Type: Article

Linking the gap from development to robust manufacturing

The complex journey of bringing a biologic to market – part 3

It is a critical shift for companies when transitioning from early development to clinical production, where you transfer from proving a concept in the lab to demonstrating that it can be reliably manufactured at scale. At this point, following preclinical work and often Phase I trials, the process should be technically defined, with early data confirming feasibility and product consistency. The focus now turns to refining that process within a manufacturing environment, ensuring it can consistently deliver high-quality drug substance under controlled conditions.

To achieve this, two key tools are essential: the qualification and/or validation of analytical methods and the execution of a non-GMP engineering batch. Together, they validate both the analytical framework and the manufacturing process on a technical scale, laying the groundwork for full-scale GMP production. Don’t underestimate the time needed, there are seldom such a thing as a “ready to go -analysis” or method. Robust analytical strategies ensure that product safety and efficacy can be accurately assessed, while the engineering batch offers a practical, hands-on test of the process in a real-world setting.

This phase demands close collaboration between sponsor and CDMO, aligning expectations, addressing potential risks, and preparing for the regulatory rigor of clinical manufacturing. It is also time-intensive, so plan accordingly and reserve calendar time in advance.

Aligning capabilities: The CDMO’s role

For CDMOs like NorthX Biologics, this phase is where collaboration deepens. It’s not just about executing a process, it’s about understanding it, challenging it, and ensuring it’s robust enough to move forward. Three focus areas anchor this phase:

1. Implementing and Qualifying Analytical Methods

During this phase, methods are transferred, verified, or developed according to a tailored transfer plan. Compendial methods are confirmed, existing validations are utilized where possible, and microbial safety testing ensures sterility. A master validation plan aligns workflows, while validated methods form the analytical foundation for drug substance release and future GMP manufacturing.

2. Running the Engineering Batch

A non-GMP engineering batch, typically at 250L scale, is executed under GMP-like conditions to simulate full production. This technical run tests the full upstream and downstream process, providing early insight into process performance and product quality. It helps to identify potential bottlenecks, refine operations, and generate data using the intended GMP analytical methods. The engineering batch serves as a critical learning step, demonstrating both process and analytics in a real-world manufacturing setting.

3. Documentation and Transparency

Clear and structured documentation is essential throughout this phase. From the initial study plan and raw material testing to batch execution and results, every step is recorded systematically. Validation and quality control data are compiled into comprehensive reports, with method validation outcomes formally documented. This ensures both internal alignment and preparedness for regulatory review.  A CDMO’s success is intrinsically tied to the sponsor’s success, and this shared objective should foster transparency in all communication.

production in action

Lipum insights: Driving alignment and readiness

From Lipum’s perspective, this phase offers an opportunity to align current analytical expectations with earlier development work, building on insights from previous method validations. The engineering batch serves as a key checkpoint, providing valuable data on process performance and product quality that informs both ongoing analytical efforts and process refinement. Material generated from the batch supports continued validation activities and helps strengthen the overall control strategy. Particular focus is placed on understanding and mitigating risks associated with critical process parameters, using analytics to ensure consistency and reliability. Additionally, one remains mindful of any regulatory or stakeholder requirements that need to be addressed at this stage to support future clinical and commercial milestones.

Looking forward: Building for long-term success

The qualifications of analytical methods and execution of a non-GMP engineering batch aren’t just procedural milestones, they’re strategic enablers. Done right, they reduce risk, refine the path to GMP, and create a stronger foundation for regulatory success.

For Lipum and NorthX Biologics, this phase represents a shift from potential to proof: the moment where science meets scale. Through close collaboration, thoughtful planning, and transparent execution, the development-to-manufacturing transition becomes not just a step forward, but a springboard toward commercialization.

About this series
This series in six parts explores key aspects and aims to provide valuable insights into the complexities of the biologic drug manufacturing journey – from early process development to commercial production – through the perspectives of both the developer and the CDMO, offering a transparent discussion on the realities of bringing a biologic to market.

Read part 1 here: The importance of collaboration
Read part 2 here: Bridging innovation and execution: The critical role of technology transfer

Viral vaccine manufacturing for clinical trials

Viral vaccines are entering a new era

Vaccines have played a pivotal role in controlling infectious diseases for more than a century. Often regarded as one of the most impactful public health innovations, they are estimated to have saved over 150 million lives in the past 50 years (1). From smallpox and polio to the rapid development of COVID-19 vaccines, the field continues to evolve through advances in technology, manufacturing, and regulation.

Today, viral vaccine development is entering a new era, marked by diverse platforms such as viral vectors, recombinant subunits, and nucleotide-based technologies—including mRNA and DNA vaccines. These platforms offer faster development timelines and more targeted immune responses, supporting both pandemic preparedness and the treatment of rare diseases.

While novel modalities receive significant attention, traditional viral vaccines—often based on live attenuated or inactivated viruses—remain vital. With well-established safety profiles and robust immune activation, they are essential for indications where long-term stability and proven efficacy are critical. For pathogens such as polio, measles, and rabies, these vaccines continue to serve as gold standards. In the case of measles, for example, two doses of an attenuated vaccine provide up to 97% protection in unvaccinated individuals (2).

Supporting public health efforts and future pandemic response requires maintaining the infrastructure, knowledge, and capacity for viral vaccine manufacturing, across both established and emerging technologies.

