Genome length determination in adeno-associated virus vectors with mass photometry.

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the adeno-associated virus (AAV) capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption, and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (coefficient of variation [%CV] < 2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV < 2.9%) considering calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with precision of %CV < 0.7% and accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their calibration materials and efforts.

Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

Systematic Studies on Stabilization of AAV Vector Formulations by Lyophilization.

Adeno-associated virus (AAV) vectors have evolved as one of the most promising delivery systems for gene therapy. The current standard for AAV vector storage is deep-freezing below -60 °C. While this allows for long-term vector storage without loss of activity, it is inconvenient and involves high costs and logistical challenges. Therefore, there is a need for AAV formulations, such as freeze-dried formulations, that allow for long-term storage at 2-8 °C. A major challenge in developing a lyophilization process for complex biological structures like an AAV vector is to minimize the stress on the capsid during the lyophilization cycle. Here, we evaluated different conditions for freeze-drying of AAV8 vectors and found that undesirable instability can be significantly reduced if secondary drying is performed at lower temperatures, kept as short as possible, and the residual moisture is kept between 1.5 and 2%. In a next step, we explored formulations with different salt concentration or excipient compositions and found that a combination of 10 mM phosphate buffer, 5.67% (150 mM) trehalose, 5% hydroxyectoine and 0.1% poloxamer with a residual moisture of approx. 1.5% provided stable long-term storage at 2-8 °C and for at least 4 weeks at 25 °C. These results pave the way for future optimizations of freeze-drying processes for AAV vector-based gene therapy products.

Towards quantification and differentiation of protein aggregates and silicone oil droplets in the low micrometer and submicrometer size range by using oil-immersion flow imaging microscopy and convolutional neural networks.

Biopharmaceutical product characterization benefits from the quantification and differentiation of unwanted protein aggregates and silicone oil droplets to support risk assessment and control strategies as part of the development. Flow imaging microscopy is successfully applied to differentiate the two impurities in the size range larger than about 5 µm based on their morphological appearance. In our study we applied the combination of oil-immersion flow imaging microscopy and convolutional neural networks to extend the size range below 5 µm. It allowed to differentiate and quantify heat stressed therapeutic monoclonal antibody aggregates from artificially generated silicone oil droplets with misclassification rates of about 10% in the size range between 0.3 and 5 µm. By comparing the misclassifications across the tested size range, particles in the low submicron size range were particularly difficult to differentiate as their morphological appearance becomes very similar.

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors.

Recombinant adeno-associated virus (rAAV) vectors have evolved as one of the most promising technologies for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in nondividing cells. rAAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. There is a high demand for efficient and scalable production and purification methods for rAAVs. This is particularly true for the downstream purification methods. The current standard methods are based on multiple steps of gradient ultracentrifugation, which allow for the purification and enrichment of full rAAV particles, but the scale up of this method is challenging. Here, we explored fast, scalable, and universal liquid chromatography-based strategies for the purification of rAAVs. In contrast to the hydrophobic interaction chromatography (HIC), where a substantial amount of AAV was lost, the cation exchange chromatography (CEX) was performed robustly for multiple tested serotypes and resulted in a mixture of full and empty rAAVs with a good purity profile. For the used affinity chromatography (AC), a serotype dependence was observed. Anion exchange chromatography (AEX) worked well for the AAV8 serotype and achieved high levels of purification and a baseline separation of full and empty rAAVs. Depending on the AAV serotype, a combination of CEX and AEX or AC and AEX is recommended and holds promise for future translational projects that require highly pure and full particle-enriched rAAVs.

Oil-Immersion Flow Imaging Microscopy for Quantification and Morphological Characterization of Submicron Particles in Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 µm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 µm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.

Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.