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.
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/