routine testing, where the logistics to produce and standardize large quantities of neutralizing reagents that are critical for the safety testing of each biological batch, is not feasible and thus the use of monoclonal antibodies is preferred. Further to the neutralization issue, toxicity of the sample matrix must also be considered (e.g., vaccine based on poxvirus vectors or herpesvirus vectors in suckling mice) when testing complex matrices. Detection of viruses in laboratory animals relies on the health of the animals with associated risks (poor quality of embryonated eggs, cannibalism of suckling mice), the ability of the test virus to induce visible signs of illness, and the innate or acquired immunity of the animal species. All of these factors play a critical role in the successful detection of an infectious adventitious agent in the test sample.

The final challenge for detection of adventitious agents using the conventional methods is the knowledge that the test method itself is no longer state-of-the-art. The circovirus contamination illustrated a significant gap in the conventional testing regime (12). It is not always possible to provide conditions in which an adventitious agent can replicate and produce an effect in vivo and/or in vitro. Additionally, while it is known that there are families of viral pathogens that are not detected or that are only partially detected by the in vivo and in vitro methods currently employed, one should also consider that there are unknown pathogens that are yet to be appreciated as being possible adventitious agents. Alternative virus detection methods have been developed and used to test many biologicals, including molecular methods for the detection of specific adventitious agents (PCRs) or for the detection of a range of viral families (degenerate PCRs)—broad-spectrum PCR combined with mass spectrometry, microarrays, PCR combined with microarrays, and next-generation sequencing (NGS) (1).

Potential Applications and Perspectives for New Molecular Methods

The use of NGS in biological safety tests provides the ability to detect a broad range of adventitious agents (both known and unknown viruses and bacteria), including those for which no assay currently exists, and the ability to test complex matrices directly without the need to neutralize or to circumvent the toxic effects of some test samples. NGS can be used directly on raw materials, cell substrates, or virus seed preparations. It may also be used following in vitro amplification to detect very low-level contaminants that may be introduced from the environment, by raw materials, or during the manufacturing process (13⇓–15). The sensitivity of the new technology can be assessed using theoretical and experimental data. This technology can provide the potential to replace or refine some of the existing conventional assays, thus contributing to the prevention of final product contamination (1).

Challenges and Opportunities for the Introduction of Alternative Broad Molecular Detection Methods

Several challenges can be anticipated with the use of new broad molecular detection methods such as NGS. The potential for exquisite sensitivity combined with broad specificity that is derived from these methods provides the perfect opportunity for detection of potential environmental and laboratory nucleic acid cross-contamination whether it is associated with noninfectious or viable/infectious microorganisms. Furthermore, this exquisite sensitivity and broad specificity results in a large amount of data generated, requiring an efficient data interpretation procedure. For this purpose, there is a need for a curated and reliable reference database as well as a clear investigation procedure in case of positive results. Finally, a significant challenge in the introduction of alternative broad molecular detection methods is the associated validation strategy. For example, the detection of adventitious viruses by NGS will include sample preparation, sequencing, processing of raw data using a bioinformatics pipeline and interpretation of results, potentially followed by an investigation. Even if the investigation step is kept outside of the overall process during the qualification/validation of the method, bringing the other remaining steps to the appropriate quality level (e.g., a good manufacturing practices environment) may be quite challenging, especially because reference data are constantly evolving. Moreover, with the breadth of detection of these new methods, the validation strategy and the comparability with existing methods may both be difficult to define.

However, in the meantime, these new methods represent great opportunities thanks to the potentially exquisite sensitivity and broad specificity that can be provided by a single assay (e.g., with NGS). In addition, one can envisage the potential reduction, replacement, or refinement of existing tests, allowing streamlining of the current adventitious agent testing package and faster turnaround time. Replacement of some existing in vivo adventitious agent tests would support ongoing 3R (Replace, Reduce, Refine the use of animals) initatives. The process to advance these new technologies into mainstream use will require a global effort through collaborative studies such as the one currently being developed with the Advanced Virus Detection Technology Users Group (AVDTUG) and/or with future potential WHO collaborative studies. From a theoretical and experimental perspective, the validation of these new technologies seems feasible providing that the appropriate, well-characterized model viruses are used. Once these methods are implemented, they will play a key role in preventing final biological product contamination.

Conclusion and Perspectives

Advances in technology have revealed the limitations of the current adventitious agent testing package. In the meantime, from a public health perspective, global vaccine and biological product supply aimed at serving all populations requires a harmonization of regulations worldwide to ensure a consistent supply of safe products to all populations around the world.

Keeping in mind that testing is only one component of viral safety, in addition to risk assessment combined with quality by design principles and viral clearance when possible, new technologies for adventitious agent detection can bring a significant improvement to the safety of all biological products.

Some pre-requisites are needed prior to the introduction of these new methods into the general testing realm: in particular, key validation data from an NIH study (10), an effort towards harmonisation and convergence of global regulations; the compilation and analysis of all available scientific rationales and data to establish a reference document, the previously mentioned collaborative/spiking studies, and the validation of the new molecular methods combined with relevant risk assessment procedures.

The expected benefits are multiple and include the implementation of a comprehensive, streamlined adventitious agent testing package based on scientific rationale and validation data; the replacement of in vivo adventitious agent tests (3Rs); the convergence of regulations and use of “standardized” methods with, at the end, an increased assurance of safety by allowing earlier and broader detection. The overall objective remains to adopt state-of-the-art technologies to continue to guarantee patients' safety and facilitate an uninterrupted global supply of high-quality biologicals.

Conflict of Interest Declaration

The authors declare that they have no competing interests. All authors are employees of Sanofi Pasteur.