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Volume 8 Issue 1 – Spring 2025
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04 Foreword TALKING POINT
06 Interview with CEO Dr. Chris Chen of WuXi Biologics
WuXi Biologics is a leading CRDMO offering end-to-end solutions. Chloe Euripides of Senglobal sits down with CEO Dr. Chris Chen, where they discuss some of WuXi’s biggest successes, recent partnerships and visions for the future. With Dr. Chen also touching on the specifics of quality assurance and the impacts of tighter regulations, technological advancements and client expectations.
REGULATORY AND COMPLIANCE
10 IDMP Readiness & FAIR Data Adoption: Where are Life Science Organisations Now?
Across pharma, the readiness and maturity surrounding implementation of ISO IDMP and FAIR data principles continues to vary from company to company. Here, MAIN5’s Michiel Stam unpacks industry progression as a result of new research developments, as well as discussing plans to adopt Pistoia Alliance’s IDMP-Ontology to optimise standardised data use.
RESEARCH/INNOVATION/DEVELOPMENT
12 Transgene Expression Methods for Viral Vector Therapeutics: A Critical Component in Drug Development
At the forefront of modern medicine lies viral vector-based therapeutics, revolutionising gene delivery in treating genetic disorders and other diseases, through its safe and efficient means of expressing therapeutic transgenes. This method, however, does not come without its challenges and thus, Alistair Michel of RSSL highlights why robust analytical strategies could help avoid these struggles and ultimately further the development and approval of viral vector therapeutics as a whole.
16 Advances in RNA Vaccine Development and Delivery: A Multifaceted Approach to Infectious Diseases and Cancer
RNA vaccines specifically tackling SARS-CoV-2 has transformed the biomedical field, furthering the scalability and accuracy of therapeutic developments. In addition to the targeted aim of infectious disease, it has now become a point of application that could be considered in oncological, genetic and autoimmune disease management. Erica Cirri and Xavier Warnet of Tebubio, thus give an overview of the latest advancements in all thing RNA and highlight the opportunities and complexities of the field.
22 The Ins and Outs About Getting to the Most Relevant 3D In Vitro Model: Facts, Trends and Expectations
In recent years the pharma industry has seen an exponential growth in technological advancements, leading to the faster progression of drug discovery and development. In light of this, Dr. Emanuela Costigliola of Newcells and Dr. Jeanne-Francoise Williamson biotech consultant discuss new approaches to preclinical studies, moving away from the use of animal models, and more toward making advancements through predictive technologies.
26 Determining the Appropriate New Generation Test for Pyrogen-free Products
When determining a test for pyrogens and bacterial endotoxin, there are many regulations, needs, limitations, and techniques to consider. Timothy Francis of Fujifilm Irvine Scientific Inc explores factors to
Contents
that must be considered in this wave of ‘new generation’ tests, from sustainability to animal-free solutions.
TECHNOLOGY
32 Navigating the Future of Pharmaceutical Oral Dose Manufacturing: Embracing the Rise of High Potency Drugs
The pharmaceutical industry is at the height of transformation, driven by technological advancements, growing patient needs and the increased demand for high potency drugs. Taking this into consideration, Tom Hegarty of Almac explores what this means for the specifics of oral dose manufacturing, stressing the importance of understanding trends shaping the industry and how these align with the growing need for high potency medications.
MANUFACTURING AND PROCESSING
36 Case Study: Development and Manufacturing of a Highly Potent OSD Product
Contract Development and Manufacturing Organisations (CDMOs) have become essential to the movements of modern pharmaceutical industry, however, before the 1990s surge these organisations were hard to come by. PCI’s David O’ Connell tells all about the evolution of CDMOs and the vast impact they have had on the industry in its entirety, from investments in technologies to scalability from clinical phases to commercialisation.
38 Building a Competitive Edge Through Tailored CMC Strategies in Drug Development
46 Building a Patient Centric Supply Chain
Patient well-being should be at the heart of all decisions made within the life sciences. Despite this, other factors often compete for decision-makers' attention and ultimately distract from the focus on patients. While much of the supply chain may seem far removed from patients, Vincent Howard of Biocair explains how this can still have a significant impact on medicine delivery and patient well-being, due to the rise in demand for specialised solutions.
PRECLINICAL SUBSECTION
48 Breath Analysis in Diagnostics: The Use of Animal Models
Volatile organic compounds (VOCs) can be key component to understanding patient health. Madeleine Ball of Owlstone Medical explains just how beneficial VOCs can be when used in animal model breath analysis and diagnostics, outlining their ability to bridge the gap between lab discoveries and clinical applications of the human patient.
52 Patient-derived Xenografts Strengthen Mouse Clinical Trials in Oncology Research
Rajendra Kumari of Crown Bioscience talks Mouse Clinical Trials (MCTs), exploring how the use of patient-derived xenograft (PDX) mouse models can benefit predicted clinical outcomes and identify potential biomarkers. Within this article Rajendra draws specific focus to the positive effects mouse models have in mimicking human cancer biology and ultimately furthering developments made to oncological studies as a whole.
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The classics season, I am talking about cycling here, is well on the way and has inspired my 11-yearold son to pick up road cycling. As an enthusiastic but admittedly slow cyclist myself, I am more than happy to support this new and hopefully lasting hobby. On Sunday afternoon my son wanted to go on a ride with me, but I had to decline because I still had to write the foreword for IBIs Spring issue. My son looked at me, the look that children give you when they think you are stuck in olden days and said: “Haven’t you heard about AI and ChatGPT? This will get your foreword done in no time, and it will be better than anything you yourself could write!”. So, I tried it. Everything you see written in italic is courtesy of ChatGPT.
The IBI Spring 2025 issue presents key developments in the biopharmaceutical industry, featuring insights into research, regulation, manufacturing, logistics, and preclinical advancements. It includes an Interview with CEO Dr. Chris Chen of WuXi Biologics, discussing industry trends and future directions.
In Regulatory and Compliance, IDMP Readiness & FAIR Data Adoption: Where are Life Science Organisations Now? by Michiel Stam (MAIN5) examines the implementation of ISO IDMP and FAIR data principles in the pharmaceutical sector, highlighting the role of the Pistoia Alliance’s IDMP-Ontology in improving standardised data use.
The Research/Innovation/Development section includes Transgene Expression Methods for Viral Vector Therapeutics by Alistair Michel (RSSL), exploring challenges in viral vector-based therapies. Advances in RNA Vaccine Development and Delivery covers new RNA technologies like mRNA, saRNA, and circRNA for infectious diseases, cancer, and genetic disorders. The Ins and Outs About Getting to the Most Relevant 3D In Vitro Model by Dr. Emanuela Costigliola (Newcells) and Dr. Jeanne-Francoise Williamson (biotech consultant) discusses the transition from animal models to predictive technologies. Determining the Appropriate New Generation Test for Pyrogen-free Products by Timothy Francis (FujiFilm Wako Chemicals U.S.A.) reviews sustainable, animal-free endotoxin testing.
The Technology section features Navigating the Future of Pharmaceutical Oral Dose Manufacturing by Tom Hegarty (Almac), addressing trends in high-potency drug manufacturing.
• Bakhyt Sarymsakova – Head of Department of International Cooperation, National Research Center of MCH, Astana, Kazakhstan
• Cellia K. Habita, President & CEO, Arianne Corporation
• Deborah A. Komlos, Senior Medical & Regulatory Writer, Clarivate Analytics
• Elizabeth Moench, President and CEO of Bioclinica – Patient Recruitment & Retention
• Francis Crawley, Executive Director of the Good Clinical Practice Alliance – Europe (GCPA) and a World Health Organisation (WHO) Expert in Ethics
• Hermann Schulz, MD, Founder, PresseKontext
• Jim James DeSantihas, Chief Executive Officer, PharmaVigilant
• Jeffrey W. Sherman, Chief Medical Officer and Senior Vice President, IDM Pharma.
In Manufacturing and Processing, Case Study: Development and Manufacturing of a Highly Potent OSD Product by David O’Connell (PCI) explores the rise of Contract Development and Manufacturing Organisations (CDMOs). Building a Competitive Edge Through Tailored CMC Strategies in Drug Development by Yoonsik Kim and Steven Lal (Samsung Biologics) discusses how CDMOs can optimise Chemistry, Manufacturing, and Controls (CMC) strategies.
The Logistics and Supply Chain section includes The Impact of Data and Academic Partnerships on Cold Chain Innovation by Niall Balfour (Tower Cold Chain) and Building a Patient-Centric Supply Chain by Vincent Howard (Biocair), emphasising patient-focused logistics.
The Preclinical section explores Breath Analysis in Diagnostics by Madeleine Ball (Owlstone), Patient-Derived Xenografts Strengthen Mouse Clinical Trials in Oncology Research by Rajendra Kumari (Crown Bioscience), and Leveraging Liquid Biopsy to Advance Metastatic Cancer Care by Dr. Corinne Renier (Vortex), highlighting novel diagnostic and cancer research approaches.
Finally, the Application Note CHO Cell Culture Process Intensification for Enhanced Production of IgG mAbs examines techniques for boosting monoclonal antibody production.
Overall, the issue provides insights into regulatory updates, innovations, and strategies shaping the biopharmaceutical industry's future.
I have to admit, my son had a point. It took me no more than five minutes to write the foreword, allowing me to spend quality time with him doing something I love. But this then left me questioning, is this form of writing respectful of the authors who dedicated their time to writing what is important to them (if they did not use ChatGPT themselves). Is this respectful of you, the reader? I would argue, no!
Regardless, from myself and Chat GPT, we hope you enjoy this really engaging issue packed full of the latest expertise within the biopharmaceutical industry.
Dr. Steven A. Watt, CBDO (Chief Business Development Officer) at A&M STABTEST GmbH
• Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation
• Maha Al-Farhan, Chair of the GCC Chapter of the ACRP
• Rafael Antunes, Vice President Business Development, Aurisco Pharmaceutical Europe
• Rick Turner, Senior Scientific Director, Quintiles Cardiac Safety Services & Affiliate Clinical Associate Professor, University of Florida College of Pharmacy
• Robert Reekie, Snr. Executive Vice President Operations, Europe, Asia-Pacific at PharmaNet Development Group
• Stanley Tam, General Manager, Eurofins MEDINET (Singapore, Shanghai)
• Stefan Astrom, Founder and CEO of Astrom Research International HB
• Steven A. Watt, CBDO (Chief Business Development Officer) at A&M STABTEST GmbH
• Steve Heath, Head of EMEA – Medidata Solutions, Inc
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Interview with CEO Dr. Chris Chen of WuXi Biologics
Quality Assurance Evolution in the Biopharmaceutical CDMO Industry – stricter regulations, technological advancements, risk management, globalisation and client expectations.
Generally Speaking, How Has Quality Assurance in Drug Manufacturing Evolved Over the Past 10 years?
In the last ten years, quality assurance in biologics drug manufacturing has greatly improved due to several important factors:
• Regulatory Evolution: Global regulatory standards have become stricter, with agencies like the FDA and EMA updating their guidelines to ensure drugs are safe and effective. This has pushed drug manufacturers worldwide to upgrade their quality systems to comply with current Good Manufacturing Practices (cGMP) and other regulatory requirements.
• Technology Advancements: New technologies, including automation and data analytics, have enhanced the precision and efficiency of quality assurance. Innovations such as single-use systems in manufacturing have also significantly lowered contamination risks and improved product consistency.
• Risk Management: There's an increased focus on risk-based quality management. Principles of Quality by Design (QbD) and risk management are now essential in both development and manufacturing, aiming to better understand and control process variables.
• Globalisation: As CDMOs go global and serve clients worldwide, they need to standardise quality assurance across different countries and regulatory frameworks. This has led to more unified global quality standards.
• Client Expectations: With biologics becoming more complex, clients are seeking higher quality and greater transparency in manufacturing. CDMOs have responded by enhancing their quality assurance systems and improving communication with clients about quality metrics.
Overall, the quality assurance in biologics manufacturing within CDMOs has become more sophisticated, compliant with regulations, and aligned with technological and market changes to meet the high standards required for biologics products.
The Practicalities of Managing a Multi-step Quality Control Process are Manifold and Complex. What Would you say is the Key to Providing Consistent and Reliable Quality Across those Steps, and Across 1 Locations Across the World? Managing a complex quality control process across extensive global sites requires a strong and coordinated strategy to maintain consistent and reliable quality. Here are the essential elements for success:
• Standardisation of Processes: It's crucial to use the same procedures and protocols at all locations. This consistency guarantees that each step of the quality control process is uniform, helping to meet international regulatory standards.
• Centralised Quality Management System (QMS): A centralised QMS improves the oversight and management of quality at all sites. It should include thorough documentation, data management, and communication tools that everyone can access, helping to keep quality practices uniform and addressing any issues quickly.
• Training and Development: Continuous, in-depth training for all staff involved in quality control is crucial. Well-trained employees across all sites maintain high-quality standards.
• Advanced Analytical Tools and Technologies: Modern analytical tools and technologies increase the precision and efficiency of quality control. Automation and digital tools minimise human error and allow for real-time data analysis, speeding up decision-making and problemsolving.
• Robust Communication Channels: Strong communication is essential. Regular meetings, updates, and reviews ensure all teams are on the same page and any problems are addressed swiftly.
• Continuous Improvement and Adaptation: Quality control should always be evolving through ongoing monitoring, evaluation, and enhancement. A culture of continuous improvement, where feedback is actively sought and implemented, greatly improves quality consistency.
• Quality Audits and Inspections: Frequent audits and inspections, both internal and external, ensure each site meets quality standards. These also identify areas for improvement and facilitate the sharing of best practices.
By focusing on these key areas, WuXi Biologics can effectively handle the complexities of a multi-step quality control process across multiple global locations, ensuring consistent and reliable product quality. To date, we have passed over 40 inspections by multiple global regulatory agencies including FDA, EMA, NMPA and received 97 license approvals, including a 100% success rate in Pre-Approval License (PAI). We continue to safeguard our data integrity to maintain our strong track record, with zero issues found regarding data integrity during regulatory inspections. Our PPQ success rate exceeds 98%, positioning us as one of the top performers in the industry and showcasing our premier and reliable quality.
How are New Technologies Driven/Continuing to Drive Advances in Quality?
New technologies are crucial in enhancing quality assurance across various sectors, including the manufacturing of biologic drugs. These innovations improve accuracy, efficiency, and adherence to regulations, ultimately boosting the quality and safety of products. Here are some key ways new technologies are advancing quality:
• Automation and Robotics: Automation minimises human errors and enhances the consistency of production processes. Robots perform repetitive tasks with high precision, essential in settings where small deviations can cause major quality issues.
• Data Analytics: Advanced data analytics provides deeper insights into quality control processes. Companies can leverage datasets to foresee potential failures and deviations, enabling proactive quality management. Real-time data analysis is crucial for making quick corrections to maintain high-quality standards.
• Internet of Things (IoT): IoT devices monitor and gather data from various stages of the manufacturing process. This connectivity allows for real-time monitoring and control, ensuring any deviations are quickly detected and corrected, thus maintaining the quality control process's integrity.
By integrating these technologies into quality assurance processes, companies can not only meet but surpass regulatory standards, cut costs related to quality issues, and boost customer satisfaction by consistently delivering high-quality products.
Your Proprietary Bispecific Antibody Platform WuXiBody™ is said to Accelerate Product Discovery by up to 18 Months; How did you Calculate/Estimate that Time Saving, and How is it Achieved with this Technology Platform?
The unique design of WuXiBody™ enhances the developability characteristics of bispecifics, facilitating an accelerated development timeline. WuXiBodyTM adopts an innovative design where the CH1/CL region of one antibody Fab is replaced by the corresponding T cell receptor (TCR) Cβ/Cα domains to promote correct heavy-light chain pairing. The characteristics of this domain can differentiate the target heterodimer from homodimers for easier removal of homodimers. The mAb-like CMC performance accelerates the drug development process for bispecifics developed by WuXiBodyTM .
In addition, our extensive experience in CMC development could further ensure the delivery of timeline by de-risking the potential rate-limiting steps such as transfection ratio study, homodimer method development, clone selection involving downstream team and CIU low concentration evaluation/ development.
Based on the widespread adoption of WuXiBodyTM platform in bispecific antibody discovery, we have upgraded it to deliver transformative and customised multi-specific antibodies, addressing the growing global demand for these complex molecules.
WuXi Biologics has Patented Number of New Technology Platforms Over the Past Couple of Years – this Suggests that Somewhere in your C-suite a Strategic Decision was Made 3 or 4 years ago to Pursue a Pioneering Path with Patented Technologies, Forming a Core Part of your Contract Development and Manufacturing Business. What triggered that Insight, which is now Yielding Clear Fruits in Terms of Market Differentiation?
Balancing speed, quality, and cost remains one of the greatest challenges in drug development. As a CRDMO, our continual launch of new technology platforms comes from our drive to push boundaries and foster innovation – all with the goal of enabling our global partners to rapidly bring more high-quality and affordable biologics. As of June 30, 2024, WuXi Biologics is supporting 742 integrated client projects, which is one of the largest portfolios of complex biologics, consisting of mAbs (316), bispecifics and multispecifics (123), ADCs (167), fusion proteins (76) and vaccines (23) as well as other proteins (37).
Our clients are investing in next-generation biologics such as bispecific antibodies and multispecific antibodies to provide patients with innovative therapies that can save their lives or improve their quality of life. In response to this demand, we have developed a universal platform, WuXiBodyTM, to enable our partners to take any mAbs and engineer these into a highlyfunctional multispecific constructs. This platform has been widely adopted by global clients, with over 40 WuXiBodyTM projects currently at different R&D stages.
In October 2024, Merck completed the acquisition of CN201, a novel investigational clinical-stage CD3xCD19 bsAb for the treatment of B-cell associated diseases, from Curon Biopharmaceutical, a client of WuXi Biologics. CN201 is one of the best examples that demonstrate how our proprietary platforms enable our client’s success. By combining our unique low-affinity antiCD3 mAb, which binds and dissociates rapidly, with the WuXiBodyTM platform, the resulting TCE molecule CN201 showed deep and sustained B cell depletion, along with reduced toxicity due to a lower level of cytokine release syndrome. Other technology platforms including TCE platform, ultra-high productivity continuous bioprocessing platform WuXiUPTM, and the high titre production CHO K1 cell line development platform WuXiaTM have also been applied in the R&D of CN201, which contribute additional value to this molecule and make it an attractive asset for the acquisition deal.
Recently, we announced an agreement with Candid Therapeutics under which Candid will have exclusive global rights to a preclinical trispecific T-cell Engager discovered at
WuXi Biologics’ proprietary universal multispecific antibody platform WuXiBody™. WuXi Biologics is eligible to obtain an upfront payment, and development and sales milestones totalling up to $925 million as well as royalties.
You have Patented Six Platforms that Accelerate Development and Manufacturing for Various Types of Biologics. What are the Comparative Demands for these Various Technologies, and do they Predict Future Trends in Biologics?
By leveraging a wide range of technology platforms in discovery (WuXiBodyTM, SDArBodyTM), development (WuXiaTM, WuXianTM, WuXiUPTM, WuXiUITM) and manufacturing, we provide reliable and flexible services tailored to accommodate our clients’ diversified requirements for different modalities and stages.
From the current industry perspective, bsAb and antibodydrug conjugates (ADCs) have the best potential growth in the biologics field over the next 5–10 years. Although the study and commercialisation of mAbs have reached a high level of maturity after decades of development, the ongoing challenge of enhancing product safety and efficacy continues to drive industry-wide efforts.
Many years ago, we had foreseen this trend and accordingly built the WuXiBodyTM and WuXiDar4TM, a platform designed to enhance DAR4 (four payload molecules per mAb) percentage in the final ADC product and improve conjugation efficiency. Currently, over 100 bsAb and 100 ADCs are under development at WuXi Biologics, accounting for approximately one-third of the total projects we served for our global clients. The increased ratio of new modalities in recent years is also a testament to our success in meeting our clients' needs and predicting future industry trends.
Continuous processing is a next-generation solution for biomanufacturing, designed to address the production difficulty and inefficiency of either labile/difficult-to-express or low-expressing-level proteins with minimised environmental impact and resource demand. Our WuXiUPTM platform employs an intensified perfusion culture process and continuous harvest, allowing for the manufacture of different types of pharmaceutical proteins with significantly higher productivity (5–15X greater) compared with traditional fed-batch and perfusion culture. The WuXiUPTM platform achieves not only higher productivity but also significantly reduces resin usage, with a smaller facility footprint. Meanwhile, our ultra-intensified fed-batch bioprocessing platform, WuXiUI™, has enhanced upstream productivity by 3–6 times compared to traditional fed-batch cultures, and has doubled the capacity for downstream purification processing while achieving comparable impurity removal. This advancement has resulted in a 50% reduction in downstream processing time and has facilitated a 30–50% decrease in the consumption of materials and consumables, thereby significantly reducing waste generation. All these factors collectively lead to substantial cost savings and reduced environmental impact on protein mass basis,which aligns with the industry trend in Environmental, Social, and Governance (ESG).
