Next Generation Sequencing

Next-Generation Sequencing (NGS) has emerged as a transformative technology in the field of biosafety testing for biological products (e.g., viral vector vaccines, CGTs, recombinant proteins) and is one of the new methodologies discussed in ICH Q5A guideline. NGS enables the comprehensive determination of nucleic acid sequences from a variety of biological samples (e.g., cell banks, virus banks, bulk harvests) without requiring prior sequence knowledge.

NGS testing provides precise and comprehensive capabilities for genetic identity confirmation, genetic stability assessment, complete nucleic acid sequencing, and virus detection, thereby enhancing the reliability and accuracy of biosafety testing. This technique provides detailed and comprehensive information with rapid turnaround times. NGS testing enables multiple analyses from a single biological sample using a single data set, surpassing traditional genetic characterization methods (e.g., qPCR, Sanger sequencing, Southern blotting, Karyotyping). Specifically, NGS has demonstrated capabilities for broad virus detection and provides defined sensitivity, reducing the need of animal use and minimizing testing time, as outlined in ICH Q5A.

Non-targeted NGS can supplement or replace traditional in-vitro cell-based methods and replace in-vivo assays, thereby promoting the global objective to replace, reduce, and refine (3Rs principle) the use of animal testing. NGS testing allows for the detection of known and unknown viruses without the need for direct head-to-head comparisons. This approach can overcome limitations of in-vitro cell culture assays (e.g., for vaccines), such as cell line susceptibility to infection and test article-mediated interference or toxicity.
ViruSure offers GMP-compliant NGS testing for genetic characterization, such as 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 (e.g., whole-genome sequencing, on-/off-targeting), as well as adventitious agent testing.

NGS testing for evaluating biologic products and samples

Our GMP-certified NGS testing service offers reliable methods for adventitious agent testing and genetic characterization, including genetic stability testing, sequence identity confirmation, cell line identity testing and mutation screening.

NGS testing for adventitious agent detection offers a highly sensitive and broad-spectrum approach to detect unwanted contaminants such as viruses, bacteria, or mycoplasmas, which may inadvertently be present in biological products or samples. Unlike cell lines with limited in-vitro susceptibility, NGS testing can identify both known and unknown agents by sequencing all genetic material in a sample. This makes NGS testing particularly valuable for detecting low-level or unexpected contaminants that could compromise product safety and efficacy. As outlined in ICH Q5A, non-targeted NGS can be used to replace or supplement in-vitro cell culture assays for detecting both known and unknown viruses. Next-generation sequencing overcomes the limitations of traditional in-vitro assays, such as the variability in cell line susceptibility to viral infections, interference, and toxicity associated with the sample or product to be tested (e.g., cell banks, bulk harvests, or viral vector vaccines, CGTs). NGS testing provides a robust method for testing live virus, addressing challenges like the need for developing antibodies to neutralize live virus and the potential loss of detection sensitivity due to high sample dilutions.

NGS testing is essential for genetic characterization, confirming the genomic identity of viruses or cell lines and detecting genetic variants, thereby assessing genetic stability.
Our GMP-compliant 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 genetic stability testing, NGS sequences the genomes of cells from e.g., the MCB and EOPCB to identify any changes or mutations in the transgene that may arise during cell expansion. Genetic variations like mutations, deletions, or rearrangements between the MCB and EOPCB can compromise product integrity, affecting its efficacy, safety, or potency.
Identity testing verifies that the genetic composition of a production cell line or viral vector matches the intended sequence, ensuring stability and confirming the absence of cross-contamination.

NGS testing aligns with the 3Rs principle (replacement, reduction, and refinement), providing a more accurate and ethical alternative to animal testing for detecting adventitious agents and characterizing genetic material. This approach supports the global initiative to replace in-vivo assays with NGS (e.g., MAP/HAP, adventitious agent testing), addressing both regulatory compliance and ethical standards increasingly valued in the industry. ICH guideline Q5A (R2) outlines that in-vivo testing may be performed based on a risk assessment considering the history and manufacturing process of the cell bank and the overall testing strategy. Non-targeted NGS is encouraged to replace in-vivo assays due to its broad detection capabilities and sensitivity, offering an alternative to the limitations of in-vivo assays, and further supporting the initiative to reduce animal testing.

