Unlocking the drug development pipeline with advanced molecular sensing

Unlocking the biopharma development pipeline with Oxford Nanopore How a molecular sensing technology is transforming biopharma Oxford Nanopore Technologies Introductory guide What are the challenges of the biopharma development pipeline? Slow and iterative processes extend timelines • Traditional biomarker discovery and target identification rely on stepwise workflows, requiring separate platforms for DNA sequencing, RNA expression analysis, and functional studies. Each method requires extensive sample preparation, data generation, and interpretation, making the process time consuming and resource intensive. • Preclinical and clinical development can involve multiple cycles of cell line engineering, compound screening, and validation assays, often requiring months to years to refine a promising drug candidate. Low-resolution or incomplete data impacts decision-making • Many current techniques rely on indirect measurements or require extensive extrapolation, rather than capturing direct molecular insights. • Short-read sequencing approaches, which require nucleic acid fragmentation and amplification, miss structural variants, epigenetic modifications, and full-length transcript isoforms, which are key to understanding drug targets, disease mechanisms, and resistance pathways. Fragmented and inflexible workflows lead to inefficiency • Traditional genomic, transcriptomic, and epigenomic methods require different instruments and expertise, often leading to data silos and a lack of cross-platform compatibility. • Many current tools require centralised laboratories and significant outsourcing, preventing real-time analysis in clinical trials, point-of-care settings, on the manufacturing floor, or in-house quality control (QC) laboratories. Multiple analytical approaches increase QC testing complexity • Biomanufacturing QC steps require numerous separate assays to release products either into clinical trials or onto the market as a commercial product. • Regulatory agencies require detailed characterisation of biopharmaceuticals according to current good manufacturing practice (cGMP) environments, yet traditional methods have been shown to suffer from analytical deficits. What you’re missing matters What if we used a molecular sensing technology to streamline drug development? The current drug development process can be long, complex, and resource-intensive, often taking 12–15 years from discovery to regulatory approval1 . Each stage — from target identification to clinical trials and manufacturing — relies on time-consuming sequential workflows, which are often costly. One major bottleneck is the use of a compendium of conventional assays (e.g. Sanger sequencing and legacy short-read sequencing methods) and analytical tools, which have inherent limitations that restrict efficiency, accuracy, scalability, and fast decision-making. As a result, there is a requirement for the industry to adopt faster, more flexible, more efficient, and more information-rich technologies to streamline processes and improve success rates. A new generation of molecular sensing technology Oxford Nanopore sequencing is a high-resolution molecular sensing technology that enables the direct analysis of native DNA and RNA molecules of any length. The single-platform solution generates ultra-rich, multiomic data — providing comprehensive genomic, transcriptomic, and epigenomic insights. This guide will explore how Oxford Nanopore sequencing can transform each stage of the biopharma development pipeline, streamlining the process from discovery to clinical development. Oxford Nanopore sequencing overcomes the challenges faced in the drug development process through: • Real-time, in-house sequencing and analysis for immediate access to actionable results • Reads of any length that detect both small genomic variants, such as single nucleotide variants (SNVs), and large complex structural variants (SVs), as well as repetitive regions and full-length isoforms • Direct sequencing of DNA and RNA for built-in methylation detection without additional steps or instruments • Scalable devices and flexible end-to-end workflows that go from sample to answer, offering GMP-like solutions as early as the analytical development phase and easing the complexity of QC GMP validation Novel biomarker discovery and target identification Comprehensive clinical research sample characterisation and analysis using functional genomics approaches Construct characterisation Sequence confirmation with whole-plasmid sequencing Viral vector characterisation Characterisation with full-length adeno-associated virus (AAV) vector sequencing QC release testing Product release testing with multi-attribute QC testing Candidate identification High-throughput screening with long amplicon sequencing Expression optimisation Native mRNA verification using direct RNA sequencing Cell line development Enhanced insertion site analysis with targeted sequencing Clinical development and post-market surveillance Patient stratification, pharmacogenomics, and recurrence monitoring with variant analysis High-throughput screening with long amplicon sequencing High-throughput screening enables the rapid identification of promising drug candidates from large libraries of potential molecules. For the analysis of drug-target interactions, long amplicon sequencing