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October 01, 2017 (Vol. 37, No. 17)

Supplement: Utility of GMP Next-Generation Sequencing (NGS) for Biosafety Assessment of Biological Products

NGS Opens a Range of Possibilities for the Analysis of Diverse DNA and RNA Populations

  • The advent of next-generation sequencing (NGS), also referred to as massively parallel or deep sequencing, affords a radically different approach to the challenge of identifying and characterizing known and unknown agents (sequences) with precision and sensitivity. By delivering significantly more data than traditional Sanger-based sequencing methods, NGS opens a range of possibilities for the analysis of diverse DNA and RNA populations.

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    Figure 2.

    NGS does not require any prior knowledge of the sample sequence; the technology is capable of detecting all sequences in a sample, whether known or not. An NGS library is constructed from sample nucleic acid and then sequenced (Figure 1). Comparison of a sequence to target sequences or to libraries of known reference sequences using bioinformatics programs reveals identities. Identification of novel sequences is made possible by virtue of homology to known elements/sequences.

    By combining custom sample preparation with tailored sequencing and bioinformatics, NGS is ideal for the characterization of biological products (e.g., viral vaccines/products, raw materials, cell lines used in biomanufacturing, and final drug products [Figure 2]) as part of a Quality by Design (QbD) approach.

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    Figure 3.

    NGS is also particularly well suited for biosafety testing, including the identification of unknown contaminants in biological samples or systems (e.g., those that result in bioreactor/fermentor failures or unexpected morphological changes/cell death during cell culture). In such instances, a rapid investigation, combined with the ability to detect contaminants without bias or prejudice, is essential and NGS can be the critical first step for contamination remediation.

    NGS continues to prove itself, not only as a key supplementary tool, but also a key alternative method to address testing requirements specific for virus-based therapeutic products. Virus-based therapeutic products (Figure 3) are viruses that are converted into therapeutic agents by reprogramming them to treat disease. They can be grouped by application:

  • • Viral vaccines—viruses designed to prevent replication and elicit an immune response
    • Oncolytic virotherapy—viruses that selectively target cancer and tumor cells and elicit an antitumor immune response
    • Viral gene therapy—viruses that deliver therapeutic genes to cells with genetic malfunctions
    • Viral immunotherapy—viruses that introduce specific antigens to a patient’s immune system

    Plenty of regulatory guidance exists around the manufacture of viral-based medicinal products. FDA and EMEA guidance documents and reflection papers outline testing strategies and recommendations. While use of NGS is not specifically defined, it may be inferred under use of “state-of-art” technologies.

  • Challenges for Traditional Testing of Viral Products

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    Figures 4 and 5.

    Traditional tests for adventitious virus are lengthy (e.g., sterility, 14 days; mycoplasma, 28 days; [Figure 4] and RCR, 28 days). Although they may detect a contaminant, they generally do not directly identify it. Often, only small lots with limited sample volumes of viral-based therapeutic products are produced, resulting in limited availability of starting material for process, product, and test-method development. Another testing challenge for these types of products is the lack of reference standards and the requirement for producing neutralizing antisera, required for many traditional assays. NGS offers opportunities for circumventing some of these challenges by offering a consistent and sensitive method compatible with a diverse range of products to test for and identify adventitious virus (Figure 5).

  • Specific NGS Applications for Virus Product Safety and Characterization

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    One key application of NGS is identity testing and variant detection, which is of great value when large or difficult-to-sequence genomes are evaluated, or when structural and rare variants are involved (Figure 6).

    Equally important is its application in contaminant detection. Here, NGS allows both detection and identification of viral, bacterial, or fungal sequence signatures, of both known and unknown agents (Figure 7).

    In summary, NGS has clearly emerged as an effective molecular tool with a wide range of applications in biosafety testing and biomanufacturing. NGS offers not only a supplementary method, but also a novel alternative testing strategy, enabling solutions where traditional testing approaches struggle or fail. Regulatory guidance driving the use of NGS for specific applications is still a work-in-progress. However, the technology can clearly be developed for, and applied in, a regulatory setting, and meet, if not exceed, the requirements and expectations.

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