Moving virus vaccines into clinical testing

Translating early-stage viral vaccine concepts into clinical-grade products involves complex technical and regulatory steps. Laboratory-scale methods often use research-grade materials and manual handling, which must be adapted to GMP standards. This includes qualifying raw materials, validating analytical methods, scaling up bioreactors, and implementing aseptic processing.

For example, virus propagation may shift from T-flasks in preclinical work to fixed-bed or suspension bioreactors at scale—each requiring different process controls and comparability assessments for regulatory compliance with EMA and FDA standards. Early CMC planning is critical to ensure successful clinical entry.

Robust quality systems, tailored to the characteristics of early-stage viral vaccines, must be in place to meet GMP requirements. This includes specialized expertise distinct from that required during discovery phases.

In many cases, biosafety-level (BSL) classified facilities are needed. NorthX Biologics have flexible capabilities for handling of virus strains up to BSL-3. For novel pathogens a conservative “+1” biosafety approach—applying higher-level protocols than officially required—is sometimes used until further safety data are available.

Balancing biosafety requirements with GMP demands presents operational challenges. Systems must simultaneously prevent product contamination and protect personnel from infectious agents—necessitating specialized equipment, validated procedures, and experienced teams.

Viral vaccine manufacturing: A multi-step process

Viral vaccine production involves a series of interconnected steps, beginning with the generation of cell and virus seed banks. These materials must meet GMP standards and align with ICH Q5A and Q5D guidelines.

A Master Cell Bank (MCB), derived from a characterized clonal population, serves as the foundation for a Working Cell Bank (WCB), which is used in vaccine production. Common cell lines include Vero, HEK293, and MDCK, with selection based on virus type, yield requirements, and regulatory considerations. Once established, these banks support scalable vaccine platforms.

Similarly, a Master Virus Seed (MVS) and Working Virus Seed (WVS) are developed for each specific pathogen. These virus seeds undergo rigorous characterization to ensure quality and consistency.

With seed banks in place, vaccine production can begin. The upstream process includes cell expansion, viral infection, and harvest. Culture conditions are tightly controlled, and the virus is harvested following replication. Downstream processing includes cell lysis or filtration, followed by purification steps such as chromatography and ultrafiltration to remove impurities.

For inactivated vaccines, the virus is rendered non-infectious using validated chemical or heat-based methods. Analytical assays confirm successful inactivation. Failures in this step have had historical consequences—such as the Cutter incident in 1955, when incomplete inactivation of poliovirus caused multiple cases of polio.

Advances in synthetic biology now allow for genetically attenuated viruses that retain immunogenicity without requiring inactivation. While traditional approaches remain in use, platforms such as subunit and mRNA vaccines are gaining prominence due to their scalability and safety profiles.

Final formulation includes combining the purified virus with stabilizers, adjuvants, or preservatives to ensure product stability and immunogenicity. Drug substance is aseptically filled into final containers under controlled conditions. Comprehensive in-process controls and final release tests—such as sterility, potency, and stability testing—ensure the vaccine meets all regulatory specifications for clinical or commercial use.

NorthX Biologics: Supporting the development of novel vaccines

NorthX Biologics provides end-to-end support for viral vaccine development, from early-stage process design to GMP manufacturing of clinical-grade drug substance and drug product. The company has contributed to multiple global initiatives, including projects with the World Health Organization and the Bill & Melinda Gates Foundation.

The company’s virus vaccine center of excellence, located in Stockholm near the Karolinska Institute, houses expert teams with experience in various modalities—including live attenuated viruses, recombinant proteins, whole-cell vaccines, and nucleotide-based platforms. During the COVID-19 pandemic, NorthX manufactured virus seed stocks and produced drug substance under BSL-3 conditions, including scale-up from adherent cultures to fixed-bed bioreactors.

The team has also supported clinical-stage arenavirus-based cancer vaccine programs in both Europe and the United States, manufacturing and releasing multiple GMP batches for ongoing trials.

Advancing safe and effective vaccines

Manufacturing viral vaccines for clinical trials involves unique challenges, particularly in balancing biosafety, regulatory compliance, and production scalability. Success requires early alignment between discovery, process development, and GMP manufacturing.

By drawing on its proven track record and cross-functional expertise, NorthX Biologics supports the advancement of safe and effective vaccines targeting both common and emerging infectious diseases. As global demand continues to grow, the company remains committed to enabling innovation in vaccine development and contributing to public health worldwide.

References:
  1. https://www.who.int/news/item/24-04-2024-global-immunization-efforts-have-saved-at-least-154-million-lives-over-the-past-50-years/
  2. https://www.cdc.gov/ncird/downloads/immunization-highlights.pdf

Automation advances for cell harvesting in biomanufacturing

In a recent article in BioPharm International, NorthX Biologics’ Agnes Zimmer and Erica Johansson explore how new technologies are revolutionizing cell harvesting. They discuss the shift from traditional, labor-intensive methods to automated systems like single-use platforms with real-time monitoring, which enhance efficiency and reduce contamination risks.

Although automation is increasingly adopted in biomanufacturing, challenges remain in implementing it at the cell harvesting step.

For those interested in the future of biomanufacturing, this article offers valuable insights.

Read the full article here.

