Abstract
Contamination of a biological product with viruses, potentially harmful for humans, is of concern for therapeutics derived from human and animal sources, i.e. plasma, and for protein therapeutics and vaccines produced in mammalian cells. More recent “Advanced Therapeutic Medicinal Products (ATMPs)”, such as gene and cell therapies, share the same concern. Tests for viral contaminants are performed at several stages of manufacturing. Current tests for detection of viruses are lengthy; cell culture-based assays and animal experiments are required. Alternative molecular tests, such as “next generation sequencing (NGS)”, provide faster results, and can eventually replace in vivo animal studies. It is noteworthy that identification of a viral contaminant that evaded detection in classical tests, by NGS, has been demonstrated. Since NGS-based virus tests yield results significantly faster than current assays, additional applications for in-process testing and viral contamination control can be envisioned.
Viral Safety of Biologics
A potential viral product contamination is a concern for therapeutics and vaccines either derived from human or animal source material, or manufactured in mammalian cells that are vulnerable to infection by viruses. Requirements for manufacturing and safety of medicinal products, including viral safety, are outlined in the Code of Federal Regulations (CFR, title 21), as well as in the US and European Pharmacopeia.
Three major pillars contribute to assurance of viral safety of products, i.e. biologics (Figure 1). The first component is “Prevention” of introduction of a viral contaminant into the manufacturing process. Preventive measures include omission of raw materials of human or animal origin from the process, audits of suppliers of consumables and raw materials, design of the manufacturing facility, and procedural controls for e.g. aseptic handling, gowning and material flows. “Testing” for viral contaminants at various stages, i.e. cell banks and cell culture harvests, is another key component. The third pillar is “Reduction”, the process’s capability to inactivate or remove potentially present viral contaminants. Purification processes, aiming to separate the product from impurities, have to include steps with validated viral clearance capacity.
Current Methods for Viral Safety Testing of Biologics
Acknowledging that products purified from human or animal material, e.g. plasma, need to be tested for viral contaminants of concern using appropriate assays, the focus of this article is virus detection methods applicable to mammalian cell culture processes, i.e. Chinese Hamster Ovary (CHO) cell processes.
Current test methods for evaluation of viral safety of biotechnology products derived from cell lines of human or animal origin are outlined in ICH Q5A, current version of guidance published 1999. A similar guidance document for viral vaccines manufactured in cell culture was published by the Center for Biologics Evaluation and Research (CBER) in February 2010.
For the vast majority of biologics manufactured in mammalian cell culture processes, the expression host is CHO cells. Table 1 lists methods for detection of virus contaminants in CHO Master Cell Banks (MCBs). A variety of tests is applied, including indicator cell-based infectivity assays, in vivo animal studies, and molecular (Polymerase Chain Reaction (PCR) based) tests. Several of the assays aim to detect adventitious virus contaminants, possibly introduced during cell bank manufacture, e.g. through the manufacturing environment, an operator, or a contaminated raw material. Tests for specific animal viruses (e.g. porcine and bovine viruses) can detect contaminants host cells have been exposed to during their cell line history. At an earlier time, CHO cells might have been cultured in media containing fetal calf serum, or when grown in monolayer culture, porcine derived trypsin might have been used to detach cells.
Rodent cell lines, such as CHO, are known to carry retrovirus like particles (RVLP); therefore, tests for detection of endogenous retrovirus are also included. As indicated in Table 1, only a subset of these assays is required at different cell ages, or stages of the process.
Limitations of Current Test Methods
A shortfall of many of the current test methods is their duration; indicator cell based infectivity assays and in vivo studies take four to five weeks. Availability of test results can become a limiting factor for release of cell banks for initiation of drug substance manufacture, and other forward processing decisions. Early stage clinical programs would especially benefit from rapid test methods with faster “turn-around time”, supporting accelerated “first in human” timelines.
