NGS testing for Genetic Characterization

GMP-certified Genetic Characterization with NGS

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NGS testing for Genetic Characterization

High-throughput sequencing (HTS; also referred to as NGS) has transformed genetic characterization by providing a comprehensive and unbiased analysis of nucleic acids without prior sequence knowledge. NGS testing can be used for critical applications like genetic stability testing, mutation screening, and identity confirmation, delivering more detailed genomic insights than conventional methods such as qPCR, Sanger sequencing, or Karyotyping. This makes NGS testing indispensable for characterizing biological products like viral vectors, CGTs (e.g., AAVs, Lentivirus, CAR-T cells, TILs, iPSCs), and cell lines used for recombinant protein production.

ViruSure offers GMP-certified NGS testing with rapid turnaround times for genetic characterization, including whole-genome sequencing (WGS), insertion site analysis, and mutation detection. This advanced tool ensures the stability and integrity of production cell lines (e.g., CHO, HEK 293, HeLa, Vero, MDCK, Sf9) and genetic constructs, while also supporting sequence identity confirmation – a critical step in the production of biological products (e.g., plasmids, viral vectors, vaccines, CGTs). With decades of expertise, we provide tailored solutions for genetic stability assessment, sequence identity verification, and genome editing evaluation.

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Genetic characterization with NGS testing

ViruSure provides fully GMP-compliant NGS testing for genetic characterization, including sequence identity confirmation and genetic stability assessments.
According to ICH Q5D, the characterization and testing of cell banks aims to confirm the identity, purity, and genetic stability of the cell substrate used directly as a therapy or for the manufacturing of biological products (e.g., recombinant proteins, CGT products like AAVs, Lentivirus, CAR-T cells, TILs, iPSCs, gene-editing therapies, stem cell therapy). These tests are essential and must be performed to ensure the quality and safety of biopharmaceutical production systems.
Manufacturers are required to perform identity testing on the MCB at least once, while stability testing during cell cultivation should be conducted for each product to be registered. Additionally, limited identity testing must also be performed once on the WCB. In certain cases, if scientifically justified, the WCB may be characterized instead of the MCB.
As stated in ICH Q5B, the expression construct must be characterized for the production of recombinant DNA protein products to ensure their quality and consistency. The protein-coding sequence of the expression construct must be verified in the MCB to confirm its integrity. If testing is not feasible on the MCB, it must be performed on the WCB.

Sequence identity confirmation

Identity testing is a critical step in the production of biological products, including plasmids, viral vectors, vaccines, and CGT products (e.g., TILs, CAR T-cells, iPSCs). GMP-certified NGS testing involves sequencing the nucleic acid of interest (therapeutic genes, vector elements) and comparing it against a reference sequence or database. For cell banks, identity testing confirms that the cells are accurately represented and distinct from other cell lines. Identity testing must be performed on the starting material (cell seeds), and is conducted on the MCB and EOPCB, with limited testing performed on the WCB. The choice of nucleic acid for NGS testing depends on the material and analysis purpose; DNA sequencing confirms species identity, genomic integrity, and therapeutic gene stability, while RNA sequencing verifies transcriptional activity, therapeutic gene expression, and the functionality of regulatory sequences in vectors.In the case of cell banks, qPCR is used to amplify a species-specific marker for confirmation of species identity. Typically, the cytochrome C oxidase is targeted for this purpose, a mitochondrial gene highly conserved within species. This enables precise differentiation among species, such as hamster, cow, pig, mouse, and human. While qPCR is a rapid and specific method, it is limited to predefined targets. To overcome these limitations, NGS testing allows for the analysis of an entire genome and comparing the data to reference sequences, thereby detecting small mutations (e.g., SNPs, INDELs) and larger rearrangements (e.g., structural variants like duplications).In CGTs or ATMPs, viral vectors (e.g., AAV or lentivirus vectors) are commonly used to deliver therapeutic genes within the human body. Identity testing for these vectors is crucial to ensure that the correct gene is produced and administered. Regulatory guidelines, such as those from the FDA, EMA, or ICH, require that the sequence of the vector is confirmed. This includes the therapeutic gene of interest, regulatory regions, and other critical elements relevant to this product.
For example, in recombinant AAV (rAAV) lot release, identity testing involves verifying the gene of interest, flanking inverted terminal repeat (ITR) regions, and other important genomic elements. NGS testing is particularly useful in this field as it can resolve complex structures, such as GC-rich and palindromic ITR regions. These regions are essential for genome replication and can influence transgene expression in therapeutic applications due to their transcriptional activity.