Bridging innovation and execution: The critical role of technology transfer

The complex journey of bringing a biologic to market – part 2

The journey from early development to commercial production in biologics is paved with complexity and few phases are more pivotal than technology transfer. This step marks the transition of early-stage development to large-scale manufacturing by Good Manufacturing Practice (GMP), and its success is vital for ensuring product consistency, regulatory compliance, and commercial readiness.

For biologic drugs, where minor deviations in process conditions can significantly affect product quality, technology transfer is not just administrative. It’s a collaborative, highly technical process involving the structured exchange of data, methods, analysis, materials, and know-how between the development site and the manufacturing partner.

From development to delivery: The CDMO’s role

NorthX Biologics is a biopharmaceutical CDMO with a rich heritage in Good Manufacturing Practice (GMP) since 1992. With a focus on scientific collaboration and expertise, NorthX Biologics is “Beyond CDMO”, working closely with biopharmaceutical developers to help advance complex biologics from early concept to clinical and commercial stages.

Technology transfer is managed through a cross-functional team that oversees planning, risk assessment, and execution. The goal is not only to reproduce the process but also to future proof it for later clinical phases by optimizing and making it scalable for GMP-compliant manufacturing.

Key steps may include:

  • Comprehensive Data Review – Evaluating process descriptions, historical batch data, and analytical methods to ensure completeness and consistency.
  • Material Transfer – Handling cell banks, reference standards, and raw materials, with a focus on verifying their integrity and suitability.
  • Confirmation Run – Conducting a small-scale production (typically 1–2 L) under NorthX Biologics conditions to test process parameters and identify any necessary adjustments.
  • Engineering Batch – A non-GMP engineering batch is executed under GMP-like conditions to simulate full production and evaluating potential scaling and/or transfer effects.
  • Documentation Finalization – Developing batch records, control strategies, and specifications aligned with GMP requirements.

Even with thorough preparation, subtle differences between development and manufacturing environments often surface during the engineering run. Early collaboration helps mitigate these by aligning expectations, refining methods, and building mutual understanding.

Preparing for the next phase

For Lipum, technology transfer is more than a procedural handoff. It’s a chance to reassess and refine the manufacturing strategy. As the process transitions from one GMP manufacturing site to another, even well-established procedures are challenged by differences in equipment, systems, and interpretation of standards, often uncovering areas for refinement and alignment.

This phase also strengthens collaboration. Clear communication, shared risk ownership, and proactive problem-solving set the stage for a smoother path to future commercialization. A thoughtful approach now helps secure a reliable and efficient manufacturing platform for the future.

Ensuring a robust and scalable process

A successful technology transfer results in a process that is both reproducible and robust, a process able to withstand the scale-up to volumes without compromising quality. This requires a deep understanding of critical process parameters (CPPs), well-defined quality attributes, and validated analytical methods.

While the confirmation and engineering runs play a vital role in uncovering issues early, the work doesn’t end there. Continuous collaboration enables iterative improvements and ensures the process evolves in tandem with clinical and regulatory milestones. Transparency, shared ownership, and a commitment to quality form the foundation of a successful long-term partnership.

Looking ahead

As the process moves toward full-scale GMP production, the lessons learned during technology transfer will continue to shape the manufacturing strategy. By focusing on precision, partnership, and proactive planning, companies can bridge the gap between innovation and execution and bring promising biologic therapies closer to the patients who need them most.

About this series
This series in six parts explores key aspects and aims to provide valuable insights into the complexities of the biologic drug manufacturing journey – from early process development to commercial production – through the perspectives of both the developer and the CDMO, offering a transparent discussion on the realities of bringing a biologic to market.

Read part 1 here: The importance of collaboration
Read part 3 here: Linking the gap from development to robust manufacturing

The importance of collaboration

The complex journey of bringing a biologic to market – part 1

Biologic drugs differ from traditional medicines as they are produced in living systems, making their development and manufacturing more complex. Every stage, from early development to large-scale production, requires precise control due to variables such as cell culture conditions, purification processes, and regulatory requirements.

One major misconception is that a process working in the lab will scale easily to commercial production. Small changes in cell culture, raw materials, or purification can impact yield and consistency. Another false assumption is that process changes can be made late in development without regulatory consequences, which can lead to costly delays.

Lipum conference room meeting

Early collaboration with a Contract Development and Manufacturing Organization (CDMO) is crucial. Engaging a CDMO during preclinical development helps refine processes, align analytical methods, and avoid unexpected issues during scale-up. Delaying this partnership may lead to difficulties in meeting clinical and commercial timelines.

Successful biologic drug development relies on strong collaboration between the developer and owner of the biologic and the CDMO. Working together from the beginning ensures smoother technology transfer, risk mitigation, and regulatory preparedness. Investors should also consider the high upfront costs of biologics, which create barriers to competition but offer long-term advantages through optimized processes and extended market exclusivity.

Case study: SOL-116

Lipum’s SOL-116, a humanized antibody targeting Bile Salt-Stimulated Lipase (BSSL), represents an innovative treatment for inflammatory diseases. Bringing it to market requires overcoming manufacturing challenges such as cell line development, process optimization, and regulatory approval.

Key strategies for maintaining product consistency include robust process control, quality attribute characterization, and stringent raw material qualification. Continuous monitoring and proactive collaboration between Lipum and NorthX Biologics will help navigate these complexities and ensure scalability.

