biopharma-introductory-guide(5c4442c627a880adb29824ba9596e17f).pdf

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1. Unlocking the biopharma
development pipeline with
Oxford Nanopore
How a molecular sensing technology
is transforming biopharma
Oxford Nanopore Technologies
Introductory guide

2. 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 approval
1
. 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.

3. 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

4. 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

5.
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
• No mutations Promoters
• Compatible with
host organism

6. 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 efficiency
4
. 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
Truncated AAV genome?ITR 1
ITR 1 Full-length AAV genome ✓ ITR 2
Truncated AAV genome ✓ITR 1

7. 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

8. 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.
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