Earlier this Year (2024) WuXi Biologics Announced a Partnership with BioNTech, and a Three-year Partnership with Medigene. Can you Give us a Little More Information about those Projects (with a little more technical detail than already provided in the
press releases) and how Novel Technology Platforms are Being Leveraged to Achieve their Goals?
We are unable to share detailed information about these projects due to stringent compliance regulations. However, we can reveal that these projects utilise our cutting-edge discovery technology platforms, the benefits of which are outlined in the Q5 and Q6 above.
Are all of these Technologies Scalable for GMP Manufacturing?
Leveraging our state-of-the-art discovery and development technology platforms, numerous clients’ projects have been advanced to GMP manufacturing with high quality. To date, we have constructed more than 800 stable cell lines for clinical and commercial production using WuXiaTM platform. WuXiUPTM processes have been successfully applied on 50 different molecules (e.g., mAbs, BsAbs, fusion proteins and enzymes) with over 20 processes scaled up to clinical and commercial GMP manufacturing, among which 11 successful INDs and 1 BLA approval have been achieved with average 7-fold productivity increase. In August 2024, we have successfully accomplished 2,000L drug substance (DS) GMP manufacturing by utilising WuXiUI™. The platform achieved a titre of 18 g/L, a 4-fold increase compared to the conventional fed-batch process.
Are you Able to give IBIJ a Scoop on any other Technologies in Development that you Might be Launching Soon?
Derived from the highly-vetted WuXia™ CHO-K1 cell line, we launched two new cell line platforms, WuXiaADCC PLUS™, a superior-performing and high-yielding mammalian cell line platform for the development and manufacturing of afucosylated antibodies, and WuXia RidGSTM, a high-yield glutamine synthetase (GS)-knockout Chinese hamster ovary (CHO) expression system platform in 2024. In 2025, we’ll launch several new cell line platforms to meet global clients’ diversified needs.
WuXi Biologics has Achieved Remarkable Success Since 2018 – it's Easy to Forget what a Young Company it is! What is your Vision for the Next Six Years?
Our commitment to our clients and patients has been the cornerstone of our success. Moving forward, we are going to intensify our efforts to expand our client base. This means not just reaching new markets, and new segments, but creating deeper, more value-driven partnership. We're pushing the boundaries of what a CRDMO can achieve by diversifying our solutions and exploring new modalities and business avenues, to empowering of clients' success and benefit patients worldwide.
Dr. Chris Chen
Dr. Chris Chen is the CEO of WuXi Biologics, a leading global Contract Research, Development and Manufacturing Organisation (CRDMO). Under his leadership, WuXi Biologics has built up a world-class open-access integrated platform, enabling over 600 global partners. Dr. Chen serves on the International Board of Directors for ISPE as the first board member from Asia. He holds Bachelor’s degrees in Chemical Engineering and Automation from Tsinghua University and a Ph.D. in Chemical Engineering from the University of Delaware.
QC -Analysis of Inhaled Antisense Oligonuclotides and mRNA
Analytical support for ASO and mRNA based therapies from early development to batch release
Antisense oligonucleotides (ASOs) and full-length mRNA -based therapeutics promise diseasespecific treatment options One of the challenges of these therapies is the tissue- or cellspecific delivery. Inhalation has been an effective means to deliver pharmaceutically active substances to the respiratory tract for centuries Today there is a growing interest in the industry to leverage on the experience with different inhalation technologies to deliver ASO and mRNAbased treatments directly to diseased respiratory tissues.
A&M STABTEST has over 15 years of experience in supporting drug development and QCanalysis of nasal and orally inhaled products Specially trained staff and dedicated laboratory facilities are the key to analyze inhaled products throughout their lifecycle with the same constantly high quality. In the past 10 years A&M STABTEST has gained in-depth experience in stability and release testing of nucleic acid -based therapies ranging from ASO to mRNA -LNP therapies. Very recently we have embarked on a project to combine our know-how of inhalable drug and nucleic acid testing to support our clients with inhaled ASO and mRNA therapies.
For QC-analysis of mRNAs formulated as lipid -nanoparticles
1: A) displays the different charge states of a 40 mer oligo in a negative mode full MS B) Deconvoluted spectrum resulting from the full MS spectrum The mass difference between the calculated mass and the observed mass is 8ppm C) Celibatarian curves from a parallel detection by UV (above) and MS (below)
A&M STABTEST has established the panel of methods described in the USP draft guidance on “Analytical Procedures for mRNA Vaccine Quality” Especially the determination of polydispersity by DLS is a valuable indicator to evaluate how well different LNP formulations respond to aerosolization Combined with our generic cell -based bioassay, we can help to predict the transfection efficiency of different LNP formulations A&M STABTEST has also developed a generic LC-MS/MS method to support early ASO development projects, offering data on quantity, purity and impurities in one analytical run
A&M STABTEST can support your early development and more advanced ASO and mRNALNP projects whether inhaled or not With our experience spanning well over two decades of providing the pharmaceutical industry with high quality analytical services, we are well suited to find analytical solutions for the industries most innovative drug development programs .
Figure
IDMP Readiness & FAIR Data Adoption: Where are
Life Science Organisations Now?
Pharma companies remain at differing levels of readiness for implementing ISO IDMP product data standards, and in their maturity around adopting FAIR data principles, geared to making data more Findable, Accessible, Interoperable, and Reusable. Here, MAIN5’s Michiel Stam unpacks the findings of new research which benchmarks the industry’s progress, as well as plans to adopt Pistoia Alliance’s IDMP-Ontology to optimise standardised data use.
Although ISO IDMP standards, designed to harmonise the way the life sciences industry records and manages data about its products, have been more than a decade in the making, companies’ state of readiness to implement and harness IDMP still varies considerably.
The same is true of their relative maturity in supporting FAIR data principles, geared to making data more Findable, Accessible, Interoperable, and Reusable. These are goals that are actively promoted by Pistoia Alliance, a nonprofit industry coalition working to lower barriers toward innovation in life science and healthcare R&D through pre-competitive collaboration. Its IDMP-Ontology (IDMP-O) project, launched in early 2024, aims to create a shared ontology (a representation of data properties and the relations between them), to encourage uniform adoption of the IDMP standards and, by extension, consistent information exchange.
With renewed momentum around EMA’s IDMP implementation in Europe, FDA’s own related plans in the US, as well as the cross-industry initiatives outlined above, MAIN5 recently partnered with Pistoia Alliance and data registry specialist Accurids to conduct new benchmark research to determine companies’ latest progress and planning around IDMP implementation.1
Silos & a Lack of Standardisation Have Compromised Companies’ Digitalisation Ambitions
Large pharma companies now generally have good awareness of the value of IDMP-based product data standardisation as part of wider process digitalisation ambitions, the survey confirmed. More than 70% of those surveyed identified IDMP’s value as an enabler of cross-functional data integration; only 11% saw compliance as the primary goal of IDMP projects.
Companies generally plan to integrate IDMP data from Regulatory, Manufacturing, Pharmacovigilance, Supply Chain, and Quality functions within the next three years. Research, (pre-) Clinical, and Commercial data integration will follow in the mid-term (within five years). This phased approach indicates that companies are initially prioritising data that supports regulatory submissions and compliance, followed by broader data integration to support product
development and commercial strategies to maximise the benefits of IDMP.
As things stand, however, product data management continues to pose a challenge for companies across the board. The benchmark study identified particular issues with manual data collection, data silos, and a lack of data integration across systems. An unclear source of truth and insufficient use of trusted external sources were also flagged as barriers to harnessing product data more strategically.
Those actively striving toward more seamless data integration across and between functions felt that a lack of resources and issues with ‘ownership’ were the main barriers to achieving this (indicated by 44% and 41% of respondents), beyond a current lack of data standardisation (the main obstacle, cited by 56%). Surprisingly, the quality of data (and therefore its usefulness) was ranked below these factors (cited by 33%).
Master Data Alignment & IDMP-O
When asked if companies currently use IDMP as the master data model for their product information, many respondents were unsure how well aligned their existing model is. Just 40% felt confident that they possess an IDMP-compatible model, although 75% use IDMP to guide product information. This is one of the gaps addressed by Pistoia Alliance’s IDMP-O project, in that it allows the exact measurement of how compatible existing data models and ambitions are with IDMP.
Promisingly, 43% of the large pharma companies taking part in the benchmark research expressed a willingness to take IDMP-O into production within their organisations within the first year of its release. (IDMP-O production release 1.0 was published in January 2024; version 1.3 is now live.) Although an encouraging observation, many of the organisations that participated in the survey are inherently closer to IDMP-O than others in the industry, so the finding may not be representative.
Respondents were then invited to express, in their own words, where they anticipated deriving the most value from IDMP-O. Their open-ended responses confirmed good awareness of the ontology’s strategic benefits, including the associated scope to enhance the integration and exchange of product data – with regulators and industry partners, among other stakeholders.
Operationally, respondents recognised that the Pistoia Alliance ontology supports cross-functional alignment on data ownership, standardisation of data definitions, and adoption of a shared data model to enable system interoperability, and improve overall data quality. These factors pave the way for improved efficiencies in data management, decision-making, submissions, and compliance. (The IDMP-O can drive and facilitate master data management, automation, and AI –
Regulatory and Compliance
positively impacting analytics, and ultimately reducing costs.) There is still work to be done before companies can harness those benefits, however.
IDMP Project Momentum Now Needs to be Reignited Where early enthusiasm around IDMP programmes had waned in response to slow progress from EMA in Europe toward clarifying specific requirements, reigniting momentum behind IDMP-based projects should be a priority now – both among life sciences companies, and the supporting vendor community.
A raft of recent developments will help companies define concrete next steps and avoid potential rework. These include the EMA’s go-live of the Product Lifecycle Management portal (with Product Management Services and electronic application forms), as well as improved clarity on implementing SPOR services and integrating with EMA systems and processes. Certainly, for companies with larger product portfolios, advanced technological capabilities will be needed to efficiently prepare data in bulk for what could be thousands of registrations. Manual updates per product by re-entering data in the PMS system is not feasible.
Defining the right strategy, implementing supportive system capabilities, recruiting and training a workforce to collect, transform, and submit data according to specific requirements is a significant undertaking that requires careful planning and execution.
The survey does suggest that many companies are now actively working toward enterprise-wide integration of data and IDMP-related processes. Harnessing Pistoia Alliance’s IDMP-Ontology offers them their best chance of cross-functional alignment on data ownership, standardisation, and adoption of a common data model to enable interoperability and improvement of data quality in line with FAIR data principles.
Ultimately, robust IDMP compliance lays the foundation for a more interconnected and streamlined regulatory landscape, benefiting pharmaceutical companies, regulatory authorities, and patients worldwide. It is an opportunity to revolutionise how pharmaceutical data is managed and used – toward a more sustainable future for healthcare.
REFERENCES
1. The IDMP bench mark survey of 18 pharma companies was conducted in Q32024 by Pistoia Alliance, MAIN5, and Accurids, and supported by the IDMP-Ontology project with participants from Abbvie, Amgen, AstraZeneca, Boehringer Ingelheim, Bayer, and Novartis
2. FAIR data principles - https://fairtoolkit.pistoiaalliance.org/why-fairdata-is-important/
5. ‘Accelerating Digital Transformation in Pharma with IDMP’https://marketing.pistoiaalliance.org/hubfs/IDMP%20Pistoia%20 Alliance%20Report%202024%20(5).pdf
Michiel Stam
Michiel Stam is a management consultant and senior regulatory expert at MAIN5 with 15 years of experience in Regulatory Information Management (RIM) and IDMP. MAIN5 is a European consulting firm specialising in digitally-enabled change for Life Sciences R&D organisations. Its customised, high-value services and solutions span the product lifecycle – from regulatory affairs and data governance, to quality management and systems validation.
Email: michiel.stam@main5.de
Transgene Expression Methods for Viral Vector Therapeutics: A Critical Component in
Drug Development
Viral vector-based therapeutics have revolutionised modern medicine, enabling precise gene delivery for treating genetic disorders, cancers, and infectious diseases. These therapies rely on the safe and efficient expression of a therapeutic transgene within target cells. However, a critical challenge in the development and regulatory approval of viral vector therapeutics is ensuring that the transgene product is correctly expressed, and functions as intended.
Regulatory agencies, including the FDA, EMA and MHRA, require detailed characterisation of transgene expression as part of the approval process for gene therapies and viral vector-based vaccines. The absence of a validated transgene expression method has previously been a significant regulatory barrier, as seen during the COVID-19 pandemic.
This column explores the essential aspects of transgene expression analysis, discusses the challenges associated and highlights why robust analytical strategies are critical for the successful development and approval of viral vector therapeutics.
Why Transgene Expression Matters
In viral vector-based therapies, the genetic material of the virus is modified to remove its ability to replicate. Instead, a therapeutic transgene is inserted, which is expressed in target cells after the vector infects them. The therapeutic effect of the treatment depends on the correct expression of this transgene, making it essential to confirm and characterise its production.
Regulatory authorities expect developers to demonstrate that their viral vector reliably produces the intended transgenic protein. Failure to do so could delay approvals, as seen with the EMA’s initial assessment of the Vaxzevria COVID-19 vaccine. A major objection during the approval process was that at the time of submission a validated method to measure transgene expression was not available. While the issue was eventually resolved, it underscored the importance of having a validated transgene expression method in place when submitting viral vector-based therapeutics for approval.1
Beyond regulatory considerations, understanding transgene expression is crucial for optimising manufacturing processes, ensuring consistent therapeutic efficacy, and identifying potential safety risks. Without reliable expression data, developers cannot confidently predict how a therapy will perform in patients.
Transduction and Its Impact on Transgene Expression
The first step in any transgene expression method is
transduction – the process of introducing the viral vector into host cells. Ensuring transduction occurs successfully is critical for subsequent expression analysis.
While optimising transduction conditions is not always essential, it can be beneficial in certain scenarios. The efficiency of transduction depends on factors such as the multiplicity of infection (MOI), or the ratio of viral particles to target cells. If the MOI is too low, transduction efficiency may be insufficient, leading to weak or undetectable transgene expression. Conversely, an excessively high MOI can result in cytotoxicity or unwanted cellular responses.
To calculate the MOI, the infectious units/mL (IFU/mL) of the viral vector needs to be known. If the IFU/mL of a sample is known, then the transgene expression method only requires one MOI to be transduced. If the IFU/mL of the viral vector is not known, then a series of MOIs would need to be transduced to ensure that transgene product can be expressed at detectable levels.
For therapies where precise dose-dependent expression is required, optimising transduction parameters can help standardise results and improve reproducibility. In other cases, it may be sufficient to use established transduction conditions without extensive optimisation.
Research / Innovation / Development
Once transduction has been performed, cells are typically cultured for a set period – often a few days – to allow the transgene protein to be expressed in sufficient quantity to enable detection.
Analytical Approaches to Confirm Transgene Expression
After the transgenic protein has been expressed, it must be harvested and processed for analysis. Several analytical methods are available to confirm transgene expression, each with its own advantages and limitations.
1. Nucleic Acid-Based Methods: PCR and qPCR
PCR-based techniques can confirm the presence of the transgene at the DNA or RNA level. Quantitative PCR (qPCR) allows for precise measurement of transgene expression by detecting transcribed mRNA. However, these methods do not confirm whether the transgene has been successfully translated into a functional protein.
While PCR-based approaches are valuable for early-stage assessments, to ensure the therapeutic protein is correctly expressed and processed, a detection at the protein level would still be required.
2. Protein-Based Immunoassays: ELISA, HTRF, and Western Blotting
Immunoassays provide direct evidence of transgene expression at the protein level, making them the preferred approach for confirming that the viral vector is producing the intended therapeutic product.
• Enzyme-Linked Immunosorbent Assay (ELISA): ELISA has historically be considered the gold standard for detecting protein concentrations due to its high sensitivity and quantification capability. It requires well-characterised antibodies specific to the transgenic protein.
• Homogeneous Time-Resolved Fluorescence (HTRF): HTRF is a more advanced immunoassay technique that provides a high-throughput alternative to ELISA. It offers enhanced sensitivity and is less prone to background noise, making it suitable for complex sample matrices.
• Western Blotting: When only identity confirmation of the transgenic protein is required, western blotting offers a simple and effective method. It allows for visualisation of protein size and provides semi-quantitative data. This method is particularly useful when working with membrane-bound or hydrophobic transgene proteins, which may be challenging to detect using ELISA-based approaches.
3. Mass Spectrometry (MS) for Protein Characterisation
While not routinely used for transgene expression analysis, MS can be valuable in cases where advanced characterisation of post translational modifications or peptide mapping of the protein is needed.
Challenges in Transgene Expression Analysis
Regardless of the analytical method employed, detecting the expression transgenic protein, presents several analytical challenges that must be carefully addressed to ensure reliable results. The choice of method depends on the nature of the transgene product, its structure, and the biological context in which it is expressed. Some key challenges include:
1. Viral Load and Protein Metabolism
One key factor affecting transgene detection is viral load and the metabolism of the expressed protein. Some transgene products, particularly those of viral origin, may undergo intracellular cleavage or degradation before they can be analysed. This means that the timing of sample harvest is critical – too early, and the protein may not yet be expressed at detectable levels;
Research / Innovation / Development
too late, and it may have undergone modifications or breakdown that complicate detection. Optimising the timing of collection ensures that the transgene product is captured in its correct and biologically relevant form.
2. Challenges with Membrane-Bound or Hydrophobic Proteins
Some transgene products, particularly membrane-associated or hydrophobic proteins, pose additional detection challenges. These proteins may be difficult to extract and solubilise without disrupting their structure or function. Standard immunoassays such as ELISA or HTRF may not work effectively if the protein cannot be properly captured or presented in an assay-compatible format. Alternative approaches, such as specialised detergents, liposome-based extractions, or cell-based assays, may be required to ensure accurate detection.
3. Antibody Specificity and Availability
The success of immunoassay-based approaches depends on the availability of high-affinity antibodies that recognise the transgenic protein. In some cases, correctly folded transgenic protein can only be detected using antibodies that recognise conformational epitopes – structures that rely on the protein being in its native, properly folded state. If the protein is denatured during sample processing, these epitopes may be lost, leading to false-negative results. Assay formats that preserve the native state, such as sandwich ELISA or specific immunoprecipitation-based techniques, may be necessary for accurate detection in these cases.
4. Assay Validation for Regulatory Compliance
Any method used to support regulatory submissions must be fully validated and so any method developed to detect the
transgenic protein must be robust and rugged enough to be used as a QC test.
Regulatory Expectations and Future Directions
As the field of gene therapy continues to expand, regulatory agencies are refining their expectations for transgene expression analysis. Developers must ensure that their analytical methods align with evolving regulatory standards.
Looking ahead, advancements in analytical techniques, such as single-cell expression profiling, multiplexed immunoassays, and next-generation sequencing could enhance the sensitivity and reliability of transgene expression analyses. These technologies may provide deeper insights into transgene function at a cellular level, helping to refine therapeutic design and improve patient outcomes.
Additionally, like in many areas, artificial intelligence and machine learning could play a role in optimising transgene expression workflows by predicting optimal transduction conditions and streamlining data interpretation.
Conclusion
Ensuring correct transgene expression is a fundamental requirement for viral vector-based therapeutics. The regulatory challenges faced during the approval of COVID-19 vaccines demonstrated the critical importance of having a validated analytical method in place. By optimising transduction conditions where beneficial and employing robust immunoassays, developers can confidently demonstrate that their therapies are producing the intended therapeutic protein.
For gene therapy developers, the message is clear: a well-defined transgene expression method is not just a technical requirement – it is a critical component of regulatory success and therapeutic efficacy. As analytical technologies continue to evolve, the future of transgene expression analysis holds the promise of even greater precision and reliability, ultimately driving the next generation of viral vector-based treatments.
Alistair Michel is an immunologist with a BSc (Hons) from the University of Edinburgh and an MSc from Imperial College London. He is a member of the British Society of Immunologists and the Royal Society of Biology. With over 20 years of experience as a bioanalytical scientist, he specialises in developing, validating, and optimising methods in GxP-compliant laboratories, focusing on ELISA and immunoassays. At Reading Scientific Services Ltd, Alistair serves as the technical lead for complex bioanalytical projects, delivering tailored solutions to address unique client challenges.