It is desirable to reduce the amount of animal studies, still part of the safety evaluation of biologics. European guidance on vaccines for human use (e.g. Ph. Eur. Chapter 5.2.3 (2014)) state that molecular methods, such as NGS, can be used to replace or substitute in vivo testing and other nucleic acid technology-based tests. Replacement of animal tests by alternative test methods is also a topic that will be addressed by an ICH (International Conference of Harmonization) working group reviewing ICH Q5A, which was initiated in 2019. Results of a study, directly comparing detection of a virus panel by animal testing and the in vitro indicator cell assay, further support moving away from in vivo testing; the in vitro adventitious virus test proved to be more sensitive and reliable.
For several vaccines and gene therapies, that are “viruses” themselves, testing in animal and cell based assays is more challenging; the product likely needs to be neutralized to avoid interference with the test. Specific reagents, such as polyclonal sera, are required.
More ATMPs are entering the market. For some gene and cell therapies, e.g. allogenic cell therapies, product stocks such as cell banks and virus stocks can be prepared; such products can be tested using a similar approach as described for other biologics. Autologous cell therapies, i.e. Chimeric Antigen Receptor - T cells (CAR-T), for which cells are harvested from a patient and modified, have a very short release timeline, and are not tested for adventitious virus contaminants.
Applications of Unbiased NGS
The shortfalls of current tests discussed above highlight the need for new molecular test methods. But by far the main event leading to eff orts to develop an NGS-based adventitious virus test was the detection of porcine circovirus DNA in a rotavirus vaccine, using NGS and a panmicrobial oligonucleotide array. The viral contaminant in the vaccine failed detection by the routine viral safety testing required for product release. The presence of infectious circovirus particles in the vaccine was also confirmed.
Of several industry initiatives for developing of NGS-based viral safety tests, the Advanced Virus Detection Technology Interest Group (AVDTIG) is worth mentioning the most. The interest group, which currently has more than 180 members, was formed as a forum for regulatory and industry scientists to discuss and exchange experiences with development of NGS-based viral safety tests. An alternative molecular test is mainly sought for products for which testing with current methods is challenging, such as vaccines and viral vectors. Like current compendia assays, an NGS-based test needs to be unbiased and capable to detect novel/unknown viruses.
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In her presentation at the BPI Europe conference in July 2020, Dr. Arifa Khan from the FDA’s Offi ce for Vaccine Research and Review (OVRR) indicated that for products for which testing with current methods is challenging - e.g. some viral vaccines when the vaccine virus cannot be neutralized and lytic viral vectors - the agency considers supplementation or substitution of current tests with unbiased NGS data on a case-by-case basis. She also mentioned that the use of unbiased NGS for rapid adventitious virus detection might be considered for COVID19 vaccine development.
NGS-based viral safety tests are gaining more acceptance for vaccines and other products for which virus detection in classic tests is difficult. In light of a changing regulatory landscape, review of ICH Q5A and mandates to reduce animal testing, one can envision that over time NGS-based viral safety tests will replace in vivo studies, and other nucleic acid technology (NAT) based tests for a wide range of therapeutic products.
Applications of Targeted Rapid Molecular Adventitious Virus Tests
Targeted NAT based tests aim to detect a panel of viral contaminants of concern. Several vendors and contract testing laboratories are currently developing such assays, specifically assays for detection of potential viral contaminants of CHO cells. The assays typically combine highly multiplexed PCR arrays with readouts by NGS or amplicon sizing by capillary electrophoresis. In contrast to unbiased NGS, such methods are at present not considered for replacement of compendia tests capable to detect novel/emerging viruses, such as the adventitious virus in vitro and in vivo tests.
However, a targeted assay might be suitable to replace in vivo tests for rodent viruses, the “Mouse Antibody Production” (MAP) test and the “Hamster Antibody Production” (HAP) test, performed for rodent cell lines. In MAP and HAP tests, mice and hamsters are injected with cell culture samples and sera of immunized animals are then screened for presence of antibodies against a panel of specific rodent viruses in immuno-assays. MAP and HAP studies are “targeted” tests; in agreement with regulatory guidance (ICH Q5A), it should be possible to replace these tests with suitable NAT based methods.
