Standardisation in the Analytical Characterization of Adeno-Associated Virus (AAV) Vectors

Summary

Adeno-associated virus (AAV) gene therapy requires well-characterized Reference Standard Materials (RSMs) to ensure accurate, reproducible analytical measurements within and across laboratories. This review examines the critical need for RSMs, evaluates available commercial and pharmacopeial standards, and provides practical strategies for in-house RSM development to advance safe and effective AAV therapeutics.

Abstract

Adeno-associated virus (AAV) has become a leading vector for in vivo gene therapy, with eight products currently holding marketing authorization. As the field rapidly evolves, the need for robust analytical methods to characterize critical quality attributes (CQAs)—including capsid titer, genome titer, capsid content (empty/full ratio), identity, and purity—continues to grow. Reference Standard Materials (RSMs) play a pivotal role by providing well-characterized, standardized AAV batches that serve as universal benchmarks. RSMs facilitate the validation of emerging analytical technologies, ensure the accuracy and reproducibility of routine assays, and enable inter-laboratory comparability. However, developing universal AAV RSMs is fundamentally constrained by the complex biology, diversity of serotypes, vector genomes, and engineered capsid variants, necessitating serotype-specific and application-specific standards. Recent advances, including the release of pharmacopeial AAV8 reference standards characterized by multiple orthogonal methods, represent meaningful progress toward measurement harmonisation. This review addresses the critical need for RSMs in AAV gene therapy, evaluates the currently available pharmacopeial and commercial standards, and outlines practical strategies for in-house RSM development. Establishing robust, serotype-specific AAV RSMs and harmonised standard operating protocols (SOPs) are essential for advancing AAV gene therapy and ensuring accuracy, reproducibility, and safety across research, development, and clinical manufacturing.

Introduction

Gene therapy offers a transformative strategy for treating genetic and acquired diseases by delivering functional genetic material into patient cells to correct or regulate gene expression. Among these, adeno-associated viruses (AAVs) stand out for in vivo applications due to their diverse tissue tropism across different serotypes, and sustained episomal transgene expression¹.​

AAVs are compact (~25 nm diameter), DNA viruses in the Dependoparvovirus genus of the Parvoviridae family. These viruses are non-pathogenic and replication-defective, requiring helper viruses such as adenovirus or herpes simplex virus for replication². The AAV genome has two genes, rep and cap, flanked by two 145-base pair palindromic repeats known as inverted terminal repeats (ITRs)³. Recombinant AAV (rAAV) vectors are generated by replacing viral coding sequences with therapeutic cassettes containing a promoter, transgene, and regulatory elements flanked by ITRs. During production, the rep and cap genes are delivered in trans, facilitating replication and packaging of the therapeutic cassette into viral capsids and yielding non-integrating, replication-deficient particles that maintain robust transduction capability while preventing wild-type viral replication⁴. Eight rAAV-based gene therapies have received marketing authorization from either the European Medicines Agency (EMA) or the U.S. Food and Drug Administration (FDA), and clinical trials with AAVs are on the rise. As of January 2025, 343 AAV clinical trials were registered globally, representing a 34% increase from 255 trials in mid-2022⁵. This rapid clinical expansion intensifies the need for robust quality control to ensure patient safety⁶.

Ensuring rAAV products are safe and effective for human use requires thorough characterization of critical quality attributes (CQAs), including quantity, potency, purity, identity, safety, and stability. However, AAV characterization remains inherently challenging due to the structural complexities of these vectors. AAVs are icosahedral particles composed of three viral capsid proteins (VP1, VP2, VP3), assembled in an approximate 1:1:10 ratio, and variations in protein assembly can affect transduction efficiency⁷. The therapeutic DNA payload must be packaged into the capsid with high fidelity; heterogeneous capsid populations are inherently generated during rAAV production, consisting of capsids containing a complete genome (full capsids), a partial genome (partially filled capsids), or no genetic material (empty capsids). Adding to this complexity, different AAV serotypes exhibit distinct packaging efficiencies, tissue tropisms, and immunological profiles, necessitating serotype-tailored assay development. Beyond these biological challenges, a critical problem exists in how measurements are performed. Different laboratories use different assays, protocols, and data‑analysis approaches to measure the same CQA. As a result, reported values for identical samples can differ by orders of magnitude between sites⁸. Even within a single laboratory, changes over time in reagents, instruments, operators, or analysis settings can lead to shifts in results from the same batch⁹. When measurements are not comparable, establishing reliable associations between CQAs and preclinical or clinical outcomes becomes challenging, thereby complicating regulatory decision-making^10,11.​

