Control of empty, full and partially filled capsids

BioPhorum establishing an industry position on this critical quality attribute


A consensus of BioPhorum member companies reject a proposed blanket minimum specification for percentage empty AAV capsids. Presently, it is premature and impractical to set a minimal specification of <30% empty capsids to apply to all AAV-based gene therapies in development.
BioPhorum calls upon industry to continue to adopt a quality by design, risk-based approach towards setting empty capsids specifications per product, based on the indication, route of administration, total capsid input (dose), immunogenicity concerns and benefit to risk ratio considerations.


Recombinant Adeno-Associated Virus (rAAV), a single-stranded DNA virus, is a widely utilized gene therapy vector that can be manufactured using a variety of different platforms. These production systems include transient transfection or infection of mammalian cells; stable producer (mammalian) cell lines, and recombinant baculovirus infection of insect cells. Despite their extensive clinical application and licensure as marketed medicinal products, further efforts are required to increase product understanding with regard to critical quality attributes (CQAs) that may have an impact on clinical efficacy, safety, or immunogenicity.

CQAs can differ depending on the nature of production and purification systems used to generate the rAAV vector. CQAs include the composition of packaged DNA within the AAV capsid and the presence of undesired empty capsids. Due to the inefficiency and potential lack of specificity of vector genome packaging into pre-formed capsids, empty AAV particles, or capsids containing a truncated vector genome, can be abundant particle types generated in the upstream cell culture phase of manufacture.

Empty capsids and capsids that are partially filled with part of the intended vector genome (a partial product) both represent product-related impurities and are technically challenging to separate from the target vector that contains the intended complete payload (i.e., the therapeutic construct including any associated regulatory elements, flanked by complete AAV inverted terminal repeat sequences) during downstream purification. 

Dependent on the exact packaged sequence, partially filled capsids, may still contribute to target cell transduction (Sihn et al 2022). However, AAV particles containing host cell and/or helper DNA are product-related impurities and may represent an immunological risk to patients (Wright 2022).

To date, health agencies have not formalized guidance to specify an upper limit for residual empty particles. This is considered to be a rational approach, since historically, different manufacturing platforms and processes have provided different levels of product-related impurities, whilst still enabling the clinical development and licensure of AAV-based products, for diseases where there is an unmet medical need.  

Despite an incomplete understanding of the clinical impact of empty capsid, when administering high numbers of empty capsids along with full packaged vectors, the potential for increasing innate or adaptive immune responses to the vector is a major concern (Verdera et al 2020). As per the FDA Advisory Committee September 2021: “The empty capsids increase overall antigenic load and potentially exacerbate capsid-triggered innate and adaptive immune responses. The empty capsids can contribute to the peptides presented by major histocompatibility complex molecules, with consequent recognition and clearance of transduced cells by capsid-specific cytotoxic T cells”.

It was further stated by the FDA in the Ad Comm how: “In addition to stimulation of innate and adaptive immune responses, AAV empty capsids may compete with full capsids for receptor binding on target cells, which could necessitate an increase in the required vector dose”.

The gene therapy field has already recommended that industry establish a total capsid titre as a CQA for safety at high vector doses (Wright 2022).

BioPhorum approach and 2023 deliverable

BioPhorum, a company-to-company collaboration, has brought together cell and gene therapy (CGT) subject matter experts from the global biopharmaceutical industry to discuss, align, and establish best practices for critical challenges within CGT. While current guidance on the control of empty, full and partially filled capsids is not yet established, BioPhorum has established a CGT Empty Capsid workstream, a cross-industry collaboration of 34 gene therapy manufacturers aligned on a common interest to increase shared understanding of empty, full and partially filled capsids characterization and best practice analytics.

The aim of this workstream is to compliment broader industry efforts in understanding of these CQAs. The BioPhorum Empty Capsid team will publish a more detailed commentary by early 2023 and this will present the findings from an extensive benchmarking exercise to highlight current industry practices and insights on emerging technologies, advocating for standardization of analytical methods and improvements in reference standards. The benefits of measuring empty capsids in process to support process development and best practice methods to improve the resolution of AAV particles filled with contaminating sequences or partial payload will also be shared.

