Crucial Aspects for Maintaining rAAV Stability

Crucial Aspects for Maintaining rAAV Stability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Crucial Aspects for Maintaining rAAV Stability Johannes Lengler, Miruna Gavrila, Janina Brandis, Kristina Palavra, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4685335/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Background The storage of rAAV vectors for gene therapy applications is critical for ensuring a constant product quality and defined amount of medication at the time of administration. Therefore, we determined the influence of different storage conditions on the physicochemical and biological properties of rAAV8 and rAAV9 preparations. Particular attention was paid to short-term storage, which plays a crucial role in both the manufacturing process and in clinical applications. Additionally, we addressed the question, of viability of rAAV8 and rAAV9 when subjected to very low-temperature storage conditions (< -65°C) or lyophilization. To determine the impact on rAAV vectors, various analyses were used, including the quantification of capsid and genome titers, as well as biopotency assessments, which are pivotal determinants in characterizing vector behavior and efficacy. Results Our data showed that freeze/thaw cycles hardly affected the functionality of rAAV9-aGAL vectors. In contrast, prolonged storage at room temperature for several days, resulted in a discernible decrease in biopotency despite consistent capsid and genome titers. When the storage temperature was further increased, the rAAV8-aGAL decay accelerated. For example, a short-term exposure of + 40°C and more, led to a reduction in the physical viral titer and to an even faster decline in efficacy determined by biopotency. However, the addition of sucrose and sorbitol to the rAAV9-aGAL and rAAV9-GAA preparations reduced the temperature sensitivity of rAAV and improved its stability. Furthermore, exposure of rAAV9-aGAL to highly acidic conditions (pH 2.5) dramatically reduced its biopotency by 70% or more. Most interestingly, a long-term storage of rAAV9-aGAL and rAAV8-FVIII vectors over 12 months and 36 months, respectively, demonstrated exceptional stability at storage temperatures below − 65°C. Also lyophilization conserved functionality for at least 10 months. Conclusions Our data showed how to maintain rAAV biopotency levels over the time without substantial loss. Storage at very low temperatures (< -65°C) preserved its effectiveness over years. Overall, pH and temperature conditions during the manufacturing process, storage and clinical application are worth considering. Consistency in the rAAV capsid titer determination did not necessarily indicate the preservation of biopotency. In conclusion, our approach determined several options for maximizing rAAV stability. Gene therapy rAAV8 rAAV9 stability biopotency Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Gene therapy represents a groundbreaking approach in modern medicine, offering the potential to treat and cure a wide range of genetic disorders by introducing functional genes directly at their source. Central to the success of many gene therapy strategies are recombinant adeno-associated virus (rAAV) vectors, which are favored for their safety profile and efficacy in delivering genetic material into host cells [ 1 ]. So far, several rAAV-based products have been commercialized: Glybera (rAAV1, 2012) [ 2 , 3 ], Luxturna (rAAV2, 2017) [ 4 ], Zolgensma (rAAV9, 2019) [ 5 ], Hemgenix (rAAV5, 2021) [ 6 ], Roctavian (rAAV5, 2021) [ 7 ], Elevidys (rAAVrh74, 2023) [ 8 ]. Therefore, it is essential to continue improving knowledge about these vector systems. The clinical success of therapies utilizing rAAVs critically hinges on the stability of these vectors throughout the storage and handling processes. Ensuring rAAV stability is paramount, as it affects various facets ranging from dosing accuracy to the ultimate therapeutic outcomes [ 9 ]. Product degradation, aggregation, and loss of efficacy during manufacturing, storage, and use are major concerns for rAAV therapies. Due to poor structural stability and suboptimal buffer conditions, many rAAV products must be stored frozen [ 4 , 7 , 10 – 13 ]. Therefore, maintaining the integrity of rAAV vectors across various environmental conditions during different stages of the workflow is crucial for maximizing their therapeutic potential and for guaranteeing consistent clinical benefits for patients [ 14 – 17 ]. Concentrated rAAV stock solutions tend to aggregate of about 1x10 13 vector genomes (vg) per ml, resulting in losses during purification and inconsistencies in testing [ 18 – 21 ]. This aggregation may alter the bio-distribution or increase the immunogenicity of the vector when administered, highlighting the importance of robust storage studies. Although immunogenicity risks are typically lower with single doses, they remain significant for immunocompromised individuals [ 21 , 22 ]. Higher concentrations lead to increased viscosity and aggregation, making it challenging to administer rAAVs to specific locations such as the central nervous system [ 23 – 26 ]. Common degradation pathways for rAAVs involve proteolysis and chemical alterations such as oxidation and deamidation. Viral vectors may unfold due to nonspecific binding to various surfaces during production and administration [ 27 – 30 ]. This degradation could directly impact the accuracy of the dosage that can be delivered to patients, which is crucial for achieving the desired therapeutic effect [ 15 , 31 ]. Therefore, improper storage could lead to degradation of the vectors, affecting their structural integrity and functional capacity. Bee JS et al. tested rAAV8 and rAAV9 subjected to five freeze-thaw cycles and various temperature conditions to assess their stability outside optimal storage. They revealed that formulations with low buffer concentrations or with 4% sucrose