Lipum conference room meeting

Building a successful partnership

An effective partnership is built on transparency, early alignment of expectations, and shared risk management. Using digital tools for process monitoring, investing in flexible manufacturing, and ensuring knowledge transfer between teams are essential for future biologic innovations. By fostering strong collaboration, companies can improve efficiency, reduce risks, and successfully bring life-changing therapies to patients.

About this series
This series in six parts explores key aspects and aims to provide valuable insights into the complexities of the biologic drug manufacturing journey – from early process development to commercial production – through the perspectives of both the developer and the CDMO, offering a transparent discussion on the realities of bringing a biologic to market.

Read part 2 here: Bridging innovation and execution: The critical role of technology transfer
Read part 3 here: Linking the gap from development to robust manufacturing

Stockholm Uppsala life science cluster

The Stockholm Uppsala Life Science Cluster is a globally recognized hub for advanced therapy medicinal products (ATMPs) and biomanufacturing. This region, encompassing major facilities in Stockholm, Uppsala, Södertälje, Strängnäs, and Solna, is renowned for its pioneering research, strong academic-industry collaborations, and significant contributions to the global biopharmaceutical market. With a robust infrastructure, extensive biobanks, and a supportive government, the cluster is at the forefront of life science innovation, making it an ideal location for companies looking to establish and expand their biomanufacturing capabilities.

Among the key players in this vibrant ecosystem is NorthX Biologics, which leverages cutting-edge technologies and extensive expertise to drive advancements in ATMPs. Supported by strategic government investments and a network of universities, hospitals, and life science companies, NorthX Biologics contributes to the region’s reputation as a leader in life science innovation. This document provides an in-depth look at the region’s strengths, key players, and the collaborative efforts driving its success in the life sciences sector.

Read the full article here.

Strengthening Sweden’s resilience through pharmaceutical manufacturing

In an increasingly unstable world, Sweden is taking decisive steps to secure access to critical pharmaceuticals during crises and war.

As part of a broader government initiative, highlighted in a directive to the National Board of Health and Welfare and the Medical Products Agency, Sweden is investing in domestic manufacturing preparedness for life-saving medicines.

As illustrated in a recent article, NorthX Biologics demonstrates how local infrastructure can serve global needs. An example is the company’s expansion in Matfors, Sweden, where cutting-edge capabilities in vaccine and advanced therapeutics production are being built. Facilities like these are crucial, not only for public health resilience during pandemics but also as part of Sweden’s national security infrastructure.

In times when resilience and supply security are critical, we believe the key isn’t to start from scratch, but to build on what already exists. With diverse competencies, decades of experience, and established GMP facilities, we are a trusted partner in ensuring Sweden’s healthcare sovereignty.

Sweden’s strategy is clear: independence in producing critical healthcare products is no longer optional, it’s essential.

Read the article in Sundsvalls Tidning (in Swedish): Inifrån svenska storsatsningen i Matfors – Sundsvalls Tidning

Manufacturing stem cells for regenerative therapies

Stem cells have revolutionized the field of regenerative medicine, offering promising solutions for various medical conditions, including difficult-to-heal skin wounds. This review focuses on the background and manufacturing processes of skin cell-based therapies, particularly keratinocytes and adipose-derived mesenchymal stem cells (AD-MSCs), as highlighted in the thesis by Hady Shahin[1]. This article provides an overview of services offered by Contract Development and Manufacturing Organizations (CDMOs) involved in cell-based solution production.

Stem cell basics

Stem cells have unique abilities to self-renew and to recreate functional tissues. They can develop into many different cell types in the body during early life and growth[2]. Researchers study many different types of stem cells, including pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) and non-embryonic or somatic stem cells (commonly called adult stem cells)[2]. Pluripotent stem cells have the ability to differentiate into cells of the 3 main germ layers in the adult body[2].  Adult stem cells are found in specific anatomical locations and can differentiate to yield the specialized cell types of that tissue or organ[2]. They serve as an internal repair system that generates replacements for cells lost through normal wear and tear, injury, or disease.

Properties of stem cells

Stem cells have the remarkable potential to renew themselves and to differentiate into various specialized cell types[2].  When a stem cell divides, the resulting two daughter cells may be both stem cells, a stem cell and a more differentiated cell, or both more differentiated cells[2]. Discovering the mechanism behind self-renewal may make it possible to understand how cell fate is regulated during normal embryonic development and post-natally, or mis-regulated during aging or in the development of cancer[2].

The skin and regenerative therapies

The skin, the largest organ of the body, serves as a protective barrier against the external environment. It consists of three distinct layers: the epidermis, dermis, and subcutaneous adipose tissue[1]. The epidermis, primarily composed of keratinocytes, plays a crucial role in maintaining skin integrity and facilitating wound healing. Keratinocytes move from the basal layer to the surface, undergoing differentiation and forming a protective barrier[1].

Difficult-to-heal wounds, such as those caused by chronic diseases, trauma, or burns, pose significant challenges in clinical practice. These wounds often result in prolonged pain, infection, and impaired quality of life[1]. Regenerative advanced therapy medicinal products (ATMPs), including cell-based approaches, offer promising solutions for enhancing wound healing and improving patient outcomes[1].