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Advances in RNA Vaccine Development and Delivery: A Multifaceted Approach to Infectious Diseases and Cancer
The emergence of RNA vaccines against SARS-CoV-2 has revolutionised the biomedical field, offering unprecedented flexibility, precision, and scalability in therapeutic development. Beyond addressing infectious diseases, RNA technology – including mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA) – is now being explored for applications in oncology, genetic disorders and autoimmune disease management.
This article gives an overview of the latest advancements in RNA vaccines against infectious diseases and cancer, including design and delivery systems, and highlights both opportunities and challenges in this rapidly evolving field.
• Messenger RNA (mRNA): A linear RNA molecule where the sequence coding for the vaccine antigen is flanked by untranslated regions (UTRs), a 5' methylguanylate cap and a 3' polyadenylate (polyA) sequence, ensuring its stability and efficient translation in the cell.
• Self-amplifying RNA (saRNA): A linear RNA molecule encoding for the vaccine antigen plus a viral replicase, composed of four non-structural proteins (nsPs). The replicase amplifies the RNA within the cell, thereby enhancing the antigen production.
• Circular RNA (circRNA): A covalently closed-loop RNA molecule that lacks a 5' cap and 3' polyA tail, relying on an internal ribosome entry site (IRES) for translation of the vaccine antigen. Although the UTRs and 3' polyA tail are not essential, they can enhance translation efficiency.
1. RNA Vaccines: A Breakthrough in Infectious Disease Control
Infectious diseases remain a major global health challenge, with lower respiratory infections among the leading causes of mortality worldwide. 2 Traditional vaccine platforms, though effective, often require lengthy development timelines, making them less adaptable during pandemics or against rapidly mutating pathogens. RNA vaccines have transformed this landscape with their modularity and rapid development capabilities. One of their key advantages is the ability to encode multiple antigens within a single molecule, enabling the targeting of multiple pathogens or strains simultaneously.
Public Health Impact of RNA Vaccines
The swift development and deployment of RNA vaccines during the COVID-19 pandemic demonstrated their potential as game-changers in global health. By leveraging synthetic RNA to encode protective antigens with unparalleled precision, researchers have expanded RNA vaccine applications to address other high-impact viral pathogens, including:
Figure 1: Structural Overview of RNA Vaccine Platforms (Adapted from1)
Research / Innovation / Development
• Respiratory Syncytial Virus (RSV): mRESVIA (mRNA-1345) is currently the only mRNA vaccine approved for a pathogen other than SARS-CoV-2. Approved in 2024 as a single-dose vaccine for adults over 60 years old, it is also in clinical trials for paediatric use.3 Other RSV mRNA vaccines are in development, including multivalent versions that combine antigens for multiple viral pathogens.3
• Influenza: Several RNA-based flu vaccine candidates are in clinical trials. Among them:
• BNT161 (mIRV, BioNTech) and mRNA-1010 (Moderna) – both in phase 3 trials – encode antigens for multiple influenza strains.2,3,4
• mRNA-1018 (Moderna) is in phase 2 trials for pandemic influenza prevention.3
• Zika Virus: RNA vaccines are being developed to provide protective immunity, particularly for high-risk populations such as pregnant women.2
• HIV-1: RNA vaccine candidates targeting multiple viral regions are under evaluation by Moderna/IAVI and Alphavax.2,3
• Ebola Virus: Preclinical studies have shown robust immune responses, suggesting promise for outbreak prevention.2
Additionally, mRNA vaccines are advancing against bacterial and protozoal pathogens, with clinical trials underway for tuberculosis, Lyme disease, and malaria.2
2. RNA Vaccines in Oncology: Pioneering Precision Medicine
RNA vaccines are transforming cancer treatment by enabling personalised immunotherapy, allowing for precise targeting of tumour antigens. Cancer vaccines can encode tumour-specific antigens (TSAs) and tumour-associated antigens (TAAs) tailored to each patient’s tumour profile.1
Clinical Achievements
Several RNA-based cancer vaccines have demonstrated promising results, particularly in high-risk melanoma:
• BNT111 (FixVac, BioNTech): Currently in phase 2, this vaccine encodes a combination of four TAAs and has shown enhanced efficacy when combined with immune checkpoint inhibitors.1,4
• mRNA-4157 (V940, Moderna): An individualised cancer vaccine encoding up to 34 neoantigens selected based on sequencing data from a patient’s tumours. The vaccine is administered only if tumour sequencing identifies the neoantigens, making it a prime example of personalised medicine. Early trials have shown improved recurrencefree survival, and it is now in phase 2/3 for non-small cell lung cancer, cutaneous squamous-cell carcinoma, renal cell carcinoma, and bladder cancer.1,3
RNA vaccine research is also advancing for other solid tumours, with clinical trials underway for glioblastoma, head and neck carcinoma, pancreatic, prostate, and colorectal cancers.
Combining therapeutic cancer vaccines with other immunotherapies – such as immune checkpoint inhibitors and CAR-T therapy – is another key area of exploration.
Innovative Approaches: Combining CAR-T Therapy with RNA Vaccines
One of the most cutting-edge strategies in oncology, combining CAR-T cell therapy with an mRNA vaccine (BNT211, CARVac), is being tested by BioNTech.4,5
While CAR-T therapies have revolutionised the treatment of blood cancers, they have struggled to achieve similar success in solid tumours. BioNTech’s approach involves:
1. Engineering patient-derived CAR-T cells to recognise the tumour antigen Claudin-6.
2. Administering an mRNA vaccine that encodes the same antigen.
This strategy aims to amplify the CAR-T response while also stimulating the patient’s own immune system, potentially overcoming key barriers to treating solid tumours.
3. Advancements in RNA Vaccine Design
RNA vaccine technology continues to evolve, with saRNA and circRNA emerging as key innovations. These advancements improve vaccine efficacy and stability, addressing limitations associated with traditional mRNA platforms.
3a. Self-Amplifying RNA (saRNA)
A major breakthrough in RNA vaccine technology, saRNA, encodes a viral replicase alongside the antigen, allowing the RNA to replicate within host cells. This mechanism significantly enhances protein expression and immune responses, while drastically reducing the RNA dose required per vaccination. In a pandemic scenario, saRNA technology could enable the production of 10 to 1,000 times more doses compared to standard mRNA vaccines.6
Key Advantages of saRNA
• Lower Doses: saRNA achieves strong immune responses with minimal RNA quantities, making it cost-effective and highly scalable.6
• Robust Immunity: Clinical trials indicate that saRNA vaccines elicit strong antibody and T-cell responses, often comparable to or exceeding mRNA vaccines.7
Clinical Progress
saRNA vaccines have demonstrated strong potential in clinical trials, particularly against COVID-19. Notable developments include ARCT-154 (Arcturus Therapeutics/CSL) and Gemcovac-19 (HDT Bio & Gennova Biopharmaceuticals) – saRNA-based COVID-19 vaccines recently approved in Japan and India, respectively.7
Challenges & Future Directions
Despite their advantages, saRNA vaccines face challenges such as:
• Larger Molecular Size: saRNA molecules are three to four times larger than mRNA, affecting production and delivery efficiency.7
Research / Innovation / Development
• Increased Reactogenicity: The self-replicating nature of saRNA triggers a stronger innate immune response, which may require fine-tuning for better tolerability. So far, modification of saRNA with pseudouridine, the most common modification used in conventional mRNA vaccines, has failed, but recent studies have succeeded with other nucleotides, such as 5-hydroxymethylcytidine, 5-methylcytidine, and 5-methyluridine.7
Current research focuses on optimising delivery vehicles, improving tolerability, and refining dose regimens for broader applications.
3b. Circular RNA (circRNA)
Another promising innovation is circRNA, a closed-loop RNA molecule that resists exonuclease degradation, offering greater stability and prolonged protein expression.
Key Advantages of circRNA
• Higher Stability: Studies suggest that circRNA has a half-life approximately 2.5 times longer than mRNA, making it a compelling option for vaccines requiring sustained efficacy.8
• Rolling Loop Translation: The circular structure may allow ribosomes to re-engage continuously, enhancing translation efficiency and protein production.9
• Adjuvant Potential: CircRNA itself can act as a vaccine adjuvant, stimulating innate immune responses and inducing long-lasting antibody protection.9
Applications of circRNA
In preclinical studies, circRNA vaccines have shown potential for targeting SARS-CoV-2 variants and other pathogens, offering longer-lasting immunity than traditional RNA platforms.9 Moreover, circRNA vaccines targeting TAAs generate strong immune responses in solid tumours, particularly malignant melanoma.9
Challenges & Future Directions
Despite its promise, circRNA technology is still in its early stages. Key challenges include:
• Optimising circRNA synthesis to increase circularisation efficiency and reduce by-products.8
• Scaling up manufacturing for clinical applications as large-scale production remains limited.8
Efficient delivery systems are crucial for RNA vaccines to achieve their full therapeutic potential. LNPs represent the most advanced non-viral RNA delivery technology, ensuring the stability, biodistribution, and cellular uptake of fragile RNA molecules.
The first LNP-based therapy, Onpattro, was approved in 2018 for transthyretin amyloidosis, marking a key milestone in LNP-based drug delivery.7 This breakthrough paved the way for the rapid development and large-scale production of SARS-CoV-2 mRNA vaccines. Today, LNPs remain the gold standard for RNA vaccine delivery.
Key Components of LNPs and Their Roles
LNPs are composed of four essential lipid components, each playing a critical role:10,11,12
• Ionisable Lipids: Essential for RNA encapsulation and endosomal escape, enabling efficient cytoplasmic delivery.
• Cholesterol: Contributes to particle stability.
• Phospholipids: Provide structural integrity and biocompatibility.
• PEGylated Lipids: Extend circulation time by preventing particle aggregation and immune system detection.
Innovations in LNP Targeting
Significant research is underway to enhance LNP targeting for specific organs and tumours. Targeting specificity can be achieved through lipid composition modifications (adjusting lipid types and ratios), surface functionalisation (attaching targeting ligands, peptides, or antibodies), or tailoring administration routes (influencing biodistribution).10
Currently, organ tropism is mostly linked to the LNP charge and protein crown in the blood stream (passive targeting). Indeed, the binding of particular serum proteins and their interaction with cell receptors are of vital importance in determining the targeting behavior of LNPs. So far, the liver remains a primary target for the accumulation of LNPs containing ionisable lipids due to the apolipoprotein E corona formed on their surfaces.10 In contrast, the presence of permanently cationic lipids facilitates lung delivery thanks to the absorption of vitronectin, while anionic or zwitterionic lipids tend to target the spleen because of their interaction with β2-glycoprotein I.10
Some innovative strategies to improve targeted delivery, based on either the conjugation of specific ligands on the surface of the LNP (active targeting) or novel lipids exploiting
Clinical Status Approved for COVID-19 and RSV Recently approved for COVID-19 Preclinical
Table 1: Comparison of RNA Vaccine Technologies This table highlights key differences between mRNA, saRNA, and circRNA platforms in terms of duration of expression, stability, scalability, and clinical progress.
Research / Innovation / Development
Figure 2: Composition of Lipid Nanoparticles (LNPs) (Adapted from13) (A visual breakdown of LNP composition, highlighting molar lipid ratios used in the Comirnaty™ (Pfizer/BioNTech) COVID-19 vaccine).
the specificities of the tumour microenvironment, have been reported in preclinical studies:
• Mannose-functionalised LNPs: Deliver to M2 microglia (therapies against acute stroke).10
• Bisphosphonate-modified LNPs: Accumulate in bone microenvironment (bone-targeted therapies).10
• Anti-CD4 antibodies: Direct LNPs toward CD4+ T cells (for immune modulation).11
• pH-sensitive LNPs: Designed for tumour microenvironment targeting, where acidic pH triggers LNP degradation and RNA release.10
• Reactive oxygen species (ROS)-responsive LNPs: Exploit high ROS levels in tumours to enhance RNA release at tumour sites.10
Alternative RNA Delivery Strategies
While LNPs dominate the RNA vaccine landscape, other innovative delivery platforms have been used or are in development:
• Lipopolyplex (LPP): A core-shell structure combining lipids, cationic polymers, and RNA.2 Used in SW-BIC-213 (Starmina Therapeutics), a SARS-CoV-2 mRNA vaccine approved in Laos (2022).
• Nano-Lipid Emulsion (NLE): A cationic nanoparticle where the RNA is adsorbed on the surface rather than encapsulated inside. 2 Used in Gemcovac-OM and Gemcovac-19 (HDT Bio & Gennova Biopharmaceuticals), approved in India (2022). Gemcovac vaccines are stable at
2–8 °C and easier to manufacture thanks to the adsorption of the RNA on the nanoparticle surface.14
• Liposome-Protamine-RNA (LPR) Nanoparticles: Utilise protamine, a positively charged protein, to condense RNA for delivery.7,9
• Exosome-Based RNA Delivery: Leverages on natural extracellular vesicles for enhanced biocompatibility and reduced immunogenicity. However, costly and low-yield isolation methods limit large-scale applications.9
Addressing Challenges in RNA Vaccine Development
Despite their rapid advancements, RNA vaccines still face several key challenges:
1. Stability and Cold Chain Requirements
Most RNA vaccines require ultra-cold storage (-70°C), complicating global distribution. Research into heat-stable formulations is ongoing, with promising developments in LNP stabilisation (addition of cryoprotectants, lyophilisation) and RNA sequence modifications.
2. Manufacturing Scalability
Large-scale high-purity RNA production remains a major bottleneck, particularly for saRNA and circRNA. New approaches in enzymatic synthesis and advanced purification methods aim to improve scalability.
3. Immunogenicity Optimisation
While RNA vaccines are designed to stimulate immune responses, excessive reactogenicity can pose safety concerns. Ongoing studies focus on nucleotide modifications and dose optimisation to balance efficacy and safety.
Conclusion
RNA vaccines have revolutionised modern medicine, offering groundbreaking solutions for infectious diseases, cancer, and genetic disorders. Innovations in saRNA, circRNA, and
Research / Innovation / Development
LNP technologies are overcoming previous limitations while unlocking new frontiers in precision medicine.
As research advances, RNA vaccines and therapeutics will play a pivotal role in shaping the future of global healthcare, making treatments more effective, scalable, and accessible than ever before.
REFERENCES
1. Yang, R., Cui, J. (2024). Advances and Applications of RNA Vaccines in Tumor Treatment. Molecular Cancer, 23, 226.
2. Lokras, A.G., et al. (2024). Advances in the Design and Delivery of RNA Vaccines for Infectious Diseases. Advanced Drug Delivery Reviews, 213, 115419.
3. www.modernatx.com
4. www.biontech.com
5. Mackensen, A., et al. (2023). CLDN6-specific CAR-T cells plus amplifying RNA vaccine in relapsed or refractory solid tumors: the phase 1 BNT211-01 trial. Nature Medicine, 29, 2844–2853.
6. Blakney, A. (2021). The next generation of RNA vaccines: self-amplifying RNA. The Biochemist, 43, 14–17.
7. Silva-Pilipich, N., et al. (2024). Self-Amplifying RNA: A Second Revolution of mRNA Vaccines Against COVID-19. Vaccines, 12, 318.
8. Cai, J., et al. (2024). Synthetic circRNA therapeutics: innovations, strategies, and future horizons. MedComm, 5, e720.
9. Zhang, Z., et al. (2024). Advances in Engineering Circular RNA Vaccines. Pathogens, 13, 692.
10. Zhang, Y., et al. (2024). Principles of lipid nanoparticle design for mRNA delivery. BMEMat, e12116.
11. Hald Albertsen, C., et al. (2022). The role of lipid components in lipid nanoparticles for vaccines and gene therapy. Advanced Drug Delivery Reviews, 188, 114416.
12. Hou, X., et al. (2021). Lipid nanoparticles for mRNA delivery. Nature Reviews, 6, 1078–1094.
13. Fekete S, Doneanu C, Addepalli B, Gaye M, Nguyen J, Alden B, et al. Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals. J Pharm Biomed Anal. 5 févr 2023;224:115174
14. https://gennova.bio/
Erica Cirri is an expert in RNA-based therapeutic development at Tebubio, with a focus on translational applications of saRNA and circRNA vaccines in personalised medicine.
Xavier Warnet
Xavier Warnet is a Project Manager at Tebubio, specialising in RNA therapeutics and advanced LNP technologies. Xavier focuses on optimising RNA delivery for oncology and infectious disease applications.
Erica Cirri
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The Ins and Outs About Getting to the Most Relevant 3D
In Vitro Model: Facts, Trends and Expectations
“What we believe in, is putting the best biology we can into our models to mimic human biology”.
In recent years, there have been significant advances in technologies and resources to support drug discovery and drug developments; still the process remains lengthy and costly due to the high attrition rate of novel therapeutic agents at clinical stage.
Encouraged by the FDA modernisation act, pharmaceutical developers are changing practices and seeking alternatives to animal preclinical studies by using new methods, also called New Approach Methodologies (NAMs), including more relevant in vitro models, improved study designs, and more informative data outputs.1,2 This legislation has catalysed the engagement of the wider community to generate more pre-clinical models and strategies for regulatory consideration. The goal of future non-clinical safety/efficacy regulatory submission packages, is to advance the use of these enabling technologies by accurately predicting and characterising human responses.3
While the idea was widely welcomed in 2022, the feasibility of replacing animal studies in regulatory packages raised questions around the state of evolving science and lack of technology-readiness.2 As a result, the FDA established a process to qualify the non-clinical testing methods resulting in the FDA Modernisation Act 3.0 bill introduced in February 2024.2 Interestingly, the review of applications that used qualified NAMs were expedited to encourage submission while accelerating data collection.
According to Dr. Jan Lichtenberg, CEO Insphero (CH), “There is a lot of drive for reducing animal models in the industry because a lot of the new modalities for drugs do not work in animals, so researchers need models they can rely on and that give them proper data and also the regulatory agencies start accepting that it doesn't make sense to force researchers to test on animal models that are not relevant”.4
Dr. Meritxell Huch, from the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden (DE) confirms that “The current debate focuses on what we can recapitulate, what we can learn, and what these cultures are not well suited to model. Their resemblance to native tissue makes them crucial for modelling human disease and investigating the principles of tissue regeneration, self-organisation, and robustness. However, the information that in vitro models provide, sometimes still lacks accuracy to fully replace in vivo studies”.5
In Vitro Models
In vitro models represent powerful laboratory-based systems mimicking the in vivo environment and providing valuable insights into biological processes and drug safety and efficacy.
Those are derived from primary cells, tissues or induced pluripotent stem cells (iPSCs) and are 2D or 3D often including multiple cell types organised similarly to in vivo tissues. They can be static or encompass flow technologies to become so called micro-physiological systems. There are many different appellations for in vitro model from organoids to microtissues or mini-tissue and tissue explants, and many such models provide sufficiently reliable information to understand the in vitro-in vivo correlation, advancing the long-term goal of reducing animal use through innovative NAMs.
There are multiple benefits from using in vitro models.
iPSC-derived models present an unlimited supply compared to tissue explants, they can be characterised and batch to batch reproducibility can be achieved through well-controlled experimental conditions such as the differentiation process required to generate organoids. iPSC-derived models have, therefore, become a tool of choice for high throughput screening, saving time and cost.
Primary cells models may sometimes recapitulate more closely human physiology, offering an enhanced capability to evaluate biological processes for testing of drugs to understand their mode of action.
What are the keys to developing and improving models to progress therapies safely and confidently into humans? According to Dr. Mike Nicholds, CEO at Newcells Biotech in Newcastle (UK), the key to in vitro model development is a detailed understanding of the biology for the specific tissue allowing detailed analyses. “What we believe in, is putting the best biology we can into our models to mimic human biology. What we are about is providing models that produce very predictive data”.6 Prof. Matthias P. Lutlof, Co-Director and Head of Translational Bioengineering at the Institute of human Biology, Roche innovation centre in Basel (CH) also believes in creating models with a purpose to understand biological processes: “As engineers, we should avoid creating complex systems “just because we can.” The focus should be on the purpose of the model, i.e., the biological process it is meant to capture and the questions we want to answer. This purpose should guide the choice of model in terms of its complexity and throughput”.5
Omics and Machine Learning Approaches
Innovative NAMs are not just limited to models but also include new methods to acquire and analyse the data from in vitro models. Improving models is one aspect of model development, but improving the readouts of the assays obtained from the model also offers a potential for new advances. Technologies such as computational approaches, omics, functional genomics, single cell technologies and high content imaging capabilities as well as machine learning, are all enabling scientists to get better and more meaningful data, and it is only the beginning.
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• expresses high levels of key renal transporters recapitulating the in vivo environment
• has been extensively validated for FDA-approved biomarkers to support regulatory submissions
Complex nephron model to gain comprehensive predictive data.