In-process testing is another possible application for rapid adventitious virus tests, i.e. targeted NGS, to either support forward processing, or for facility contamination control. Such testing differs from regulatory requirements for release of cell banks and products, and serves business decisions and risk mitigation. A targeted assay that might return results in two days or less, and can be implemented in a Quality Control laboratory environment, is more suitable for in-process testing than more complex unbiased NGS.
Figure 2 shows process steps at which rapid test methods can help to accelerate timelines via faster forward processing, e.g. faster release of cell banks for drug substance (DS) manufacture and faster forward processing of DS to drug product (DP) conversion. Acceleration of project timelines is especially important for early stage clinical projects, speeding up “first in human” timelines and bringing promising medicines sooner to patients.
A targeted assay might be used for viral contamination control. In more than 30 years of manufacturing biologics in mammalian cell culture, about 20 viral bioreactor contaminations have been reported. With the exception of Minute Virus of Mice (MVM), viral bioreactor contaminants could be tracked back to animal derived raw materials (ADRMs). MVM found in ADRMs-free processes is shed in feces of ever-present mice, is environmentally stable and might not be detected by established pest/ rodent control measures. While bioreactor contaminations are extremely rare, the consequences of a viral contamination event can be significant for a firm, including facility down time, shortage of marketed medicines, and damage to the company’s reputation. If a viral contaminant is identified by routine pre-harvest testing, it is likely that the entire manufacturing facility and process have been contaminated at that time, and that several batches of DS are impacted. Since MVM is the “highest risk” viral threat for ADRMs-free cell culture processes, many firms have implemented an in-process test at production bioreactor stage (see Figure 2), preventing forward processing of an MVM contaminated harvest, and limiting the scope of a potential facility contamination. Use of a targeted NGS assay at this process step would allow detection of a wider range of potential viral contaminants, including viruses identified in previous bioreactor contamination events, and viruses shown to infect CHO laboratory cell lines. It would also improve current controls, and further mitigate the risk of a viral facility contamination.
Conclusions
Viral safety testing is important for the assurance of product quality and patient safety. The aim of this review was to outline opportunities for novel test methods, i.e. unbiased NGS and targeted rapid tests. Unbiased NGS methods are receiving increased acceptance for replacement of compendia tests, i.e. assays able to detect novel/emerging viruses, for products for which testing using current methods is challenging, such as vaccines and viral vectors. Based on future changes in regulatory guidance, e.g. resulting from revision of ICH Q5A, unbiased NGS might replace animal testing for a wider range of therapeutic products. Targeted assays might be used to replace release/animal tests, such as the MAP and HAP assays, that are also targeted tests. Furthermore, rapid targeted tests can be used for in-process testing, supporting accelerated timelines via faster forward processing, and for testing in context of viral contamination control.
References:
- International Conference of Harmonization (ICH), Tripartite Guideline Q5A (R1): Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (1999).
- Guidance for Industry. Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications. FDA, Center for Biologics Evaluation and Research (2010).
- European Pharmacopeia: Chapter 5.2.3. Cell substrates for the production of vaccines for human use (2014).
- Gombold J et al. Systematic evaluation of in vitro and in vivo adventitious virus assays for detection of viral contaminants of cell banks and biological products. Vaccine. 2014;32(24):2916-2926.
- Victoria JG et al. Viral nucleic acids in live-attenuated vaccines: Detection of minority variants and an adventitious virus. J. Virol. 2010;84(12):6033-6040.
- Barone PW et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nature Biotechnology 2020;38: 563-572.
- Berting A et al. Virus susceptibility of Chinese Hamster Ovary (CHO) cells and detection of viral contaminations by adventitious agent testing. Biotechnology and Bioengineering 2010;106:598-607.
Author Biography
Jürgen Müllberg received a Ph.D. in Biology from RWTH Aachen in Germany. He studied molecular mechanisms of inflammation and immune modulation in both, academia and biotech. His work in biopharmaceutical firms focused on expression of therapeutic proteins and vaccines in mammalian cell culture. Currently Jürgen serves as network viral safety SME at Bristol Myers Squibb, and provides guidance on adventitious agent control and safety of manufacturing processes and cell substrates.