minimized DNA release from rAAV particles, indicating improved viral stability during these stress cycles [ 32 ]. Therefore, it is essential to address storage challenges to ensure that rAAV-based treatments are safe and effective, as variations in storage conditions, such as temperature fluctuations and exposure to light, can alter the potency of the rAAV vectors, thereby influencing their therapeutic effectiveness. Both, short-term and long-term storage studies are indispensable for the development and successful deployment of rAAV-based gene therapies. Short-term studies ensure that rAAVs are stable through the distribution and immediate post-distribution phase, whereas long-term studies guarantee that rAAVs retain their therapeutic properties throughout their intended usage period, aligning with regulatory, logistical, and clinical needs. Together, these studies build a comprehensive understanding of the product’s behavior over its entire lifecycle, from manufacture to administration [ 33 – 36 ]. In this study, we evaluated the stability of rAAV9 vectors under various conditions to optimize their storage and handling. We assessed the impact of freeze-thaw cycles, pH exposure, and both, short-term and long-term storage at differing temperatures and conditions to understand how these factors influence rAAV9 biopotency. Additionally, we compared it to historical long-term stability data of the rAAV8 serotype. Although there are less data available for rAAV8. it confirmed observations obtained for rAAV9 Materials and Methods rAAV Manufacturing Process The production and purification process of rAAV particles was adapted from methods outlined previously [ 37 – 40 ]. In brief, human embryonic kidney 293 (HEK293, #CRL-1573, ATCC) cells grown in FreeStyle™ F17 media (ThermoFisher, NY, USA) were cultured in suspension under controlled conditions (+ 37°C and 5% CO 2 ). rAAV8 and rAAV9 vectors were produced by triple transfection of plasmids harboring a helper containing Adenovirus 5 genes, the packaging genes rep2/cap8 or rep2/cap9, and the added transgene of interest. rAAV vectors incorporate the transgene of a correct DNA sequence version of either 1) coagulation factor IX [ 41 ], which is deficient in hemophilia B disease, here termed as AAV8-FIX, or 2) the coagulation factor VIII, which is deficient in hemophilia A disease, here termed as AAV8-FVIII, or 3) the alpha-galactosidase (aGal) gene, which is deficient in Fabry disease, here termed as AAV9-aGAL [ 42 ], or 4) the alpha-glucosidase (GAA) gene, which is deficient in Pompe disease, termed here as rAAV9-GAA [ 43 ], or 5) the iduronate sulfatase (I2S) gene, which is deficient in Huntington’s disease, here termed as AAV9-I2S [ 44 ]. Transfection plasmids were complexed with polyethylenimine (PEI, Polysciences) according to the supplier's protocol. For rAAV8 serotypes, rAAVs released in the supernatant were harvested after five days post transfection. For rAAV9, three days post-transfection, cells were disrupted using an in-line disperser device to access the intracellular rAAVs. The suspension was diafiltrated and rAAVs were purified by ion exchange chromatography steps and ultracentrifugation as described elsewhere [ 37 , 38 ]. Conditioning of rAAV Samples The following sections describe the conditions that were applied to the rAAV8 and rAAV9 to verify their durability, stability and efficacy. For this purpose, a series of freeze/thaw cycles, pH exposure screening, and short-term storage and long-term storage were performed. Freeze/Thaw Cycles 1 ml of rAAV9-aGAL vector was frozen at <-65°C and then thawed at room temperature (n = 2). Subsequently, a 50 µl aliquot stored in the refrigerator for a maximum of 8 hours at approx. +5°C pending subsequent use. The remaining vector was refrozen. This cycle was repeated up to ten times. Afterwards, the functionality of the treated vector aliquots was determined via the biopotency assay. pH Exposure rAAV9 samples were titrated with hydrochloric acid and adjusted to pH values of 8.5, 8.0, 6.0, 4.0 and 2.3. rAAV9-aGAL vectors derived from two different manufacturing processes underwent treatment. The vectors were then neutralized in cell culture medium and subsequently measured via biopotency assay. Short-Term Storage of rAAV Vector material was stored and analyzed at four distinct temperatures for a duration of up to 12 weeks. For this purpose, AAV9-aGAL vectors were filtered at 0.22 µm (Thermo Fisher Scientific, Austria) to prevent microbial growth at higher temperatures, divided into aliquots, and maintained at the specific temperatures for the duration of the respective study period. To assess the effect of incubation at ambient temperature, + 40°C, and + 5°C, an aliquot was taken for each point in time and tested for capsid titers in anti-AAV9 ELISA, vector titers in droplet digital PCR (ddPCR) analysis, and functionality in biopotency assay. For incubations at + 37°C, vector material was taken from the ultra-low temperature freezer in a more close-meshed manner over 72 hours, according to the same sampling scheme, and analyzed for its efficacy in biopotency. Long-Term Storage of rAAV For long term shelf life in the frozen liquid state, stability samples were stored in qualified, monitored storage areas at a temperature not exceeding − 60°C for a duration of up to 36 months. The initiation of the studies was defined by the set-down date of the samples in the stability chambers. The testing timepoints adhered to ICH guidance [ 33 , 34 ], encompassing assessments at least every 3 months during the initial year, every 6 months in the subsequent year, and annually thereafter. At the designated testing timepoints, the samples were removed from the stability chambers, equilibrated at room temperature, aliquoted, and subjected to comprehensive analysis including physicochemical attributes and biopotency. Lyophilized study samples were maintained at + 2 to + 8°C (monitored and