Current Testing Methods and Challenges for Detection of Adventitious Viruses

Sources of Adventitious Virus Contamination and Mitigation Strategies

There are various sources of adventitious virus contamination in biologics (1): (a) the source materials (such as virus seed, cell substrate, or biological raw materials for cell culture, e.g., serum and trypsin) may contain naturally occurring exogenous or endogenous viruses or may have become infected with viruses if the donor species was naturally exposed to infectious agents or possibly from animal vaccines; (b) viruses may be acquired during cell culture passage by cross-contamination from infectious materials in the laboratory or other cell production facilities, environment, or by use of nonqualified animal-derived raw materials, as well as arise de novo by recombination; and (c) potential introduction of viruses can also occur during product manufacture due to handling, equipment, or environmental exposure, and due to possible activation of endogenous retroviruses or reactivation of latent DNA viruses by manufacturing conditions and reagents used to enhance product yield.

A comprehensive detection and mitigation strategy for product safety can consist of a three-prong approach aimed at (a) minimizing the risk of introduction of virus contamination in the starting materials for production, (b) assessing the potential for introduction of virus contaminants during product manufacture, and (c) maximizing virus clearance by including steps for effective inactivation and/or removal of any contaminating viruses.

The risk of virus contamination in the upstream processes may be reduced by use of qualified critical biological raw materials. This can be done by extensive testing of the biological starting materials such as cell substrates, vaccine virus seeds, vector virus preparations, donors/tissues, and others for known and unknown agents and by use of certified/tested animal-derived biological materials required for cell growth, differentiation, and selection (e.g., serum, trypsin, antibodies, media/media components, and growth factors).

Evaluation for virus contamination during manufacturing by developing a comprehensive plan for in-process testing of known and novel viruses may provide assurance to a great extent for demonstrating the absence of adventitious viruses in bulk/production lots.

The risk of virus contamination in downstream manufacturing can be mitigated by designing efficient validated processes to (1) reduce potential adventitious virus load, (2) inactivate potentially contaminating viruses in the final product, and (3) demonstrate reduction of residual cell substrate materials.

Detection of Adventitious Viruses

Testing for virus contamination is critical for demonstrating product safety. This is particularly important for novel cell substrates, which may contain unexpected and unindentified agents, and for demonstrating the safety of non-inactivated products, since there are no virus inactivation and removal steps in their manufacturing. Regulatory authorities generally recommend extensive adventitious agent testing at different stages of the manufacturing process targeting steps of potential introduction of virus contamination, and using various sensitive and broad detection assays (2–13). The routine tests for adventitious viruses currently recommended by CBER are detailed in Table I, and examples of some of the viruses detected in the different assays are indicated in Table II. These include general and specific virus detection assays for testing different virus families. However, the use of novel cell substrates and new technologies for product development, and discoveries of previously unknown viruses pose challenges for these conventional assays (Tables III and IV). Therefore, additional tests may be recommended to demonstrate the safety of novel cell substrates (14). These include chemical induction assays using drugs with different mechanisms of virus activation to evaluate latent and occult viruses in novel cell substrates (15, 16) and in vivo oncogenicity assays to evaluate oncogenic viruses in tumorigenic cell substrates. Some general features of these assays are described in Table V. Recently, new, sequence-based technologies have emerged for broad virus detection (17–21). The methods used to discover novel viruses and sequences of previously unknown viruses—massively parallel or deep sequencing, virus microarrays, or long-range polymerase chain reaction (PCR) with mass spectrometry—warrant further consideration and evaluation for their applicability to the testing of biological products (Table VI).

Conflict of Interest Declaration

The author declares no competing interests.