Which biological products can be tested with NGS?

NGS testing allows for the assessment of biological products such as vaccines, CGTs, and recombinant proteins by identifying contaminants, verifying genetic stability, and ensuring genetic integrity. At ViruSure, we offer GMP-certified NGS testing for various biological products to address specific challenges often associated with either traditional methods, or new biological product types.

• Viral vector vaccines (e.g., HPV, Hepatitis B, influenza, malaria) are typically produced in cell lines like Vero, MDCK, HEK 293, or pMK and require extensive testing to confirm the absence of adventitious agents. Traditional in-vitro adventitious agent testing poses challenges for virus seeds due to the interference of neutralizing antibodies that can hinder the detection of potential viral contaminants. The development of neutralizing antibodies or antisera is necessary to prevent cytopathic effects of the vaccine virus on cell cultures. However, this process is both expensive and time-consuming and does not guarantee success. Additionally, the requirement for high dilution levels during testing lowers the detectable concentration of contaminants, thereby reducing the sensitivity and reliability of the in-vitro infectivity assay. Moreover, the ethical concerns associated with in-vivo testing for vaccines highlight the importance of developing alternative methods. As described in ICH Q5A, NGS testing addresses these challenges by offering highly sensitive and unbiased approaches for detecting both known and emerging contaminants.

• Cell and gene therapy (CGT) products (e.g., TILs, CAR T-cells, iPSCs, AAVs, Lentivirus, oncolytic viruses like T-VEC) represent significant advancements in addressing previously unmet medical needs. However, their complexity presents new challenges for product release testing, particularly in ensuring sterility, safety, and efficacy. One of the primary challenges in CGT products is the inability to perform downstream processing steps like viral clearance, as these procedures would destroy the cells or vectors. This limitation makes it crucial to thoroughly source and test raw materials and reagents to detect any potential adventitious agents early in the process. Next-generation sequencing offers a solution by enabling rapid sterility testing (RMM), which is essential for the timely administration of these therapies to patients. NGS testing is not only critical during the development phase but also plays a vital role in release testing by confirming both on-target and off-target effects and verifying the correct integration of viral sequences into target cells.
  Traditional in-vitro cell-culture based assays for detecting contaminants in CGT products are often problematic due to potential matrix interference effects and the high volumes of samples required, which can be costly. In contrast, NGS testing allows for the detection of contaminants with low sample volumes, making it a more efficient and reliable approach for testing CGTs.
  For cell therapies (e.g., CAR T-cells, TILs) viral clearance steps cannot be applied, as they would damage the cells. For gene therapies (e.g., AAVs, lentivirus, CRISPR), the ability to perform viral inactivation or removal depends on the type of viral vector used. Non-enveloped viral vectors like AAVs can be inactivated with detergents or low pH treatments. However, enveloped viral vectors like lentiviruses are incompatible with these methods, as such treatments would compromise the integrity of the vector. Given these complexities, extensive sourcing and rigorous testing of raw materials and reagents are critical to maintaining the safety and quality of CGT products.

• Recombinant proteins (e.g., mAbs, recombinant vaccines, blood clotting factors, hormones, enzymes, interferons, growth factors) continue to be prominent in the biotech industry. These therapeutics are produced using engineered cell lines (e.g., CHO, BHK, avian and insect cells) that express the target gene. It is essential to maintain genetic stability after multiple expansions of the MCB or WCB to the EOPCB and to ensure the absence of contamination in large-scale production systems. NGS is a powerful tool that enhances the quality control of recombinant protein production by detecting even minor genetic variations or contaminations that could affect the production process. Additionally, NGS can replace in-vivo adventitious agent testing for recombinant proteins, aligning with the 3Rs principle by minimizing the use of living organisms. NGS testing improves precision and efficiency in identifying contaminants and verifying the integrity of recombinant proteins at a molecular level, thereby reducing the need for animal testing.

Which biological samples can be tested with NGS?

NGS testing can be applied to various biological samples (e.g., cell banks, virus seeds, bulk harvests, raw materials containing nucleic acids) for the detection of adventitious agents to assure stock purity, rapid identification of contaminants, or for the evaluation of the genetic stability. ViruSure offers GMP-certified NGS testing tailored to your specific needs, ensuring the highest standards of quality, safety, and efficacy for biopharmaceutical samples.