RNA Sequencing

To confirm the accurate transcription of a genetic insert into mRNA, cDNA is typically generated and compared to a reference sequence using NGS testing. However, the Oxford Nanopore platform, i.e., long-read technology, enables direct mRNA sequencing, eliminating the need for additional RNA processing that can introduce biases. This method is particularly valuable for mRNA vaccines, where the integrity and accuracy of the mRNA sequences are critical. Direct RNA sequencing allows for the analysis of full-length transcripts in a single read, ensuring that the mRNA utilized in vaccines, such as those developed for COVID-19, is complete and unaltered. Errors in the mRNA sequence could impair the vaccine’s ability to trigger an immune response.
Moreover, these full-length transcripts are suitable for the accurate characterization of isoforms and their abundance.

Genetic stability assessment

Genetic stability assessment using GMP-compliant NGS testing is essential for verifying the
integrity of genetic inserts throughout the production cycle, particularly in production cell lines (e.g., CHO, HEK 293, HeLa, Vero, MDCK, Sf9) and in CGT products (e.g., AAVs, Lentivirus, CAR-T cells, TILs, iPSCs, gene-editing therapies, stem cell therapy).The stability of the cell substrate ensures consistent production of the desired product and maintains production capacity during storage under specified conditions. To evaluate the stability of the cell substrate, testing should be carried out at a minimum of two points: Testing will first be conducted on cells with minimal sub-cultivation (e.g., MCB), followed by testing on cells at or beyond the limit of in-vitro cell age (LIVCA). This evaluation is typically conducted once for each product marketing application. For recombinant cell lines (e.g., mammalian, insect, avian and microbial expression systems), verifying the coding sequence of the expression construct (expression vector containing the coding sequence of the recombinant protein), including its regulatory elements (e.g., promoters, enhancers), is crucial at these stages to confirm genetic integrity. One primary application is comparing the genomic DNA of cell banks (e.g., EOPCB to MCB) to ensure that the correct coding sequence of the product has been incorporated into the host cell and is maintained during the entire production process. When multiple copies of the expression construct are integrated into the host genome, it is likely that not all copies are transcriptionally active. In such cases, analyzing the transcriptional product directly, via mRNA or cDNA analysis, provides more relevant insights than genomic DNA analysis. NGS also allows for genome-wide detection of off-target editing events, which could have negative impacts on patient safety, such as oncogenesis.

NGS testing approaches: targeted and non-targeted sequencing

Two Next Generation Sequencing approaches can be used for genetic characterization: targeted and non-targeted. Targeted sequencing focuses on specific regions of interest (e.g., genes), enabling detailed and precise genetic analysis with high accuracy. In contrast, non-targeted sequencing, like whole genome sequencing (WGS), provides a comprehensive overview of the entire genome. WGS is particularly useful for detecting unexpected genetic variants and assessing off-target editing events, offering a broad and unbiased view of genetic integrity. Both approaches play a key role in enhancing genetic characterization of biopharmaceutical products (e.g., viral vector vaccines, CGTs like AAVs, CAR-T cells, stem cell therapies).

Differences between targeted and non-targeted sequencing

Targeted and non-targeted approaches differ in their methodologies for genetic analysis.
Targeted sequencing focuses on specific, known reference sequences during bioinformatic analysis, enabling precise characterization of specific genes or genomic regions of interest. This method can be enhanced through techniques like amplicon enrichment or hybridization-based enrichment, which increase the representation of specific sequences and allow for high sequence coverage, making it ideal for studying specific genetic features.

Non-targeted sequencing provides a comprehensive view of the entire genome or a significant portion of it, without focusing on specific genes or regions. This includes methods such as whole genome sequencing (WGS), which captures both coding and non-coding sequences, and whole exome sequencing (WES), which focuses on the protein-coding regions of the genome, collectively known as exome. The unbiased nature of non-targeted sequencing allows for the discovery of unexpected genetic variants (e.g., structural variations like deletions or duplications, copy number variations, off-target insertions) and provides a more complete understanding of genetic diversity.