Standardisation aims to address this issue by ensuring measurement comparability. In pharmaceutical analytics, standardisation encompasses two key aspects: the use of common physical reference standard materials (RSMs) and adherence to harmonised detailed written standard operating procedures (SOPs), ensuring that different laboratories measure titer, identity, quality, potency, and purity consistently¹². RSMs are well-characterized samples that act as benchmarks. By validating assays against the same RSM, laboratories anchor their measurements to a common reference point. This reduces variability between laboratories and allows results from different studies, products, and manufacturing sites to be reliably compared¹³. Furthermore, it also serves as an in‑house control to monitor assay performance and monitor consistency over time¹⁴. SOPs further ensure that analytical methods are executed consistently across laboratories, minimising variability arising from differences in sample pretreatment, instrumentation, calibration strategy, data processing, and operator practice^8,9,12. Together, these two components form the foundation of a robust analytical standardisation framework.

This review systematically addresses the key aspects of standardisation in AAV analytics. We examine the types of RSMs and their analytical applications, evaluate currently available AAV-specific RSMs for their fitness for purpose, and provide consolidated practical guidance for in-house RSM production, purification, characterisation, and storage. We further identify prevailing manufacturing challenges and analytical limitations. Recognizing that RSMs alone are not sufficient, we also discuss harmonised SOPs as a complementary and equally important standardisation component. Together, this review is intended as a practical reference for analytical scientists, quality professionals, and manufacturers seeking to advance measurement harmonisation and support science-based regulatory decision-making in AAV gene therapy.

Protocol

1. RSM: definitions and need

RSMs are organized hierarchically, with two main types: primary and secondary RSMs. According to ISO Guide 30, a primary standard is defined as a “standard that is designated or widely acknowledged as having the highest metrological qualities and whose value is accepted without reference to other standards of the same quantity, within a specified context”. For example, RSMs from the United States Pharmacopeia (USP), European Pharmacopeia (Ph. Eur.), British Pharmacopeia (BP), etc., that have been extensively characterized through multi-laboratory studies qualify as primary RSM. Individual manufacturers subsequently use these primary RSMs to calibrate and qualify their own proprietary working reference materials, which serve as secondary RSMs for routine quality control testing^12,15. Hence, ISO defines a secondary RSM as a “standard whose value is assigned by comparison with a primary standard of the same quantity”. This hierarchical approach ensures traceability: in-house measurements trace back to primary standards, which in turn are characterized using state-of-the-art analytical methods¹⁶. Using RSMs provides common benchmarks that enable manufacturers to calibrate and validate their analytical methods to the same reference point, thereby leading to more accurate measurements. Regulatory authorities, therefore, encourage the use of primary RSMs to calibrate in-house RSMs and validate test methods, improving consistency throughout AAV manufacturing^17,18. This allows standardisation across varying analytical techniques, leading to better comparison of rAAV characterization results across laboratories and different products^17,19. The improved comparability enhances understanding of safety risks potentially associated with high doses or product-related impurities, such as empty and partially filled capsids. Ultimately, these insights can inform science-based regulatory policy formulation, such as a minimum threshold of full capsids, analogous to the ≥70% viable cells criterion for somatic cellular therapies, thus contributing to safer and more reliable gene therapy products^13,20,21.​

For rAAV, several CQAs clearly illustrate the need for standardisation. Vector genome titer directly determines the patient dose; inaccurate titers risk under- or over-dosing. Quantitative PCR (qPCR) and digital PCR (dPCR) are widely used to measure titer, but results can differ for the same sample if different in-house standard curve calibrants or protocols are used¹¹. Capsid content (the ratio of full, partially filled, and empty capsids) is another critical CQA, as empty and partially filled capsids can contribute to immunogenicity or reduce efficacy⁶. More than ten different techniques are used to measure capsid content, including analytical ultracentrifugation (AUC), Size-Exclusion Chromatography coupled with multi-angle light scattering (SEC-MALS), mass photometry (MP), charge detection mass spectrometry (CDMS), electron microscopy (EM), anion-exchange (AEX) chromatography, capillary isoelectric focusing (cIEF), ultraviolet-visible spectroscopy (UV/Vis), etc. In addition, capsid content is often estimated indirectly using the ratio of vector genome titer to viral particle titer, determined by qPCR or dPCR and ELISA, respectively (PCR/ELISA)²². Each technique measures different physical properties and can report different capsid content values for the same samples. A recent NIST interlaboratory study comparing UV/Vis, AUC, SEC-MALS, and PCR/ELISA demonstrated substantial inter-method and inter-laboratory variability, with PCR/ELISA showing particularly poor reproducibility and AUC exhibiting significant between-laboratory divergence despite being widely regarded as a reference method⁸. These findings indicate that reducing variability in AAV analytics requires both shared RSMs and harmonised analytical workflows, as discussed in Section 5.