The forthcoming paper will also provide the opportunity for BioPhorum members to contribute to the broader discussion on setting specifications for empty capsids. Results from the team’s benchmarking survey will support a quality by design, risk-based approach towards setting specifications for AAV empty capsids per product (per ICH Q8(R2), ICH Q9 and ICH Q10). Such specifications for empty AAV capsids must be justified and supported by pre-clinical and clinical safety and efficacy data that also considers the following: the route of administration; capsid serotype; total capsid input and vector genome doses; immunogenicity considerations; the indication; and the benefit to risk ratio. The application of a blanket theoretical empty capsid limit proposal, which may or may not be reliably met, will not be considered.

The survey data has also highlighted the diversity of methods utilized across industry to determine empty, full and partially filled capsids in AAV products. One of the aims of the publication is to promote harmonization and alignment within the industry and articulate recommendations for the best practices for analysis of this CQA.


Setting specifications for the percentage of full payload capsids in rAAV vectors is recognized as one of the critical challenges for the industry, as factors such as phase of development, indication, route of administration and dose levels can influence the requirements. The target percentage of empty capsids is also dependant on the capability of the manufacturing process and can vary from product to product. Currently there is no formal regulatory guidance governing or recommending an acceptable level of empty and capsids filled with a truncated vector genome in viral vector drug products. The BioPhorum Empty Capsids team continues to promote a quality by design approach to specification setting taking into consideration the limiting factors known to cell and gene therapy products.

BioPhorum members wish to promote new and emerging technologies as an area of particular interest. The survey data shows an industry shift in the usage of the newer platforms and/or software, with many CGT companies investing in exploratory and/or qualification activities. By sharing learnings to benefit all, BioPhorum members aim to raise awareness of the benefits of emerging new technologies to support wider industry efforts as it strives to ensure the safe and effective delivery of gene therapy products to patients.

About BioPhorum

BioPhorum’s mission is to create environments where the global biopharmaceutical and device industry can collaborate and accelerate its rate of progress, for the benefit of all.  

Since its inception in 2004, BioPhorum has become the open and trusted environment where senior leaders of the biopharmaceutical industry come together to openly share and discuss the emerging trends and challenges facing their industry.  

Growing from an end-user group in 2008, BioPhorum now comprises over 135 manufacturers and suppliers deploying their top 6,000 leaders and subject matter experts to work in ten focussed Phorums, articulating the industry’s technology roadmap, defining the supply partner practices of the future, and developing and adopting best practices in drug substance, fill finish, process development and manufacturing IT. In each of these Phorums, BioPhorum facilitators bring leaders together to create future visions, mobilise teams of experts on the opportunities, create partnerships that enable change and provide the quickest route to implementation, so that the industry shares, learns and builds the best solutions together.  


Food and Drug Administration (FDA) Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) Meeting #70. Toxicity Risks of Adeno-associated Virus (AAV) Vectors for Gene Therapy (GT) September 2-3, 2021. https://www.fda.gov/media/151599/download

Sihn CR, Handyside B, Liu S et al. Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers. Mol Ther Methods Clin Dev. 2021 Dec 21;24:142-153. https://pubmed.ncbi.nlm.nih.gov/35036471/

Verdera HC, Kuranda K, Mingozzi F. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther. 2020 Mar 4;28(3):723-746. https://pubmed.ncbi.nlm.nih.gov/31972133/

Wright JF. AAV vector manufacturing process design and scalability – Bending the trajectory to address vector-associated immunotoxicities. Mol Ther. 2022 Jun 1;30(6):2119-2121. https://pubmed.ncbi.nlm.nih.gov/35594866/


Cell & Gene Therapy

Maximizing the impact of the many advanced therapies under development requires that the industry comes together to improve understanding of how to develop, control and manufacture these life changing therapies.