from Oxford Nanopore can be used to target specific genes or genomic regions associated with disease pathways and rapidly assess mutations or variants in druggable targets (e.g. cell surface receptors or signalling proteins). This targeted approach ensures that the drug candidates with the highest potential for efficacy are taken forward in development — resulting in a more focused and efficient lead selection process. Comprehensive clinical research sample characterisation and analysis using functional genomics approaches By sequencing native DNA, Oxford Nanopore technology enhances the identification of disease-specific variants and epigenetic markers that contribute to disease susceptibility and progression. Like with short-read approaches, high-accuracy nanopore reads provide the ability to detect SNVs; however, they additionally provide the ability to detect SVs, short tandem repeat (STR) expansions, and methylation patterns in a single assay — without amplification bias. Furthermore, nanopore sequencing expands on the transcriptomic insights provided by legacy short-read technologies by simultaneously delivering epitranscriptomic data. By capturing full-length RNA transcripts and RNA modifications, nanopore technology enables deeper understanding of gene regulation, alternative splicing, gene fusions, and expression dynamics in disease-relevant cell types. Oxford Nanopore offers a range of simple and rapid end-to-end workflows for clinical research sample characterisation, which start with a recommended sample extraction method and go all the way through to data analysis. These workflows enable the discovery of novel biomarkers and therapeutic targets with unprecedented depth and resolution. With Oxford Nanopore technology, you can perform: • Human multiomic variant calling • Large cohort sequencing • Single-cell transcriptomics • Bulk transcriptomics • Direct RNA sequencing • Tumour-normal sequencing Novel biomarker discovery and target identification Candidate identification ‘Oxford Nanopore sequencing presents an alternative to Illumina and PacBio sequencing, offering theoretically unlimited amplicon size, cost-effectiveness and minimal capital requirements’ McFarlane, G.R., Polanco, J.V.C., and Bogema, D.2 Additionally, for candidate identification of biologics, such as monoclonal antibodies, long amplicon sequencing can be used to comprehensively analyse B cell repertoires. Oxford Nanopore reads cover the full-length of the amplicons, providing detailed characterisation of antibody heavy- and light-chain diversity. This enables the selection of lead antibodies with high specificity and affinity, ensuring they effectively bind to the intended target. Going beyond antibody discovery, Oxford Nanopore technology is also able to capture T cell receptor genes in single reads, which is key for the development of effective CAR-T cell therapies. Oxford Nanopore offers a fast and simple end-to-end long amplicon sequencing workflow that uses rapid barcoding to prepare up to 96 amplicon samples, without the need for primers. This workflow has been optimised to sequence amplicons from 500 bp to 5 kb in length and uses the intuitive data analysis software EPI2ME™ for variant calling and generation of de novo consensus sequences. Oxford Nanopore technology directly sequences native DNA and RNA molecules of any length Construct characterisation Expression optimisation Sequence confirmation with whole-plasmid sequencing After identifying a promising drug candidate, it is critical to ensure that the expression constructs used for production or functional studies contain the correct sequence. Any errors in the plasmids, such as mutations, truncations, or rearrangements, can compromise the accuracy of downstream analyses and the success of the therapeutic candidate. Traditional Sanger sequencing has many limitations, including the exclusion of the plasmid backbone and the inability to resolve repetitive regions, dimers, and deletions, plus the requirement for vector-specific primers. As the reads generated by Oxford Nanopore technology are unrestricted in their length, whole plasmids can be covered in single reads — allowing full confirmation of expression construct identity. Oxford Nanopore offers an end-to-end workflow that uses rapid barcoding to prepare up to 96 plasmid samples for sequencing. This method is PCR free, preserves base modifications, and requires a short preparation time — making it the ideal option to go from sample to answer in applied settings. In addition, the nanopore-only microbial isolate sequencing solution (NO-MISS) — a flexible and rapid approach for whole-genome sequencing of bacterial isolates — can be used to ensure plasmid production strains are well-characterised, stable, and free from mutations and contamination. Oxford Nanopore sequencing provides: Complete plasmid sequence • Full-length, high-accuracy sequence confirmation to verify that the plasmid contains the intended lead sequence without errors • Detection of SVs, including insertions, deletions, and rearrangements that could impact gene expression • Rapid turnaround time, with real-time data generation allowing for quick validation before proceeding to cell line engineering or further functional testing • De novo assembly and annotation via the EPI2ME wf-clone-validation workflow, generating a fully annotated plasmid map Native mRNA verification using direct