2. rAAV RSM landscape

Efforts to establish RSMs for AAV have resulted in two sources providing primary AAV RSMs. Among pharmacopeial sources, the USP is currently the only provider of primary AAV RSM. Beyond USP, primary AAV RSMs are also available from the American Type Culture Collection (ATCC). A growing range of commercial secondary RSMs for multiple serotypes is also available, reflecting ongoing community efforts to address the lack of standardisation inherent to AAV characterisation. Table 1 summarizes the currently available AAV RSM materials and their key characteristics.

1. ATCC standards
   The first international effort to establish RSMs for rAAV began in 2010 when the AAV Reference Standard Working Group (AAVRSWG) produced primary RSMs for rAAV2, followed by rAAV8 in 2014. This volunteer organization included members from five industrial organizations, 13 universities, the Food and Drug Administration (FDA), National Institute of Health (NIH), and Williamsburg Bioprocessing Foundation (WilBio), representing four countries: the US, France, Germany, and Japan. The primary RSMs were intended to serve as community-wide standards to facilitate inter-laboratory comparison of doses across nonclinical and clinical studies. Researchers are encouraged to report titers in relation to this primary RSM when publishing or submitting data to health authorities. Production aimed to achieve a final titer of 1 × 10¹² vector genome per milliliter (vg·mL^-1) and to yield approximately 5,000–10,000 vials containing 0.5 mL each¹⁹. The materials were characterized for vector genome titer (qPCR), particle titer (ELISA), transducing titer (fluorescence microscopy), infectious particle titer (TCID₅₀), purity (sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)), and vector identity (gel electrophoresis) through a multi-laboratory study involving 16 laboratories worldwide. Additionally, mycoplasma, bioburden, sterility, and endotoxin were evaluated to ensure product safety^23,24,25. An in-depth overview of AAV analytical characterization, including the key assays and the information each assay provides, is presented in Section 4.
   Although ATCC RSMs were pioneering materials in the AAV field, their utility is constrained by the fact that they were produced more than a decade ago, before many current analytical approaches for AAV characterization became widely adopted. For example, the original characterization did not include empty/full capsid profiling, which limits their direct applicability for standardizing capsid-content analysis, now considered an important CQA for rAAV. In addition, substantial inter-laboratory variability was reported for vector genome titer, capsid titer, and infectious titer despite the use of shared protocols and reagents, underscoring that common materials alone do not ensure harmonised measurements, and more mature assays with detailed SOPs are equally important. According to current supplier information, the availability of these materials is also restricted, which may further limit broader implementation¹¹. According to current supplier product information, availability of these materials is restricted to 3 vials per customer per year for AAV2 (ATCC VR-1616), with supply noted as pending replenishment, and 12 vials per customer per year for AAV8 (ATCC VR-1816). These restrictions necessarily limit the broader and routine use of these materials as community-wide RSMs^26,27.
2. USP standards
   In terms of pharmacopeial initiatives, the USP is currently the only organization that has produced primary RSMs for AAV. The following characterisation data is based on USP application notes^28,29,30. In 2025, the USP released AAV8 RSM for full and empty capsids in an effort to standardize capsid content analytics. To achieve this, the USP established RSMs with high concentrations (> 3.0E+12 viral particle titer per milliliter (vp·mL^-1)) and substantial volumes (300 µL), enabling their application across various capsid content analytical approaches. A comprehensive multi-laboratory collaborative study was performed to fully evaluate the USP AAV RSM and ensure measurement accuracy and precision. This study involved five independent laboratories and employed several capsid content analytical techniques: AUC, SEC-MALS, CD-MS, MP, and UV/Vis. Additional biophysical characterization methods were applied to the AAV samples, including peptide mapping, next-generation sequencing (NGS), dPCR, and capillary gel electrophoresis (CGE), providing further particle characterization data²⁸.