We connect strategic thinkers in drug development and contract manufacturing organizations with the aim of ensuring harmonization and alignment around issues such as potency assays, phase-appropriate guidance for critical quality attributes, operator safety, regulatory guidelines, and C&GT-specific validation issues, as well as working toward resolving the current challenges to commercializing C&GT products. Discover more and learn about the outputs here.

Development Group

The ever-increasing need to reduce product development lead times and the opportunities to exploit innovative technologies in development are two key industry themes that remain at the forefront of our work. The Phorum successfully connects process development organizations and provides a space where subject matter experts can work on issues such as quality by design and process characterization, cell line technologies, and molecule selection. Read some of the outputs and discover how you could benefit by getting involved.

Drug Substance

Accelerating improvement, exploiting opportunities offered by innovation, and identifying best practice approaches across all aspects of the manufacturing process – these are the essential objectives

Data and digital capabilities are at the core of many of these industry challenges. Work in BioPhorum Drug Substance is informed by BioPhorum Technology Strategy, which has summarized our industry’s market drivers and trends. Capacity, flexibility, sustainability and pace all feature as requirements to succeed in a rapidly changing environment.

Find out more about what our members are currently discussing and learn about some of the successful implementations of our output.

Fill Finish

Quality and demonstrating compliance are two priorities of BioPhorum Fill Finish. Representatives of the world’s top sterile filling operations come together to develop solutions to some of the most intractable technical and regulatory challenges.To address these challenges in drug product aseptic filling (‘fill’) and secondary packaging (‘finish’) operations, our community of industry experts is working relentlessly to continually improve the pharmaceutical quality system (PQS). Together they continue to strive towards developing and implementing safe, predictable, lean, and agile processes in drug product operations. Find out more about the Phorum output here. 

Information Technology

IT and digital advances are crucial in responding to the pharmaceutical industry’s challenges around speed, cost, quality, flexibility, and sustainability. Unlocking and realizing the value in the industry’s IT opportunities is the core mission of the IT Phorum. 

The digital manufacturing facility of the future needs to consider technology advances alongside organizational perspectives while maintaining foundational security requirements. All elements must be considered in the long term to deliver systemic change toward the digital factory vision. Our members collaborate in a format that stimulates and supports industry innovation and implementation. 


BioPhorum Regulatory CMC is affording professionals the opportunity to shape their own collaboration program focused on challenges and opportunities across strategy, science, operations, submissions and compliance. Following a discovery program of work with Heads of Regulatory CMC, in which we identified some key topics of collaboration, we are giving you the opportunity to participate and help us to shape the final collaboration program. This will focus on, filing and review, interactions with Health Authorities, and harmonization. Our mission is not just to discuss, but to deliver applicable outputs and publications that will have an impact – learn more about how you can get involved.

Supply Chain to Patient

The pharmaceutical outbound supply chain was built on a blockbuster model and doesn’t fit the needs of the changing pipeline and new modalities currently being developed. The geopolitical challenges of recent years have shown that the outbound supply chain needs to evolve to become more agile, and resilient, and address a more expectant end-user bying on patient-centricity. Industry collaboration can accelerate improvements and transform performance in a way not possible when working independently. This Phorum is founded on the belief that learning from each other and challenging current thinking to re-imagine the supply chain can reduce costs and increase efficiency.

Supply Partner

BioPhorum SP members want the inbound supply chain for the biomanufacturing industry to be recognized as one of the best-understood and managed global supply chains. They intend to be not only a trusted advisory group but also the engine that drives change for the industry. Working together, we aim to achieve supply chain transformation that provides:

  • an enhanced robustness and resilience
  • accelerated capability and capacity to effect technology adoption
  • reduced cost of quality, and
  • industry-wide alignment on operational implementation

There’s an extensive list of Phorum successes and tangible benefits that have been delivered to define industry best practise.