• Cell banks (e.g., MCB, WCB, EOPCB): Cell banks can be analyzed using NGS testing to ensure genetic integrity and stability. NGS allows for a comprehensive examination of the MCB, confirming the accuracy of the genetic sequence and the absence of contaminants such as viruses, bacteria, or cross contamination with other cell lines. NGS testing ensures that the WCB remains genetically stable with the MCB and is free from any adventitious agents or mutations that could potentially arise during cell culture. The EOPCB is analyzed to evaluate the stability of the cell line following extensive use and passaging of the MCB. NGS testing is employed to verify that the cells have maintained their genetic integrity over time during cell expansion, and to detect any unexpected genetic changes (e.g., mutations, deletions, insertions), or contaminations. According to ICH Q5A, testing for adventitious viruses is required for the MCB, WCB and cells cultured up to or beyond the LIVCA (EOPCB). NGS testing may be used as an alternative to traditional in-vitro and in-vivo assays for detecting adventitious viruses.

• Virus seeds (e.g., MVS, WVS): NGS can be used to verify the MVS´s (stock, lot, or bank) identity, purity, and genetic stability, ensuring it is free from unintended mutations or viral contaminations that could impact the safety or efficacy of the final product. NGS testing of the WVS confirms that it maintains the genetic characteristics of the MVS and has not acquired any contaminants or mutations during storage and handling. As stated in ICH guideline Q5A, NGS can be used as an alternative assay for virus testing at applicable stages, particularly when the viral vector or its derived product is not amenable to neutralization and/or inactivation/removal. Testing should be conducted on both the virus seed and the unprocessed bulk harvest. Virus testing should consider the origin of the cell line and the raw materials and reagents used in the virus seed preparation to ensure that no adventitious or replication-competent viruses are present.

• Bulk harvests: NGS testing can identify potential contaminants in bulk harvests, ensuring that the production process has not introduced impurities that could impact the safety or quality of the final product. As outlined in ICH Q5A, adventitious virus testing should be routinely performed on each unprocessed bulk harvest lot. Testing of unprocessed bulk should consider different factors like the nature of the cell lines used, the extent of virus testing performed during cell line qualification, cultivation methods, sources of raw materials and reagents used. If the unprocessed bulk is toxic or interferes with virus detection in traditional assays (e.g., as for viral vaccines), NGS testing can be employed as an alternative analysing approach.

• Raw and in-process materials: NGS testing is essential for evaluating both raw materials (e.g., bovine serum, porcine trypsin, human platelet lysate) and in-process materials (e.g., cell culture media, buffers, reagents, intermediate harvests, filtrates) used in cell-based manufacturing processes, with rapid turnaround times. Effective quality control of these materials is essential to prevent contamination and ensure the safety and efficacy of biological products. Technologies like short-read sequencing are limited in their ability to distinguish between live-replicating viruses and inactivated viral particles that may remain after irradiation of animal-derived materials (e.g., bovine serum, porcine trypsin). In contrast, long-read sequencing technologies provide more accurate contamination detection by reducing background noise, making it possible to detect even low titer contaminants in smaller data sets, thereby lowering the risk of false-positive hits. Moreover, long-read sequencing can capture complete genomes of potential contaminants in a single read, offering more definitive evidence of a contamination. While irradiation is commonly used to inactivate viruses in raw materials, this method can also degrade essential components, compromising their functionality. Therefore, alternative testing approaches, such as NGS, are necessary to provide a more precise and comprehensive assessment of raw and in-process materials in cell-based manufacturing.

The advantages of Oxford Nanopore´s long-read sequencing technology

ViruSure uses Oxford Nanopore´s long-read sequencing technology for its advanced capabilities over traditional short-read sequencing methods. Oxford Nanopore sequencing utilizes flow cells with nanopores embedded in a membrane. Each nanopore is connected to an electrode that measures electrical current disruptions caused by DNA or RNA passing through it. These disruptions create unique patterns that are decoded in real-time to determine the sequence of the genetic material. This technology can analyze both short and ultra-long DNA or RNA fragments directly, without the need for amplification. Oxford Nanopore’s long-read sequencing provides real-time data and effectively analyzes repetitive regions, structural variations, and isoform distinctions compared to traditional methods. It also allows for the detection of base modifications, including methylation.

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