   Furthermore, USP developed an AAV5-based quantitative PCR control containing cytomegalovirus (CMV) enhancer, chicken β-actin (CBA) promoter, green fluorescent protein (GFP), simian virus 40 (SV40) polyadenylation signal, intended for use as a standard curve calibrant or assay control in genome titer measurements. The capsid content was determined by SEC-MALS, while the viral genome titer was quantified by dPCR targeting multiple sequences. A multi-laboratory dPCR study, including 4 laboratories, revealed that targeting SV40 yielded lower inter-laboratory variability (CV 11.2%) compared to ITR targeting (CV 36.1%), likely due to ITR secondary structure interference. Consequently, the assigned reference value is based on SV40 targeted titer and equals 7 x 10¹⁰ vg·mL^-1,32. With respect to reference materials for capsid titer assays, USP provides capsid titer reference materials for AAV1, AAV5, AAV8, and AAV9. These materials, characterized by SEC-MALS for absolute capsid particle quantification and ranging from 1x10¹¹ to 1x10¹² vg·mL^-1, can serve as standard curve calibrants or positive controls in capsid titer assays³⁰.
3. Commercial standards
   Several commercial companies offer AAV full and empty RSMs for multiple serotypes, e.g., PROGEN, AAVnergene, and Revvity; the following characterization data and product specifications are based on supplier-provided information, including product sheets and application notes^{31,32,33,34,35}. As secondary RSMs, these materials are frequently benchmarked against ATCC RSMs. Compared with pharmacopeial RSMs, these RSMs cover a broader range of serotypes, with certain suppliers supplying AAV1–13 and engineered variants. Commercial RSMs are typically characterized for basic quality attributes such as capsid titer (by ELISA or SEC-MALS), vector genome titer (by qPCR or dPCR), and capsid content (often by a single analytical method).
   However, some of these commercial materials are only available in small quantities, restricting their use for extensive method development of different assays or multi-site studies. Furthermore, an extensive characterisation through a multi-laboratory collaborative study involving multiple orthogonal techniques, including stability assessment, is lacking, thereby limiting confidence in assigned values and inter-laboratory applicability. Despite these limitations, commercial RSMs play an important role in assay development, validation, and process optimization, and can serve as interim RSMs when primary RSMs are unavailable for specific serotypes.
   Some suppliers are working toward more rigorous characterization protocols and increased transparency to enhance the utility of their RSM offerings^36,37.
4. In-house standards
   Individual laboratories can also develop in-house RSMs, a strategy often employed for unique AAV products, such as capsid-engineered serotypes or specific viral genome constructs. In-house RSMs may also be favored to reduce costs, as primary and commercial secondary reference materials can be expensive. When feasible, these in-house RSMs should be benchmarked against primary RSMs to subsequently be used for assay development, validation, routine testing during manufacturing, or quality control. Several important considerations apply when using in-house RSMs: these materials should be thoroughly characterized, sufficient quantities should be reserved, and protocols should be established to monitor stability over time. Moreover, planning for the production of new RSM batches is critical, including retaining enough material from the previous batch to bridge measurements and ensure continuity throughout product development²⁰. In section 3, a detailed overview of strategies for in-house RSM production, covering upstream and downstream processes as well as analytical characterization, is provided. Together, these approaches offer a practical framework for generating in-house RSMs.

3. RSM manufacturing strategies:

Producing high-purity AAV RSMs presents inherent challenges. Achieving 100% empty capsid preparations is complicated by the fact that "empty" capsids may contain residual genetic material. Similarly, obtaining pure full capsid preparations proves difficult because empty and partially filled capsids share similar physical characteristics and co-purify with full capsids during separation processes¹⁰. Despite these challenges, significant advances in production technology now enable enrichment of predominantly full or predominantly empty capsid populations. The following section provides a comprehensive overview of current production and separation technologies, as well as characterisation methods and formulation conditions that may be employed for producing, characterizing, and storing AAV RSMs.

