Full/empty capsid ratio in AAV products: A critical quality attribute under regulatory scrutiny (ENG)
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Scientific Journal
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Pharmaceutical Affairs
Introduction
Gene therapy has emerged as a major area of interest within the scientific and medical communities, with viral vectors playing a central role in therapeutic development. Among these, Adeno-Associated Viruses (AAVs) are currently one of the most widely used platforms. To date, around 10 AAV-based therapies have been granted marketing authorization, and more than 230 are under clinical investigation [1,2].
Recombinant adeno-associated virus (rAAV) is a viral vector engineered from the non-pathogenic adeno-associated virus to deliver genetic material into cells. It consists of a protein capsid enclosing a DNA genome [1]. The first AAV-based gene therapy was approved in Europe in 2012 [3], highlighting the relatively recent emergence of these products. Consequently, the regulatory framework governing AAV-based therapies is still evolving and remains an area of active discussion.
One critical quality attribute for AAV products is the ratio of full to empty capsids in the final drug product. This parameter is increasingly scrutinized due to its potential implications for product quality [4–6].
This article provides a concise overview of current regulatory expectations regarding the AAV full/empty capsid ratio, summarizes the positions of major health authorities, and highlights key challenges related to its analytical assessment.
Figure 1. Schematic of different AAV capsid populations.
Discussion
AAV preparations typically consist of a heterogeneous mixture of capsid species, including empty, partially filled, and full particles, depending on the DNA content encapsulated within the viral capsid (Figure 1). Empty capsids, which lack a packaged nucleic acid genome, can represent between 50% and 90% of total particles produced in cell culture systems [4,7,8].
Given this heterogeneity, the proportion of empty versus full capsids is a critical parameter to evaluate during process development and product characterization. Empty capsids may negatively impact efficacy by competing with genome-containing particles for binding to target cells, potentially reducing transduction efficiency and increasing the required therapeutic dose [9,10].
Conversely, some studies suggest that empty capsids may act as decoys, protecting functional AAV particles from immune clearance and thereby enhancing overall gene transfer efficiency [11,12]. From an immunological perspective, capsid-derived peptides can be presented by major histocompatibility complex (MHC) molecules, leading to recognition and elimination of transduced cells by capsid-specific CD8+ T cells. While such immune responses have been documented in humans, they are not solely attributable to empty capsids, as total capsid load also plays a significant role [13].
Taken together, these sometimes-conflicting observations highlight the complexity of defining an optimal full/empty capsid ratio and underscore the importance of its careful control as a critical quality attribute. This scientific uncertainty, combined with its potential impact on both efficacy and safety, has led regulatory authorities to increasingly focus on the characterization and monitoring of this parameter. However, the regulatory framework addressing the full/empty capsid ratio remains relatively non-prescriptive.
In Europe, the general monograph on Gene Therapy Medicinal Products (Ph. Eur. 3186) states that full and empty particles should be quantified either indirectly – through the ratio of capsid titre to vector genome titre – or via suitable analytical methods such as analytical ultracentrifugation (AUC) or size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALLS). Acceptance criteria are defined on a product-specific basis [14].
Similarly, the 2018 EMA guideline on gene therapy products requires the determination of total and functional viral particles but does not define specific thresholds for acceptable full/empty ratios [15]. In the United States, the FDA considers empty capsids as product-related impurities that should be quantified and controlled.
While the agency does not prescribe specific analytical methods or acceptance criteria, it emphasizes the importance of monitoring ratios such as full-to-empty particles or total particles-to-infectious units [16,17]. Table 1 shows extracts of the different regulations mentioned above.
Table 1. Extracts from EU and US regulatory guidelines regarding full and empty AAV particles.
As an initial overview, several orthogonal analytical methods are currently employed to assess the full-to-empty capsid ratio in rAAV preparations [18,19,20], each with distinct advantages and limitations in terms of resolution, throughput, and quantification accuracy. A concise summary of these approaches is presented in Table 2.
Given the diversity of available methods and the absence of a universally accepted standard, a combination of complementary analytical techniques is often recommended to achieve a robust and comprehensive characterization of rAAV particle heterogeneity. The reader is referred to the cited literature for more detailed methodological comparisons and practical considerations [18,19,20].
Table 2. Analytical methods for evaluating the full-to-empty capsid ratio in AAV preparations: advantages and limitations.
Conclusion
Despite increasing regulatory attention, current guidelines from both European and US authorities remain non-prescriptive, leaving significant flexibility to sponsors. Regulatory agencies consistently emphasize the importance of minimizing empty capsids and implementing robust manufacturing and purification processes, but no universal acceptance criteria have been defined. Instead, expectations are assessed on a case-by-case basis, taking into account factors such as dose, route of administration, and clinical indication.
At the same time, important scientific and technical challenges remain unresolved. In particular, the diversity of analytical methods currently used to assess full/empty capsid introduces significant variability in results. Questions regarding method comparability, accuracy, and suitability for regulatory decision-making remain largely open, contributing to an additional layer of uncertainty beyond the definition of acceptable specifications.
In this context, further alignment between regulatory authorities, along with continued methodological and clinical investigations, will be essential to support the development and standardization of AAV-based therapies.
The growing complexity and lack of harmonization in analytical strategies suggest that the evaluation of full/empty capsid ratios may, in itself, warrant dedicated discussion, as it remains a critical yet insufficiently resolved aspect of AAV product characterization.
C. Lemaigre is a Pharmacist and PhD with experience in contract development and manufacturing organization (CDMO), specializing in gene therapy and viral vector development. Her expertise focuses on chemistry, manufacturing and controls (CMC), with particular interest in analytical characterization and regulatory considerations for AAV-based products.
At Strand, we support our partners through project staffing, project and service management, outsourcing solutions, and tailored training, helping them meet regulatory, operational, and business challenges efficiently.
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