• Renal safety data
• Functional and efficacy studies
• Renal transporter uptake analysis
• DDI evaluations
• Cross-species comparison
Visit www.newcellsbiotech.co.uk to find out more.
Research / Innovation / Development
Additional improvements rely on improving compatibility and integration in the industry. By improving scalability, liquid handling, integration with imaging system and workflows, in vitro systems will be adopted more widely to produce endpoints of interest.
Limitations to Overcome Complexity
The complexity of a model is a property researchers and developers need to balance carefully. If the model it too simple, the answers are not sufficiently informative and reliable. On the other hand, if a model is too complex, the model is difficult to interrogate and the data hard and costly to analyse. In vitro models, therefore, need to be sufficiently complex to be interrogated whilst allowing the generation of predictive data. In his articles in In vitro models, J. Miguel Oliveira also emphasises “As a starting point, the in vitro systems should fit a specific research question to be addressed. Other important “Do” and “Don’t” tips and features should be also considered… Don’t complicate your model, i.e., a simple and reproducible model can make the adoption by other researchers easier”.7
Scalability
Scalability is another hurdle, which upcoming technologies, for example bioprinting, are aiming to address. “Bioprinting offers assembly of biologically relevant building blocks, such as stem cells, organoids, and extracellular matrix-like biomaterials. We are moving toward large-scale fabrication of functional human tissues/organs for drug testing, preclinical experimentations of therapy development, and regeneration. However, the complex, time-dependent deformation of biomaterials poses a challenge for a reproducible process.” explained Dr. Rui Yao, from the Institute of Zoology, Chinese Academy of Sciences, Beijing, (CN).5
Standardisation
In parallel to the regulatory drivers to establish standardised processes to qualify NAMs, the International Council for Harmonisation (ICH) has also published guidelines in relation to this matter. Scientists, drug developers in biotech and pharmaceutical industry experts are encouraged to contribute to NAMs though consortia participation, peer-reviewed publications and documenting the reduction of animals in studies and programmes.2,8 The scientific community has worked hard towards establishing consistent protocols for NAMs across different laboratories to facilitate comparison and there are several consortia brining key subject matter experts across various disciplines from distinct companies and industries together to work through challenges and develop broadly useful methods. These include the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), the IQ consortium, the National Center for Advancing Translational Sciences (NCATS), the Center for Alternatives to Animal Testing (CAAT), the European Partnership for Alternative Approaches to Animal Testing (EPAA), Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), The NAMs Methodology hub and many more.
Through standardisation of methods, strategies, processes, computational approaches, in vitro models, in vivo study designs, knowledge sharing and even policies, the incorporation of NAMs into regulatory packages will be facilitated, playing a vital role in working towards the goals set out in the FDA
Modernisation Act 3.0 – the eventual elimination of animals in pharmaceutical development.
“Perhaps, the big advance now is the introduction of immunology components in the models, because many diseases affect the immune system directly or indirectly”
Trends and Future Developments
“The trend of talking about very complex models continues. We see a lot more functional validation of complex models in vitro compared to a few years ago. There are also trends towards combining different microtissue types into one, through microphysiological systems or other similar types of approaches, to see the interaction of various organs with each other. We’ll wait and see where it takes us” said Dr Valeria Chichagova, Director of Technology, Newcells Biotech (UK).9
In vitro models are now becoming more readily available not only for a range of organs (kidney, retina, lung, liver, pancreas, central nervous systems, intestine, heart, skin, bone etc.,) but also for a range of diseases, perhaps one day offering a rapid means to evaluate personalised medicines. The push to develop disease models of complex pathologies is also driven by funding bodies including the NC3Rs (UK), which opened a call for funding on the topic in October 2024 with the MRC, to address a lack of understanding of the mechanisms of complex human diseases. The goal of the call is to help overcome current model limitations by representing human physiopathology and disease heterogeneity more accurately.10 The 3R collaborative has also organised work groups, such as the MPS initiative, which has shared insights into the roles of developers, end-users and regulators in forwarding the use of microphysiological systems.11
“In terms of the disease areas, oncology is going to be a big market. Specific diseases like fibrosis will play an important role across a range of different tissue types. On the safety side, we'll see kidney, cardio, and neuro mainly. There will be more need for diversity as patients have different mutations, ethnic backgrounds, age, and sex. If we can generate this information in vitro, it could be helpful to stratify clinical trials and help bring these drugs successfully into the clinic.” Jan Lichtberg, CEO insphero (CH).12 This, however, comes at a cost: “The generation of in vitro models from patients comes with a range of additional hurdles including reproducibility” explained Prof. Dr. Jeffrey Beekman from the regenerative medicine centre in Utrecht (NL). “What is crucial for patient-derived organoids models, is to be able to maintain robustness in culture as this robustness is essential for standardisation and applications in clinical settings. Whether a model exactly recapitulates the in vivo tissue composition is not too important for clinical applications. The validity of these models comes from directly showing that organoid phenotypes in vitro associate with and predict patient phenotypes in vivo”.5
The next steps to create better models focus on combining models, either as organs on a chip, or by creating multicellular models and including an immune component or a vascular component. The incorporation of microenvironmental cues is another avenue for new developments.
Research / Innovation / Development
Dr. Colin Brown, CSO Newcells Biotech confirm this trend: “The first trend in the field, is to make more physiologically relevant models meaning models that are more predictive. The other trend is to try and make models more complex. Perhaps, the big advance now is the introduction of immunology components in the models, because many diseases affect the immune system directly or indirectly.”13
Through a deeper understanding of biology, and by combining existing models, scientists will in the coming years open new avenues. By limiting the use of animal models, drug discovery and development can be accelerated, and improved in vitro models and assay readouts will unravel a deeper understanding of cellular interconnectivity within tissue, allowing significant advances to be made for the treatment of diseases.
Explore more about in vitro models at newcellsbiotech.co.uk
REFERENCES
1. FDA modernisation act 2.0 (https://www.congress.gov/bill/117thcongress/senate-bill/5002, n.d.) and FDA modernisation act 3.0 (https://www.congress.gov/bill/117th-congress/senate-bill/5002, n.d.)
2. Carratt SA, Zuch de Zafra CL, Oziolor E, Rana P, Vansell NR, Mangipudy R, Vaidya VS. An industry perspective on the FDA Modernization Act 2.0/3.0: potential next steps for sponsors to reduce animal use in drug development. Toxicol Sci. 2025 Jan 1;203(1):28-34. doi: 10.1093/toxsci/kfae122. PMID: 39298459.
3. Williams M. Improving Translational Paradigms in Drug Discovery and Development. Curr Protoc. 2021 Nov;1(11):e273. doi: 10.1002/ cpz1.273. PMID: 34780124.
5. Lutolf, MP. et al., In vitro human cell-based models: What can they do and what are their limitations? Trends in Biotech, 2024 Volume 42, Issue 12, 1577 - 1582
7. Oliveira, J.M. Complex in vitro models: do not complicate it. In vitro models 2, 67–68 (2023). https://doi.org/10.1007/s44164-023-00060-1
8. Baker, Thomas K. et al., The Current Status and Use of Microphysiological Systems by the Pharmaceutical Industry: The International Consortium for Innovation and Quality Microphysiological Systems Affiliate Survey and Commentary. Drug Metabol and Disposition, 2024 , Volume 52, Issue 3, 198 – 209, 10.1124/dmd.123.001510
11. LaFollette MR, Baran SW, Curley JL, et al. The Use of MPS in Three Rs and Regulatory Applications: Perspectives From Developers on Stakeholder Responsibilities. Alternatives to Laboratory Animals. 2025;53(1):26-41. doi:10.1177/02611929241310566
Dr. Emanuela Costigliola is the Chief Marketing Officer at Newcells. With nearly 20 years in the life science and biotechnology industry, she has held senior roles at GE Healthcare, Qiagen, Life Technologies, and AstraZeneca. She was Head of Marketing at the Cell and Gene Therapy Catapult and has consulted on commercial capabilities in immuno-oncology. Dr. Emanuela holds a Ph.D. in Immunology and Molecular Pathology.
Dr. Jeanne-Françoise Williamson
Dr. Jeanne-Françoise Williamson is a senior consultant in biotech, pharma and healthcare experienced at understanding innovative ecosystems and helping clients bring their innovations to market. Jeanne holds a D.Phil in molecular biology and retrovirology.
Determining the Appropriate New Generation Test for Pyrogen-free Products
When determining a test for pyrogens and bacterial endotoxin, there are many regulations, needs, limitations, and techniques to consider. The original pyrogen test, the Rabbit Pyrogen Test (RPT; USP <151>, formerly EP 2.6.8, and JP 4.04) and the original endotoxin test, the Bacterial Endotoxin Test (BET; USP <85>, EP 2.6.14, and JP 4.01) served, and continue to serve, the quality control needs of pyrogen-free injectable pharmaceuticals, medical devices, accessory items. These will be referred to as “traditional” tests.
The next generation of pyrogen and endotoxin tests fulfil the need for sustainable, in-vitro, and animal-free tests solutions. These currently include the Monocyte Activation Test (MAT) (EP 2.6.30) for pyrogens, recombinant Factor C (rFC; EP 2.6.32 and USP <86>) for endotoxins, and recombinant cascade reagent (rCR; USP <86>) for endotoxins. These will be referred to as “new generation” tests.
When choosing the appropriate test method, several factors must be considered in choosing a next generation test method to replace a traditional method. These include pharmacopeial acceptance, the need for endotoxin-specific or general pyrogen testing, time of test and variability considerations, and interference considerations.
Firstly, it must be pointed out that there are advantages to the traditional tests. These tests are well-established, compendial, and harmonised across the pharmacopeia. This year, the RPT is being phased out in the European Pharmacopoeia, but it is still de facto international standard for pyrogens. Additionally, as an in-vivo test, the RPT has no need for considerations of interference as pyrogenicity is being directly measured. Finally, as new technologies, the new generation tests do not have universal acceptance in the pharmacopeia or harmonisation. Depending on the client’s location and product distribution, the best recommended method may vary. For example, the rCR reagent virtually replicates the traditional BET method. In the US, this reagent is now compendial and so provides an excellent new generation testing method. However, in locations where this is not published, the path to adoption is currently challenging. These factors are valid concerns, but they can be overcome by adopting the new generation tests.
Secondly, certain users will need to consider whether pyrogen testing, or endotoxin testing is required. Currently, most products allow for endotoxin tests as opposed to pyrogen testing. However, there are a few products that have pharmacopeial monographs requiring a pyrogen test. Two factors require the need for pyrogen testing that led to these requirements. First, pyrogen testing detects non-endotoxin pyrogens (NEPs). Although endotoxins are the primary cause of pyrogenic shock, there are other pyrogenic molecules,
including lipoteichoic acids, peptidoglycan, and other bacterial molecules. Although rare, if any of these molecules have a risk of contaminating a sample to pyrogenic levels, a pyrogen test will be required for the sample. Second, pyrogen tests will overcome causes of direct interference to the proteins in the endotoxin reagents. This includes many vaccines and protein-containing samples. However, the ability of the BET reagents to overcome interference has greatly increased with the quantitative traditional reagents and the new generation reagents. USP <151> does allow for the endotoxin test to be adopted as an alternative to the pyrogen test if method suitability is demonstrated. In many cases, samples that traditionally required the RPT may be suitable for any of the new generation methods.
Third, test-time and variability considerations will play a crucial role in selecting the new generation method of choice. The standard assay time to result for the RPT is 3 hours and for the BET is 1 hour or less. For endotoxin testing, the new generation methods have time to result that is comparable to that of traditional methods. However, ELISA-based MAT requires incubation and assay times significantly longer than the RPT incubation time. Additionally, MAT based on whole blood or PBMC pooling may exhibit natural variations. Cell line cultures will reduce variability, and alternative reporting methods can reduce the incubation and assay times of conventional MAT tests. An example is FUJIFILM Wako’s lumiMAT™ pyrogen test which utilises a luciferase reporter gene added to a cell line; allowing for luciferase to be detected by a luminescence reader which directly correlates to the NF-KB activation caused by pyrogens binding to the pyrogen receptors on the monocytic cells. This test method allows the user to obtain test results in approximately four hours – comparable to the time required of the traditional pyrogen test. Endotoxin tests traditionally exhibit lot-to-lot variability due to the natural source of the lysate reagent. However, the new generation methods reduce this variability due to the controlled manufacturing producing the reagent. rCR reagent uniquely mimics natural LAL reagent as the entire protein cascade is replicated. An example is FUJIFILM Wako’s PYROSTAR™ Neo+ reagent.
Finally, interference considerations play a role in determining the new generation test of choice. As mentioned previously, the MAT has the potential for interference as opposed to the traditional pyrogen test. But, MATs have unique resilience over certain products that contain interfering factors to the LAL reagent proteins found in endotoxin test reagents. Those products that utilised the traditional pyrogen test due to protease interference that targeted Factor C of LAL will continue to see good success with the MAT. However, The endotoxin test reagents have an advantage over all pyrogen tests in Limit of Detection (LOD). For example, PYROSTAR™ Neo+ can detect endotoxin down to 0.001 EU/mL, over 10-times more sensitive than the LOD of 0.0125 EE/mL of lumiMAT. Products that contain interference to the MAT may be more suitably tested by the
Research / Innovation / Development
BET. In the new generation of pyrogen testing, products which were tested by the pyrogen test may be better suited to the endotoxin test if interference persists with MAT testing that may be overcome by BET testing.
Many factors come into play in determining the correct pyrogen or endotoxin test for a product. The good news is that information on these new generation tests continues to be released to enable the user to make the best decision. FUJIFILM is committed to advancing the safety and sustainability of pyrogen testing in the future. PYROSTAR Neo+ endotoxin test reagent and lumiMAT pyrogen test were created to effectively navigate users from the traditional methods to the new generation methods as effectively as possible. As these methods continue to be established, the acceptance and harmonisation enjoyed by the traditional test users will transfer to this new generation of test methods.
Timothy Francis is the Senior Technical Specialist for the LAL Division of FUJIFILM Wako Chemicals U.S.A. Corporation. He holds a B.S. in Biochemistry and a M.S in Science Education. He comes into the Technical Specialist role with 5 years of experience teaching the natural sciences at a college level. He is proficient at taking the complex, technical aspects of a topic and breaking them down into clear, understandable pieces that all connect back to the big picture. He draws upon this experience to provide professional technical support and training for the PYROSTAR™ line and to help you with your technical needs.
CHO Cell Culture Process Intensification for Enhanced Production of IgG mAbs
Increased demand for higher product yield in biologics requires optimal use of existing manufacturing capacity. Process intensification is one way a facility can meet increased demand as it allows for fewer or smaller batches to provide the same amount of product in the same timeframe. Approaches for cell culture intensification may include addition of perfusion in seed train or production stages and modification of growth and production media to support cell densities not achievable in a standard fed-batch process. Perfusion requires specialised equipment such as tangential or alternating tangential flow (TF or ATF) controllers which require additional training and increase manufacturing costs.
Installation of this equipment may be limited by existing manufacturing site capabilities and could require costly expansion to accommodate their use. For facilities designed for fed-batch processes, the implementation of perfusion is too costly or impractical. Alternatively, significant gains in cell growth and productivity can be achieved by modification of media and feeds, capturing many of the benefits of perfusion without the additional costs related to equipment, facility upgrades, and training.
This poster shows a significant gain in growth and yield with similar product quality is achievable by modifying media and feeds. Intensification of the seed train was first assessed in these studies to allow for seeding a production vessel at a higher initial cell density. Multiple designs of experiments were executed. First, to intensify the seed train to deliver sufficient cell density for the production stage. Second, to evaluate the impact of production stage parameters on an intensified cell culture process in terms of growth, productivity, and product quality. Harvest studies were performed to ensure feasibility of processing the intensified cell culture at the manufacturing scale.
Objective
Create a process intensification workstream aimed at increasing
the titer of CHO processes by 20–50%, adhering to the following constraints:
• No effect on the process timeline
• Maintains comparable product quality attributes
• No need for additional equipment purchases or training
• Compatible with all manufacturing facilities within Thermo Fisher’s network.
Materials and methods
The intensification workstream was divided into two stages. The first stage defined the enriched media composition and operating parameters that enable achieving viable cell densities at passage sufficient to inoculate a production bioreactor at a seeding density ≥5.0 x 106 VC/mL. The media composition identified in this experiment would also function as the basal medium for the intensified production bioreactor. The second stage defined the operating parameters and feed strategy of intensified production bioreactor.
Cell Lines
Two CHO K1 cell lines were utilised to produce two recombinant human monoclonal antibodies, Rituximab and Herceptin. Intensification development was performed using the cell line expressing Rituximab. The intensified process developed using rituximab was then applied to the cell line expressing Herceptin to show response in an alternative cell line without optimisation.
Media and Feeds
Chemically defined catalog media and feeds were formulated per vendor instructions and then blended per experimental design to the specified formulations.
N-1 DOE
JMP statistical software was used to generate and analyse a custom DOE design evaluating the following factors: feed % in medium, seeding density, medium powder concentration, feed start day, and daily feed percentage. Conditions were run in parallel in 2 x 48 vessel Ambr™ 15 microbioreactor systems (Figure 1).
Figure 1. The N-1 fed-batch design space on an Ambr 15 system
JMP software was used to construct a model of the intensified N-1 step showing the optimal parameters to maximise viable cell densities (VCD). This model provided a robust set of parameters to achieve a VCD >20 x 106 VC/mL in 4 days with no adverse impact to viability (Figure 2).
N-stage Production Vessel Intensification Development
JMP statistical software was used to generate and analyse a custom DOE design evaluating the following factors: seeding density, feed start day, feed amount per cell, and temperature shift target. Conditions were run in parallel in 1 x 24 vessel Ambr™ 250 bioreactor system (Figure 3).
Results and Discussion
N-1 DOE
Figure 5 shows cell line expressing Rituximab that was scaled via the standard platform (blue) vs. the intensified process (red). Compared to the standard platform process, the intensified N-1 step process generated more than 4x as many cells to inoculate the production vessel.
Figure 6 shows cell line expressing Herceptin that was scaled via the standard platform (blue) vs. the intensified process (red). Similar results were observed, with higher VCD and viability observed from cells produced using the intensified process.
JMP software was used to construct a model of the intensified production step showing the optimal parameters for titer and specific productivity. This model provided a robust set of parameters to achieve 7 g/L in a standard 14 day fed batch (Figure 4).
Figure 2. A model of the intensified N-1 step using JMP software
Figure 5. Effect of standard platform vs. the intensified process on VCD and viability from cell line expressing Rituximab
Figure 6. Effect of standard platform vs. the intensified process on VCD and viability from cell line expressing Herceptin
Figure 3. The N-stage production intensification design space on an Ambr 250 system
Figure 4. A model of the intensified production step using JMP software.
N-stage Production Vessel Intensification Development
Rituximab
Figure 7 shows the VCD, viability, titer, and specific productivity of cell line expressing Rituximab produced using the standard platform process (blue) vs. the intensified fed batch process (red).
Herceptin
The same intensified process developed for the Rituximabexpressing cell line was applied to the Herceptin-expressing cell line. Higher titers were achieved while maintaining comparable product quality attributes (Figure 8).
Table 1 shows that the intensified process delivered comparable product quality for cells expressing Rituximab compared to the standard platform process.
Figure 7. Effect of standard platform vs. the intensified process on cell line expressing Rituximab. (A) VCD, (B) viability, (C) titer, and (D) specific productivity
Figure 8. Effect of standard platform vs. the intensified process on cell line expressing Herceptin. (A) VCD, (B) viability, (C) titer, and (D) specific productivity
Table 1. Comparison of product quality attributes of Rituximab-expressing cells produced using the intensified vs. standard platform process
Table 2 shows that the intensified process delivered comparable product quality for cells expressing Herceptin compared to the standard platform process.
Conclusions
We successfully developed a workflow for intensifying CHO processes to increase titers from 20% to 100% while retaining comparable product quality attributes. The intensified process can be executed within our existing manufacturing platform and facilities. The intensified processes increased titers up to 100% when optimised for specifically for a molecule as shown in the case of Rituximab. An application of a general intensified process achieved a 20% increase in titer for a Herceptinexpressing cell line. No additional equipment, training, raw materials, or consumables were needed.
Thermo Fisher Scientific Pharma Services
Thermo Fisher Scientific provides industry-leading pharmaceutical services solutions for drug development, clinical trial logistics, and commercial manufacturing. With more than 60 locations worldwide, the company offers integrated, end-to-end capabilities across all phases of development. Pharmaceutical and biotech companies of all sizes gain instant access to a global network of facilities and technical experts. Thermo Fisher delivers integrated drug development and clinical services tailored to fit each drug development journey, ensuring high quality, reliability, and compliance as a leading pharmaceutical services provider.