Whole genome sequencing

Whole genome sequencing (WGS) offers a comprehensive genetic analysis with high-resolution and a base-by-base coverage of the genome and enables the detection of both large and small variants that may be overlooked by targeted sequencing. By generating ultra-long reads (greater than 4 Mb), nanopore sequencing can capture complex structural variants and repetitive regions that are typically challenging to access with short-read technologies. This capability enhances the ability to examine genomic regions that often cannot be resolved by other sequencing technologies. Additionally, nanopore sequencing can directly sequence native DNA (and RNA) without amplification, enabling the detection of epigenetic modifications (e.g., DNA methylation, histone modification, non-coding RNA expression) alongside the nucleotide sequence for deeper genomic insights.

WGS for allogenic cell-based therapies

For allogenic cell-based therapies (e.g., allogenic CAR-T cell therapies; iPSC therapies), including cultured donor cells or combination products, comprehensive testing is crucial to ensure safety. The risk of viral or microbial contamination and genetic changes leading to tumorigenic (cancer-like) cells increases during the manufacturing process, particularly when cells are extensively cultured. To mitigate these risks, thorough donor screening and testing are essential. WGS is recommended for cell banks derived from continuous or genome-edited cell lines, as these cells may accumulate mutations over time, including in genes like p53, which are associated with tumorigenic potential.
WGS should be conducted with a read depth of at least 50X to ensure sufficient sensitivity and accuracy. The sequencing results should be compared to a database of cancer-related mutations to identify any mutations of concern. It´s important to justify the sequencing method, read depth, and safety conclusions drawn from the analysis.

For highly expanded clones of genetically modified cells, WGS is used to assess both off-target and on-target genome editing effects, detect vector integration events, and identify mutations of concern. This comprehensive analysis ensures the genetic integrity of the final product and supports patient safety by identifying any genomic alterations that could pose a risk.

Evaluating on-target and off-target genome editing events with WGS

Off-target genome editing refers to unintended genetic modifications that can occur when using e.g., engineered nuclease technologies (e.g., TALENs, ZFNs, CRISPR-Cas9). This occurs when the nuclease used acts on untargeted genomic sites within the host genome, causing cleavages that could lead to potentially harmful genetic changes. NGS testing methods, such as WGS, are effective for assessing off-target mutation rates. Traditional methods, while technically less complex and faster, are limited in their ability to detect multiple off-target sites efficiently and require significant resources to screen broader regions. In addition to WGS, target enrichment techniques, such as hybridization capture and amplicon sequencing, can be employed to focus on specific regions of interest. While these methods are useful for assessing on-target sites, unbiased WGS remains crucial for identifying potential off-target effects and understanding the overall impact on genome editing.
Let´s break down some FAQs!
For AAV genome analysis, why is long-read sequencing the preferred method?

NGS long-read sequencing is the preferred technology for rAAV products because it enables full-length sequencing of the entire viral genome, including complicated regions such as inverted terminal repeats (ITRs). The complex, GC-rich, palindromic ITR regions, critical for viral replication and packaging, pose sequencing challenges for short-read sequencing methods. With the advantage of full-length reads, Nanopore´s long-read sequencing offers deeper insights into both the viral genome and its structural components, resulting in a more precise characterization of rAAV products.

What is NGS testing for identity confirmation?

NGS testing is used for confirming the genomic identity of biological products by sequencing entire genomes and comparing them to reference sequences, offering a comprehensive and high-resolution approach. Compared to traditional techniques like Sanger sequencing or qPCR, NGS testing enables faster turnaround times and broader sequencing applications. For viral vectors and recombinant proteins, NGS ensures that genetic material matches the respective reference, minimizing the risk of unintended and potentially harmful mutations.

What is NGS testing for genetic stability analysis?

NGS testing verifies the integrity of genetic inserts throughout the production cycle, especially in production cell lines. NGS enables the detection of genetic changes, including mutations, insertions, deletions, rearrangements, SNPs, and INDELs, by comparing samples at different stages (e.g., MCB versus EOPCB), leveraging full genome coverage and rapid sequencing of large inserts. Additionally, off-target genome editing events can be analyzed, thereby minimizing the risk of unexpected or harmful genetic alterations.

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