controlled) for 10 months and were reconstituted at defined intervals. Lyophilization was performed as stabilizing process in which the substance in the liquid formulation is first frozen and then the quantity of the solvent is reduced first by sublimation (primary drying) and then by desorption (secondary drying) to values that will no longer support biological growth or chemical reactions [ 45 ]. First freezing was conducted at -60°C to solidify the water content, followed by primary drying where a vacuum is introduced to reduce the pressure within the drying chamber, leading to direct sublimation of ice crystals from solid to vapor, bypassing the liquid phase. In this experiment, the shelf temperature was maintained at -55°C under a pressure of 1.6 Pa. Once most of the water has been removed, the sample underwent secondary drying to further remove any remaining bound water molecules. This involved raising the shelf temperature to a target of + 25°C and reducing the chamber pressure to 1.1 Pa. Finally, reconstitution of the lyophilized samples was performed at 0 M (months), 1 M, 2 M, 3 M, 6M, and 10 M testing timepoints by adding WFI (water for injection) to the dried material, which represented the volume prior lyophilization. The sample was then rehydrated under gentle agitation until the drug product was fully dissolved and subsequently subjected to stability testing. Analytical Methods Droplet Digital PCR For vector genome quantification, a Bio-Rad based ddPCR method was used, applying the fully automated QX One System or semi-automated QX 200 AutoDG system. The key steps of this analytical method involved partitioning of the sample into as many as 20,000 oil droplets, allowing each droplet to function as an independent compartment for PCR reaction. Following PCR amplification using fluorescent-probe-based method, the fluorescence was determined by a droplet reader. Droplets containing the target sequence were identified through fluorescence and categorized as positive, while droplets lacking fluorescence were categorized as negative. Poisson statistical analysis of the counts of positive and negative droplets enabled the absolute quantification of the target sequence.Sample preparation: To remove extraneous DNA, rAAV samples were treated with 4U DNase I (2000 units/mL, New England Biolabs, Ipswitch, MA, USA) for 60 min at + 37°C. This reaction was stopped with 0.5M EDTA pH 8.0 (Art. No. E177, VWR Life Science, Austria). In order to enhance the efficiency of DNase I activity, samples from intermediate production steps were pre-diluted at a ratio of 1:500 in a dilution buffer containing 0.1% of 10% Pluronic F-68 (Art. No. 24040032, Poloxamer 188 Non-ionic Surfactant (100X), Thermo Fisher Scientific, Austria), 2 ng/mL Salmon Sperm DNA, sheared 10 mg/mL (Art. No. AM9680, Thermo Fisher Scientific, Austria) and 1x GeneAmp PCR Buffer (Art. No. 4379876, 10X PCR Buffer, Thermo Fisher Scientific, Austria). The dilution buffer described was essential for ensuring an even distribution of rAAV capsids in droplets. It was also utilized for further diluting the samples following DNase I treatment. The dilution factor was determined based on the sample concentration to achieve droplets containing capsids, as well as droplets without. For an expected sample concentration of 1.00E + 13 vg/mL, the optimal total sample dilution, including all sample preparation steps prior to PCR, was determined to be 5.00E + 07 vg/ml. Droplet generation, ddPCR cycling and readout: Mastermix for ddPCR was prepared by using 2X ddPCR Supermix for Probes (no dUTP) (Art. No. 1863025, Bio-Rad, Austria), 900 nM forward and reverse primer each (Suppl. Table 1), 200 nM probe (Microsynth AG, Austria) and 2 µL of sample preparation in 20 µL total volume for each replicate. Droplets were generated automatically in the Bio-Rad Droplet Generator. A three-step PCR was carried out with a reduced ramp rate of 2°C per second, initiating with a single cycle at a temperature of + 95°C for 10 minutes to facilitate the degradation of the capsids and enable DNA amplification. Subsequent PCR denaturation steps were performed at + 95°C for 30 seconds and PCR extension steps at + 72°C for 15 minutes. The first 5 PCR cycles were performed with an annealing temperature of + 65°C, followed by 42 cycles of + 60°C for 60 seconds respectively. The PCR was completed with one step at + 98°C for 10 minutes. Plate reading was performed according to Bio-Rad instructions. Vector genome concentration was calculated by the appropriate Bio-Rad software and the vector genome titer (vg) per milliliter (mL) of the rAAV sample was determined. ELISA of rAAV8 and rAAV9 Antigens The quantification of rAAV8 capsids (cp) was conducted using an AAV-8 Titration ELISA Kit (Art. No. PRAAV8, Progen, Heidelberg, Germany) on a TECAN robotic system. Initially, microtiter strips were coated with a monoclonal antibody (ADK8), specifically targeting a conformational epitope on the assembled rAAV8 capsids. This coating facilitated the capture of rAAV8 particles. The detection of these captured rAAV8 particles was carried out in a two-step process. First, a biotin-conjugated monoclonal antibody, designed to specifically bind to the ADK8 antibody, was introduced to form an immune complex with the rAAV8 particles. Then, streptavidin peroxidase conjugates were added. These conjugates reacted with the biotin on the monoclonal antibody, forming a complex. For the detection an anti-AAV8 antibody (clone ADK8, Progen Germany) labelled with HRP (abcam HRP conjugation kit, ab102890 used as instructed by manufacturer) was added. To visualize the results, a peroxidase substrate solution was then applied. This addition initiated a chromogenic reaction, the intensity of which directly correlated with the quantity of rAAV8 particles present. Finally, The intensity was measured at a wavelength of 450, providing an estimate of the rAAV8 capsids concentration in the sample (cp/ml). For quantification of rAAV9 capsids, a microtiter plate was coated with anti-AAV9 (clone ADK9, Progen Germany) overnight in PBS pH 7.4 at + 4°C. After four washing steps with PBS + 0.1% tween20, pH 7.4 (= PBST), samples and a rAAV9 standard of defined concentration were incubated in the plate for 1 hour at + 37°C. Again, after four washing steps, the detection antibody anti-AAV9 (clone ADK9, Progen Germany) labelled with HRP (abcam HRP conjugation kit, ab102890 used as instructed by manufacturer) was added, and incubated under the same conditions. Finally, the plate was washed 5x with PBST and TMB (3,3’m5,5’-tetramethylbenzidine, ThermoFisher Scientific, Austria) solution was added. Color development was stopped after approximately 10 min using 0.25 M sulfuric acid. The plate was measured at 450 nm and corrected for 620 nm absorbance. Samples were quantified relative to a 4-parametric fit of the rAAV9 standard curve. In Vitro Biopotency Assay The FIX in vitro biopotency assay was performed as described previously [ 46 ]. In brief, rAAV8-FIX vectors were quantified by ddPCR. Subsequently, the respective rAAV8 amount of each test item was used to infect the human liver cell line HepG2. During incubation, protein was expressed and released into the supernatant. In a second step, the activity of the FIX protein secreted into the supernatant was directly measured by a Rox Factor IX kit (Rossix, Moelndal, Sweden). The measurements of rAAV8-FIX samples are given as a percentage relative to a purified internal rAAV8-FIX vector standard material. The FVIII in vitro biopotency assay was performed similarly as described above. Differing from the FIX assay, viral rAAV8-FVIII vector was used to infect HepG2 cells in the presence of 15 µg/mL VWF (von Willebrand factor), Takeda, Austria) and 7.5 µM EIPA (5-(N-Ethyl-N-isopropyl) amiloride, Sigma, Austria) supplemented in the cell supernatant to stabilize the expressed and secreted FVIII protein. Subsequently, the FVIII in the supernatant was used as cofactor for FX activation and its activity was measured in a Coatest® SP Factor VIII chromogenic assay (Chromogenix, Sweden). Similarly, potency assays were used for rAAV9 therapeutic Fabry- and Pompe-vectors. These assays determine the metabolic activity of the transgenes aGAL, and GAA, respectively. Again, the potency is expressed relative to a purified internal vector standard material. Size Exclusion Chromatography rAAV9 samples were analyzed for aggregate formation by size exclusion chromatography (SEC). Analysis was performed on an Agilent 1260 HPLC system (Agilent, Waldbronn, Germany), consisting of a degasser, binary pump, autosampler, column oven coupled with a fluorescence detector, excitation at 280 nm and emission at 340 nm. For chromatographic separation an Agilent Bio SEC-5, 5 µm, 1000 Å, 7.8*300 mm (5190 − 2536) and a 1.47 mM KH 2 PO*2H 2 O, 2.68 mM KCl, 8.09 mM Na 2 HPO 4 , 350 mM NaCl, 0.02% NaN 3 , pH 7.4 running buffer was used. All chemicals were purchased from Sigma Aldrich (Saint Louis, MO, USA). Samples were transferred to an Agilent 300 µL high recovery, amber and after the column was equilibrated at 1 mL/min with run buffer, 4E + 11 rAAV9 capsids (based on ELISA) were injected. Aggregates were detected and integrated in a range of 5.5 to 8.5 min for signal intensity, and in the range of 8.5 to 10 min for monomer capsids. Aggregates % were calculated in relation to the total integrated area of aggregates plus monomer in each sample. Differential Light Scattering (DLS) Sample preparations were analyzed using the Wyatt PR III device (Wyatt Technology Corporation, Santa Barbara, CA, USA) and standard settings provided by the supplier. The rAAV particles were diafiltered and buffered in various buffers listed in Table 1 . All samples were measured at a concentration of 1E + 13 cp/mL, which corresponds to reported dosages in pre-clinical and clinical studies ranging from 1E + 11 to 1E14 vg/ml [ 47 – 50 ]. With a theoretical molecular mass of 3729 kDa per empty capsid, the concentration of rAAV is approximately 0.064 mg/ml. This concentration was sufficient for DLS measurement due to the large size of rAAV capsids, compared to reference proteins such as BSA or lysozyme, which require higher minimum concentrations for detection. A data filter was employed to automatically exclude unsound measurements from the calculation of hydrodynamic radius (Rh). Table 1 Hydrodynamic ratio and thermal stability of rAAV8 and rAAV9 in various buffers. This table illustrates the impact of different buffer compositions on the hydrodynamic diameter (measured in nanometers, nm) of recombinant adeno-associated virus serotypes 8 and 9 (AAV8-FIX and rAAV9-I2S). The data highlights variations in particle size due to changes in the buffer environment, reflecting potential influences on stability and bioactivity relevant to gene therapy applications. The temperatures reflect the onset points where significant changes in the structural stability of the viral capsids are observed, indicating their thermal tolerance in different buffer environments. Code Buffer Description rAAV8 (nm) rAAV9 (nm) rAAV8 (°C) rAAV9 (°C) B1 10mM Histidine, 50mM glycine, 5% trehalose,0.005% Tween80, pH 7.0 12.1 17.2 65.1 73.2 B2 10mM Histidine, 5mM sucrose, 0.005% Tween80, pH 7.0 13.6 15.7 65.8 68.2 B3 50mM Histidine, 0.005% Tween80. pH 7.0 13.5 15.5 65.8 71.7 B4 10mM Na 3 Citrate, 0.005% Tween80, pH 7.0 13.8 16.1 64.1 70.4 B5 80mM Glycine, 450mM NaCl, pH 5.5 14.5 13.6 63.3 69.8 B6 80mM Glycine, 150mM NaCl, pH 5.5 13.5 13.7 67.1 61.7 B7 80mM Glycine, 150mM NaCl, pH 2.5 13.8 33.6 < 55.0 < 55.0 B8 80mM Sodium Acetate, 150mM MgCl 2 , pH 5.5 13.3 13.5 64.8 69.6 B9 80mM Sodium Acetate, 450mM MgCl 2 , pH 5.5 13.1 13.6 64.8 68.0 B10 80mM Sodium Acetate, 1400mM MgCl 2 , pH 5.5 13.2 14.4 68.8 65.2 B11 80mM Glycine, 150mM, 150mM MgCl 2 , pH 3.5 13.3 13.8 58.9 61.4 B12 80mM NaAcetate buffer, 0.05% Tween80, pH 6 − 0 10.0 16.1 66.1 < 55.0 B13 1mM HCl, pH 3.0 14.1 44.8 67.1 n.a. B14 40mMTRIS, pH 8.0 14.2 n.a. 61.6 n.a. B15 40mM TRIS, 100mM NaCl, pH 8.0 13.6 n.a. 64.8 n.a. B16 40mM TRIS, 450mM NaCl, pH 8.0 14.1 n.a. 65.6 n.a. B17 40mM TRIS, pH 10.0 14.3 n.a. 61.2 n.a. B18 40mM TRIS, 100mM NaCl, pH 10.0 14.2 n.a. 60.9 n.a. B19 40mM TRIS, 450mM NaCl, pH 10.0 14.2 n.a. 60.4 n.a. The samples were stored in either Eppendorf tubes or Falcon tubes. Prior to measurement, liquid samples were briefly vortexed to ensure even distribution, but further vortexing was avoided to prevent aggregation. Instead, samples were centrifuged at 10,000 rpm for 5 minutes to remove airborne contaminants gently, and then pipetted into well plate. Results Factors Influencing rAAV9 stability Numerous conditions require consideration when working with rAAV vectors. For example, freezing and thawing enable resource-saving handling, while pH adjustments and sucrose are additives that can contribute to the purification processes, provided that the viral titers and functionality are preserved. In this study, we focused on (i) the change in physical state, (ii) pH variations, (iii) sucrose, and (iv) other matrix compounds. Changes of the Aggregate State Evaluation of the impact of freeze/thaw cycles on the rAAV9 vector can simplify analysis and save materials, facilitating a comprehensive understanding of its stability. To simulate this scenario, AAV9-aGAL vectors underwent multiple freeze-thaw cycles, following assessment of their functionality via biopotency assays. It could be shown that up to ten freeze-thaw cycles have no detrimental effects on the effectiveness of the vector material. The biopotency of AAV9-aGAL remained consistently stable throughout this procedure (Fig. 1 A). pH Conditions Certain manufacturing process steps necessitate alterations in pH conditions. Consequently, it was imperative to quantify the extent to which pH irreversibly influenced the functionality of rAAV9 vectors. Therefore, pH titrations were performed utilizing rAAV9 and subsequently the biopotency was determined to track product quality. Across the range of pH 8.52 to pH 4.0, biopotency remained consistent. However, in a very acidic environment, biopotency abruptly dropped to less than one third of its original value (Fig. 1 B). Consequently, stability of rAAV9-GAL was demonstrated to at least pH 4.0. While process steps involving lower pH values offered the possibility of more complete column cleaning, they may adversely impact the effectiveness of the rAAV9-GAL vector purification. Sucrose We next sought to evaluate rAAV9 thermal stability. The Tm denoting the point at which half of the full rAAV9 capsids are denatured was found to be + 77.0°C in PBS using Differential Scanning Fluorimetry (DSF) [ 51 ]. We thus subjected rAAV9 capsids of two different projects (rAAV9-aGAL and rAAV9-GAA) to selected temperatures, and then measured vector genome integrity (ddPCR, Fig. 2 A), capsid integrity (ELISA, Fig. 2 B), % of aggregates (Fig. 2 C), and biopotency (Fig. 2 D). As expected, at + 80°C no biological activity, capsid or vector genome integrity could be detected. Already at + 50°C vector genome integrity and biopotency were affected, and a slight increase in aggregates was observed. We then used a fraction of AAVs from an ultracentrifugation process in 52% sucrose /TBS (tris buffered saline solution, pH = 7.4), subjected it to the same heat treatment. The high sucrose concentration was protective as the AAV9 capsids did keep their protein and vector genome integrity as well as their biopotency. In addition, no detectable increase of aggregates was observed up to + 60°C. It was also striking to still detect intact capsid and vector genome at + 80°C with the addition of sucrose. To confirm this effect, we diluted the rAAV9 sample material of rAAV9-GAA 1:20 in either 50% sucrose/ or 50% sorbitol/ PBS (phosphate saline buffer a, pH = 7.4). Again, the material maintained its capsid and vector genome integrity up to + 60°C with no detectable increase in aggregates (Suppl. Figure 1), However, no biopotency was recorded under these conditions. Buffer Compounds DLS was utilized to evaluate the impact of various buffers on rAAV9 stability. We analyzed how these buffers influenced the Rh and their effects on temperature onset, providing insights into how buffer composition affects the physical stability of AAV9-I2S under different temperature conditions (Table 1 ). Additionally, historical data sets with rAAV8-FIX were included to enrich the analysis. In our analysis, rAAV9 displayed a significant increase in Rh in buffer B13, reaching a value of 44.8 nm compared to smaller sizes in contrast to other buffers. This could indicate that buffer B13 (low HCl) may cause substantial aggregation or conformational changes in rAAV9, making it appear much larger. Similarly, buffer B7 (Glycine buffer with medium ionic strength at low pH) increased rAAV9's radius to 33.6 nm. In contrast, rAAV8 showed less variability across buffers, with its smallest radius at 10.0 nm in buffer B12 (Sodium Acetate with high Tween80), a decrease from the typical 13 nm up to 14 nm range observed in other buffers. Upon reviewing the temperature onset values for rAAV8 and rAAV9 in various buffers, it was observed that significant differences in stability existed. Buffer B10 (Sodium Acetate with high MgCl 2 ) provided the highest stability for rAAV8, recording a temperature onset of + 68.8°C. In contrast, Buffer B7 presented the lowest stability for both, rAAV8 and rAAV9, with onset temperatures below + 55.0°C. For rAAV9, the most stable condition was found in Buffer B1 (histidine, glycine, trehalose, low Tween80), which achieved the highest onset temperature of + 73.2°C, indicating significant enhancement in the vector's thermal stability. Effects of Short-Term Storage of rAAV9 at Different Temperatures Two insights are to be gained from short stability studies. First, is it feasible to