1. Upstream processing
   rAAV, like other biotherapeutics, must be produced in living cells, most commonly using either transient transfection or viral infection-based platforms. The dominant approach for clinical-grade material is triple-plasmid transient transfection in HEK293 suspension cells, using three separate plasmids for the ITR-flanked transgene, the rep/cap genes, and helper functions; this flexible, helper virus-free system supports rapid serotype and transgene interchangeability and is used for most clinical programs and approved products³⁸. However, plasmid DNA costs increase substantially as production scales up, representing a significant disadvantage. To reduce raw material costs, production systems have been streamlined from three plasmids to either two (rep/cap/helper genes on one plasmid, transgene on another) or one (with all genes on a single plasmid)^39,40. HEK293 cells can be cultured in adherent or suspension formats with comparable productivity, though suspension culture is preferred for its scalability⁴¹.
   Alternative production platforms include the Baculovirus Expression Vector System (BEVS) in Sf9 or other insect cells, stable producer cell lines, herpes simplex virus-based systems, and inducible expression systems. Notably, different production platforms can yield significantly different empty/full ratios even for the same serotype, with reported %full ranging from 8% to over 39% depending on the platform and specific AAV serotype employed⁴².​
   Selecting the appropriate production platform and optimizing upstream conditions for RSM generation requires careful consideration of the target capsid population. With respect to HEK293 transient transfection, the most widely used production system for clinical material and currently available RSMs (Table 1), various process parameters can be optimized to enhance both yield and the empty/full capsid ratio. For full capsid RSMs, upstream process optimization aims to maximize yield and genome packaging efficiency to minimize partial or empty capsid formation, as the initial empty/full ratio in crude lysate directly impacts the downstream empty/full ratio. Key parameters include cell density at transfection, plasmid molar ratios, the ratio of transfection reagent to DNA, harvest timing, small molecule additives (e.g., cell cycle arrest agents), and transgene cassette design^{40,43,44,45,46}.
   Empty capsid RSMs are typically produced by omitting the transgene plasmid during transfection, preventing DNA packaging after capsid assembly. In practice, however, these “empty” preparations often still contain contaminating DNA, making them compositionally heterogeneous and highlighting the need for thorough analytical characterisation^47,48.
2. Downstream processing
   The downstream process (DSP) includes harvesting, clarification, and purification of the vector to obtain a highly pure AAV product while ensuring both high vector yield and an optimal empty/full ratio. Downstream processing begins 72 hours post-transfection with cell lysis to recover intracellular rAAVs⁴⁹. Although viral egress varies by serotype, cell lysis ensures complete intracellular recovery⁵⁰. Lysis is achieved mechanically via freeze-thaw cycles or chemically using detergent-based buffers (e.g., Triton X-100, Tween-20/80, CHAPS), with chemical methods preferred for improved scalability^49,51. Nuclease treatment (e.g., Benzonase) during lysis degrades uncapsidated vector genomes and contaminating plasmid DNA, preventing bias in genome titration and reducing DNA viscosity for further DSP steps. A common lysis buffer for HEK293-derived rAAV is 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, Benzonase 25–50 U.mL^-1,51. The lysate is then clarified by depth filtration or centrifugation to remove cellular debris. Tangential flow filtration (TFF) then concentrates the harvest and exchanges buffer for downstream purification, though this step can be omitted if the downstream purification step can handle large volumes. Further purification proceeds via either ultracentrifugation (UC) or chromatography-based methods⁵².
   
   Gradient density UC, most commonly through cesium chloride (CsCl) gradient or iodixanol, has been traditionally employed for rAAV purification, providing high yield and effective separation of full capsids from process and product-related impurities⁵³. While UC offers serotype independence and high purity, it has significant shortcomings, including manual handling, limited scalability for large volumes, and labour-intensive processing. Column chromatography has emerged as a preferred alternative, offering scalability, faster processing times, and automation capability, and has been successfully applied for clinical-grade rAAV and rAAV RSM production. Column chromatography for rAAV is typically performed in two sequential steps: capture and polishing^52,54.
   
