NGS testing for identification of contamination events

Next-Generation Sequencing (NGS) is a powerful tool for identifying contamination events in the manufacturing of biopharmaceutical products, including cell and gene therapies or ATMPs (e.g., AAVs, CAR-T cells, TILs, iPSCs, gene editing therapies, and stem cell therapies). Unlike traditional assays, NGS testing does not require prior knowledge of a contaminant’s genetic sequence, enabling the detection of both known and unknown adventitious agents down to the species level. With its high sensitivity and broad detection capabilities, NGS testing offers a rapid and reliable solution for contamination identification. Its fast turnaround time allows manufacturers to take immediate corrective actions, minimizing production delays, mitigating losses, and ensuring process continuity.

Contamination events in bioreactor cultures present serious risks to biopharmaceutical manufacturing, including lost batches, costly investigations, decontamination efforts, and regulatory compliance challenges. These contaminants can originate from multiple sources, such as inoculum, handling errors, sterilization failures, environmental exposure, or issues with gas supplies, reagents, and cleaning processes. To mitigate these risks, rapid and precise identification of contaminants is critical. NGS testing helps minimize disruptions, safeguard product integrity, and ensures timely corrective actions, supporting the overall reliability of biopharmaceutical production.

NGS testing for rapid identification of contamination events

In the event of an unexpected contamination, NGS testing provides a rapid and effective solution for identifying the contaminant. Given the time-sensitive nature of such incidents, time-critical adjustments can be made to accelerate the testing process, often omitting limit of detection (LOD) determination since contaminants are typically present in high concentrations. This allows for adapted sample preparation strategies, reduced sequencing runtimes, and shortened bioinformatics pipelines to deliver results faster. To further enhance turnaround times, smaller reference databases and more rapid classification tools can be employed, enabling quicker data processing to obtain results faster, supporting risk-based decision-making and timely implementation of corrective actions.

The broad detection capabilities of NGS testing are especially valuable for identifying contamination sources, such as raw materials, equipment, personnel, or the manufacturing environment. By providing detailed genetic information, NGS helps pinpoint these sources and supports the implementation of effective corrective actions. Its rapid turnaround time, delivering results in just a few days, makes NGS essential during bioreactor or bulk harvest contamination events, where quick identification is crucial to minimize production delays, costly investigations, and prevent large-scale spread.

Detection and identification of a viral contaminant using NGS: a case study

ViruSure successfully identified a viral contaminant in a biopharmaceutical product through NGS testing. The sample in question was a bulk harvest from a bioreactor culturing hamster cells expressing a recombinant protein. At ViruSure, this sample underwent GMP-compliant biosafety testing, including a cell culture-based infectivity assay for adventitious agent detection, which is required for all bulk harvests as per ICH Q5A.
In-vitro adventitious agent testing involves inoculating the sample onto indicator cell lines to detect a broad range of potential human and animal viruses. These assays typically include cells from the species of origin, human diploid cells (e.g., MRC-5), and monkey kidney cells (e.g., Vero). The sample to be tested is inoculated onto permissive cells and incubated for a 28-day period, with one subpassage after 14 days.

After four days of incubation, a cytopathic effect (CPE) was observed in the Vero cells, resulting in an out-of-specification (OOS) investigation. Given the positive result from the in-vitro adventitious agent test, multiple methods were considered for follow-up analysis to identify the contaminant. Traditional techniques, such as qPCR and TEM testing, were evaluated, each with their own advantages and limitations. While qPCR can identify known viral contaminants based on sequence information, it requires prior knowledge of potential agents and may miss unknown contaminants. Transmission electron microscopy provides structural insights but lacks the sensitivity and specificity for definitive identification.

To address the issue comprehensively, NGS testing was chosen for its capability to identify a broad range of contaminants without requiring prior knowledge of their genetic sequences. A combination of DNA and RNA sequencing was employed to capture a broader spectrum of potential agents. DNA analysis was performed with PCR amplification due to the low DNA concentration in the sample, while RNA analysis was used to detect any viral RNA present.
The NGS analysis generated 13 million reads, initially revealing only retroviral sequences. However, these retroviral hits were also present in the negative control (supernatant from Vero cells without the test sample), suggesting that they were not the cause of the cytopathic effect observed. A second run of RNA sequencing provided more promising results.

To enhance the dataset and achieve more reliable results, optimizations were made, including an additional step to denature dsRNA to ssRNA, improving the overall dataset size. This resulted in 65,000 reads, with approximately 8% classified as EHDV.
To confirm the presence of EHDV, follow-up analysis was performed by mapping the dataset against the respective reference genome, achieving high coverage of nearly all ten genome segments. Confirmation PCR assays verified the presence of EHDV with high titers (approximately 8 log10 gc/mL).

EHDV, the identified contaminant, is a reovirus that infects cattle and is endemic in most parts of the world, except for New Zealand. Reoviruses can lead to high contamination levels in pooled serum from a single infected cow, posing a risk whenever bovine components (e.g., bovine serum) are used in cell culture media. This risk can be mitigated through careful sourcing, testing of raw materials, and appropriate inactivation procedures (e.g., heat inactivation, gamma-irradiation).

NGS testing was invaluable for quickly identifying the contaminant and ensuring reliable detection.
This case highlights the critical role of NGS testing in ensuring the safety and efficacy of biopharmaceutical products, particularly in the context of complex therapies like cell and gene therapies, which cannot (or only limited) undergo conventional viral clearance processes. NGS enables early detection and resolution of contamination issues, safeguarding the production process and the quality of the final product.