Autologous vs allogenic skin ATMPs

Keratinocytes —the most abundant cell type in the epidermis— are instrumental in the re-epithelialization process during wound healing. They proliferate and migrate to cover the wound bed, forming new epidermal layers[1]. An autologous therapeutic approach involves harvesting skin biopsies from a patient’s healthy donor sites, isolating keratinocytes, expanding them, and reapplying them to the same patient after thorough characterization, quality control, and safety testing. The classical method for culturing keratinocytes includes enzymatic digestion of the epidermis, followed by expansion in culture media [1]. However, a major challenge in this process is the use of animal-derived products, which poses regulatory hurdles [1]. To address these challenges, Shahin’s thesis proposes a xeno-free workflow for keratinocyte isolation and expansion. The study validates the use of a xeno-free workflow to manufacture human keratinocytes as ATMP [1]. This approach ensures the production of keratinocytes that comply with regulatory standards, making them suitable for clinical applications [1].

The allogeneic approach, on the other hand, involves Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) as a promising alternative to address the scalability challenges associated with keratinocytes, which are mature cells. AD-MSCs are multipotent stem cells isolated from adipose (fat) tissue. They possess the ability to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes[1]. AD-MSCs are particularly attractive for regenerative therapies due to their ease of isolation, high yield, and immunomodulatory properties[1].

In the context of wound healing, AD-MSCs contribute to tissue repair by promoting angiogenesis, reducing inflammation, and enhancing collagen synthesis[1]. Shahin’s thesis explores the potential of AD-MSCs as an alternative to keratinocytes for treating difficult-to-heal wounds[1]. The study highlights the differentiation of AD-MSCs into keratinocyte-like cells through direct co-culture with keratinocytes[1]. This approach leverages the paracrine signaling between the two cell types to enhance the differentiation process[1].

Manufacturing cell therapies for clinical use

The manufacturing of stem cells for clinical applications involves several critical steps, including cell isolation, expansion, and quality control. Ensuring compliance with Good Manufacturing Practice (GMP) guidelines is essential to produce safe and effective cell-based therapies[1].

Every cell therapy product is special, NorthX Biologics adopts a flexible manufacturing operation where we together with the client tailor a manufacturing and testing process meeting the specific product’s exact requirements.

Proposed workflow for manufacturing a cell-based ATMP for wound healing

1. Cell isolation

Keratinocytes are typically isolated from skin biopsies using enzymatic digestion. Shahin’s thesis validates the use of a completely xeno-free keratinocytes extraction method, ensuring the production of GMP-compliant keratinocytes in a timely manner[1]. AD-MSCs are isolated from adipose tissue through enzymatic digestion and centrifugation[1]. The high yield of AD-MSCs from adipose tissue makes them a viable option for large-scale production[1].

2. Cell expansion

The expansion of keratinocytes and AD-MSCs requires optimized culture conditions to maintain cell viability and functionality. Shahin’s study demonstrates the use of xeno-free culture media for keratinocyte expansion, eliminating the need for animal-derived products[1]. For AD-MSCs, the co-culture with keratinocytes enhances their differentiation into keratinocyte-like cells, providing a scalable approach for producing epidermal cells [1].. Rigorous quality control is needed to ensure such in-vitro cell manipulation is safe and does not compromise the properties of the cells. Therefore, thorough characterization and stability testing are needed for cell-therapies to be considered safe for clinical use and to fulfil stringent regulatory requirements for ATMPs.

At NorthX Biologics, we provide an extensive range of testing services tailored to the advanced products we manufacture, to support designing a comprehensive analytics panel for your ATMP (consulting on release criteria with the regulatory bodies).

3. Cell transportation

As cell therapy manufacturing for clinical use must be conducted under strict control in a GMP facility, the final cell solution often needs to be transported from the production site to the treatment site, which may be several hours away. Shahin’s study demonstrated that the cell solution can be transported for up to 24 hours under controlled conditions while maintaining cell functionality and characteristics.

This finding allowed the research team to establish human serum albumin as the preferred carrier solution for keratinocytes in clinical treatments, ensuring their viability and functionality during transport. Additionally, it was validated as the final formulation solution for administration.

At NorthX Biologics, we offer tailored fill & finish solutions with integrated analytical support.

4.  Ensuring quality and safety in cell-based ATMP manufacturing

Ensuring the quality and safety of cell-based ATMPs is paramount. Shahin’s thesis emphasizes the importance of thorough characterization of keratinocytes and AD-MSCs, including the assessment of cell viability, differentiation potential, and functionality[1]. The use of cell and molecular characterization methods, including but not limited to immunophenotyping, gene and protein expression analyses are instrumental tools for monitoring and ensuring the quality of the produced cells [1].

Conclusion

The thesis by Hady Shahin offers valuable insights into manufacturing cell-based regenerative solutions for skin healing. The proposed xeno-free workflows and co-culture techniques present promising methods for producing GMP-compliant cell therapies. These advancements pave the way for effective treatments for difficult-to-heal wounds, ultimately improving patient outcomes.

Hady Shahin, PhD
Production Scientist
NorthX Biologics

Hady is a Production Scientist at NorthX Biologics, providing bioprocess and CMC support for GMP manufacturing of drug substances (DS), contributing to the production of advanced biologics for clinical trials. Biotechnologist by training and SME in scaling up cell therapy solutions, with over 15 years of experience in regenerative medicine and stem cell research. Hady holds a PhD in Cell and Molecular Biology and an MSc in Biomedical Science, with a strong background in ATMP development and GMP manufacturing.

