General Chapter 2.6.41 of the European Pharmacopoeia (EP), namely High-throughput Sequencing (HTS, also referred to as NGS) for the Detection of Adventitious Viral Agents, will come into official force on April 1, 2026. NGS technology has officially become a compliant method for the quality control of adventitious viruses in biological products. The industry has moved beyond the discussion of "whether to adopt NGS" and entered the practical implementation stage of "how to make NGS methods compliant and robust".

This article focuses on the core key point of NGS methodological validation implementation — virus standard panels for validation, and discusses what kind of standard panels meet international regulatory requirements, and how to complete the standardized preparation and full performance validation of standard panels.

In January 2024, the World Health Organization (WHO) officially published the article A Collaborative Study to Evaluate the Proposed First WHO International Reference Panel for Adventitious Virus Detection in Biological Products by High-throughput Sequencing (HTS) Technologies. Based on viral genome type, presence/absence of envelope and physicochemical resistance, WHO recommended 7 viruses as the standard panel for NGS adventitious virus detection (Table 1).

Table 1： WHO First Edition Standard Panel for NGS Adventitious Virus Detection

Combined with the latest EP 2.6.41 chapter and ICH Q5A(R2) guideline, this paper aims to clarify the selection strategy and preparation key points of model viruses for NGS adventitious virus detection reference materials, and provide practical application guidelines for standard panels in method validation.

01 Selection Strategy and Preparation Criteria of Validation Virus Standard Panels

EP 2.6.41 specifies that "to ensure the validity of HTS tests, concurrent external or internal positive controls are required for each HTS assay. Especially for Genomics or Viromics Approach analytical methods, the control viruses set shall include a certain number of DNA and RNA viruses or virus-like particles spiked into a suitable matrix". Prior to formal experiments, it is necessary to validate the NGS adventitious virus detection method to prove that the assay has sufficient sensitivity and specificity.

1.1 Selection of Spiking Materials

In general, model viruses are spiked into the detection system for Genomics or Viromics Approach, while infected cells are required for the Transcriptomics Approach. The selection of spiking materials shall be based on risk assessment, taking into account factors such as sample type, analytical strategy and intended use of the detection method. Validation spiking materials may derive from different sources such as model viruses or infected cells, and key information including traceability, composition and purity shall be focused on to properly evaluate the performance of the method.

If the Transcriptomic approach is used to test cell substrates/cell banks, cells infected with different viruses serve as the optimal spiking materials. For cells infected with model viruses, parameters such as the number of infected vs. uninfected cells, confirmation of viral gene expression, viral sequences and host background shall also be annotated.

1.2 Selection of Reference Materials

Section 3.2 SELECTION OF SPIKING MATERIAL FOR VALIDATION clearly states that for analytical methods adopting genomics and viromics approaches to test cell substrates/cell banks, virus seeds and harvests, the corresponding reference materials shall be representative model viruses. Full consideration shall be given to viral characteristics, including genome type (single/double-stranded, DNA/RNA, linear/circular, genome size), physical properties (particle size, presence/absence of envelope) and chemical properties (low, medium and high resistance).

1.3 Selection of Standard Panels

Taking the WHO international standard panel as an example, it is designated for NGS adventitious virus detection of biological products, consisting of 7 viruses covering diverse types (REO, FeLV, RSV, PCV, EBV, hCoV and MVM). It is recommended to be used as the minimum standard panel of model viruses for validation. The above model viruses shall be annotated with parameters such as genome copy number, infectious titer, viral genome sequence (including any variants) and other background signals.

02 Preparation Process and Equivalence Validation of SHENTEK^® NGS Virus Standard Panel

Based on its proprietary exogenous risk factor quality control platform and in-depth understanding of the domestic biopharmaceutical industry and customer demands, Huzhou Shenke has launched the self-owned intellectual property SHENTEK^® NGS adventitious virus detection system. Meanwhile, referring to international standards, it has established virus standard panels complying with the quality control requirements of biological products.

• The standard panel consists of 7 model viruses (REO, MLV, RSV, PCV, EBV, BVDV and MVM) (Table 2).