Table 2. Comparison of product quality attributes of Herceptin-expressing cells produced using the intensified vs. standard platform process
Navigating the Future of Pharmaceutical Oral Dose Manufacturing:
Embracing the Rise of High Potency Drugs
The pharmaceutical industry is undergoing a transformative period, driven by advancements in technology, evolving patient needs, and the increasing demand for high potency drugs. As we look towards the future, it is essential to understand the trends shaping pharmaceutical oral dose manufacturing and how they align with the growing need for high potency medications.
Oral solid dosage forms (OSDs) have long been the cornerstone of pharmaceutical delivery, favoured for their convenience, stability, and patient compliance. However, the landscape of oral dose manufacturing is rapidly evolving. Industry leaders predict a continued focus on improved adherence through user-friendly dosage forms and designs. This shift is driven by the need to cater to diverse patient demographics and preferences, ensuring that medications are not only effective but also easy to administer. According to a recent market analysis, the oral solid dosage forms market is expected to grow at a compound annual growth rate (CAGR) of 6.4% through 2027, highlighting the increasing importance of this segment in the pharmaceutical industry.
Technological advancements are significantly influencing this evolution. Innovations such as automation and continuous manufacturing are transforming production methods, providing unmatched efficiency, consistency, and scalability. These developments enable manufacturers to respond swiftly to market demands, reduce production costs, and maintain high-quality standards. Automation reduces human error and enhances precision in the manufacturing process, ensuring that each dosage form meets stringent quality standards. Continuous manufacturing allows for the uninterrupted production of medications, which is particularly advantageous in responding swiftly to market demands and reducing production costs. As we move forward, the integration of advanced technologies will further enhance the capabilities of oral dose manufacturing, paving the way for more personalised and precise treatments.
One of the most noteworthy trends in the pharmaceutical industry is the rising demand for high potency drugs. These medications, recognised for their potent biological activity at low doses, have become crucial in treating severe and life-threatening conditions such as cancer. The pharmaceutical sector is witnessing a substantial shift towards high potency drugs due to their efficacy in addressing these critical health issues. High potency active pharmaceutical ingredients (HPAPIs) are increasingly prevalent, with approximately one-third of all drugs in the pharmaceutical pipeline classified as HPAPIs. This trend is projected to persist, with the market size for HPAPIs expected to grow at a compound annual growth rate (CAGR) of 8–10% through 2025.
High potency drugs offer several advantages, including targeted therapy and reduced dosage requirements. This precision in treatment minimises adverse effects and enhances patient outcomes. As a result, high potency drugs are gaining prominence across various therapeutic areas, including oncology, hormonal imbalances, and neurology. The ability to deliver potent therapeutic effects at low doses is particularly beneficial in oncology, where the goal is to target cancer cells while sparing healthy tissues.
While the demand for high potency drugs presents significant opportunities, it also brings unique challenges to the manufacturing process. The production of drug products using HPAPIs requires specialised facilities, equipment, and stringent safety protocols to prevent cross contamination and ensure operator safety. This is achieved through the use of advanced containment technologies, such as isolators, glove boxes, ‘split butterfly’ containment valves and wash in place technologies which provide a controlled environment for handling HPAPIs. Regulatory agencies, such as the FDA and EMA, have established guidelines for the safe production of high potency drugs, and manufacturers must comply with these standards to ensure product quality and safety.
Despite these challenges, the pharmaceutical industry is well-positioned to capitalise on the growing demand for high potency drugs. Continuous advancements in containment technologies and manufacturing processes are enabling safer and more efficient production of HPAPIs.
Contract Development and Manufacturing Organisations (CDMOs) play a crucial role in advancing pharmaceutical oral dose manufacturing. CDMOs provide specialised expertise and state-of-the-art facilities, enabling pharmaceutical companies to develop and produce high potency drugs efficiently and safely. Collaboration between pharmaceutical companies and CDMOs fosters innovation and accelerates the development of new therapies.
By way of an example, Almac Pharma Services implemented advanced containment technologies to safely manufacture a high potency oncology drug. By utilising state-of-the-art isolators and containment systems, we ensured the safety of our operators while maintaining the integrity of the product. This approach not only met regulatory standards but also enhanced production efficiency and reduced downtime.
As the industry evolves, there is a growing emphasis on patient-centric approaches and personalised medicine. High potency drugs, with their ability to deliver precise and targeted treatments, align perfectly with this trend. Personalised medicine aims to tailor treatments to individual patients based on their genetic makeup, lifestyle, and specific medical conditions. This approach not only improves treatment efficacy but also enhances patient adherence and satisfaction.
The development of flexible dosage forms is enhancing patient adherence and satisfaction. Sprinkle formulations, mini tablets, and orally disintegrating tablets are designed to cater to diverse patient populations, including paediatric and geriatric groups. These dosage forms are easier to administer and can be tailored to individual patient needs, improving compliance and health outcomes. For example, orally disintegrating tablets dissolve quickly in the mouth, making them ideal for patients who have difficulty swallowing traditional tablets.
Indeed, there is a significant growth in the use of mini tablets for paediatric treatment. Mini tablets offer a flexible and user-friendly option for administering medications to children. These small tablets can be easily swallowed or mixed with food, making the process of taking medicine less daunting for young patients. The precise dosing and ease of administration provided by mini tabs enhance adherence and ensure that children receive their prescribed treatments effectively. As pharmaceutical companies continue to innovate, the popularity
and utilisation of mini tabs in paediatric care are expected to rise.
Looking ahead, the future of pharmaceutical oral dose manufacturing is bright, with numerous opportunities for growth and innovation. The continued integration of advanced technologies, will further enhance manufacturing processes, enabling real-time monitoring and optimisation. This digital transformation will drive efficiency, reduce costs, and improve product quality, ensuring that patients receive the best possible treatments.
Moreover, the collaboration between pharmaceutical companies, CDMOs, and technology providers will be crucial in navigating the complexities of high potency drug manufacturing. By leveraging each other's strengths and expertise, the industry can overcome challenges and accelerate the development of innovative therapies.
In conclusion, the trends shaping pharmaceutical oral dose manufacturing are closely intertwined with the rising demand for high potency drugs. As we embrace these changes, it is essential to remain focused on patient needs, technological advancements, and collaborative efforts. The future of pharmaceutical manufacturing holds great promise, with the potential to deliver more effective, personalised, and patientcentric treatments.
Tom Hegarty
Tom has significant experience in the pharmaceutical industry working for companies such as Allegan and Actavis. He has held a range of positions spanning formulation, technical services, manufacturing, packaging and engineering in the areas of solid oral dose, liquid oral dose, creams, gels and ointments, sterile injectables and drug device combination products. He is a Lean Six Sigma Black Belt with extensive experience in delivery of product optimisation and remediation projects. Tom, who holds a BSc (Hons) in Chemistry, joined Almac Pharma Services in 2020 and is responsible for Formulation & Process Development, IMP and Commercial Manufacturing Operations, Technical Support Engineering Maintenance and Capital Projects.
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talkfuture@pci.com
Building
High potent solutions include:
• High potent pharmaceutical development, clinical trial and commercial manufacture
• Containment down to an OEL of 0.01µg/m3
• Xcelodose® microdosing technology and roller compaction
• Tablets, capsules, powders, liquids, suspensions, semi-solids and drug-in-capsule/vial
• Analytical laboratory and QP services
• Clinical and commercial packaging
Manufacturing & Processing
Case Study: Development and Manufacture of a Highly Potent OSD Product
In the modern pharmaceutical industry, Contract Development and Manufacturing Organisations (CDMOs) have become integral players. However, this was not always the case. Before the CDMO surge in the 1990s, these organisations were relatively rare. Emerging initially in the 1980s as an industrial solution to address capacity challenges, the CDMO sector is now conservatively expected to surpass $340 billion by 2033.1 This rapid growth has naturally driven advancements in service offerings, such as expertise in handling complex and highly potent dosage forms, scalability from early clinical stages to full commercial launch, and continuous investment in cutting-edge technologies and state-ofthe-art facilities.
While there are now over 500 CDMOs operating globally, only a small fraction of these organisations are equipped to act as long-term strategic partners for their clients.2 A truly strategic CDMO goes beyond the role of a basic service provider. It serves as a consultative partner, offering a range of non-core, value-added services in addition to the core functions of development, manufacturing, and packaging. By doing so, it becomes an extension of its sponsor organisation, embedding itself deeply into the supply chain and enabling greater collaboration and efficiency.
With data indicating that approximately 41% of drug compounds are classified as highly potent – requiring an Occupational Exposure Limit (OEL) of 10 µg/m³ or lower – the ability to develop and manufacture such challenging dosage forms has become a highly sought-after capability in the CDMO space.3 This article delves into a real-world case study, demonstrating how a CDMO leveraged its global network and decades of expertise to successfully tech transfer, develop, and manufacture a highly potent solid oral drug product, ensuring a seamless transition from clinical supply to commercial launch.
Drug Product Overview
The drug product at the centre of this case study is a selective sphingosine 1-phosphate (S1P) receptor modulator, approved for the treatment of moderately to severely active ulcerative colitis (UC).
At the time of initial tech transfer, the Active Pharmaceutical Ingredient (API) was classified with an Occupational Exposure Limit (OEL) of 0.1 µg/m3. As is often the case in the development of highly potent drug products, additional data emerged over time, enabling the CDMO to re-evaluate the potency classification. Following this assessment, the API was reassigned an OEL of 0.2 µg/m3 – a slight adjustment that, while notable, remained well within the high-potency category. This classification necessitated the use of engineered containment measures throughout all stages of development and manufacturing to uphold both operator safety and product integrity.
NPI/Technical Transfer
When working with highly potent molecules, a critical component of New Product Introduction (NPI) is conducting a comprehensive COSHH (Control of Substances Hazardous to Health) assessment. This process evaluates each new molecule for its Occupational Exposure Limit (OEL) and Permitted Daily Exposure (PDE) before it is accepted at the site. By defining the molecule, its mechanism of action, and the specific handling requirements, the COSHH assessment ensures that environmental and operator safety is upheld at all times.
To fulfil the requirements of a COSHH assessment, a detailed toxicological and pharmacological evaluation must be carried out for every molecule entering the site. This assessment not only safeguards the scientists and operators handling the highly potent molecule but also supports the development of an appropriate cleaning assessment and verification parameters. These measures are critical in eliminating the risk of cross-contamination to subsequent products. Following the COSHH evaluation, a GMP Failure Mode and Effect Analysis (FMEA) is created, supported by data from safety assessments, licensing requirements, equipment reviews, and premises evaluations, ensuring the product can be processed safely.
A recommended strategy for technical transfer involves producing an initial placebo batch. This step allows the CDMO to define and optimise process parameters before manufacturing batches containing the active drug substance, thereby minimising the risk of wasting costly API. For highly potent APIs, this approach also facilitates a safe breach of containment to investigate potential issues or observe the product – something that would not be feasible with an active product. In the case of this drug product, the initial placebo batch was manufactured at the proposed commercial scale of 100 kg.
Once the process parameters were defined, the next stage involved producing a small-scale active batch of 2.5 kg. This batch was used to conduct a cleaning feasibility trial, aimed at establishing a robust cleaning process for the equipment exposed to the highly potent API. Although this step is not always required, it is particularly beneficial when working with highly potent molecules, as it helps mitigate the risk of cross-contamination in a multiproduct facility.
Given the time-sensitive nature of drug development projects, performing each stage sequentially would result in unacceptable delays. To avoid this, analytical method development was carried out parallel to the manufacture of the placebo and small-scale active batches. By the time the cleaning method and process parameters were finalised, the CDMO was able to proceed with manufacturing an active large-scale batch, which was then evaluated for uniformity of content, dissolution, assay, and impurities.
Manufacturing & Processing
Development Challenges
Formulation development is inherently a dynamic and experimental process. Even in strong, collaborative relationships between a sponsor and a CDMO, challenges occasionally arise that demand focused problem-solving to overcome.
For this particular drug product, one of the recurring challenges observed during the development stages involved punch sticking during tablet compression. Punch sticking occurs when powder material adheres to the punch face, leaving a defect on the tablet surface or transferring material to subsequent tablets, resulting in similar defects. This phenomenon is often caused by the adhesive nature of the API or other components in the formulation, or by inadequate lubrication. The problem can be further intensified by tooling design, particularly embossing configurations.
In the early stages of development, this issue was mitigated by increasing the amount of lubricant used during manufacturing. However, when the tooling design was modified to introduce a debossing effect for the final commercial tablet image, punch sticking reoccurred. Since excessive lubrication can negatively affect product dissolution, the issue was resolved by adjusting the tooling embossing and incorporating chromium nitridecoated tooling in collaboration with the tooling manufacturer. Lubricant levels were subsequently reassessed during pre-validation trials to ensure the robustness of the process prior to commercialisation.
A history of punch sticking highlights a critical consideration when selecting tablet debossing designs. Certain characters or shapes are prone to creating areas on the tooling where material can accumulate during batch compression. For example, characters such as 0, P, A, and 4 are particularly susceptible to
material sticking at their centre portions. Tooling suppliers can often recommend alternative designs or modifications to minimise this risk, underscoring the importance of cultivating strong partnerships with suppliers.
Another challenge encountered during the bottling process stemmed from the unusually small size of the tablets, which caused miscounting errors. The tablets did not align properly with the feed system of the existing bottling equipment, resulting in discrepancies of +/- 4 tablets per bottle. To address this, specialist change parts were developed to accommodate the unique dimensions of the tablets. Once again, strong supplier relationships were instrumental in resolving the issue. Fortunately, the site’s packaging facility had been designed with diverse drug product requirements in mind, incorporating features to manage size-related challenges. Testing and qualification of the equipment were conducted using placebo tablets, ensuring the system could reliably handle the product and ultimately eliminate miscounting errors.
Summary
This case study highlights the essential role of strategic planning, thorough testing, and strong supplier partnerships in addressing the challenges associated with developing and manufacturing highly potent oral solid dosage forms. From resolving punch sticking issues during tablet compression to overcoming bottling inaccuracies caused by unique size constraints, each stage of the process required customised solutions and advanced engineering expertise. By employing meticulous testing protocols, such as the use of placebo batches, and leveraging specialised equipment, the CDMO successfully maintained product integrity, ensured operator safety, and optimised process efficiency. This achievement underscores the CDMO's ability to navigate the complexities of pharmaceutical development, seamlessly transitioning from clinical supply to commercial launch.
David O’Connell is the Director of Scientific Affairs at PCI Pharma Services. After graduating from Glasgow Caledonian University with a BSc. in Applied Bioscience, David spent seven years as a Supervisory Scientist working for Aptuit in Edinburgh before moving to Penn Pharma as Head of Formulation Development in 2009. Here he played a vital part in the design of the potent Contained Manufacturing Facility (CMF), which won the ISPE Facility of the Year award for Facility Integration (2014). In 2013 David took on the role of Director, Pharmaceutical Development at the PCI site in Tredegar and in 2017 became PCIs Director of Scientific Affairs.
Manufacturing & Processing
Building a Competitive Edge through Tailored CMC Strategies in Drug Development
The healthcare industry’s demand for safe, effective, and accessible advanced therapeutics is rising rapidly. Drug developers are becoming more competitive in the race to develop and expand their drug pipelines, especially for complex molecules, to meet this demand and remain viable in this fast-evolving industry.
As a result of the competition toward biologics innovation, drug developers expect contract development and manufacturing organisations (CDMOs) to provide tailored services, especially in the field of chemistry, manufacturing, and controls (CMC).
As a strategy for maintaining consistent quality from drug development to commercialisation, CMC nowadays has to be specifically designed to accommodate the unique characteristics of complex molecules, like bispecific antibodies (bsAb) and antibody-drug conjugates. CDMOs can provide customised CMC guidance by enhancing their service flexibility and client-centric communication.
Building a Framework to Design a Customised CMC Strategy CMC is a fundamental framework that dictates early drug discovery to final delivery to patients, meaning it must be designed and executed with preemptive plans to respond promptly to challenges that may arise. Drug developers that want to achieve clinical milestones in time should embrace CMC as the guiding principle of drug development success.
Consistent product quality is a critical criterion that the U.S. Food and Drug Administration (FDA) evaluates during its review of clinical applications. The FDA’s approval threshold becomes more stringent should molecules concern multi-targeting antibodies or conjugation. By maintaining consistent quality, a nascent molecule derived from a biological organism stays pure, potent, and stable until it enters the human body and generates the desired therapeutic effect.
Building a robust CMC framework requires heterogeneous elements to work in harmony: comprehensive knowledge of candidate molecules, innovative technologies, advanced platforms, and experienced people who know how to execute each project stage right the first time. According to an industry report, at least 1,500 biologics are in trials.1 Whether a monoclonal antibody, bsAb, or Fc-fusion protein, each molecule’s distinctive characteristics require a custom CMC approach to maintain its efficacy until it is aseptically filled into a vial. Developing a tailored CMC strategy, however, is resource-intensive work, which can impact drug developers with limited capital. Therefore, drug developers partner with CDMOs, expecting to be provided with customised CMC plans for their pipelines. To ensure client satisfaction, CDMOs must develop a standardised, flexible CMC strategy that is tailored to a molecule’s characteristics or client-specific requirements.
A customised CMC strategy can prepare drug developers to overcome unpredictability at any stage of the project – the beginning, middle, or end – with the help of CDMOs that can offer three fundamental CMC frameworks.
The three CMC frameworks can be categorised into simplified, comprehensive, and enhanced.
• Simplified: Time is a crucial resource for drug developers. The journey to Investigational New Drug (IND) approval often includes time-sensitive challenges. The simplified framework allows drug developers to leverage CDMO resources – such as accumulated project data, problemsolving expertise, and analytical method development – to overcome challenges and file IND applications in time. At the core of this framework are readily available clinical infrastructures, such as a molecule-specific design of experiment, that drug developers can leverage to expedite process development and release testing of their molecules. The foundation of the framework is data-driven calculations and assessment of regulatory risk in the planning phase, which ensure drug developers use resources efficiently at each stage while minimising waste.
• Comprehensive: Multifaceted approaches are required to ensure the efficacy of biologic molecules. The comprehensive framework creates an optimised environment in which difficult-to-express proteins can generate the desired characterisation of a molecule. This framework enables drug developers to select the best development tools to maximise efficacy and maintain consistent quality. Additionally, a comprehensive and thorough testing plan is at the heart of this framework. The plan comprises development methods, such as host-cell DNA/protein impurity analysis and residual protein A detection for leaching or modulation of glycans and size/ charge variants. The comprehensive CMC framework also
Manufacturing & Processing
provides in-depth analyses during process development and tailored characterisation tests. Alongside process development, the framework includes processes for screening molecules for surfactants, pH/buffers, and other impurities to minimise risk, pre-assess stability, and maximise the likelihood of approval.
• Enhanced: This framework embraces the characteristics of the previous two, offering robust contingency plans at all stages of a drug development project and late-phase process redevelopment. It includes problem-solving tools, such as quality-by-design models, and sciencebased reasoning to create strategic plans to overcome development and regulatory challenges. Thorough studies of forced degradation, structure-activity relationships, process characterisation, and other relevant elements enable drug developers to understand their molecules’ behaviors and determine the robustness of their process
development methods. Whether the challenge is redesigning a cell line, improving drug purity, or reducing a technology transfer process timeline, leveraging this framework will increase the chance of derisking drug development projects.
The incremental nature of the three CMC frameworks prepares CDMOs to be versatile in their services. By offering a diverse range of technology, platform, and facility options, CDMOs can guide drug developers in building a bespoke CMC strategy tailored to their unique molecules.
Thorough planning should be followed by thorough execution. As a reliable partner, CDMOs should offer flexibility in execution and facilitate transparent communication with their clients. When flexibility and client-centric communication merge and build on a robust CMC foundation, the therapeutic potential of molecules can be fully achieved.
Manufacturing & Processing
Flexibility and Communication: Key for Fully Leveraging a CMC Framework
A well-designed CMC framework works best in a one-team environment where drug developers and CDMO partners proactively share knowledge and expertise.
One important reason drug developers work with CDMOs is to leverage the rich resources from their partners to advance drug candidates in time. The two should share the same goals and knowledge to ensure the best outcomes with the available resources. Misaligned communication and goals may jeopardise project quality. CDMOs and drug developers should work together and use their strengths to mitigate the others’ weaknesses to achieve their shared goals of drug approval and commercialisation.