   Affinity chromatography serves as the primary capture step, using immobilized ligands on resins to bind AAV capsids while removing most process-related impurities. Single-domain antibody fragments (V_HH) have recently become the preferred choice of ligand due to versatility across serotypes and high specificity. Commonly used antibody-based columns include POROS CaptureSelect AAVX, AVB Sepharose High Performance, and Capto AVB⁵¹. POROS CaptureSelect AAVX offers the broadest specificity, binding AAV1–9, rAAV-10, and engineered variants, making it suitable for serotype-agnostic purification⁵⁵​. It is commonly employed for most commercial rAAV RSM purification (Table 1). Since affinity columns cannot differentiate between full and empty capsids, as the epitopes are present on both variants, a further polishing step is employed. The polishing step typically employs anion exchange chromatography (AEX) for separating full and empty capsids. This method exploits the slight isoelectric point (pI) difference between full (~5.9) and empty (~6.3) capsid populations⁵². Three stationary phase formats are commercially available: packed resins, monoliths, and membrane columns⁵³. Comparative performance data across all three formats for AAV is limited. However, monoliths and membrane columns support higher convective-based flow rates, making them preferred at larger scales where reduced processing time is critical. Packed resins rely on diffusion-limited mass transfer and higher back pressure, restricting flow rates^56,57. A list of commercially available AEX columns can be found in a review from Jungbauer et al⁵¹. For elution, stepwise salt gradients are increasingly preferred over shallow continuous salt gradients because fixed salt steps are less sensitive to instrumental variations (pump mixing, flow rate) than continuously changing gradients, improving reproducibility while reducing buffer consumption⁵². Optimised AEX protocols can achieve >90% full capsid purity across multiple serotypes⁵⁸. However, unlike UC, AEX has not yet demonstrated reliable separation of partially filled capsids, and each serotype requires optimization as the charge profile varies by serotype⁵¹. Notably, most commercial RSM purification processes combine affinity capture chromatography with UC (iodixanol or CsCl) as a polishing step, leveraging the high purity advantages of UC following the initial affinity-based impurity removal (Table 1).
3. Formulation
   AAV formulation optimisation is important for ensuring the stability of RSM and its shelf life. Formulation design involves excipient selection, osmolarity regulation, buffer, surfactant selection, and control of ionic strength to minimise aggregation. The most common buffer systems include phosphate-based, tris, acetate, citrate, and lactate, typically operating within a pH range of 7.4 ± 0.3 across different serotypes. Excipients are stabilising agents commonly found in AAV formulations, including NaCl, KCl, poloxamer 188 (Px188), MgCl₂, CaCl₂, sucrose, PS20, sorbitol, mannitol, glycerol, and human serum albumin. Px188 at ~0.001% is commonly used to reduce interfacial and mechanical stress, thereby limiting capsid rupture and titer loss during handling and freeze-thaw⁵⁹. Osmolarity is an important consideration for in-human administration, but is less relevant for research-grade RSMs⁶⁰. Most commercial RSMs use PBS-based buffers, occasionally supplemented with additional salts. Suppliers recommend storage at -80 °C and avoiding multiple freeze-thaw cycles, as this can promote capsid rupture at ice-water interfaces, potentially leading to a loss in titer^34,61.

4. Analytical characterization: Defining the standard

Once the RSM has been produced, purified, and formulated, a comprehensive analytical characterization is performed. This step ensures the quality of the RSM and its applicability as a reference across different analytical methods. Employing orthogonal analytical techniques to assess the same quality attributes is particularly important to ensure robustness and is therefore highly recommended. The key quality attributes specifically for AAV RSM can be divided into three categories: titer, identity, and purity, as listed in Table 2.