• The standard panel incorporates BVDV (common bovine-origin contaminating virus) and MLV (common murine-origin contaminating virus) in the biopharmaceutical industry, which is more in line with the demands of the biopharmaceutical sector.

• Fully performance-validated in accordance with EP 2.6.41 and ICH Q5A(R2) guidelines, achieving equivalent performance to the WHO standard panel.

Table 2 SHENTEK^® NGS Validation Virus Standard Panel

The SHENTEK^® NGS validation virus standard panel is prepared from high-concentration viral stock solutions produced with traceable seed viruses. After ultracentrifugation and tangential flow concentration and buffer exchange, it features high purity and low host residue, meeting the requirements of NGS adventitious virus detection for biological products.

Preparation Process of Virus Reference Standards

1. Establishment of virus bank system Seed viruses introduced from authoritative domestic and international culture collections (e.g., ATCC) are used to establish a three-tier virus bank, including Seed Virus Bank (SVB), Master Virus Bank (MVB) and Working Virus Bank (WVB).

2. Virus amplification and stock harvest Referring to the preparation criteria of the WHO virus standard panel, working passage viruses are amplified in susceptible cells, followed by repeated freeze-thaw and low-speed centrifugation. The supernatant is collected as viral harvest fluid.

3. High-purity virus collection via density gradient centrifugationThe viral harvest fluid is subjected to density gradient ultracentrifugation, and target bands are collected to obtain high-purity viral fluid with impurities removed.

4. Ultracentrifugation medium removal and concentrationHigh-purity viral fluid contains high concentrations of ultracentrifugation medium. To minimize impact on subsequent operations, tangential flow filtration (TFF) or chromatographic purification is adopted to remove the medium. Viruses are preserved in preservation solution (containing 0.5% BSA, 10 mM Tris-HCl, 135 mM NaCl, 5% trehalose) or PBS, with elution volume controlled at 1 CV to further increase the concentration of virus reference standards.

5. Quantification and release of virus reference standardsVirus reference standards are quantified via digital PCR (dPCR), subpackaged, quality-inspected and warehoused for release.

Performance Validation of Virus Standard Panel

Referring to the validation criteria of the WHO virus standard panel, Huzhou Shenke has conducted systematic performance validation on the self-developed NGS validation virus standard panel, mainly including system suitability, limit of detection, specificity and detection range.

• System suitability validation: Key steps in the workflow (nucleic acid extraction, library construction, bioinformatic analysis) are validated and quality-controlled to evaluate system performance.

• Limit of Detection (LOD) validation: Viral reference standards of different types are serially diluted 10-fold down to the theoretical detection limit (1E5, 1E4, 1E3, 1E2 VGC/mL), and spiked into Ad5 at 1E9 VGC/mL for testing. The LOD reaches up to 10³ GC/mL under the set detection criteria.

• Specificity (and detection range) validation: Multiple viral reference materials are mixed into matrix samples to verify the discrimination capability when multiple viruses coexist. The results show all mixed viruses are stably detected with no false positives.

Table 3:COA of SHENTEK^® NGS Validation Virus Standard Panel

03 Precautions for Using Validation Virus Standard Panels

From the perspective of industry demands and current regulatory requirements, the NGS adventitious virus assay is applicable to the qualification of cell banks and virus banks, as well as intermediate release testing of cell and gene therapy products. ICH Q5(R2) clearly states that NGS methods can be used to replace in vivo assays and supplement/replace in vitro assays for adventitious virus detection without head-to-head comparison. Nevertheless, full validation or confirmation of NGS methods is still required prior to formal testing.The purpose of adding virus reference materials for NGS adventitious virus detection method validation is to verify whether the entire experimental workflow of NGS detection has sufficient specificity, sensitivity and sample applicability.

Therefore, for specific scenarios of adventitious virus detection, the addition of virus standard panels enables whole-process quality control of NGS detection, covering sample preparation, nucleic acid extraction, library construction, high-throughput sequencing and bioinformatic analysis. In addition to high-quality virus standard panels, stable detection of standard panels in performance validation can only be guaranteed by reasonable spiking methods of virus reference materials, efficient nucleic acid extraction and library construction strategies, as well as validated bioinformatic analysis workflows.