If a drug developer needs to file an IND application in an expedited timeline but its molecule has an unstable cell line that needs to be redeveloped, a responsible CDMO would first strive to understand the client’s molecule: how it was previously characterised, how it can be expressed better, how it can be developed with low impurity, and which quality parameters the client wants to maintain. Through client-centric communications, CDMOs learn to strategise and execute from the client’s perspective, enabling them to suggest the right CMC strategy. To support this process, drug developers should proactively share rich scientific and technical knowledge of the molecules with their CDMO partners. Close-knit collaboration between the teams can ensure that a development challenge, such as cell line redevelopment, can be overcome without delaying the timeline.
Trust, forged through open discussions and mutual respect, is vital for close-knit collaboration. So, CDMOs must drive such discussions with their clients.
When a project requires fast process development, an impurity issue in the purification stage could risk delaying the IND filing, thereby requiring additional time and resources. In a time-sensitive scenario, CDMOs should establish real-time communication channels with the client to brainstorm solutions, pooling knowledge and expertise as a team. During this process, CDMOs should provide data and analyses from past project track records to serve as a base for ideating solutions.
The unique nature of drug development projects, especially for complex molecules, creates challenges for CDMOs wanting to standardise their drug development platforms. No single CMC strategy can be suitable for developing all hundreds of distinctive molecules, underscoring the need for customised CMC plans. CDMOs must be flexible and engage in transparent communication to readily adapt to change requests and take on challenges.
Overall, CDMOs must provide a root-cause analysis of the situation and work with clients to resolve the issue. Robust CMC infrastructures can improve a CDMO’s reliability and help them become trusted partners that can ensure client satisfaction by leveraging their flexibility and client-centric communication to advance their client’s molecules to their full potential.
Yoonsik Kim is an associate director of method development analytics as Samsung Biologics. Before joining Samsung Biologics, he spent seven years developing analytical methods for a range of molecules with a focus on physico-chemical and biochemistry techniques at Genexine. Kim holds a master's degree in Life Sciences from Seoul National University.
Steven Lal
Steven Lal is a lead scientist in non-GMP analytics at Samsung Biologics. Before joining Samsung Biologics, he worked as an associate principal scientist and group leader within analytical and development services, with a focus on extended characterisation techniques at Lonza in the United Kingdom. He holds both master’s and doctorate degrees in Chemistry from Imperial College London.
Logistics and Supply Chain
The Impact of Data and Academic Partnerships on Cold Chain Innovation
Cold chain logistics are vital in the pharmaceutical and life sciences industries, where temperature-sensitive products such as vaccines, medications, and biological samples must be transported and stored under strict temperature controls. Ensuring the integrity of these products is essential, as their efficacy often relies on precise temperature regulation. However, managing the distribution of such sensitive goods presents significant challenges. With growing sustainability demands, the industry is increasingly relying on innovative solutions that reduce carbon emissions while upholding product integrity.
One such solution has been the collaboration between academic institutions and industry stakeholders to optimise cold chain logistics, with a growing focus on sustainability. A notable example is the partnership between Cardiff University's Business School and Tower Cold Chain (part of Cold Chain Technologies), aimed at utilising academic expertise to enhance operational efficiency while prioritising sustainable practices.
The Environmental Challenges of Cold Chain Logistics in the Pharmaceutical Industry
The pharmaceutical industry's cold chain logistics operations are highly energy-intensive, primarily due to the stringent temperature requirements needed to preserve the efficacy of drugs and biological materials. For example, vaccines typically need to be stored between 2–8°C (35–46°F), while gene therapies and biologics may require sub-zero or even cryogenic temperatures. Maintaining these conditions over long distances and periods can be technologically challenging and environmentally costly.
Traditional cold chain solutions often involve single-use packaging and active cooling systems, which require constant electricity and result in significant waste and carbon emissions. Additionally, the need for global logistics networks exacerbates the environmental impact. The pharmaceutical sector’s increased demand for reliable cold chain logistics, particularly during emergencies like the COVID-19 pandemic, has put further strain on the industry’s environmental footprint.
As the global demand for temperature-sensitive medicines rises, so does the need for a wider variety of solutions. This scenario poses a dual challenge: companies must meet regulatory standards for product preservation while minimising waste and emissions.
The Shift Toward Reusable Temperature-Controlled Containers
To address these challenges, reusable, passive containers have become a viable solution for maintaining temperature control without the need for continuous power or disposable cooling
agents. These containers are engineered to preserve specific temperature ranges for extended durations, sometimes up to and beyond 120 hours, without relying on active refrigeration. This innovation allows pharmaceutical companies to lessen their dependence on single-use materials and energy-intensive cooling systems, providing a reusable approach that still ensures precise temperature management.
Equipped with advanced insulation and phase-change materials (PCMs), these containers absorb and release thermal energy, maintaining stable temperatures. With capabilities to sustain temperatures from -80°C to +20°C, they support the wide range of temperature-sensitive products in the pharmaceutical industry. These containers are also built for durability, often lasting up to 15 years, which reduces the environmental footprint tied to the production, shipping, and disposal of single-use alternatives.
The shift to reusable containers addresses key concerns in the industry. By decreasing the need for manufacturing and disposing of single-use containers, it helps reduce overall waste. Additionally, these containers contribute to a lower carbon footprint by minimising energy consumption. However, transitioning to this sustainable solution requires a comprehensive review of supply chain operations to ensure the approach is cost-effective and operationally efficient.
Evaluating the Total Cost of Ownership
While the sustainability advantages of reusable containers are clear, their implementation requires careful consideration of the total cost of ownership across the entire supply chain. The initial cost of reusable containers is often higher than that of single-use options, but the long-term savings can be significant. These savings stem from reduced waste, fewer repair costs, lower shipping costs, and extended lifespans.
However, to realise these savings, companies must assess the broader logistics and distribution strategies they employ. The transition to reusable containers must be accompanied by a thorough analysis of how to streamline inventory management and shipping practices. Proper inventory management ensures that containers are available when and where needed, reducing delays and ensuring compliance with regulatory requirements.
Logistics networks must also be optimised to accommodate the new containers. For instance, organisations need to assess the most efficient routes and distribution methods to minimise transportation distances and reduce the environmental impact.
Offering an extensive, regional hub network and the use of a space-efficient conditioning system are also key factors in reducing carbon emissions. By offering shorter distances for servicing and redeployment, combined with lower energy requirements for container conditioning, logistics providers can
Logistics and Supply Chain
help to decrease environmental impact while supporting more efficient operations.
The integration of reusable containers into any supply chain requires close coordination between all stakeholders to identify the best strategies for improving efficiency and reducing costs.
The Role of Academic Collaboration in Advancing Sustainability
The collaboration between academic institutions and industry partners plays a crucial role in driving innovation and efficiency in cold chain logistics. Academic research provides valuable insights into operational optimisation, helping industry stakeholders make informed decisions about logistics strategies and container use. This collaboration is particularly important in ensuring that sustainability goals are met without compromising operational requirements.
For example, Cardiff University’s Business School has worked with logistics providers to analyse demand patterns, shipping routes, and inventory management practices. By leveraging data analytics, the university’s research helps identify opportunities for optimising container usage across various global locations. This data-driven approach enables companies to streamline their supply chains, minimise inefficiencies, and reduce unnecessary carbon emissions.
One of the critical challenges identified in this research is the phenomenon of "empty leg" journeys, where containers travel to and from distribution centres without cargo. These journeys
result in additional costs and unnecessary carbon emissions. By analysing data on shipping routes and container usage, Cardiff University’s research has helped cold chain providers reduce empty leg trips and increase the efficiency of transportation networks.
Through the partnership, research has also focused on resource allocation, ensuring that reusable containers are positioned in locations where they can be most effectively utilised. This approach minimises transportation distances and increases the frequency of fully loaded trips, further reducing environmental impact and costs.
Sustainable Practices and Compliance with Regulations
As environmental regulations become increasingly stringent, pharmaceutical companies are under pressure to demonstrate their commitment to sustainability. The integration of sustainable practices into cold chain logistics not only helps companies comply with these regulations but also supports their broader sustainability objectives. By prioritising the use of reusable containers and optimising logistics processes, companies can reduce their reliance on single-use packaging and cut down on waste.
This shift aligns with the broader industry trend toward adopting circular economy principles. By extending the lifespan of containers and reducing waste, pharmaceutical companies can significantly reduce their environmental impact. Furthermore, the adoption of sustainable practices often leads to long-term cost savings. Reduced packaging waste, improved logistics efficiency, and more effective resource allocation
Logistics and Supply Chain
contribute to a more cost-effective and environmentally responsible supply chain.
The Future of Pharmaceutical Cold Chain Logistics
The future of cold chain logistics in the pharmaceutical sector is moving toward greater sustainability. However, achieving this goal requires ongoing innovation and collaboration between industry players, academic researchers, and regulatory bodies. Initiatives like the partnership between Cardiff University and Tower Cold Chain serve as examples of how data analytics, operational efficiency, and sustainable technologies can work together to create a more environmentally responsible logistics landscape.
By integrating academic research with industry practice, these collaborations help to address the complexities of cold chain logistics, from regulatory compliance to operational optimisation. As pharmaceutical companies increasingly prioritise sustainability, they will need to embrace innovative solutions, such as reusable containers and data-driven optimisation strategies, to meet both environmental and operational goals.
This collaborative approach underscores the critical role of data analytics in transforming the pharmaceutical cold chain industry. By making informed decisions based on comprehensive data analysis, companies can reduce their environmental impact while ensuring that temperature-sensitive products are safely and efficiently transported. Moreover, the use of sustainable practices not only helps meet regulatory requirements but also aligns with the growing demand for corporate responsibility in the face of climate change.
The future of pharmaceutical logistics will likely continue to evolve as sustainability becomes a central focus. As data analytics, technology, and academic research continue to shape
the industry, pharmaceutical companies will be well-positioned to meet modern challenges while contributing to a more sustainable and resilient global supply chain.
Conclusion
The path toward sustainability in pharmaceutical cold chain logistics is an ongoing process, but collaborative efforts between industry stakeholders and academic institutions provide a promising blueprint for the future. By optimising logistics operations, adopting reusable containers, and leveraging data to drive efficiency, the pharmaceutical industry can significantly reduce its environmental impact. The integration of these practices into everyday operations will not only contribute to a more sustainable future but will also help pharmaceutical companies meet the evolving demands of the global healthcare landscape. As the industry continues to innovate and embrace new technologies, sustainability will undoubtedly become an increasingly integral part of the pharmaceutical cold chain.
Tower Cold Chain’s Chief Executive Officer, Niall Balfour, is a highly experienced and versatile director operating in the life science logistics and pharmaceutical supply markets. Joining Tower in 2015 as CEO, Niall brings 30+ years of industry knowledge to the company, with previous positions including Vice President, Commercial Operations, Biocair, Vice President Sales, Europe and Asia Pac, Marken, and Director, EU Sales & Marketing, Quest Diagnostics Clinical Trials. Niall is committed to driving Tower’s mission in being a dynamic and profitable company that creates value for all its stakeholders through innovation, regulatory compliance, and sustainability.
Niall Balfour
PYROSTAR™ Neo+
Recombinant Endotoxin Detection Reagent, Plus...
PYROSTARTM Neo+ is based on a genetically engineered approach to produce reactive factors required for endotoxin detection at pharmaceutical and medical equipment manufacturing sites. With greater stability of the negative control and better
endotoxin recovery in heparin and heparin-based compounds, Neo+ facilitates the same sensitivity of endotoxin detection as our traditional limulus amebocyte lysate (LAL) reagent, through a more sustainable and environmentally friendly method.
• Colorimetric method, can be used with an absorbance plate reader
• 3-Factor system mimics the same cascade reaction as traditional LAL
• Endotoxin-specific reagent eliminates the risk of false positives from (1-->3) ß-D-Glucan
• 100% free of horseshoe crab blood
• Quantitative range: 0.001 to 50EU/mL
• High sensitivity with less lot-to-lot variation
• Stable storage after dissolution (4 hrs. at 2-8°C and 2 weeks at -30°C)
Recombinant Protein-Reagent
Building a Patient Centric Supply Chain
Patient well-being should be at the heart of all decisions made within the life sciences. Despite this, other factors often compete for decision-makers' attention and ultimately distract from the focus on patients. While much of the supply chain may seem far removed from patients, it can have a significant impact on medicine delivery and patient well-being.
A patient-centric supply chain is one which puts the needs of the patient first. To support the rise of more bespoke treatments and fully personalised medicines, patient-centric supply chains must be developed. This presents new challenges to logistics specialists as they endeavour to ensure patients receive the right care, at the right time, and in the right condition.
But a patient centric approach should not just focus on medicine delivery, it needs to encompass the entire process from discovery, to development, to clinical trials and manufacturing. As a truly global industry, materials need to cross borders and align with different regulations throughout their journey, increasing demands on the supply chain. Logistics experts connect all stages and locations of this complex network and play a major role in delivering a renewed patient focus.
Understanding Traditional Healthcare Supply Chains
Traditionally, the pharmaceutical industry has taken a broadbrush approach to medicines, developing treatments designed to treat huge numbers of people in the same way. However, as our understanding of genetics has grown, so has our appreciation for the benefits of treatments that are tailored to treat individuals.
Traditional supply chains worked in a similar way. With millions of patients to treat all over the world, the main drivers were cost, efficiency and delivering the correct stock levels. The move to a more personalised model raises challenges around inefficiencies, driven by a lack of flexibility, poor communication between stakeholders, and the failure to anticipate patientspecific needs. This can ultimately negatively impact patient care with delays in treatment, decreased patient satisfaction and potential harm. A patient-centric approach to logistics shifts the focus to personalisation, outcomes, and ultimately the patient experience.
Key Principles of a Patient-centric Supply Chain
To build a comprehensive supply chain that meets patients’ needs, the main priorities are:
• Flexibility: A one-size fits all approach will not work. Each solution requires a tailored approach, guided by logistics expertise and the unique needs of each organisation.
• Data-Driven Decision Making: Using advanced analytics, AI, and real-time data can help experts anticipate logistics needs and optimise supply chain efficiency.
• Collaboration: Ensuring that all stakeholders work together seamlessly. An agnostic approach to technology and packaging partners can help to ensure this flexibility.
• Transparency: Offering clear visibility across the supply chain assisted by adopting new tracking technologies.
• Agility: Rapidly adjust to unforeseen circumstances like supply shortages or global logistics challenges.
• Experience: Employing people with experience in the scientific and pharma industry in logistics roles can aid in understanding and overcoming challenges.
• Risk mitigation: Taking proactive steps to assess and reduce risk through lane mapping and packaging validation, enabling rapid responses to any issues.
• Sustainability: Considering sustainability in all processes to support industry goals and future proof supply chains.
In order to deliver life-changing treatments to patients who need them, organisations need to build robust supply chains throughout the drug development process. Logistics experts can help to ease the burden on scientists so that they can focus on the research that improves patients’ lives.
Technology
as an Enabler
Recent technology developments are already having a significant impact on supply chains and will be essential in ensuring greater patient focus. Leveraging artificial intelligence and machine learning can help to predict demand, optimise routes, reduce waste and anticipate supply disruptions.
For personalised medicines and cell and gene therapies, chain of custody and condition monitoring are essential to ensure product safety and comply with regulations. GPS tracking and advanced temperature sensors allow for real-time tracking, improving shipment visibility.
This is further facilitated by cloud connectivity and supply chain management tools that allow real-time visibility and coordination of supply chains. Integration of systems such as Trakcel’s OCELLOS platform ensures tracking of both chain of identity and chain of custody for cell and gene therapy supply chains. Implementation of blockchain can further help to track chain of custody and ensure security and transparency throughout the supply chain.
Logistics and Supply Chain
Personalised Supply Chains for Autologous Cell Therapies
A US-based cell therapy developer needed to transport cells to the manufacturing facility in Europe and then back to patients. As living drug products, cell therapies have complex demands, including short life spans and narrow treatment parameters. It was therefore essential to provide real-time information regarding a shipment’s location and condition to ensure safe delivery of these critical materials.
Delivering cells successfully can often be a complex, multi-step process. This particular application included a number of manufacturing and patient sites across a variety of locations globally. To protect against potential incidents such as flight delays and cancellations, expert logistics planning was required, including devising and monitoring alternative supply routes. To meet these challenges, the latest temperature-controlled packaging and monitoring technologies were used, as well as chain of custody tracking.1
Challenges Remain
The integration of new technologies can dramatically enhance supply chains, but challenges remain. Data privacy and security are paramount in any industry, but particularly within life sciences due to the sensitive nature of private healthcare information. Any new technologies incorporated into the supply chain must also protect this private data.
In many cases, these new technologies are not designed for interoperability, making integration of diverse systems difficult. There are also cost implications to any technology, and with healthcare systems straining to support an aging population, cost remains a leading issue. Partnering with logistics experts can help to overcome these challenges, while also ensuring regulatory compliance and protection of product quality.
Problem-solving in Supply
Rocket Pharmaceuticals, a US-based cell and gene therapy developer, needed to rapidly transport time- and temperature-sensitive biospecimens between a hospital in central London and a medical genomics lab based in the Netherlands. Following Brexit, the shipping lane was located outside the European Union’s single market and customs union, whereas the destination was within the EU. This added increased regulatory compliance and customs clearance challenges, including greater risk of administrative delays, to an already tight timescale.
Precise route planning, pilot shipping and expedited regulatory approval through an experienced life science logistics provider was required to solve this problem. By thinking creatively and working closely with the agencies involved, these potential delays were overcome.2
The Future of Patient-centric Supply Chains
The benefits of a patient focused supply change are evident, but all stages of the process need to align. The shift towards personalised treatments will require tailored transportation solutions, with new technologies being key to implementing
these strategies. But challenges remain, and to ensure technologies are implemented in an integrated way that supports efficient medicine development you should connect with a trusted life science logistics specialist to develop supply chain solutions tailored to your needs.
REFERENCES
1. https://www.biocair.com/case-studies/supply-chains-for-autologouscell-therapies?utm_campaign=Global%20%27Always%20 On%27%20Brand%20Campaign&utm_source=Feature&utm_ medium=JCS&utm_content=Building_a_patient_centric_supply_ chain (Accessed 5 February 2025)
2. https://www.biocair.com/case-studies/skills-ensure-rapid-deliveryof-vital-child-biospecimens-0?utm_campaign=Global%20 %27Always%20On%27%20Brand%20Campaign&utm_ source=Feature&utm_medium=JCS&utm_content=Building_a_ patient_centric_supply_chain (Accessed 5 February 2025)
Vincent Howard
Vincent Howard is Biocair's CEO, joining in 1993 as the company’s first science graduate. With extensive experience in global operations and sales, he has built strong relationships with key clients, offering tailored logistics solutions. Passionate about customer service, Vincent believes it starts with care for the people you work alongside. This belief carries through in how Biocair serves clients and, ultimately, the patients at the end of every shipment.
Breath Analysis in Diagnostics: The Use of Animal Models
Developing breath analysis methodologies in mouse models has significant benefits for clinical research and in practice for a range of therapeutics.
Sampling mediums such as blood and urine are routinely collected from patients in clinical practice to measure biomarkers and inform on physiological processes that may have been altered as part of an underlying disease. Exhaled breath can also be collected for similar purposes, as it contains volatile organic compounds (VOCs) that can originate from deeper within the body as opposed to the immediate respiratory system (Figure 1). VOCs can similarly provide insights into physiological processes that are relevant for understanding disease processes in the body. VOCs are a novel group of molecules related to metabolic processes (including microbial metabolism) with applications where other biomarker technology may have failed to be specific, or sensitive enough to apply to medical practice. As such, candidate VOC biomarkers have been identified for a variety of conditions, such as diabetes, respiratory diseases, cancer, gastrointestinal diseases, and infections.1
Due to the unique properties of breath as a sampling medium, and the chemical attributes of VOCs, breath VOC biomarkers have many advantages for clinical use. Exhaled breath can be collected in a completely pain-free non-invasive manner, and is convenient for subjects providing samples. As breath is almost continually released from the body, it is available in an unlimited supply, and a large sample volume can, therefore, be collected to enable the pre-concentration of lower abundance metabolites for subsequent analysis. Breath can also be collected serially at frequent intervals allowing for time course analysis. This unique attribute can be exploited to improve the power of breath testing for clinical benefit, for example, by administering an exogenous volatile organic compound (EVOC®) probe such as limonene and observing the changes in breath composition over time (Figure 2). A dynamic breath testing approach using limonene has been trialled to diagnose liver cirrhosis, with a sensitivity and specificity of 0.83 ± 0.07 and 0.9 ± 0.06 achieved respectively.2 Limonene is thought to be metabolised less efficiently when the liver is diseased. The CYP450 enzymes CYP2C9 and CYP2C19 in the liver metabolise limonene to perillyl alcohol, trans-carveol, and trans-isopiperitenol slower, leading to higher levels of limonene
Figure 1. Exhaled breath is a mixture of exogenous, and endogenous volatile organic compounds.