utilize vector material at room temperature without complex handling, and can it also be stored in the refrigerator for a few days? Second, short-term storage above the freezing point allows rapid conclusions about the shelf life of the vector material, without a long-term stability study. Therefore, the stability of rAAV9-aGAL vector material was investigated over days and weeks in a liquid state. Upon storage at room temperature, capsid and vector genomic titers were constant for 4 weeks. However, the biopotency decreased significantly and continuously over that period to half of its initial activity (Fig. 3 A). It is noteworthy that the decline in biopotency activity within the first week was small at just 8%, hence hardly less than the baseline activity. As expected, potency decreased much faster at the higher temperature of + 40°C and was already less than 10% of the initial activity after one week (Fig. 3 B). In contrast, the capsid and vector genomic titers remained unchanged. Under both temperature conditions, room temperature and + 40°C, it is noticeable that the physical titers were proven to be stable, whereas the potency dropped. In contrast, shelf-life including biopotency was maintained over at least twelve weeks at + 5°C (Fig. 3 C). A detailed potency assessment was performed at + 37°C (Fig. 3 D). For this purpose, AAV9-aGAL vector was incubated for up to 72 hours. A continuous, approximately linear decrease in potency was observed, reaching half the efficacy at 70 hours. It should be noted that the activity decreased by only 3% or less within the initial eight hours. This showed that short-term stability was guaranteed for several hours, allowing uncooled handling. Long-Term Stability of rAAV8 and rAAV9 Vectors The long-term stability of recombinant AAV materials is of utmost importance, due to the cost-intensive nature of production and the extended treatment periods, especially for rare diseases. In our longest study involving the recombinant AAV8-FVIII vector, the capsid titer was regularly determined by anti-AAV8 ELISA alongside with the vector potency. For this purpose, five different vector lots were placed on stability. There was a certain degree of variability in the individual determinations (Suppl. Figure 2). For the ELISA, this resulted in a relative standard deviation of 7–12% for the respective time point. In case of the potency, the CV was significantly higher between 5% and 35% (Fig. 4 A). Nevertheless, the mean values did not show a downward trend and thus showed physical and functional stability over 36 months. The recombinant AAV9-aGAL vector also finds application in gene therapy programs. For this purpose, we could revert to stability data on the genome and capsid titer, as well as on biopotency over one year. None of these lot productions indicated a downward trend (Fig. 4 B). Together, they show a notable level of both, physical and functional stability, for this serotype for at least one year. Ultra-low temperature freezers are not necessarily available in the clinical field and a material shipment of rAAV, as well as its ultra-low temperature storage, is always associated with increased costs. Therefore, AAV9-aGAL vector was lyophilized and tested for its stability at 4°C. The material for the capsid and ELISA titers was stable over the observation period of ten months (Fig. 4 C). Biopotency also remained stable. Lyophilized AAV8-FIX shelf-life confirmed this data (Suppl. Figure 2). In both analyses, however, a variable measurement of the starting point was observed. Nevertheless, lyophilization opened an opportunity to store rAAV vectors over a longer period. In summary, AAV8-FIX and rAAV9-aGAL could be stored in ultra-low temperature freezers for at least 12 months without losing its functionality. Its conversion into a lyophilizate enables bypassing the deep cooling. Discussion The use of rAAVs in gene therapy requires their stability and storage, which can affect their efficacy. In this manuscript, it was shown that the manufacturing conditions, such as the pH value, temperature, and duration of storage, could be used within certain limits without suffering a major loss of effectiveness. Furthermore, rAAV was physically stable and its potency could be conserved for years at low temperatures. We observed that a very acidic pH caused an immense drop in rAAV9 efficacy, as measured by in vitro biopotency. Such low pH values are commonly used in column elution, such as immunoaffinity chromatography. The melting temperatures of rAAV9, but also of other serotypes determined by DSF, also indicated pH-dependent destabilization, especially at low pH values [ 52 ]. In addition, a decrease in transduction efficiency was measured for the serotypes AAV1, AAV2, AAV5 and AAV8 (rAAV9 was not part of this study) [ 53 ]. Taken together it is clearly beneficial to avoid very low pH exposure of rAAV. Once the freeze-thaw process was described to affect rAAV stability. In that study, it was shown that after ten freeze-thaw cycles, the AAV2 hydrodynamic particle radius swelled [ 21 ]. However, in another study of Bee JS et al., rAAV8 and rAAV9 revealed minimal DNA release after several freeze-thaw cycles at low buffer concentrations or formulations including sucrose [ 32 ]. We did not observe any significant decrease in in vitro biopotency for rAAV9 with similar matrix and treatment. At least for rAAV9, it appears that the vector can be reused after several freeze-thaw cycles without any loss of functionality. rAAV vectors are weakened in their potency by short-term exposure to high temperature. The higher the temperature, the more this accelerates the vector decrease in transduction efficiency and biologic effectiveness(Figs. 2 and 3 , [ 15 ]. By adding high concentrations of sucrose and sorbitol, we could show enhanced thermal stability of AAV vectors. The stabilizing effect of sucrose is well documented on proteins and viruses [ 54 , 55 ]. An