The BioPhorum Sustainability program is jointly designed, governed, and delivered by a membership drawn from licence holders, contract manufacturers, major suppliers and niche suppliers. This dynamic partnership means that we can quickly focus on the areas that have the greatest industry impact, and we can leverage learning from each other and other sectors – the chemical industry and the built environment for example.

BioPhorum’s unique ability to build a strong coalition for change for an entire industry is now being applied to the greatest challenge of our generation. Driven by evident industry needs, Phorum members are working to develop agreed industry benchmarks, establish the ROI of sustainability investments and to increase the efficacy of implementation of members’ sustainability strategies.

Technology Strategy

With a mission to define strategies to accelerate technology transformation and support the adoption of innovative technologies, BioPhorum Technology Strategy evolved from BioPhorum Technology Roadmapping, strives to realize the technology roadmap vision 2.0.

This is being achieved through a multi-stakeholder team of member companies with collective capability and influence who are committed to driving forth industry goals associated with: Speed to market, Quality, Value, Agility and On-Demand Supply and Sustainability.

Building on industry feedback to standardize plasmid release specifications

Plasmids release specifications are critical to the manufacture of many cell and gene therapy (CGT) products, but current regulatory... read more

How to predict, optimize and analyze high-concentration biologic therapeutic formulations

Developing high-dose biologic drugs for subcutaneous injection often requires high-concentration formulations. It also needs to optimize... read more

A media fingerprinting toolbox that comes with a host of benefits

Cell culture media raw materials can contain many chemical substances, each with its own properties. While simple substances can be... read more

Why 100% visual inspection does not mean 100% defect detection

There is a common misunderstanding that 100% visual inspection (VI) of biopharmaceutical products should mean 100% detection of defects.... read more

How to bridge the data integration gap between sponsors and contract organizations

Bringing BioPhorum’s unique perspective to continuous manufacturing in biologics

Continuous manufacturing (CM) has been a goal for the pharmaceutical industry for several years. While small molecule drug substances and... read more

Supply Chain to Patient has launched!

Have your outbound supply chain costs increased? Do you want to leverage data and digital technologies more effectively to reduce... read more

Electronic data exchange – the foundation for an efficient, resilient inbound supply chain

The COVID-19 pandemic, geopolitical unrest, and military action across the world have fundamentally disrupted the assumption and practice... read more

How are you improving your environmental sustainability performance?

Climate change and global warming resulting from greenhouse gas emissions are widely recognized as the biggest threats to global health.... read more

Adding value for analytical instrument partners

Analytical instrument vendors have been integral to developing and publishing our technology roadmaps and developing product... read more

Introducing MediPhorum

The medical technology (MedTech) industry is a critical and complex sector. As highlighted during the Covid-19 pandemic, the MedTech industry provides a wide array of products and technologies that are vital to the safe and effective provision of care to many. Also, as seen over recent years, there is often the requirement for flexibility in supply, rapid development, and the introduction of new products to keep pace with global public health demands.

Advanced Wound Care and Wound Management

Advanced wound care (AWC) is the medical treatment for wounds that have not healed properly using conventional wound care products such as gauze, bandages, plasters, and wadding.

AWC involves treating chronic or complex wounds using products that are not as easily obtained over the counter. AWC can be divided into three main categories: wound healing, infection prevention, and restoration.

Drug Delivery

Drug delivery products are therapeutic and diagnostic products combining drugs, devices, and/or biological products, which enable safer and more efficient treatment due to careful and precise drug targeting, local administration, and tailored treatment.

Introducing the new MediPhorum

MediPhorum by BioPhorum has been launched to bring together senior business leaders and subject matter experts across multiple MedTech... read more

Introducing the new MediPhorum

MediPhorum by BioPhorum has been launched to bring together senior business leaders and subject matter experts across multiple MedTech... read more

Introducing the new MediPhorum

MediPhorum by BioPhorum has been launched to bring together senior business leaders and subject matter experts across multiple MedTech... read more
BioPhorum Deliverables Report
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