Viruses present a different problem. They may be associated intrinsically with the cell substrate or enter adventitiously through raw materials, reagents or manufacturing activities. The revised ICH Q5A(R2) explicitly considers viruses potentially present in master cell banks as well as adventitious viruses introduced during production [2]. 

Consequently, biosafety cannot sensibly depend upon one analytical test. What is required is a layered contamination-control strategy built around prevention, detection, risk assessment and an ability to respond effectively when something goes wrong.

Controls: Creating multiple barriers to contamination

The strongest approach to mycoplasma and viral control begins before the first production culture is initiated. Prevention starts with understanding the provenance of the cell substrate and each biological material to which it will subsequently be exposed.

This means establishing well-characterised master and working cell banks, protecting their integrity and maintaining traceability. Cell substrate qualification should consider source, history, growth characteristics and susceptibility to adventitious agents. Biological raw materials and ancillary reagents require similar scrutiny. The US Food and Drug Administration (FDA) guidance, for example, specifically considers serum, trypsin and other biological reagents alongside cell-bank qualification and testing at different stages of manufacture [3]. 

Where practicable, reducing dependence on animal-derived components removes potential contamination pathways. Likewise, supplier qualification should extend beyond conventional chemical quality attributes to the biological origin and manufacturing history of critical materials.

Physical and procedural segregation is another important defence. Different cell lines and infectious materials should not be manipulated simultaneously within the same working area where cross-contamination is possible. Closed processing and single-use systems offer opportunities for reducing open manipulations. Changes in technology also need an appropriate assessment of the risks introduced by components, materials and their supply chains. Testing provides the next layer.

For mycoplasma, traditional culture-based and indicator-cell techniques are increasingly complemented by Nucleic Acid Amplification Techniques (NAAT), particularly PCR-based assays. Molecular techniques have the potential to provide sensitive and rapid detection [1]. Mycoplasma control nevertheless needs to be systematic. Testing cells only once is inadequate where subsequent handling provides opportunities for recontamination. Viral detection presents a broader analytical challenge because there is no universal viral equivalent of a bacterial sterility test. Conventional approaches have combined cell-culture assays, virus-specific molecular tests and, historically, animal-based assays [2,3]. 

This is an area undergoing considerable technological change. Next-Generation Sequencing (NGS), also termed High-Throughput Sequencing (HTS), can provide broad detection of viral nucleic acids. Recent validation work by Bova and colleagues demonstrated an agnostic NGS approach for detecting RNA viruses and actively replicating DNA virus contaminants in cell banks. The authors reported limits of detection in the range of 10³ to 10⁴ genome copies for the model viruses evaluated, depending on virus type [4]. 

Importantly, ICH Q5A(R2) reflects the evolving scientific environment surrounding viral safety assessment. The guideline was adopted at ICH Step 4 in November 2023 and became effective in the European Union on 14 June 2024 [2,6]. This regulatory development makes NGS more than an interesting research technology - appropriately validated genomic techniques are increasingly relevant to modern adventitious-agent testing strategies. 

Risk assessment: Understanding where contamination can enter 

Testing generates data. Risk assessment determines what should be tested, where and why. A useful mycoplasma and viral risk assessment begins by mapping the complete lifecycle of the cell line. Five broad questions provide a useful framework:

1.    What is the origin and history of the cell substrate?
2.    Which biological raw materials contact the cells?
3.    Where can personnel, equipment or other cultures introduce contamination?
4.    At which process stages would contamination be most readily detectable?
5.    What downstream barriers could remove or inactivate an agent if contamination occurred?

This moves the exercise away from simply compiling a list of tests towards understanding the complete contamination pathway. The distinction between hazard and risk is especially important. The theoretical ability of a virus to occur is not equivalent to its probability of being introduced, surviving and ultimately reaching the product. The risk assessment should therefore integrate source history, raw-material exposure, cell susceptibility, process conditions and downstream clearance. ICH Q5A(R2) captures this wider concept through its consideration of cell-line qualification, testing for viruses and the evaluation of virus removal and inactivation rather than treating testing as the sole determinant of viral safety [2]. 

NGS also creates an interesting risk-assessment issue. Greater analytical sensitivity does not necessarily make interpretation easier. A detected sequence can potentially represent infectious virus or residual non-infectious viral nucleic acid. Research by Cheval and colleagues highlighted this difficulty and investigated sequencing of newly synthesised RNA as an approach for differentiating cell infection from carry-over viral nucleic acids [5]. 

The emerging challenge is therefore no longer simply ‘Can we detect something?’, but ‘What is the biological significance of what we detected?’ This places increasing importance on validated bioinformatics pipelines, suitable controls and databases, confirmatory approaches and scientifically justified investigation strategies.

Remediation: When prevention fails

Detection of mycoplasma or an unexpected viral signal should trigger a controlled investigation rather than an immediate assumption about either its significance or its source. The immediate priorities should include securing relevant samples and data, confirming the validity of the result, containing potentially affected cultures and assessing the extent of potential impact. Orthogonal confirmation is particularly important where unexpected findings arise from highly sensitive molecular methods.

With mycoplasma, eradication using antimicrobial approaches is technically possible and published literature describes strategies for both prevention and eradication [1]. However, successful suppression of detectable organisms does not automatically restore confidence in the biological integrity of a cell line. Mycoplasma infection may have altered cellular characteristics before it was discovered. 

For a valuable research culture for which no uncontaminated stock exists, treatment followed by repeated testing and characterisation may sometimes be scientifically defensible. For a controlled production cell bank, by contrast, recovery from an authenticated and demonstrably uncontaminated source provides a stronger assurance strategy where such material is available.

Viral contamination requires even greater caution. The appropriate response depends upon the identity of the agent, whether there is evidence of replication, the materials potentially exposed and, for processes where applicable, the ability of downstream manufacturing stages to remove or inactivate viruses. For biotechnology products within its scope, ICH Q5A(R2) considers viral clearance studies as part of the overall viral-safety framework [2]. Such studies should not, however, become justification for accepting avoidable contamination upstream. Clearance complements prevention - it does not replace it. 

Following either a confirmed mycoplasma or viral event, remediation should extend into a formal root-cause investigation. This may require examination of cell-bank genealogy, raw-material lots, culture operations, equipment and potentially associated activities. Corrective and preventive action should ultimately address the identified or plausible contamination pathway rather than simply adding further end-product testing.

From testing towards biological assurance

The most important shift is conceptual. Mycoplasma and viral safety should not be viewed simply as a sequence of release tests. Rather, protection of a cell line arises from a network of mutually reinforcing controls.

Emerging technologies strengthen that network. A rapid Nucleic Acid Test (NAT) offers increasingly useful approaches to mycoplasma detection, while NGS/HTS expands viral interrogation beyond predefined targets [1,4]. Research has also demonstrated how specialised HTS approaches can assist with the difficult distinction between viral sequence detection and evidence indicative of active infection [5]. Yet increasingly powerful detection does not eliminate the fundamentals of biosafety. Indeed, it arguably makes them more important. The objective should therefore remain straightforward: prevent contamination where possible, detect it where it matters, understand its significance through risk assessment and maintain a scientifically credible remediation strategy for circumstances where controls fail. 

The safest cell line is not simply one that has produced a negative test. It is one for which the origin is understood, exposure pathways have been controlled, testing is scientifically justified and the biological history remains demonstrably trustworthy.