Preclinical Subsection
in the breath for longer.3,4 Such probes are also a promising mode of early cancer detection, with clinical trials currently in progress for lung cancer targeting cancer-specific altered enzyme activity.5
In order for new cancer treatments, disease diagnostic tools, or medical devices to be translated into clinical use, they must be thoroughly tested during clinical trials. It can take over a decade for a potential new drug treatment to undergo all the phases of clinical trials to be approved for medical use. This length of time doesn’t only affect the speed at which innovative treatments become available to patients in need but is also associated with high cost.
Animal Models to Quantify and Validate Biomarkers
Before clinical trials can progress to humans, pre-clinical research involving in vivo work with animal models is an essential step in the current drug discovery workflow. To gain a full understanding of a potential new drug therapy, relevant physiological responses are often used to measure how the drug works, including how long it takes to be absorbed, when and where it is excreted, whether it is safe, and how effective the drug is at treating the target disease. Information can be gathered from these samples by analysing and quantifying biomarkers from sampling mediums such as blood, giving insights into the physiological changes the body has undergone in response to the drug. Developing breath biomarkers as diagnostic and monitoring tests in the clinic can be expedited by establishing reliable animal models of breath. Novel probe
technology is being actively developed in mouse models for optimal sensitivity and specificity of breath tests, enabling a non-invasive method for detecting cancer early. 6,7 The underlying principle of synthetic probe technology for boosting the signal of VOC breath biomarkers can be applied to several disease contexts where an appropriate target can be identified, and so is an important avenue of research to pursue.
The importance of appropriate, and accurate animal models cannot be overstated. In 2006, a phase I clinical study was conducted to test a new therapeutic, a CD28 superagonist antibody known as TGN1412 that directly stimulates T cells.8 Despite a dose being given that was 500 times smaller than was found to be safe in animal studies, all six human volunteers ended up having an extremely life-threatening immunological reaction known as cytokine release syndrome (CRS). Since this disaster, the way we conduct clinical trials has changed. These changes particularly focus on the pre-clinical in vitro and animal model studies conducted, as inappropriate inference between the animal and human drug target was a key factor in the incorrect dose selection. Establishing a reliable method for breath analysis in mouse models can enable key pre-clinical experiments to be conducted that mechanistically link VOCs present in the breath to physiology, taking advantage of controlled lab settings to reduce variability and better experimental control. Coupling mouse models with human breath research studies is critical in understanding the ability to extrapolate results between systems, and boost the effectiveness of mouse research.
Figure 2. Use of limonene as an EVOC probe for monitoring of liver cirrhosis.
3. A schematic showing an intubated system for collecting breath from mice (image from Taylor et al 2024).
Novel Techniques for In Vivo Breath Models
Several papers have previously studied the breath of mice to establish them as a reliable model, and range from clean mice allowed to freely roam in ventilated cages, to mice in nose-only inhalation tubes.9,10,11 However, collecting VOCs from unrestrained animals in a cage can include VOCs from the skin, fur, urine, and faeces – rather than specifically the breath. A recently developed method can accurately analyse the VOCs in the breath of intubated mice using GC-MS, and reliably separate these from background contamination from inhaled air and breath sampling equipment quantitatively (Figure 3).12 Collection and analysis of breath from intubated mice can ensure that VOCs analysed are truly exclusively from the breath, and improving the signal-to-noise ratio can provide complementary data to advance the understanding of the mouse breath matrix.
Using the method developed in this study, it is also possible to simultaneously collect lung function data via the flexiVent used to mechanically control breathing and correlate this to breath VOC composition. This is of particular benefit in respiratory disease research, as VOCs detected in the breath of a respiratory disease mouse model can be monitored for changes alongside physiological lung function data in response to new drug treatments supporting the identification of mechanistic markers of drug activity, and physiology underpinning the disease for diagnostic markers.
Establishing Physiological Relationships Between the Gut Microbiome and Disease
A key area of study is the gut microbiome, which has been implicated in many important roles in health and disease development such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), metabolic dysfunctionassociated fatty liver disease (MAFLD), cardiometabolic diseases, and cancer. 13,14,15,16 Many microbial metabolites already have strong links to specific species, pathways, and processes, many of which happen to be volatile, and therefore could be monitored in the breath. Many VOCs in the breath in mice that are in common with human breath are also thought to originate from the gut microbiome, including Trimethylamine
(TMA), which has been associated with kidney disease and colorectal cancer.17,18
Studies have demonstrated a strong, significant, relationship between the composition of the intestinal microbiota and the VOCs that are detectable in exhaled breath.19 Intubated mouse models have recently been used to study the connection between the composition of the gut microbiome, and VOCs in breath.20 In this study, the breath VOC profile of 27 healthy children was compared to the microbiota species detectable in paired stool samples, and a gnotobiotic mouse model was developed to enable robust control of the gut microbiome to establish causal relationships between the gut microbial species and breath composition. Using this mouse model, the VOCs emitted from cultured gut microbes grown under anaerobic conditions were comparable to the VOCs detectable in the breath of mice colonised with the same bacterial strains. For example, E. coli, which is present in more than 90% of an individual’s gut flora, was associated with the breath VOCs benzothiazole, ethyl acetate, and octanal.20 Mechanistically linking a unique pattern of breath VOCs with specific infectioncausing pathogens, therefore allows non-invasive breath tests to be utilised for the diagnosis, monitoring, and transmission tracking of infectious diseases.
Bringing Breath Testing to Clinical Practice
Breath VOCs have been identified in association with a broad range of disease areas, and are positioned as promising diagnostic and monitoring biomarkers for next-generation non-invasive tools. The inherent non-invasiveness of the collection of breath and ease of repeat sampling in particular can be paired with probe-based methods for better diagnostic and monitoring tools that could detect diseases such as cancer earlier. However, the number of breath tests that have been successfully developed for clinical use remains limited, partially due to the lack of consistent methodologies across the breath research literature. Studying breath VOCs in mouse models is invaluable for advancing the field of breath research, providing a controlled environment for superior control of experimental variables, and a platform to gain pre-clinical mechanistic data
Figure
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linked to drug and disease mechanisms to support the funding of larger, more targeted human clinical trials. Mouse models in particular, can fast-track the progression of breath-based technology into the clinic, bridging the gap between pre-clinical models and human clinical trials, and ultimately expediting the identification, validation, and translation of breath tests for clinical use.
REFERENCES
1. Chou H, Godbeer L, Allsworth M, Boyle B, Ball ML. Progress and challenges of developing volatile metabolites from exhaled breath as a biomarker platform. Metabolomics. 2024 Jul 8;20(4):72.
2. Ferrandino G, Ricciardi F, Murgia A, Banda I, Manhota M, Ahmed Y, et al. Exogenous Volatile Organic Compound (EVOC®) Breath Testing Maximizes Classification Performance for Subjects with Cirrhosis and Reveals Signs of Portal Hypertension. Biomedicines. 2023 Nov;11(11):2957.
3. Miyazawa M, Shindo M, Shimada T. Metabolism of (+)- and (−)-Limonenes to Respective Carveols and Perillyl Alcohols by CYP2C9 and CYP2C19 in Human Liver Microsomes. Drug Metabolism and Disposition. 2002 May 1;30(5):602–7.
5. Owlstone Ltd. Diagnostic Accuracy Study for OWL-EVO1 as a Lung Cancer EVOC® Probe [Internet]. clinicaltrials.gov; 2024 Mar [cited 2024 Jan 1]. Report No.: NCT06193239. Available from: https:// clinicaltrials.gov/study/NCT06193239
6. Wang ST, Anahtar M, Kim DM, Samad TS, Zheng CM, Patel S, et al. Engineering Multiplexed Synthetic Breath Biomarkers as Diagnostic Probes [Internet]. bioRxiv; 2025 [cited 2025 Feb 6]. p. 2024.12.30.630769. Available from: https://www.biorxiv.org/conten t/10.1101/2024.12.30.630769v2
7. Labuschagne CF, Smith R, Kumar N, Allsworth M, Boyle B, Janes S, et al. Breath biopsy early detection of lung cancer using an EVOC probe targeting tumor-specific extracellular β-glucuronidase. Journal of Clinical Oncology [Internet]. 2022 Jun 2 [cited 2023 Jun 19]; Available from: https://ascopubs.org/doi/pdf/10.1200/JCO.2022.40.16_ suppl.2569?role=tab
8. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006 Sep 7;355(10):1018–28.
9. Kistler M, Szymczak W, Fedrigo M, Fiamoncini J, Höllriegl V, Hoeschen C, et al. Effects of diet-matrix on volatile organic compounds in breath in diet-induced obese mice. J Breath Res. 2014 Feb;8(1):016004.
10. Hintzen KFH, Smolinska A, Mommers AGR, Bouvy ND, Schooten FJ van, Lubbers T. Non-invasive breath collection in murine models
using a newly developed sampling device*. J Breath Res. 2022 Feb;16(2):027102.
11. Jonasson S, Magnusson R, Wingfors H, Gustafsson Å, Rankin G, Elfsmark L, et al. Potential exhaled breath biomarkers identified in chlorine-exposed mice. J Anal Toxicol. 2024 Mar 28;48(3):171–9.
12. Taylor A, Blum S, Ball M, Birch O, Chou H, Greenwood J, et al. Development of a new breath collection method for analyzing volatile organic compounds from intubated mouse models. Biology Methods and Protocols. 2024 Nov 14;bpae087.
13. Afzaal M, Saeed F, Shah YA, Hussain M, Rabail R, Socol CT, et al. Human gut microbiota in health and disease: Unveiling the relationship. Front Microbiol. 2022;13:999001.
14. Tripathi A, Debelius J, Brenner DA, Karin M, Loomba R, Schnabl B, et al. The gut-liver axis and the intersection with the microbiome. Nat Rev Gastroenterol Hepatol. 2018 Jul;15(7):397–411.
15. Aron-Wisnewsky J, Clément K. The gut microbiome, diet, and links to cardiometabolic and chronic disorders. Nat Rev Nephrol. 2016 Mar;12(3):169–81.
16. Kim S, Yin X, Prodhan MAI, Zhang X, Zhong Z, Kato I. Global Plasma Profiling for Colorectal Cancer-Associated Volatile Organic Compounds: a Proof-of-Principle Study. J Chromatogr Sci. 2019 May;57(5):385–96.
17. Bain MA, Fornasini G, Evans AM. Trimethylamine: metabolic, pharmacokinetic and safety aspects. Curr Drug Metab. 2005 Jun;6(3):227–40.
18. Grabowska-Polanowska B, Faber J, Skowron M, Miarka P, Pietrzycka A, Sliwka I, et al. Detection of potential chronic kidney disease markers in breath using gas chromatography with mass-spectral detection coupled with thermal desorption method. J Chromatogr A. 2013 Aug 2;1301:179–89.
19. Smolinska A, Tedjo DI, Blanchet L, Bodelier A, Pierik MJ, Masclee AAM, et al. Volatile metabolites in breath strongly correlate with gut microbiome in CD patients. Analytica Chimica Acta. 2018 Sep 26;1025:1–11.
20. Hernandez-Leyva AJ, Berna AZ, Liu Y, Rosen AL, Lint MA, Whiteside SA, et al. The breath volatilome is shaped by the gut microbiota. medRxiv. 2024 Aug 8;2024.08.02.24311413.
Madeleine Ball
Madeleine Ball is a scientific content writer at Owlstone Medical and is responsible for writing and editing written content for a variety of purposes, including peer-reviewed publications, informative articles, blogs, and more. She has an MPhil in Translational Biomedical Research and a PhD in Physiology, Development, and Neuroscience from the University of Cambridge, and joined Owlstone in early 2023.
Patient-Derived Xenografts Strengthen Mouse Clinical Trials in Oncology Research
Patient-Derived Xenograft (PDX) mouse models involve implanting human tumour tissues into immunodeficient mice, preserving the original tumour's genetic and histological characteristics to closely mimic human cancer biology. Mouse Clinical Trials (MCTs) leverage these models in large, statistically powered cohorts to evaluate drug efficacy, predict clinical outcomes, and identify potential biomarkers, enhancing the translational relevance of preclinical oncology studies.
Oncology drugs generally have higher failure rates than other therapies, emphasising the need for better drug development. A major challenge is the lack of clinical efficacy despite promising preclinical results. Oncology drugs fail most in Phase II, where patient efficacy is first tested, which highlights the urgent need for more reliable preclinical models.1
This gap may result from the type of model used and its accuracy. Traditional tumour models use immortalised cell lines grown in 2D and implanted in mice (Figure 1). These models help with early drug discovery, including pharmacology testing, by linking in vitro and in vivo data. However, growing tumour cells in 2D alters them, reducing their clinical relevance.2 Culturing heterogeneous tumour cells on plastic also favours uniform cell populations that no longer reflect the original tumour.
In contrast, PDX models are created by implanting patient tumours directly into immunocompromised mice without in vitro manipulation (Figure 1). Unlike cell line models, they avoid artificial selection pressures, making them more clinically relevant. PDX models are extensively characterised for pathology, growth, response to treatments, and genomic profiling using next-generation sequencing (NGS) technologies. Studies confirm that PDX models maintain the original tumour’s genomic integrity and closely match patient treatment responses.3,4,5 Retrospective analyses show that
PDX models reliably predict tumour responses, making them superior to cell line models.6 They also better reflect patient diversity and tumour heterogeneity, providing more accurate in vivo models and offering predictive preclinical data before clinical trials.
Extensive biobanks of PDX models, enriched with genomic, molecular, and phenotypic data – including tumour growth, standard treatment responses, histopathology, and patient information – support patient-focused research and clinical studies.7 By maintaining the key characteristics of patient tumours, PDX models provide more clinically relevant data, enhancing drug development and clinical translation. These models bridge the gap between preclinical research and clinical success, giving oncology drug developers a more reliable tool for accurate predictions and improved treatment outcomes.
Mouse Clinical Trials Using PDX Models
Historically, novel agents have been assessed for activity using a small number of xenograft models, with a large number of subjects in each arm. Instead, MCTs can assess a large number of models with a small number of subjects in each arm. MCTs serve as human surrogate trials by testing cohorts of PDX models in a randomised, controlled, and statistically powered setting. Each PDX model mirrors the pathology of its original patient, acting as a "patient avatar," while the combined cohort represents the diversity of the human population. This approach provides predictive data on responder and non-responder subgroups, helping refine clinical strategies and patient stratification.
In human trials, diverse patients with heterogeneous diseases receive the same treatment, and outcomes like Progression-Free Survival (PFS) or Overall Survival (OS) are measured. MCTs replicate this process using many distinct PDX models, each with a small number of subjects, ensuring that each model represents a unique patient and better simulates clinical trials (Figure 2). As a result, MCTs can be used to explore various hypotheses, such as targeting a specific mutation across multiple cancer types or assessing different mutations within
Figure 1: Traditional tumour cell line models vs PDX models
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3: Step-by-step guide for MCTs design
a single cancer type (Figure 4). A landmark study by Gao et al. at Novartis validated this method, testing over 60 treatment regimens in more than 250 models using a 1x1x1 design – one mouse per model per treatment group.8
Guide for MCTs
MCTs are widely used for preclinical oncology drug development to improve clinical trial translatability. As such, the design, execution, analysis and application of MCTs needs expertise and experience to ensure successful outcomes.9 Figure 3 outlines the steps in planning a MCT as well as analysing and interpreting the results.
There are multiple types of MCTs, each with distinct designs, utilities, and limitations, making the selection of an appropriate method crucial for any drug development programme (Figure 4). Guo et al. published an in-depth guide on the statistical framework for MCT, providing essential insights into optimal study design and analysis.10
The design requires key aspects of evaluation, including assessing drug effects on tumour size, adverse effects, and
survival, identifying outliers, applying Response Evaluation Criteria in Solid Tumours (RECIST) criteria to differentiate responders from non-responders, exploring and validating drug mechanisms of action, identifying genetic features associated with drug response or resistance, and analysing biomarkers. Deep learning techniques are employed to translate MCT findings into clinical trials, facilitating the identification of potential response biomarkers, patient stratification for precision medicine, and providing strategic guidance for future clinical trials, drug positioning or repositioning, and drug combination strategies.12
The MCT design allows for large studies across multiple tumour types in 1+1 format while limiting the number of models used. The study structure can be extended based on the number of arms required, such as 1+1+1. An MCT with n>1 per group can also be designed to accommodate additional n + n MCTs. The foundation of any MCT lies in enrolling PDXs from different patients, ensuring each model represents an individual case and collectively captures the heterogeneity of the clinical population. Each model undergoes the designated treatment, and relevant outcomes are measured.
Figure 2. A representative PDX MCT design
Figure
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Figure 4. Design of a MCT based on study objectives
In a standard 3+3 MCT design (depicted in Figure 2), each model includes a control or comparator arm with three mice per group from the same PDX. The comparator arm typically consists of a standard-of-care (SoC) therapy, while the treatment arm involves SoC combined with a novel agent, simulating Phase II trials. The presence of a comparator arm facilitates response assessment. Additionally, a 3+3 design can be employed for endpoint sampling, PK/PD analysis, evaluation of intra- and inter-tumour heterogeneity, or enhancing statistical accuracy when model availability is limited.
For instance, achieving 80% statistical power requires a minimum of 28 PDX models in a 1+1 design, whereas a 3+3 design necessitates only 11 PDX models, or three models per group. The greater the number of PDX models, the more comprehensive the representation of inter-tumour heterogeneity and the greater the potential for biomarker discovery.10,11 Furthermore, fewer PDX models are required when a therapy exhibits a potent response. For example, in a 3+3 design with a 0.05 significance level, 40 models are needed to achieve 10% efficacy, whereas only five PDXs are necessary to reach 30% efficacy.10
Indication-Driven MCT
An indication-driven MCT evaluates whether an agent works in only one specific type of cancer, which may be driven by a range of different mutations (Figure 4). This type of MCT provides a framework for biomarker discovery within one disease indication, with responders and non-responders within the single cancer type identified. Another benefit of an indicationdriven MCT, is that it allows the assessment of the dependence
of your agent on a specific target in the case of target-negative (or target-low) PDXs. The drawbacks of an indication-driven MCT are that, inherent in the study design, it does not allow cross-indication exploration. This approach might also require a higher number of models, or of animals, if target incidence is low.
Target-Driven MCT
A target-driven MCT is independent of cancer type. This approach provides robust target validation - evaluating whether a target/common genetic mutation is present across a range of cancer types, whether the target is engaged, and if there is a downstream effect (Figure 4). Another benefit of a target-driven MCT is that it provides a framework for exploration of resistance mechanisms in the case of target-positive PDX models where expected activity is not seen. One drawback of this MCT method is it is not likely to support robust indication selection, given that the target of interest may be across multiple different cancer types.
MCTs Facilitate Biomarker Discovery
MCTs offer added value beyond efficacy readouts by enabling sample collection for biomarker identification. By utilising patient-derived material, MCTs allow classification of treated samples into responder and non-responder populations, facilitating genomic and proteomic comparisons that provide insights into drug mechanisms of action and potential resistance phenotypes.
Ready-to-use immunoassays and standard biomarker analyses across various tissue types streamline this process, along with
flexible multi-omics methodologies and advanced technologies tailored to specific research and drug development needs. MCTs also leverage patient-to-patient variability in preclinical settings, mirroring the heterogeneity observed in clinical practice. Identifying a biomarker during drug development can significantly enhance the likelihood of clinical success.
Figure 5 illustrates the probability of investigational agent success in clinical trials across all indications, comparing trials with and without biomarker selection. Studies incorporating biomarkers demonstrate a higher success rate, particularly during the challenging transition from Phase II to Phase III, where the inclusion of a biomarker nearly doubles the chances of a successful trial.
MCTs serve as a valuable tool for retrospective biomarker analysis, as PDX models capture the heterogeneity observed in patient populations. Data analysis from these models can generate robust prospective inclusion and exclusion criteria for clinical trials, improving trial planning and control to enhance success rates. Additionally, MCTs offer a cost-effective approach for exploring translational hypotheses.
Summary
Enhancing the efficiency and translation of preclinical oncology research is crucial for reducing drug failure rates by improving clinical prediction, patient stratification, and biomarker discovery. PDX models preserve the genomic integrity and heterogeneity of the original patient tumour and closely mimic clinical outcomes. Properly designed and executed PDX MCTs offer critical insights into clinical responses. Data analysis can offer valuable prospective inclusion/exclusion criteria for clinical trials, enabling better planning and control to potentially enhance success rates. PDX MCTs are changing the way preclinical data is viewed, providing a cost-effective approach to explore translational hypotheses and how compounds are progressed to clinical trials.
