Emerging methods for the testing of adventitious agents.
There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we know we don’t know. But there are also unknown unknowns. There are things we don’t know we don’t know.” Donald Rumsfeld’s statement, uttered during a US Department of Defense briefing about weapons of mass destruction, also outlines the problems for biosafety testing of biopharmaceuticals.
A biopharmaceutical, also known as a biologic, or biological, is any pharmaceutical drug product manufactured using a cellular substrate, which could be of animal, insect, plant, or microbial origin. These include vaccines, blood products, therapeutic proteins or even whole cells. Since these substrates have the potential to be contaminated with disease-causing adventitious agents (including bacteria, fungi, viruses), either within the Master Cell Bank or introduced during production, the International Conference on Harmonisation (ICH) has several guidelines covering the technical requirements for registration of pharmaceuticals for human use. One such guideline, ICH Q5D, states: “Virus testing of cell substrates should be designed to detect a wide spectrum of viruses by using appropriate screening tests and relevant specific tests, based on the cultivation history of the cell line, to detect possible contaminating viruses.” ICH Q5A(R1) suggests selecting and testing cell lines and other raw materials, including media components, for the absence of undesirable viruses which may be infectious and/or pathogenic for humans, and testing the product at appropriate steps of production for absence of contaminating infectious viruses.
No single approach will necessarily establish the safety of a product. The type and extent of viral tests required at different steps of production will depend on various factors and should be considered for each product, with testing adjusted as related to their specific product and its production process. The World Health Organization (WHO) has recently updated their recommendations for cell substrate evaluations and indicated that “there may be as yet undiscovered microbial agents for which there is no current evidence or means of detection” and when new testing methods are developed and validated, they should be considered by manufacturers and regulatory agencies for their applicability to the characterization and control of new animal cell substrates.
These general guideline statements, even those written in the 1990s, allow flexibility in testing strategies and allow for future technological improvements to be incorporated into a testing system, as long it can be justified. The type and extent of viral tests could fall into the three categories of Known Knowns, Known Unknowns and Unknown Unknowns.
The Virological Divide
Known knowns apply to characteristics of the biological cell substrate used to produce biologicals. This is illustrated by Chinese hamster ovary cells (CHO), a well-characterized, commonly used cell line used to generate biologics. All CHO cell lines generate non-infectious retrovirus-like particles, derived from endogenous retroviral sequences within the chromosome, which contain reverse transcriptase, a marker used to detect infectious retrovirus. Since this marker is known to be present in CHO cells, appropriately modified testing for infectious retroviruses can be used to avoid false positive results. The WHO document uses the CHO example to state that “risk versus benefit must be considered when determining the suitability of a cell substrate for the production of a specific product. Further, risk-mitigation strategies during production, including purification (removal) and inactivation by physical, enzymatic, and/or chemical means, should be implemented whenever appropriate and feasible.”
Known unknowns apply to a list of adventitious agents based on historical data that may potentially contaminate a biological product such as porcine circovirus from trypsin used in cell culture, Cache Valley virus from bovine serum used in cell culture, and human adenovirus that may have been introduced by production personnel. These types of agents are currently being screened by multiple methods including in vivo testing, cell-based infectivity testing and specific PCR or immuno-based testing for known nucleic acid or protein targets, respectively.
Unknown unknowns are new, undocumented or latent and occult viruses that may be undetected using traditional existing infectivity assays or targeted PCR or immuno- assays. This is the most worrisome to those in the biopharmaceutical and regulatory arena who want to ensure the safety of products manufactured with cellular substrates and who want to improve the safety testing process by incorporating new technologies. So industry experts are exploring new technologies that can be useful complementary tools to existing assays.
New tools for a new day
For example, in 2010, published analysis of a rotavirus vaccine demonstrated detection of an adventitious virus using deep sequencing of vaccine samples. While follow-up studies by the manufacturer and other laboratories generated data suggesting that this particular contaminant posed an insignificant risk, the article generated a great deal of discussion about the potential for adventitious agent testing using massively parallel sequencing (MPS) or other next generation sequencing (NGS) methods.
According to an article from FDA researchers, “the U.S. Food and Drug Administration (FDA) anticipates that the use of NGS data to support regulatory submissions will continue to increase as the scientific and clinical communities become more familiar with the technologies and identify more ways to apply these advanced methods to support development and evaluation of new biomedical products”. One goal is to standardize tools, data formats and protocols across the industry.
A technical report put out by the Parenteral Drug Association (PDA) outlined several new technologies that may increase the ability to detect unknown virus targets, incl
uding broad-range PCR followed by mass spectrometry (PCR-MS), virus microarrays, and MPS or other NGS. The advantage of these methods is that they are broad range, a plus if your goal is to maximize contamination risk reduction without having a known adventitious agent target. The microarrays and the PCR-MS do rely on nucleic acid sequence conservation of virus families but NGS has an unlimited detection range for detection of known or unknown sequences, with the potential limitation coming after the sequence detection. In other words, can the detected sequence be identified as a potential viral pathogen? Downstream analysis, including high quality bioinformatics and sequence assembly, are necessary to identify sequence data as being derived from true pathogens.
These new technologies will expand the biosafety net for biopharmaceuticals but will work best in a multi-modal testing strategy that utilize existing technologies for result confirmation and follow-up studies that can delineate non-viable/non-infectious detected viral nucleic acids from viable/infectious virus particles.
To quote Donald Rumsfeld one more time, “You go to war with the army you have, not the army you might want or wish to have at a later time.” Global regulatory agencies and the biopharmaceutical industry do not find this concept acceptable. In the battle for biopharmaceutical safety, rock, paper and scissors need to merge with new technologies to help thoroughly investigate vaccines and other biologics for adventitious agents that could pose a risk to patients. This is more important than ever with the globalization of disease, as global authorities face intense pressure to manufacture vaccines not just for the usual suspects like measles or influenza, but for emerging viruses like Zika and Ebola.
This is the third in an occasional series about the intersection between biomedical science and politics. You can find the complete list of articles here.
How to cite:
Hantman, Michael and Ruvolo, Brian. Rock, Paper, Scissors, Lizard, Spock, Zika. Eureka blog. July 25, 2016. Available: https://eureka.criver.com/rock-paper-scissors-lizard-spoke-zika/