RNA sequencing Oxford Nanopore direct RNA sequencing is the only available technology that directly reads native RNA molecules. This amplification-free approach can be used to assess mRNA sequence and processing; even if the plasmid construct has been validated and the DNA sequence is correct, errors can arise during transcription, which can impact mRNA stability and function. Direct RNA sequencing can confirm that transcribed mRNA accurately reflects the gene construct with no unwanted sequence changes, verify transcript fidelity by detecting incomplete or degraded mRNA, and capture post-transcriptional information, such as RNA base modifications (e.g. N1-methylpseudouridine), polyadenylation, and alternative splicing. By sequencing RNA molecules directly, without requiring cDNA conversion, Oxford Nanopore technology provides a detailed, unbiased view of transcript identity and integrity. This is particularly valuable for mRNA-based therapeutics, where ensuring sequence identity, structural integrity, and poly-A tail length of mRNA molecules are essential for therapeutic success. Resistance genes • Correct sequence • Not already present in host organism Backbone • No mutations • Suitable for storage and future use Gene insert • Correct orientation Promoters • No mutations • Compatible with host organism Cell line development Viral vector characterisation Characterisation with full-length adeno-associated virus (AAV) vector sequencing AAV vectors are widely used in cell line engineering and gene therapy development, enabling the delivery of genes into host cells. Therefore, ensuring that correct, error-free AAV genomes are packaged into capsids is crucial, as truncated, rearranged, or incorrectly packaged sequences can compromise efficacy and safety. However, due to the inherent limitations of legacy short-read sequencing, critical features such as highly structured inverted terminal repeats (ITRs) are not resolved. Enhanced insertion site analysis with targeted sequencing Engineered cell lines are fundamental to biopharmaceutical development, serving as production systems for high-value drugs, such as biologics, gene therapies, and cell-based treatments. A critical aspect of this process is ensuring that transgene insertions are precise, stable, and do not disrupt essential cellular functions. Oxford Nanopore sequencing provides a powerful solution for enhanced insertion site analysis to verify both on- and off-target editing events, enabling a comprehensive assessment of genetic modifications in engineered cells. Nanopore sequencing is compatible with CRISPR-Cas9- based target enrichment. This capability, coupled with long reads, means that nanopore technology provides comprehensive identification of transgene copy numbers, orientation, concatemers, truncations, and inverted repeats — all of which may be missed with legacy methods, such as targeted locus amplification (TLA), that rely on short-read sequencing. Additionally, Oxford Nanopore technology simultaneously basecalls nucleotides alongside modified bases, and in Chinese hamster ovary (CHO) cells, methylation differences in production hosts have been associated with variability in antibody productivity and assembly efficiency4. Therefore, the technology delivers high-resolution analysis of engineered cell lines, and this approach ensures that therapeutic cell lines are genetically well-characterised, reproducible, and optimised for consistent, high-yield expression, accelerating the path to regulatory approval and commercial-scale manufacturing. Oxford Nanopore sequencing Long sequencing reads deliver complete AAV coverage Short-read sequencing Ambiguous data caused by incomplete AAV coverage ‘[Oxford] Nanopore sequencing is a state-of-the-art method for comprehensive, in-depth rAAV vector batch analysis during all stages of gene therapy development’ Dunker-Seidler, F. and Breunig, K. et al.3 The any-length reads delivered by Oxford Nanopore sequencing can span entire AAV genomes — from ITR to ITR — enabling full-length AAV vector validation, including the identification of truncated and rearranged genomes and any plasmid DNA or host cell contamination. This complete and accurate picture of AAV genomes ensures that the full transgene and regulatory elements, such as promoters, are present and that no structural alterations, such as deletions or insertions, have occurred during vector production. By offering an end-to-end workflow, Oxford Nanopore sequencing provides a simple method to characterise full-length native AAV vectors, enhancing production and QC. ITR 1 Full-length AAV genome? ITR 2 ITR 1 Truncated AAV genome? ITR 1 Full-length AAV genome ✓ ITR 2 ITR 1 Truncated AAV genome ✓ QC release testing Clinical development and post-market surveillance Patient stratification, pharmacogenomics, and recurrence monitoring with variant analysis Selecting the right treatment for each patient is critical for improving therapeutic outcomes. Oxford Nanopore sequencing provides a real-time, high-resolution solution for variant analysis, which could be used in the future to support patient stratification, pharmacogenomics, and improved recurrence monitoring* . Oxford Nanopore is actively developing and optimising methodologies with partners to provide solutions to support this research. The technology has the potential to improve clinical trial efficiency, support real-time decision-making, and