REFERENCES
1. BIO Industry Analysis. (2021). Clinical Development Success Rates 2011–2020.
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2. Gillet, et al. (2013). The Clinical Relevance of Cancer Cell Lines. JNCI: Journal of the National Cancer Institute, 105(7), 452-458.
3. Hidalgo, et al. (2014). Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discovery, 4, 998-1013.
4. Bertotti, et al. (2011). A molecularly annotated platform of patientderived xenografts (“xenopatients”) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer. Cancer Discovery, 1, 508-523.
5. Corcoran, et al. (2015). Combined BRAF and MEK inhibition with Dabrafenib and Trametinib in BRAF V600-mutant colorectal cancer. Journal of Clinical Oncology, 33(34), 4023 4031.
6. Guo, et al. (2016). Molecular pathology of patient tumors, patientderived xenografts and cancer cell lines. Cancer Research, 76(16), 4619-4626.
7. Crown Bioscience. (n.d.). Databases. https://www.crownbio.com/ databases, visited on 19 Feb 2025.
8. Gao, et al. (2015). High-throughput screening using patient derived tumor xenografts to predict clinical trial drug response. Nature Medicine, 21(11), 1318-1325.
9. Xie, Z. and Mao, B., (2024) How to Leverage Bioinformatics to Optimize Mouse Clinical Trials: From Study Design to Biomarker Discovery – Crown Bioscience. https://blog.crownbio.com/how-toleverage-bioformatics-to-optimize-mouse-clinical-trials, visited on 19 Feb 2025.
10. Guo, et al. (2019). The design, analysis and application of mouse clinical trials in oncology drug development. BMC Cancer, 19(1), 718.
11. Zhang, L., et al. (2013). A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. Scientific Reports, 3, 2992.
12. Mao, et al. (2023). Statistical Assessment of Drug Synergy from In Vivo Combination Studies Using Mouse Tumor Models. Cancer Research Communications, 3(10), 2146-2157.
As Executive Director of Integrated Solutions, Rajendra currently leads the cross-disciplinary drug discovery solutions within Crown Bioscience Inc. She has > 20 years of oncology research experience with a PhD in Molecular Pharmacology, holding positions such as Assistant Professor in Preclinical Oncology (University of Nottingham), COO/co-founder of PRECOS Ltd, CSO/General Manager (Crown Bioscience UK) and Global Head of Scientific Communications.
Email: rajendra.kumari@crownbio.com
Rajendra Kumari
Figure 5: Clinical trial success with or without biomarkers
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Leveraging Liquid Biopsy to Advance Metastatic Cancer Care
Liquid Biopsy: A Non-Invasive Approach to Tumour Profiling
Metastasis is responsible for 90% of cancer-related deaths, yet most treatments focus on primary disease. Despite its critical role in cancer progression, metastasis remains largely unaddressed therapeutically, with limited targeted interventions and no effective prevention strategies. Understanding metastatic spread is essential for developing early interventions.1,2
Liquid biopsy is a non-invasive method for real-time tumour monitoring. Compared to tissue biopsy, it allows serial sampling for disease monitoring and treatment decisions.3 In metastatic cancer, liquid biopsy is crucial for tracking clonal evolution and resistance mechanisms over time.4,5 Beyond metastasis, it holds promise for early cancer detection, minimal residual disease (MRD) monitoring, and refining treatment strategies to reduce relapse risk.6,7,8,9 Circulating tumour DNA (ctDNA), a key component of liquid biopsy, has rapidly emerged as a non-invasive biomarker for cancer diagnosis, monitoring, and treatment guidance. It provides real-time insights into tumour mutations, supporting personalised treatment strategies.10 Despite its promise and the ease of sample preparation and scalability, ctDNA is yet to reach its full potential in cancer care.
Current use of ctDNA in Clinical Applications
Due to its ease of collection and scalability of analysis, ctDNA has become the predominant analyte in liquid biopsies in clinical practice. Next-generation sequencing (NGS) technologies, traditionally applied to tumour tissue, can also analyse ctDNA from a simple blood draw, facilitating the identification of oncogenic drivers and resistance mechanisms while enabling repeated monitoring.
Currently ctDNA is used in two key applications: real-time disease monitoring and treatment guidance. In monitoring, ctDNA enables tracking of treatment response and emerging resistance mutations.11,12 It is also used for MRD detection after surgical resection, helping tailor adjuvant therapies and identify patients at risk of recurrence particularly in lung and colorectal cancer.13,14
For treatment guidance, ctDNA informs therapy decisions by identifying druggable and resistance mutations. In EGFR-mutant non-small cell lung cancer (NSCLC) ctDNA facilitates early detection of resistance to tyrosine kinase inhibitors, allowing adjustments in treatment plans.15 Additionally, ctDNA dynamics can guide clinical decisions on continuation, escalation, or de-escalation of immunotherapy, potentially improving treatment outcomes.16,17 By detecting resistance mutations quickly ctDNA enables adjustments in treatment regimens, ensuring patients receive the most appropriate care.
FDA-Approved ctDNA-Based Liquid Biopsy Tests
FDA approval of liquid biopsy tests like Guardant360® CDx and FoundationOne® Liquid CDx has accelerated ctDNA use.
Guardant360® CDx, the first FDA-approved comprehensive liquid biopsy test, uses NGS to analyse 74 cancer-related genes from plasma samples. It is primarily used for advanced cancer patients unable to undergo traditional biopsies, helping identify actionable mutations such as EGFR in NSCLC and ESR1 in breast cancer.18 Similarly, FoundationOne® Liquid CDx employs high-throughput hybridisation-based capture technology to detect genetic alterations in 311 genes. In addition to common mutations, it identifies complex rearrangements, such as BRCA1/ BRCA2 fusions. Like Guardant360®, it serves as a companion diagnostic for targeted therapies in advanced cancers.19
Emerging ctDNA Applications in Early Cancer Detection
While ctDNA is widely used in advanced cancer management, its role in early detection remains under investigation. Tumours in early stages, shed minimal ctDNA into the bloodstream making detection challenging.
The Galleri® Test (GRAIL) aims to address this limitation by leveraging bisulfite sequencing and AI-driven methylation profiling to detect over 50 cancers. This multi-cancer early detection (MCED) approach aims to detect cancers before symptoms arise, providing an opportunity for earlier intervention and improved survival rates. However, concerns about false positives, detection sensitivity at early stages, and lack of definitive diagnostic capability limit its current clinical utility.20,21
Freenome’s multi-omics strategy, for example, combines ctDNA, cell-free microRNA (cfmiRNA), and circulating proteins, to improve colorectal cancer detection and potentially other cancers.22 AI-driven analysis of genomic, methylomic, transcriptomic, and proteomic data offers a broader view of tumour biology. Ongoing trials, such as PREEMPT CRC (colorectal cancer screening) and PROACT LUNG (early lung cancer detection), will further evaluate this approach.
Limitations of ctDNA
Despite its promise as a biomarker for cancer detection and monitoring, several limitations hinder the widespread clinical implementation of ctDNA-based assays.
Limited Insight into Tumour Heterogeneity and Spatial Resolution
A primary challenge of ctDNA is its inability to fully capture tumour heterogeneity or provide spatial resolution of mutation origin. Derived from necrotic and apoptotic cells, ctDNA offers a fragmented snapshot of the tumour genetic landscape rather than a comprehensive view of active cancer cell behaviour and adaptation. This lack of spatial and temporal resolution means ctDNA may not reflect clonal evolution or the diversity of mutations across tumour areas. Bulk analysis of genetic fragments
limits the assessment of co-occurring mutations within the same clone, reducing its predictive power for tumour behaviour and treatment response. Moreover, ctDNA cannot provide insights into metastatic live cells in circulation or functional markers like gene expression and proteins, crucial for understanding the tumour functional state. A multi-analyte strategy, combining circulating tumour cells (CTCs) with ctDNA, significantly strengthens liquid biopsy capabilities.
Low ctDNA Shedding
Cancers with low vascularisation or minimal cell turnover may shed insufficient ctDNA for detection. For instance, poorly vascularised brain tumours release minimal ctDNA into the bloodstream, increasing the risks of false negatives. Similarly, slow-growing tumours with low metabolic activity may not release detectable ctDNA levels, reducing the utility of liquid biopsy assays.23
Challenges in Early Detection and MRD Monitoring
Early-stage cancers and MRD pose specific challenges for ctDNA analysis. Early-stage tumours may release ctDNA at levels below assay detection thresholds, leading to false negatives. Likewise, distinguishing residual tumour DNA from normal background cfDNA remains difficult in patients undergoing treatment. Overcoming these barriers will require more sensitive technologies capable of detecting ultra-low ctDNA concentrations in patients with low-burden disease.23
Incomplete Genomic Profiling for Metastatic Progression
While ctDNA is effective for detecting known mutations, it does not capture transcriptomic alterations, protein expression, or other functional markers that play key roles in metastasis and therapeutic resistance. This limitation restricts the ability of ctDNA assays to provide a comprehensive view of tumour progression, particularly in metastatic disease.
Combining ctDNA and CTCs for Comprehensive Tumour Profiling
Although ctDNA-based liquid biopsy tests have begun to revolutionise advanced cancer treatment, challenges in capturing tumour heterogeneity, early detection, and MRD monitoring persist. Overcoming these hurdles with advanced technologies and complementary multi-analyte approaches is essential to fully harness liquid biopsy potential. Recognising the limitations of ctDNA alone, a multi-analyte strategy looking at the “circulome” incorporating CTCs alongside ctDNA and other analytes will further enhance liquid biopsy capabilities. While ctDNA reveals genetic mutations and methylation changes, CTCs provide critical insights into mRNA expression and protein markers, offering a more comprehensive view of tumour progression.21,24
CTCs in Research and Clinical Applications
CTCs are rare, intact cancer cells shed into the bloodstream, providing a more comprehensive biological profile than cell-free DNA. Unlike ctDNA, which consists of fragmented genetic material released from dying cells, CTCs reflect viable, potentially metastatic tumour cells. Their intact structure enables morphological assessment, while transcriptomic and proteomic analyses offer insights into gene expression, signalling pathways, and therapeutic targets. This multidimensional approach allows for a deeper analysis of tumour heterogeneity, clonal evolution
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and treatment resistance, providing valuable information beyond genetic mutations alone.25
However, their relative scarcity – sometimes only a few cells per tube of blood in metastatic disease – makes detection challenging, requiring advanced technologies to enrich them from the far more abundant blood cells. Various technologies have been developed to isolate and analyse CTCs, based on their unique biological and physical properties. CellSearch (FDA approved), established the clinical relevance of CTCs, demonstrating that higher CTCs counts correlate with shorter progression-free and overall survival, while lower counts indicate a more favourable prognosis in metastatic breast, prostate, and colorectal cancer.26,27,28
In contrast, newer label-free, microfluidic-based approaches are expanding CTC applications beyond enumeration to functional analysis. Unlike label-based methods, microfluidic approaches isolate CTCs based on their physical properties (size, deformability), preserving their native state for downstream genomic, transcriptomic, and proteomic profiling. For instance, platforms such as Vortex VTX-1® and Angle Parsortix® enable unbiased CTC enrichment without relying on surface markers, making them particularly valuable for studying metastasis and therapy resistance.29,30,31 Notably, Parsortix® has received regulatory approval for CTC enrichment in metastatic breast cancer.32,33
With advancements in label-free CTC capture technologies, research has expanded beyond detection and enumeration. In the era of precision medicine, identifying actionable druggable targets is crucial. CTCs provide valuable insights into tumour biology. However, fully harnessing their potential requires integrating functional studies with multi-omics approaches, including genomic, transcriptomic, and proteomic profiling. Such strategies can uncover therapeutic targets to disrupt CTC-driven disease progression. Single-cell multi-omics technologies have already revealed key molecular traits of treatment-resistant and metastasis-prone CTCs.34
Moreover, recent proof-of-concept experiments using multi-dimensional morphology analysis, have shown the potential to study tumour heterogeneity at single-cell resolution. By leveraging AI and microfluidics, this approach enables high-resolution profiling of unlabelled cells based on morphological features, offering insights beyond traditional biomarker-based approaches. This approach has demonstrated its potential in distinguishing malignant cells, identifying drug-resistant populations, and differentiating between cancer types, highlighting its potential for CTC analysis and furthering our understanding of tumour biology.35,36
CTC Clusters:
A Window into Metastasis and Cancer Progression
While ctDNA reflects tumour burden and provides valuable genomic insights, it lacks spatial, phenotypic, and functional context, making it insufficient for understanding tumour cell behaviour. In contrast, analysis of whole-cell CTC – particularly of clusters – offers several advantages.
CTC clusters are groups of CTCs that travel together in the bloodstream. They can be homotypic (composed of cancer
Preclinical Subsection
cells only) or heterotypic (containing both malignant and non-malignant cells such as stromal or immune cells).37 These clusters possess distinct biological properties that enhance survival in circulation and promote metastasis. They exhibit stem cell-like properties, including tumour-initiating capacity and proliferation, conferred by increased cell-cell junction activity. Compared to single CTCs, clusters show significantly higher metastatic potential and are associated with poor prognosis in breast, colon, and non-small-cell lung cancer patients.37,38,39,40
Gkountela et al. showed that targeting CTC clusters with FDA-approved Na+/K+-ATPase inhibitors can break them apart into single cells, leading to DNA methylation changes and reduced metastasis in a xenograft model.38 In a follow-up trial, first-in-human data from metastatic breast cancer show that digoxin reduces CTC cluster size and induces molecular changes that weaken metastatic traits, including the downregulation of cell cycle and adhesion genes. 41 These findings underscore the translational significance of CTC clusters, linking their biological role to a therapeutic opportunity that could reshape metastasis management. These findings highlight the importance of studying CTC clusters. Their functional properties provide crucial insights into the mechanisms driving metastasis and treatment failure. Targeting CTC clusters offers a promising therapeutic strategy to intervene in metastatic progression early, potentially preventing the formation of clinically detectable lesions. By advancing CTC cluster analysis as both a biomarker and a therapeutic target, we move toward a more refined, predictive, and actionable liquid biopsy strategy that extends beyond enumeration and genomic profiling.
Clinical implementation of CTC-Based Liquid Biopsy Tests
The integration of CTCs in the clinical practice has been limited, however several LDT are available to patient and their clinicians. One such example is the Oncotype DX AR-V7 Nucleus Detect test, a CTC-based laboratory-developed test (LDT) developed by Epic Sciences.42,43 This CLIA-certified test identifies patients with metastatic castration-resistant prostate cancer (mCRPC) who are resistant to androgen receptor signaling inhibitors (ARSi). As a predictive and prognostic test, it helps determine whether patients are unlikely to benefit from further ARSi therapy and should consider alternative treatments such as taxane chemotherapy.
In a different approach for metastatic settings, DefineMBC is a comprehensive blood-based LDT that combines CTC and ctDNA analysis from a single draw to profile metastatic breast cancer.44 It detects CTCs, quantifies ER and HER2 protein expression, assesses ERBB2 gene amplification via single-cell sequencing, and analyses ctDNA for Microsatellite Instability (MSI), Tumour Mutational Burden (TMB), and genomic alterations in 56 relevant genes using NGS to guide treatment decisions.
As research into CTC profiling advances, the clinical application of these technologies could become increasingly valuable for treatment response monitoring and resistance detection. By leveraging the insights gained from CTC-based tests, clinicians will be able to make more informed, individualised treatment choices that can significantly impact patient outcomes.
The Future of CTCs and Liquid Biopsy
Advancements in single-cell and rare-cell capture technologies like Vortex VTX-1®, Angle Parstortix®, and CellSearch® are paving the way for clinical adoption of whole-cell analysis. Ongoing research into CTCs aims to enhance personalised cancer care through deeper biological insights at the single-cell level. AI-driven analysis and multi-omic integration hold the potential to revolutionise cancer detection, monitoring, and treatment, leading to more individualised therapies and improved patient outcomes.
The clinical use of CTC-based diagnostics is expanding, with applications in early cancer detection, treatment monitoring, and recurrence prediction. However, while significant progress has been made, certain applications – especially early detection and MRD monitoring – still present unique challenges.
CTC levels in the blood are particularly low in early stage and MRD settings compared to metastatic disease, and CTC heterogeneity can further complicate detection. Overcoming these limitations will require improvement in both the sensitivity and specificity of current detection methods.8,45 Addressing these challenges is essential for unlocking the full potential of CTC as reliable indicator of cancer recurrence, as seen in recent studies like that in early-stage lung adenocarcinoma.46 These challenges and opportunities emphasise the need for ongoing research and innovation as reviewed by Pantel.8,45
At the same time, ongoing technological advancements and decreasing costs in rare cell capture and analysis are gradually improving the accessibility of CTC diagnostics. As sensitivity and reliability continue to improve, the market for CTC diagnostic is becoming more accessible for broader clinical adoption. This trend is fostering interest from both biotech companies and healthcare providers, creating a competitive landscape ripe for innovation.47 One notable example is the FirstSight™ Blood Test (CellMax Life) for early colorectal cancer (CRC) and precancerous advanced adenomas, which combines cfDNA (methylated septin 9 mutation profiling via NGS) with CTC counts and AI-driven analysis, showcasing the power of multi-omic liquid biopsy.48
CTC-Derived Data and AI-Enhanced Diagnostics
When combined with AI-driven analysis, CTCs enhance liquid biopsy capabilities. Machine learning models are trained to detect subtle morphological and molecular patterns in CTCs, linking them to disease progression and treatment response. Platforms like DeepCell and AxonDx integrate AI with high-resolution imaging to refine single-cell morphology analysis, strengthening multi-omic approaches for precise, individualised insights.35,36,49
Conclusion
Looking ahead, the future of CTCs in liquid biopsy lies in multi-omics; integrating and combining multiple analytes such as CTCs, ctDNA, miRNA, exosomes and others to provide a comprehensive molecular and cellular landscape of cancer.50 By leveraging AI-driven analysis and these multi-omic approaches, CTC-based innovations hold the potential to revolutionise how we detect, monitor, and treat cancer. The future vision points to deliver truly individualised therapies, ultimately improving patient outcomes and transforming metastatic cancer care.
Preclinical Subsection
A B
Figure 1. Functional Properties of CTC Clusters in Metastasis and Treatment Resistance, CTC clusters provide insights into metastasis mechanisms and treatment resistance, highlighting their potential as biomarkers and therapeutic targets for early intervention. (A) Representative images of single CTCs and homotypic CTC clusters isolated from a metastatic triple-negative breast cancer (mTNBC) patient. The majority (~80%) of isolated CTCs formed homotypic clusters, consistent with the aggressive nature of the patient’s cancer. (B) Disease progression and treatment timeline of the mTNBC patient.
Figure 1. Functional Properties of CTC Clusters in Metastasis and Treatment Resistance CTC clusters provide insights into metastasis mechanisms and treatment resistance, highlighting their potential as biomarkers and therapeutic targets for early intervention.
(A) Representative images of single CTCs and homotypic CTC clusters isolated from a metastatic triple-negative breast cancer (mTNBC) patient. The majority (~80%) of isolated CTCs formed homotypic clusters, consistent with the aggressive nature of the patient’s cancer. (B) Disease progression and Homotypic
doi:10.1001/jamaoncol.2022.4457
11. Klocker EV, Hasenleithner S, Bartsch R, et al. Clinical applications of next-generation sequencing-based ctDNA analyses in breast cancer: defining treatment targets and dynamic changes during disease progression. Mol Oncol. Published online June 12, 2024. doi:10.1002/1878-0261.13671
Figure 2. CTCs as a Platform for Functional Analysis of Metastasis and Drug Response
Circulating tumor cells (CTCs) generate tubulin-based protrusions, known as microtentacles (McTNs), which promote reattachment, retention at distant sites during metastasis, and tumor cell cluster formation. A microfluidic cell tethering chip (TetherChip technology) enabled functional analysis of metastatic phenotypes in CTCs. Computational tracking of McTNs (white arrows) allowed drug response measurements within one hour of CTC isolation from blood, enabling ultra-rapid tumor cell phenotyping. Adapted from Thompson KN et al., 2022.51
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Dr. Corinne Renier
Dr. Corinne Renier, Director of R&D at Vortex Biosciences, is an accomplished scientific leader with over 25 years of experience in academia and biotech startups. She holds a Ph.D. in Molecular Genetics from the University of Rennes and furthered her training in pharmacogenomics and cancer biology as a postdoctoral fellow at Stanford School of Medicine. Dr. Renier leads the development of microfluidicbased platforms for isolating and characterising Circulating Tumour Cells (CTCs), integrating innovative technologies to advance cancer detection and personalised medicine, with the goal of improving patient outcomes.
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