1. Titer
   RSM characterization encompasses measuring titer, quantified as vector genome titer and capsid particle titer, providing information on the number of vector genomes and capsids present. Genome titer is typically measured by qPCR or dPCR targeting the transgene, ITRs, or other transgene cassette elements, with assay design and primer/probe placement significantly influencing results. ITR-based assays are serotype-independent and broadly applicable, but the hairpin structure of ITRs can hinder primer binding, and such assays may overestimate titers when partially filled capsids retaining ITR sequences are present; ITR-targeting qPCR has been shown to overestimate genome titers for AAV2 and AAV8 RSMs relative to expression cassette-targeting assays¹¹. A further source of bias in qPCR is the use of double-stranded plasmid DNA calibrants, which exhibit different amplification kinetics than single-stranded rAAV genomes²⁹.
   dPCR addresses both limitations through absolute quantification via Poisson statistics, eliminating standard curve dependency, and has been shown to be less susceptible to ITR secondary structure interference⁶². Notably, qPCR can achieve equivalent accuracy when calibrated against an rAAV-based rather than plasmid-based calibrant, offering a more cost-effective alternative⁶³. Appropriate sample pretreatment is equally important. DNase treatment removes external DNA to ensure only encapsidated genomes are quantified, and must be followed by inactivation prior to capsid disruption, using proteinase K or heat treatment^64,65,66. An optimized and validated protocol for ddPCR-based genome quantification in purified vector samples has been published by our research group⁶⁷.
   The capsid particle titer is commonly determined using an ELISA or SEC-MALS. ELISA offers high specificity and robustness to matrix effects but is serotype-specific, standard curve-dependent, and labor-intensive. SEC-MALS is serotype-independent and faster but requires specialised instrumentation²².
2. Identity
   Assessment of identity aims to confirm that the produced AAV conforms to the intended serotype and genomic sequence, excluding unintended alterations at both the protein and DNA levels. Capsid protein identity refers to serotype identification. Common methods include ELISA and western blot (WB) using serotype-specific antibodies. More recently, mass spectrometry coupled to high-performance liquid chromatography (LC-MS) has emerged as a higher-order, complementary approach for serotype identification and capsid composition analysis because of its high reproducibility, accuracy, and robustness, although it is generally not used for routine in-house RSM characterization^6,22. Vector genome identity can be assessed using sequencing methods, such as nanopore sequencing. This approach provides long-read coverage of complete AAV genomes, allowing accurate determination of sequence identity²².
3. Purity
   A third important quality parameter for AAV RSMs is purity, encompassing protein purity, capsid and genome integrity, capsid content, and aggregation. Capsid integrity is evaluated by verifying VP stoichiometry (VP1:VP2:VP3 ≈ 1:1:10) using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Capillary Electrophoresis Sodium Dodecyl Sulfate (CE-SDS), or WB, with WB additionally providing capsid protein identity confirmation²².
   Genome integrity, whether the transgene is full-length or truncated, can be assessed by Agarose Gel Electrophoresis (AGE), multiplex ddPCR, or sequencing-based approaches such as nanopore sequencing. While AGE is straightforward, it offers limited resolution and specificity. Multiplex ddPCR provides quantitative, region-specific data that gel-based methods cannot, albeit at the cost of more extensive assay development^22,68.
   Capsid content is often estimated by dividing the genome titer by the capsid titer; however, this relies on two independent assays, which can introduce additional variability and is therefore not recommended for RSMs characterization. More suitable techniques for this attribute with lower variability include AUC, SEC‑MALS, MP, CD‑MS, and UV/Vis^{8,69,70,71,72,73}. Recent developments have further increased the value of AUC and SEC‑MALS as multi-attribute methods. In particular, multi-wavelength AUC (MWL-AUC) enables not only discrimination of empty, partially filled, and full capsids, but also assessment of genome titer, capsid titer, and aggregation^71,74. Similarly, SEC‑MALS can provide, within a single assay, information on capsid titer, genome titer, purity, aggregation, and full-to-empty capsid ratios^73,75. Aggregation can also be monitored using dynamic light scattering (DLS). Notably, only AUC, MP, and CDMS have traditionally offered sufficient resolution to quantify partially filled capsids. However, recently an enhanced TEM method has been developed, which can also quantitate partially filled capsids accurately compared to SV-AUC and MP⁷⁶. As no single method has emerged as a gold standard for capsid content, and these techniques rely on different analytical principles, using multiple complementary approaches is recommended to obtain a comprehensive assessment of overall purity¹⁰.
4. Stability
   Long-term stability is critical for RSMs, as reference values must remain constant to ensure reliable calibration over time. AAV RSMs are typically stored at ≤ -70 °C to maintain capsid and genome integrity. A multi-laboratory stability study of AAV8 RSM (ATCC VR-1816) demonstrated stable vector genome titer, infectious titer, and capsid titer for at least two years at < -70 °C⁷⁷. Procedures for stability testing can follow ICH Q1A guidelines, assessing key attributes at defined intervals: every 3 months (year 1), every 6 months (year 2), and annually thereafter ⁷⁸. Short-term stability at accelerated storage conditions (2–8 °C) is typically evaluated at 1, 2, and 4 weeks⁷⁹. Critical stability-indicating attributes include genome titer, capsid titer, capsid content, capsid protein integrity, and aggregation. RSMs should also be evaluated for freeze-thaw sensitivity⁸⁰.