advance the future of precision medicine. *Oxford Nanopore Technologies products are currently for research use only (RUO). The Plasmid Identity QC Test Pack and mRNA Identity QC Test Pack allow for: • Whole-plasmid sequencing — including verification of construct identity, linearisation efficiency, restriction enzyme site mapping, contamination, and homologous regions, such as long terminal repeats (LTRs) and ITRs • Direct mRNA sequencing — including verification of sequence identity, integrity, and poly-A tail length estimation in multivalent formats, and N1-methylpseudouridine base modifications • Single-platform efficiency — reduction on the reliance of multiple QC assays by offering full-length, native sequencing in a single workflow Product release testing with multi-attribute QC testing Historically, numerous analytical methods have been necessary for routine QC testing of drugs produced within cGMP environments. Oxford Nanopore sequencing enables comprehensive QC tests that can measure multiple critical quality attributes (CQAs) with less complexity than conventional methods and legacy sequencing technologies — all in one assay and with faster results. The QC Test Packs from Oxford Nanopore include the required consumables, analysis pipelines, and documentation to perform multi-attribute QC testing — delivering the results within hours and generating a detailed report with pass/fail criteria based on CQAs, such as identity, integrity, and key aspects of purity. In addition to product characterisation and release testing, nanopore sequencing enables bioprocess monitoring to detect contamination from adventitious viral agents (AVA). Traditionally, AVA testing comprises a complex list of compendial methods that require significant investment in infrastructure, large capital equipment, and labour. Oxford Nanopore sequencing is a fast, accurate, and high-throughput alternative that can replace/supplement compendial methods with a single assay. With a long history of use for pathogen surveillance, Oxford Nanopore technology is the ideal solution for detecting viral contaminants. Patient stratification • By analysing genetic variants in patient research samples, nanopore sequencing could be used to identify likely responders and non-responders, supporting the selection of the most suitable candidates for targeted therapies Pharmacogenomics • Oxford Nanopore sequencing can accurately resolve complex pharmacogenomic variants, including those in highly homologous or repetitive genes (e.g. CYP2D6), which are difficult to analyse with short-read sequencing methods Recurrence monitoring • For long-term disease management, nanopore sequencing has the potential to be a powerful, non-invasive tool for detecting early signs of recurrence and tracking minimal residual disease (MRD) through cell-free DNA methylation analysis References 1. Singh, N. et al. Front. Drug Discov. 3:1201419 (2023). DOI: https://doi.org/10.3389/fddsv.2023.1201419 2. McFarlane, G.R., Polanco, J.V.C., and Bogema, D. BMC Res. Notes 17(1):205 (2024). DOI: https://doi.org/10.1186/s13104-024-06861-1 3. Dunker-Seidler, F. and Breunig, K. et al. Mol. Ther. Methods Clin. Dev. 33(1):101417 (2025). DOI: https://doi.org/10.1016/j.omtm.2025.101417 4. Chang, M. et al. Biotechnol. Bioeng. 119(4):1062–1076 (2022). DOI: https://doi.org/10.1002/bit.28036 About Oxford Nanopore Technologies Founded in 2005, Oxford Nanopore Technologies has developed a new generation of molecular sensing technology. The goal of Oxford Nanopore is to enable the analysis of anything, by anyone, anywhere. The company offers the only sequencing technology to combine scalability with real-time data delivery and the ability to elucidate accurate, rich biological data through the analysis of short to ultra-long fragments of native DNA or RNA. The technology is used in over 120 countries worldwide to deliver rapid, comprehensive genomic insights to users across academic, healthcare, environmental, and industrial settings. The company is headquartered in Oxford, UK, with satellite offices around the world. Nanopore sequencing delivers: • Accurate, ultra-rich data — for comprehensive insights • Any read length — from short to ultra long (>4 Mb) • PCR-free data — no amplification bias • Built-in methylation detection — no additional bisulfite or enzymatic conversion • Native RNA information — modified base calling alongside nucleotide sequence • Real-time analysis — immediate access to actionable results • Scalable devices — portable to ultra-high throughput www.nanoporetech.com Information correct at time of publication. May be subject to change. Oxford Nanopore Technologies, the Wheel icon, EPI2ME, GridION, MinION, and PromethION are registered trademarks or the subject of trademark applications of Oxford Nanopore Technologies plc in various countries. Information contained herein may be protected by patents or patents pending of Oxford Nanopore Technologies plc. All other brands and names contained are the property of their respective owners. © 2025 Oxford Nanopore Technologies plc. All rights reserved. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. BR_1296(EN)_V1_16Apr2025 phone +44 (0)845 034 7900 email support@nanoporetech.com oxford-nanopore-technologies @nanopore @nanoporetech.com Contact us today to discuss your drug development requirements, or visit nanoporetech.com/biopharma for more information.