HTS/NGS - Draft Ph.Eur. Chapter "High-Throughput Sequencing for detecting Viral Extraneous Agents"

NGS stands for "Next-Generation Sequencing" (also known as High Throughput Sequencing/HTS, Massively Parallel Sequencing/MPS, or Deep Sequencing) and refers to a series of high-throughput-based methods for the rapid and cost-efficient sequencing of DNA or RNA. In contrast to traditional Sanger sequencing, NGS enables the parallel sequencing of many DNA fragments, resulting in a massive acceleration and scalability of the sequencing process.

NGS has a wide range of applications in various fields, including genomics, transcriptomics, epigenetics, metagenomics and biological systematics. It is used for a variety of applications, including the study of genetic variations, the identification of disease causes, personalised medicine, the study of evolution and the discovery of new biological relationships.

The technology has significantly reduced sequencing costs and shortened sequencing times, leading to broader access to genomic data and accelerated research in the life sciences. NGS has also contributed to significant advances in the diagnosis and treatment of disease by enabling a deeper understanding of the underlying genetic mechanisms.

Areas of application

NGS can be used in a variety of ways in the pharmaceutical industry:

• Drug development and validation: NGS is used to identify potential drug targets, validate drug candidates and evaluate the safety and efficacy of drugs. By analysing genome variants in target genes and their association with diseases, potential biomarkers and therapeutic approaches can be identified. These genetic markers are used to predict the efficacy of drugs and to investigate adverse drug reactions (see below).
• Pharmacogenomics: NGS enables a comprehensive analysis of genetic variations in populations, leading to the identification of genotypes that may influence the response to drugs. This enables personalised medicine, where drugs are prescribed based on the individual genetic profiles of patients.
• Pharmacovigilance: NGS enables a comprehensive investigation of the genetic variations that may be associated with drug side effects. This helps to identify potential risk factors and improve drug safety.
• Microbiological safety: NGS is also used for microbiological quality control of drugs. By sequencing microorganisms in intermediate stages of medicinal products, companies can ensure that their products are free from viral contamination, for example, and meet quality standards. This is particularly important in the manufacture of biological drugs, vaccines and other products that may contain microbial contamination.
• NGS also enables comprehensive identification and characterisation of microorganisms in samples, including potential contaminants such as bacteria, viruses, fungi and other pathogens. This offers a higher resolution compared to traditional microbiological methods using cultivation.
• By using NGS in microbiological quality control, companies can detect potential contaminants early, ensure product integrity and improve patient safety.

Overall, NGS helps to accelerate drug development, improve the safety and efficacy of drugs and enable personalised medicine based on the individual genetic profiles of patients.

Draft of new Ph. Eur. chapter 2.6.41. HIGH-THROUGHPUT SEQUENCING FOR THE DETECTION OF VIRAL EXTRANEOUS AGENTS

There is also a reaction to such developments at regulatory level. For example, the EDQM published the draft of the Ph. Eur. general chapter "2.6.41 High-Throughput Sequencing for the detection of viral extraneous agents" for comment in Pharmaeuropa 36.2.

With this draft, the EDQM addresses one aspect of the NGS application, namely the detection of viral contaminants. These can enter or be introduced into biological products at various stages of the manufacturing process. Based on the principles of Ph. Eur. chapter 5.1.7 on viral safety risk assessment, tests as described in the general chapter 5.1.7 may be necessary to ensure the quality and safety of biological products. These may include testing for viral contamination of starting materials such as cell banks or virus seeds or raw materials of animal or plant origin. These NGS-based tests may be necessary at different stages of a manufacturing process, depending on where cell banks or virus seeds are used or, for example, where proteins or viruses are harvested. They can complement test methods, for example to fill existing gaps identified during risk assessment, as a replacement for in vivo tests or in vitro methods using cell cultures. A particular benefit could be to improve the testing of new cell lines for the detection of known and unknown viruses or to test the absence of viruses from virus seeds or proteins obtained, especially when conventional tests are not suitable or subject to interference due to lack of neutralisation of the vaccine/vector virus or due to toxicity of the sample. The introduction to the new Ph. Eur. chapter states:

"HTS can be used to detect all genomic viral nucleic acids (DNA and RNA) (genomics), viral RNAs (transcriptomics) or encapsulated viral genomes (viromics). The analytical approach can be selected depending on the type of sample to be analysed. HTS can be more complex for the detection of retroviruses in cases where the host sequences containing endogenous retroviral sequences are filtered out by bioinformatics. In such cases, a specific analysis strategy for retrovirus detection should be developed. ....
The design of the method can enable
-detection of a broad spectrum of viral contaminants (known and unknown viruses) with a non-targeted approach using a comprehensive database of viral sequence diversity, or
-detection of a spectrum of known viruses (or unknown viruses related to known viruses) with a targeted approach using selective capture or amplification of viral sequences or using reference viral genomes for bioinformatic analysis."

The present draft of the new Ph. Eur. chapter is therefore concerned with the description of NGS methods that are used to test for viral contamination in biological products; e.g. vaccines for human and veterinary medicine, recombinant proteins, viral vectors for gene therapy and cell-based preparations for cell therapy. In addition, assistance is to be provided for the validation of NGS-based detection methods. Further guidance concerns the requirements for the replacement of in vivo and in vitro tests for contamination and impurities by NGS methods. These recommendations are based on the revision of the ICH guideline Q5A(R2) published in January 2024, which deals with the viral safety assessment of biotechnology products derived from cell lines of human or animal origin.

The currently published draft of the new Ph. Eur. chapter 2.6.41 is open for public comment until 30 June. It can be accessed, after free registration, on the Pharmeuropa website. The ECA Pharmaceutical Microbiology Group has set up an NGS Task Force for comments.

Please note the NGS workshop on 25 June in Munich, which will take place as part of the European Microbiology Conference. The new Ph. Eur. chapter 2.6.41 and the comments of the NGS Task Force on the draft of the new Ph. Eur. chapter will be presented there as well as ICH Q5A(R2) will be presented and discussed.