LinkedIn

References
  1. Shahin, H. (2023). Keratinocytes and Adipose-derived mesenchymal stem cells: The heir and the spare to regenerative cellular therapies for difficult-to-heal skin wounds. Linköping University Medical Dissertation No. 1880.
    https://liu.diva-portal.org/smash/get/diva2:1810734/FULLTEXT01.pdf
  2. National Institutes of Health. (2021). Stem Cell Basics. Retrieved from https://stemcells.nih.gov/info/basics/stc-basics.

Driving therapeutic innovation with OMVs and EVs

Outer membrane vesicles (OMVs) and extracellular vesicles (EVs) are unlocking new possibilities in vaccines and therapeutics. OMVs, with their natural adjuvant properties and capacity to carry antigens, enhance immune responses, while EVs, as immune-silent carriers, offer a versatile platform for delivering a range of drug substances. NorthX Biologics is at the forefront of manufacturing these nanoscale technologies, addressing key manufacturing and analytical challenges to accelerate their clinical potential.

Authors: Isa Lindgren, Ph.D., Head of Analytics, and Ola Tuvesson, Chief Technology Officer, NorthX Biologics

Expanding applications of vesicle-based therapeutics

Outer membrane vesicles (OMVs) derived from gram-negative bacteria and extracellular vesicles (EVs) from mammalian cells are emerging as promising platforms for vaccines and therapeutics. Their unique characteristics make them highly versatile tools for addressing diverse medical needs.

OMVs are naturally released by gram-negative bacteria to aid in their survival, delivering virulence factors and DNA to host cells.1–3 These nanoscale vesicles mimic bacterial surfaces, incorporating antigens and pathogen-associated molecular patterns (PAMPs) without pathogenicity. Their small size promotes cellular uptake and effectively triggers both innate and adaptive immune responses. As a result, OMVs are gaining attention as both adjuvants and standalone vaccines.

Historically, four OMV-based vaccines targeting meningitis-causing bacteria have been approved in the United States and Europe, though only two remain commercially available due to declining infection rates and reduced demand.1 Today, research is expanding into new candidates targeting pathogens such as COVID-19, invasive non-typhoidal Salmonella, Neisseria gonorrhea, Shigella, and Hemophilus influenzae type b (Hib), with several currently in phase I or II clinical trials. Of particular interest is VAXELIS, a hexavalent pediatric vaccine under development by Merck & Co., which combines OMV-based actives with toxins and inactivated viruses. Advances in genetic engineering have also enabled OMVs to target pathogens other than their bacterial sources, achieving high safety and immunogenicity.

Beyond vaccines, OMVs are being explored as carriers for proteins and nucleic acids. While loading heterologous proteins onto OMV surfaces remains a technical challenge, progress in chemical conjugation, molecular engineering, and glycoengineering is helping to overcome these barriers.1 The dense expression of antigens on OMV surfaces has been shown to significantly enhance immune responses.2 Recent innovations also include alternative delivery routes for OMV-based vaccines, such as intranasal administration, which has demonstrated localized and systemic immunity in animal studies with excellent safety profiles.

EVs, on the other hand, have no approved therapeutic or vaccine products yet, but they are the focus of a rapidly growing number of clinical trials — outpacing those for OMVs. These nanoscale vesicles, produced by mammalian cells, serve as intercellular messengers, carrying cellular components such as surface receptors, signaling proteins, transcription factors, enzymes, extracellular matrix proteins, nucleic acids, and lipids.5 Their involvement in numerous signaling pathways allows them to mediate both physiological and pathological processes, including cancer metastasis and viral propagation.6

EVs’ natural presence in bodily fluids has also spurred interest in their use in diagnostic applications. Furthermore, their ability to transport bioactive molecules across biological barriers with minimal immunogenicity makes them highly attractive as potential targeted drug delivery vehicles.6 Common sources of EVs include immune cells, mesenchymal stem cells (MSCs), cancer cells, and human kidney embryo (HEK) cell lines.

Isa Lindgren, Head of Analytics

As of 2023, nearly 50 active clinical trials were investigating EV-based therapeutics across a broad spectrum of indications, including pain management, hair and bone loss, dementia, infectious diseases, autoimmune conditions, cancers, and gastrointestinal disorders.6 This breadth highlights the growing recognition of EVs’ therapeutic potential in both established and emerging medical fields.

Overcoming limitations in drug delivery with vesicle technology

The delivery of genetic material for vaccines or therapeutics has traditionally relied on viral vectors owing to their inherent efficiency. Viruses are naturally optimized for cellular infection and the delivery of nucleic acid cargo. Adeno-associated viral (AAV) vectors, for example, can leverage a variety of serotypes to achieve targeted delivery to specific cell types in vivo, making them highly effective in many contexts.

Despite their advantages, viral vectors come with notable risks and limitations. Pre-existing immunogenicity, where the immune system recognizes and neutralizes viral carriers before they can deliver their payload, remains a significant hurdle. Additionally, viral vectors are typically limited in their ability to deliver substances beyond nucleic acids, which restricts their versatility.

In contrast, vesicles such as OMVs and EVs present a highly adaptable alternative. These nanoscale carriers are capable of transporting not only nucleic acids but also recombinant proteins, antigens, and a variety of other drug substances, offering significantly broader therapeutic potential. Vesicles also avoid the issue of pre-existing immunogenicity, a key drawback of AAVs, and can be further engineered to enhance specificity and functionality.