5. Harmonised SOPs as a second pillar of AAV analytical standardisation

Beyond the availability of well-characterised RSMs, meaningful standardisation in AAV analytics also requires harmonised, sufficiently detailed SOPs. Even when the same analytical method is applied, results may differ substantially across laboratories due to differences in sample pretreatment, instrumental settings, calibration strategy, data processing, and interpretation criteria, factors that have been shown to contribute to interlaboratory variability independently of the analytical platform itself^8,9.

This is illustrated by a collaborative NIST, National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), and USP interlaboratory study in which six laboratories evaluated the same AAV5 and AAV8 samples for capsid content using four methods. Optical methods, including SEC-MALS and UV/Vis, showed the strongest interlaboratory agreement, whereas PCR-ELISA showed poor reproducibility and was considered unsuitable for quantitative use without further harmonisation. Despite being widely regarded as a gold-standard method, SV-AUC showed greater interlaboratory variability than SEC-MALS, and follow-up efforts to develop standardised SV-AUC procedures have been identified as an important next step^8,81.

A similar conclusion has emerged for genome titer determination by dPCR, where sample pretreatment, extraction strategy, instrument platform, dilution conditions, capsid lysis timing, and freeze-thaw history can all affect reported values. Importantly, tighter procedural control was shown to reduce variability substantially, further underscoring the value of harmonised SOPs⁹.

RSMs and SOPs should therefore be viewed as complementary pillars of AAV analytical standardisation. RSMs provide the measurement anchor required for traceability and calibration, whereas SOPs reduce procedural variability and help ensure that assigned values are reproduced consistently across analysts, instruments, and laboratories.

Results

rAAV gene therapy is advancing faster than the field's ability to consistently characterize quality attributes. This review demonstrates that the primary barrier to comparable AAV characterization is not a lack of analytical tools, but rather the absence of shared measurement anchors and harmonised workflows. RSMs address this challenge by transforming method-dependent measurements into traceable, comparable values. RSMs enable method validation, improve inter-laboratory reproducibility, and provide ongoing controls to detect assay drift. However, producing high-quality AAV RSMs remains technically demanding due to the difficulty of obtaining pure empty and full capsid populations, the need for comprehensive orthogonal characterization, and the requirement for long-term stability under defined storage conditions. Recent advances in manufacturing and purification technologies have enabled the production of predominantly empty and full capsid preparations, which can be used to generate in-house RSMs, and current offerings from pharmacopeial bodies (USP), collaborative consortia (ATCC), and commercial suppliers represent meaningful progress. However, developing universal AAV RSMs is fundamentally constrained by the diversity of serotypes, vector genomes, and increasingly, engineered capsid variants. Unlike monoclonal antibodies, AAV's heterogeneity demands serotype-specific and application-specific RSMs. This complexity is further amplified by hybrid and/or engineered capsids, which require developers to maintain proprietary internal primary RSMs, thereby fragmenting the measurement landscape. Further, other quality attributes like post-translational modifications (PTM) have been highlighted as potential CQA and lack well-characterized RSMs for PTMs, creating a critical gap in understanding this vector attribute. Beyond RSM availability, harmonised SOPs are equally critical. The field would benefit from compendial methods—standardized, detailed protocols published by pharmacopeial bodies—that ensure all laboratories execute consistent and controlled testing for AAV CQAs. Written guidance documents detailing best practices for commonly used methods would further reduce inter-laboratory variation and harmonize analytical workflows. By establishing traceable AAV measurement RSMs and implementing consistent, accurate characterization practices, the community can strengthen the scientific foundation linking CQAs to safety and efficacy, support confident regulatory decision-making, and ultimately deliver safer, more reliable gene therapies to patients.

Table 1: Currently available rAAV RSMs and their characteristics

This table summarizes available rAAV reference standard materials and their key characteristics. Please click here to download this Table.

Table 2: Recommended suite of analytical methods for in-house RSM characterisation

This table summarizes analytical methods recommended for in-house RSM characterization. Please click here to download this Table.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
POROS™ CaptureSelect™ AAVX Affinity Resin	Thermo Fisher Scientific	A36740	AAV affinity chromatography resin
AVB Sepharose™ High Performance	Cytiva (formerly GE Healthcare Life Sciences)	28411202	Affinity chromatography resin
Capto™ AVB	Cytiva	17372203	Affinity chromatography resin / AAV capture resin