NGS for Adventitious Agent Screening

Introduction

Contamination is the invisible enemy of biologic products, threatening their safety and efficacy. To tackle this, pharmaceutical manufacturers have long relied on animal-based tests, cell cultures, and PCR techniques to detect these adventitious agents—viruses, bacteria, and other microorganisms that sneak into the production process. The need for stringent virus detection protocols can be traced back to the early 20th century. In 1901, the deaths of 22 children from contaminated diphtheria antitoxin prompted the U.S. government to pass the Biologics Control Act of 1902. Since then, regulations have evolved significantly to ensure the safety of biologics. Next-Generation Sequencing (NGS) has emerged as a transformative tool enabling the pharmaceutical industry to circumvent limitations of traditional methods. By sequencing the entire genetic material in a sample, NGS can identify both known and unknown agents, offering unprecedented sensitivity and speed. Regulatory bodies like the International Council for Harmonization (ICH) have begun to endorse this technology, as reflected in the updated Q5A (R2) guidelines.

Limitations of Traditional Methods

Traditional adventitious agent testing methods often rely on the replication of infectious agents in cell cultures, animal models, or specific media. These approaches are time-intensive, taking weeks to yield results, and may produce false negatives if the system cannot support the growth of certain contaminants. PCR-based assays, while faster, require prior knowledge of the target sequence and may miss unknown agents.
For biologic products like cell and gene therapies, where shelf life is limited and production scales are small, the inefficiencies of traditional methods pose significant challenges. Rapid and comprehensive testing solutions, like NGS, are therefore critical.

Advantages of NGS in Adventitious Agent Screening

NGS is versatile and applicable to various biologic products and manufacturing stages: The fermentation stage in biologics production is a key point for contamination risk. NGS has proven its ability to identify viruses that traditional methods missed. A striking example is the detection of porcine circovirus type 1 (PCV1) in a licensed rotavirus vaccine (Victoria et al., 2010). Another remarkable case involved the discovery of a novel rhabdovirus in Sf9 insect cells used for baculovirus-expressed products (Ma et al., 2014). These findings underscore the importance of NGS in ensuring the safety and quality of biological products.
NGS’s sensitivity and broad detection capability make it an excellent candidate for supplementing or even replacing some of the current assays, such as in vivo animal testing and PCR assays.
Consider the fermentation stage in biologics production. This phase—where cells grow and express the desired product—is also a vulnerable point for contamination. Testing unprocessed bulk harvest samples with NGS provides the best chance of catching any adventitious agents before they’re removed downstream.
NGS offers a powerful alternative for adventitious agent testing, with distinct advantages:

• Broad Detection Capabilities: Unlike PCR, which is target-specific, NGS can identify a wide range of contaminants, including viruses, bacteria, fungi, and mycoplasma, without requiring prior knowledge of their genetic sequences.
• Sensitivity and Specificity: NGS detects low-level or unexpected contaminants that may be missed by traditional assays.
• Efficiency: By enabling multiple analyses from a single sample and reducing testing time, NGS accelerates drug development timelines.
• Ethical and Regulatory Compliance: NGS aligns with the 3Rs principle (replacement, reduction, refinement) by minimizing the need for animal testing and addressing global initiatives to adopt more ethical testing methods.
• Comprehensive Genetic Characterization: Beyond contamination screening, NGS supports applications like sequence identity confirmation, genetic stability testing, and mutation screening.

Challenges and Considerations

The adoption of NGS for adventitious agent screening, while transformative, presents several challenges and considerations that must be addressed. One significant hurdle is the need for bioinformatics expertise, as analyzing the vast amounts of sequencing data generated by NGS requires specialized tools and skilled personnel. Additionally, the lack of universal protocols and reference materials poses a challenge to standardization, complicating the validation process and hindering regulatory acceptance. These challenges highlight the importance of addressing technical, standardization, and regulatory complexities to fully leverage the potential of NGS in adventitious agent screening.

Future Directions

Advancements in NGS technology, combined with artificial intelligence and machine learning, promise even greater sensitivity and efficiency in adventitious agent detection. While these innovations require thorough validation before widespread adoption, they represent the next frontier in ensuring the safety of biologics.

Key Steps Forward

1. Developing Reference Materials – Standardized virus stocks for benchmarking NGS workflows.
2. Refining Bioinformatics – Enhancing databases; eg: Reference Virus Database (RVDB) for accurate virus identification.
3. Harmonizing Guidelines – Finalizing the ICH Q5A(R2) revision to provide clear regulatory pathways for NGS adoption.

Conclusion

NGS is revolutionizing adventitious agent testing by offering a robust, efficient, and ethical alternative to traditional methods. Its ability to detect a broad spectrum of contaminants, streamline testing workflows, and meet regulatory requirements positions NGS as an indispensable tool in pharmaceutical manufacturing. As technology continues to evolve, NGS will remain at the forefront of ensuring the safety and efficacy of biological products.

Strand’s NGS Capabilities

At Strand we simplify the complexity of genome sequencing, thus making it accessible to everyone. We started this journey as a spin off from the Indian Institute of Science in the year 2000. We know that hundreds of thousands of lives can be saved if genome sequencing becomes universal and our ~500 strong team comprising engineers, scientists and clinical personnel works tirelessly towards this goal. We combine our bioinformatics, NGS assay development, and clinical network to solve problems in genomics.

Our India commercial research labs based in Bengaluru have expanded our reach, collaborating with numerous Indian academicians, industries, and pharmaceutical companies. In the past year, our lab has successfully processed over 11,000 samples across various NGS applications, including whole-genome sequencing (WGS), whole-exome sequencing (WES), RNA sequencing, Shotgun metagenomics, Amplicon sequencing, Microarray, and Spatial transcriptomics. We’ve worked with a diverse range of species and sample types.
Additionally, our NGS testing for viral vector genomic identity provides comprehensive verification of the vector sequence by sequencing the entire genome and comparing it to the reference sequence to confirm the correct genetic insert and detect any unintended modifications. In addition to the aforementioned services, we also provide genetic stability testing (e.g., copy number determination), sequence identity confirmation (e.g., for vaccines, viruses, bacteria, gene therapy vectors), cell line identity determination, and mutation screening.

References

1. ICH, Q5A(R2) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Step 2b version (2022).
2. Beurdeley-Fehlbaum, P; Pennington, M; Hégerlé, N.; et al., Evaluation of a Viral Transcriptome Next Generation Sequencing Assay as an Alternative to Animal Assays for Viral Safety Testing of Cell Substrates. Vaccine 2023, 41 5383-5391. https://doi.org/10.1016/j.vaccine.2023.07.019
3. Victora CG, de Onis M, Hallal PC, Blössner M, Shrimpton R. Worldwide timing of growth faltering: revisiting implications for interventions. Pediatrics. 2010 Mar;125(3):e473-80. doi: 10.1542/peds.2009-1519. Epub 2010 Feb 15. PMID: 20156903.
4. Ma H, Galvin TA, Glasner DR, Shaheduzzaman S, Khan AS. Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines. J Virol. 2014 Jun;88(12):6576-85. doi: 10.1128/JVI.00780-14. Epub 2014 Mar 26. PMID: 24672045; PMCID: PMC4054387.