This flexibility positions OMVs and EVs as ideal candidates for applications requiring customized and diverse payload delivery, while their immunological profile provides an edge in scenarios where viral approaches may be less viable.

Tackling manufacturing complexity for vesicle-based therapeutics

Producing OMVs and EVs involves unit operations similar to those used for other biologic drug substances. However, the nanoparticle nature of these vesicles introduces unique challenges, particularly during downstream purification, where consistency and scalability are critical.

OMVs are typically produced through bacterial fermentation, employing processes similar to those used in protein production. When using anaerobic bacterial strains, strict control measures are needed to minimize exposure to air, adding a layer of complexity to the manufacturing process. To ensure sufficient yields, genetically engineered cell lines are often utilized. OMVs are secreted as nanoparticles into the supernatant, allowing for separation from cells through ultracentrifugation at small scales or microfiltration and tangential flow filtration (TFF) at larger scales. Further purification steps, such as chromatography, are employed to isolate the vesicles, with additional steps required for viral inactivation, removal of contaminants, and final formulation.

Scaling up these processes remains a significant hurdle for manufacturers. Ultracentrifugation, while effective at the lab scale, is impractical for large-scale production owing to its labor-intensive nature and low throughput. Similarly, size-exclusion chromatography — commonly used for vesicle purification — has limited capacity compared to other chromatography methods, presenting challenges as demand grows.

Innovative solutions are being explored to address these scalability issues. Efforts are underway to streamline vesicle purification and adapt existing bioprocessing technologies for larger-scale applications. By investing in scalable platforms and refining production techniques, innovative manufacturers are steadily working to bridge the gap between early-stage research and commercial manufacturing of OMV and EV-based therapeutics.

Addressing analytical uncertainty

The inherent complexity and diversity of vesicle-based vaccines and therapeutics present significant challenges for comprehensive characterization and quality control. A foundational aspect of analysis is determining particle size distribution, a critical parameter given that OMVs and EVs exist as nanoparticles. However, even this relatively straightforward step can become complicated, as the appropriate target size must first be established to assess whether a product’s size distribution aligns with specifications.

The composition of OMV and EV particles is far more challenging to analyze. These vesicles are complex structures, often containing diverse biological components, making it difficult to define their profiles and ensure consistency. The difficulty is heightened in OMV formulations that include a mix of vesicles, membrane particles, and other substances. In some cases, the incorporated proteins offer an analytical “handle,” simplifying certain evaluations. However, these formulations still demand rigorous methods to characterize their heterogeneity and ensure compliance with specifications.

Consistency is critical, given the variability between batches. Methods must focus on ensuring uniform particle size distribution and consistent levels of incorporated antigens and naturally occurring proteins. Achieving this requires the use of cell lines capable of producing vesicles with reproducible profiles across batches.

Common analytical techniques include dynamic light scattering (DLS) for particle size analysis, high-performance liquid chromatography (HPLC) for separating components, capillary electrophoresis for profiling proteins, and assays for total protein quantification. However, developing assays that are both accurate and sensitive can be particularly challenging when vesicles require high detergent levels for lysis during testing. This variability often complicates the measurement of components like total protein content.

Potency assays, which are essential for determining the functional efficacy of vesicle-based products, represent another bottleneck in GMP manufacturing. As with cell therapies, these assays must be tailored to each specific product, requiring significant time and resources to develop. The lack of standardized potency assay frameworks adds another layer of complexity, contributing to delays in advancing vesicle-based products through clinical and commercial stages.

Ola Tuvesson, Chief Technology Officer

Overcoming safety challenges for OMVs and EVs

As biologic products, OMVs and EVs are subject to stringent safety requirements, ensuring they meet regulatory standards for microbial contamination, endotoxins, and adventitious agents. These measures are critical to protect patient safety and to demonstrate consistent quality across production batches.

OMVs, commonly used as adjuvants, require extensive pyrogen testing due to their inherently pyrogenic nature. Depending on the OMV source organism, the use of standard endotoxin quantification methods may prove insufficient, and non-endotoxin pyrogens have to be evaluated. Safety assessments for OMVs not originating from gram-negative bacteria need to be performed using non-endotoxin pyrogen detection systems, such as monocyte activation testing (MAT). With high intrinsic pyrogenicity, absolute quantification of pyrogens may not be possible, and analysis must instead rely heavily on demonstrating batch-to-batch consistency, ensuring that the pyrogenic

activity remains uniform and predictable. The true safety of OMVs is ultimately validated through clinical studies, where their immunogenicity and other safety parameters are closely monitored.

In contrast, EVs are less inherently immunogenic, simplifying some safety assessments. Non-endotoxin pyrogens associated with EVs can often be both defined and quantified, enabling more straightforward analytical approaches. However, rigorous testing is still essential, particularly as EVs advance toward therapeutic applications. Maintaining consistency in the presence of potential contaminants or immunogenic components is key to ensuring the safety and efficacy of EV-based products.

For both OMVs and EVs, the development and implementation of robust safety protocols tailored to their unique properties are essential to support their advancement as vaccines and therapeutics.

Breaking barriers in early-stage vesicle development

The relative immaturity of the OMV and EV fields presents significant challenges for developers aiming to bring new vaccines and therapeutics to market. Chief among these is the lack of regulatory clarity, which creates uncertainty around development pathways and compliance expectations. This ambiguity is particularly daunting for smaller, emerging firms that often lead innovation in this space but face limited access to funding. Typically, significant financial investment only becomes available after the generation of phase I clinical data, requiring developers to absorb the substantial upfront costs needed to reach this milestone.

On the production side, one of the biggest hurdles is developing high-yielding manufacturing processes tailored to these complex vesicle products. Achieving reproducible outputs while maintaining consistency and scalability requires careful optimization. Compounding this challenge is the need to establish robust analytical methods that can accurately characterize vesicle composition and behavior.

Balancing process development is another key issue. Overdevelopment at early stages can exhaust limited resources, while insufficient effort may result in suboptimal processes that hinder progress. Developers must carefully identify and validate the right set of critical quality attributes (CQAs) for each specific product, ensuring they are both scientifically rigorous and commercially viable. Additionally, creating a fit-for-purpose analytical package capable of generating accurate and consistent data across development stages is critical for regulatory approval and downstream success.

Navigating these early-stage challenges demands strategic decision-making and innovative problem-solving, as well as close collaboration between developers, contract development and manufacturing organizations (CDMOs), and regulatory bodies to accelerate the maturation of the OMV and EV fields.

NorthX Biologics: advancing OMV and EV innovation

NorthX Biologics stands out as one of the few CDMOs with substantial experience in nanoparticle-based vaccine development, actively supporting several OMV projects and engaging in discussions with companies developing EV-based therapeutics. This depth of expertise positions NorthX Biologics as a leader in a rapidly evolving field.

As a smaller, agile CDMO, NorthX Biologics excels at tailoring its approach to meet the specific needs of each client. Viewing client relationships as true partnerships, the company provides not only manufacturing services but also scientific consultation and innovative problem-solving. This flexibility enables NorthX Biologics to work with specialized bacterial strains that may fall outside the standardized platform technologies used by larger CDMOs, ensuring bespoke solutions for even the most niche requirements.

At NorthX Biologics, we offer end-to-end support, from early-stage development to clinical manufacturing, leveraging robust capabilities in adherent cell culture using fixed-bed and hollow-fiber bioreactors. Our comprehensive suite of analytical methods is designed to be adaptable to the unique demands of each project. Furthermore, NorthX Biologics continues to invest heavily in manufacturing and analytical technologies to deepen our understanding of OMV and EV products, ensuring that we deliver the highest level of support. Our expertise in

nucleic acid therapies further strengthens our ability to assist clients seeking to use OMVs and EVs as delivery vehicles.

One standout example of NorthX Biologics’ collaborative approach is its partnership with Abera Biosciences. Together with Testa Center, another Swedish Innovation Hub, NorthX Biologics has supported Abera in developing a pneumococcal OMV-based vaccine platform. This partnership seeks to develop a scalable, GMP-compliant manufacturing process and the development of robust analytical methods. As a result, Abera’s candidate is on track to enter clinical trials, exemplifying the success of NorthX Biologics’ integrated and innovative approach.

Significant progress on the horizon

With a growing number of OMV and EV candidates advancing through clinical development, the field is poised for transformative progress in manufacturing and analytical technologies over the coming years. At NorthX Biologics, we anticipate playing a key role in driving these advancements, building on our expanding expertise and experience.

We are actively developing a robust OMV analytical platform designed to address the shared characteristics of the diverse bacterial strains used in OMV production. This platform will embody a plug-and-play approach, enabling streamlined analytics by employing standardized technologies wherever possible. By simplifying and accelerating the analytical process, this platform aims to reduce development timelines and support the scalability of OMV-based products.

Looking ahead, we are equally excited to leverage our expertise in OMVs to establish a similar analytical platform for EV-based therapeutics. Drawing parallels between these vesicle types, NorthX Biologics aims to extend our innovative solutions to encompass EV development, ensuring comprehensive support for clients as the industry evolves. This dual focus on OMV and EV platforms will position NorthX Biologics at the forefront of vesicle-based therapeutic innovation, meeting the needs of clients across diverse therapeutic areas.

References

Ola Tuvesson
Chief Technology Officer
NorthX Biologics

As CTO, Ola is leading NorthX Biologic’s development and project organization, focusing on delivering technologies and strategies to ensure high-end services within bioprocessing and analytics. He has more than 20 years’ experience from the pharma and biotech industry, ranging from early development to commercial GMP manufacturing. Ola has worked in several fields, including ATMP products, vaccines, and other biologicals. He has the essential knowledge to take a product from early pre-clinical development into clinical trials and to the market.

Isa Lindgren, Ph.D.
Head of Analytics
NorthX Biologics

Isa Lindgren, Ph.D., is Head of Analytics at NorthX Biologics, leading the QC and Analytical Development teams across the Matfors and Stockholm sites. With a background of 15+ years in life sciences research and experience from preclinical work at Chiesi Pharma in biologics and ATMPs, Isa has extensive expertise in analytics. Six years in the US have equipped her with valuable international experience for global communication and high-level customer care. Known for her technological acumen, she ensures NorthX Biologics remains a front-runner in analytics to deliver biologics at the highest quality. 

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Read the full article by Peter Boman (NorthX Biologics), and  Zach Hartman (Cytiva), in Cell & Gene Therapy Insights here.