enhanced thermal stability could open an alternative process for the inactivation of unwanted enveloped viruses. Currently the AAV and similar manufacturing processes rely on solvent detergent for viral inactivation measures. It however needs to be shown that enveloped viruses like HIV and RSV, which usually denature at + 40 to + 60°C [ 56 ] are still inactivated at 60°C or less with a high concentrations of the protective agent present. As expected, temperature played a major role in the storage of rAAV. Our long-term storage of the rAAV8 and rAAV9 indicated that it can be stored for up to three years at temperatures lower than − 65°C. However, conducting such studies is expensive and time consuming, especially if they are required for each biologically different rAAV. For this reason, the physical and functional titers of the rAAV materials were also studied at higher temperatures. In this manner, we obtained a quick impression of its stability through accelerated conditions. We observed that rAAV is subject to a temperature-dependent decrease. It turned out that the physical titers remained quite stable up to 40°C for at least one week, although the biopotency decreased. Potential factors contributing to this phenomenon are deamidation and oxidation [ 29 ]. Also, the vectors may unfold due to nonspecific binding to surfaces during storage [ 27 – 30 ]. This would explain why the capsid antigen determination and vector genome titer analysis did not yet show any effect, but the infectivity and ultimately the biopotency were substantially impaired. In a comparable study, similar observations on their infectivity were made for AAV1, AAV2, AAV5 and AAV8 [ 53 ]. Another aspect of long-term studies the preservation of biopotency over such periods of time. In vivo biopotency is usually subject to large fluctuations, as is in vitro biopotency when measured absolutely, e.g. as FIX after AAV8-F9 infection of HepG2 cells [ 57 , 58 ]. A remarkable decrease in the variation of the in vitro biopotency was achieved by normalizing it against an internal rAAV standard material. While a WHO or external rAAV control was not available, it raises the question whether the potency of the control decreases in a similar manner as observed in the stability test. Although a decline in efficacy cannot fully be ruled out, the standard vector material operated in a narrow range and did not show any drift of biopotency due to the rAAV control material. (Suppl. Figure 3). In a previous work, we demonstrated that our lyophilization formulation for rAAV vectors enabled a cheaper way to easily store material in the refrigerator for long periods of time without major loss of efficacy [ 59 ]. Other studies go in the same direction [ 13 ] offering opportunities for further exploration of the field, particularly concerning rAAV9. Short-term storage of rAAV9 in the liquid state in the refrigerator for days was shown to not be associated with titer decline and loss of efficacy. This observation is consistent with that of other serotypes, at least for physical parameters [ 51 ] (Benett et al., 2017, Thermal Stability.), and is a prerequisite for future clinical application. Conclusion This manuscript explored approaches for working with rAAV without jeopardizing its stability. Particular attention was directed towards the vector genome or capsid titers, as well as the vector biopotency, which was the primary focus of the analysis. We show that physical stability is not necessarily in line with the preservation of full efficacy of rAAV viral vectors. Besides that, rAAV had a shelf life of years at temperatures below − 65°C foryears. Abbreviations Abbreviation Definition 6FAM 6-Carboxyfluorescein, fluorescent dye aGAL Alpha-galactosidase AUC Analytical Ultracentrifugation. BPU Biopotency Unit cp Capsid ddPCR Droplet digital Polymerase Chain Reaction DLS Dynamic Light Scattering DSF Differential Scanning Fluorimetry dUTP Deoxyuridine triphosphate EIPA 5-(N-Ethyl-N-isopropyl) Amiloride. ELISA Enzyme-Linked Immunosorbent Assay. Fabry The correct gene used in this work for treating Fabry's disease FIX Factor IX FVIII Factor VIII GAA Alpha-acidic glucosidase HEK293 Human Embryonic Kidney 293 cells HemA FVIII HemB FIX HepG2 Humane Hepatocyte Cell Line HRP Horseradish Peroxidase Huntington The correct gene used in this work for treating Huntington's disease M Months MGBNFQ Minor groove binder non fluorescent quencher PCR Polymerase Chain Reaction PBS Phosphate-Buffered Saline Pompe The correct gene used in this work for treating Pompe’s disease rAAV Recombinant Adeno-Associated Virus rAAV8 Recombinant Adeno-Associated Virus Serotype 8 rAAV9 Recombinant Adeno-Associated Virus Serotype 9 PBST Phosphate-Buffered Saline with Tween ROX FIX Rox Factor IX (a specific reagent) TMB 3,3’,5,5’ Tetramethylbenzidine VIC VIC phosphoramidite, fluorescent dye vg Vector Genome VWF Von Willebrand Factor. Declarations Ethics approval and consent to participate Not applicable Consent for publication All authors of this manuscript consent to its publication in this journal Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests Johannes Lengler, Miruna Gavrila, Janina Brandis, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus and Juan A. Hernandez Bort are all employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owner of stock options. Juan A. Hernandez Bort conducted the research presented in this manuscript while employed at Baxalta Innovations GmbH, a part of Takeda companies. Between the completion of the research and the publication of this manuscript, he has transitioned to an additional affiliation with the University of Vienna, which is now listed as his correspondence address. Funding The presented work was funded by Baxalta Innovations GmbH, a part of Takeda companies. Authors' contributions Conceptualization JL, MG, JB, KP, SU, FF, BK & JAHB; Methodology: JL, MG, KP, FD, SU, FF; Visualization: JL,MG, JB,KP,FF & JAHB; Writing – Original Draft Preparation: JL, JB, KP, FF, JAHB; Writing – Review & Editing: All co-authors Acknowledgements The authors would like to express their gratitude to former project collaborators: Falko G. Falkner, Hanspeter Rottensteiner, Josef Mayrhofer, Eva Fritscher and Theresa Pfisterer Authors’ information All authors are affiliated with Baxalta Innovations GmbH, a part of Takeda companies: Department Gene Therapy Process Development, Uferstraße 15, 2304, Orth an der Donau, Austria Johannes Lengler, Miruna Gavrila, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus, and Juan A. Hernandez Bort Department Drug Product Development Europe, Industriestrasse 72, A-1221, Vienna, Austria Janina Brandis In addition, Juan A. Hernandez Bort is affiliated to the University of Vienna, Austria References Srivastava A: Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy . Mol Ther Nucleic Acids 2023, 32 :949-959. Miller N: Glybera and the future of gene therapy in the European Union . Nat Rev Drug Discov 2012, 11 (5):419. Yla-Herttuala S: Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union . Mol Ther 2012, 20 (10):1831-1832. Keeler AM, Flotte TR: Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here? Annu Rev Virol 2019, 6 (1):601-621. Urquhart L: FDA new drug approvals in Q2 2019 . Nat Rev Drug Discov 2019, 18 (8):575. Mullard A: 2021 FDA approvals . Nat Rev Drug Discov 2022, 21 (2):83-88. Philippidis A: BioMarin's ROCTAVIAN Wins Food and Drug Administration Approval As First Gene Therapy for Severe Hemophilia A . Hum Gene Ther 2023, 34 (15-16):665-668. Hoy SM: Delandistrogene Moxeparvovec: First Approval . Drugs 2023, 83 (14):1323-1329. Au HKE, Isalan M, Mielcarek M: Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings . Front Med (Lausanne) 2021, 8 :809118. Croyle MA, Cheng X, Wilson JM: Development of formulations that enhance physical stability of viral vectors for gene therapy . Gene Ther 2001, 8 (17):1281-1290. Grossen P, Skaripa Koukelli I, van Haasteren J, A HEM, Durr C: The ice age - A review on formulation of Adeno-associated virus therapeutics . Eur J Pharm Biopharm 2023, 190 :1-23. Zhi L, Chen Y, Lai KN, Wert J, Li S, Wang X, Tang XC, Shameem M, Liu D: Lyophilization as an effective tool to develop AAV8 gene therapy products for refrigerated storage . Int J Pharm 2023, 648 :123564. Rieser R, Menzen T, Biel M, Michalakis S, Winter G: Systematic Studies on Stabilization of AAV Vector Formulations by Lyophilization . J Pharm Sci 2022, 111 (8):2288-2298. Erratum to "Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors" [Journal of Pharmaceutical Sciences 110 (2021) 2609-2624] . J Pharm Sci 2021, 110 (9):3324. Srivastava A, Mallela KMG, Deorkar N, Brophy G: Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors . J Pharm Sci 2021, 110 (7):2609-2624. Fu Q, Polanco A, Lee YS, Yoon S: Critical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing . Biotechnol Bioeng 2023, 120 (9):2601-2621. Snyder RO, Audit M, Francis JD: rAAV vector product characterization and stability studies . Methods Mol Biol 2011, 807 :405-428. Cashen P, McLaughlin, K.: Overview of Current Downstream Processing for Modern Viral Vectors . In: Bioprocess and Analytics Development for Virus-based Advanced Therapeutics and Medicinal Products (ATMPs). Edited by Gautam S, Chiramel, A.I., Pach, R.: Springer, Cham; 2023. Xie Q, Hare J, Turnigan J, Chapman MS: Large-scale production, purification and crystallization of wild-type adeno-associated virus-2 . J Virol Methods 2004, 122 (1):17-27. Wright JF, Qu G, Tang C, Sommer JM: Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector . Curr Opin Drug Discov Devel 2003, 6 (2):174-178. Wright JF, Le T, Prado J, Bahr-Davidson J, Smith PH, Zhen Z, Sommer JM, Pierce GF, Qu G: Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation . Mol Ther 2005, 12 (1):171-178. Wright JF: Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment . Biomedicines 2014, 2 (1):80-97. Rodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM: Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye . Pharm Res 2018, 36 (2):29. Puhl DL, D'Amato AR, Gilbert RJ: Challenges of gene delivery to the central nervous system and the growing use of biomaterial vectors . Brain Res Bull 2019, 150 :216-230. Hocquemiller M, Giersch L, Audrain M, Parker S, Cartier N: Adeno-Associated Virus-Based Gene Therapy for CNS Diseases . Hum Gene Ther 2016, 27 (7):478-496. Zhou K, Han J, Wang Y, Zhang Y, Zhu C: Routes of administration for adeno-associated viruses carrying gene therapies for brain diseases . Front Mol Neurosci 2022, 15 :988914. Jia L, Sun Y: Protein asparagine deamidation prediction based on structures with machine learning methods . PLoS One 2017, 12 (7):e0181347. Zhou Y, Wang Y: Direct deamidation analysis of intact adeno-associated virus serotype 9 capsid proteins using reversed-phase liquid chromatography . Anal Biochem 2023, 668 :115099. Giles AR, Sims JJ, Turner KB, Govindasamy L, Alvira MR, Lock M, Wilson JM: Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function . Mol Ther 2018, 26 (12):2848-2862. Xiang YS, Hao GG: Biophysical characterization of adeno-associated virus capsid through the viral transduction life cycle . J Genet Eng Biotechnol 2023, 21 (1):62. Francois A, Bouzelha M, Lecomte E, Broucque F, Penaud-Budloo M, Adjali O, Moullier P, Blouin V, Ayuso E: Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls . Mol Ther Methods Clin Dev 2018, 10 :223-236. Bee JS, Zhang YZ, Phillippi MK, Finkner S, Mezghebe T, Webber K, Cheung WD, Marshall T: Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9 . J Pharm Sci 2022, 111 (5):1346-1353. Williams HE, Bright J, Roddy E, Poulton A, Cosgrove SD, Turner F, Harrison P, Brookes A, MacDougall E, Abbott A et al : A comparison of drug substance predicted chemical stability with ICH compliant stability studies . Drug Dev Ind Pharm 2019, 45 (3):379-386. Gonzalez-Gonzalez O, Ramirez IO, Ramirez BI, O'Connell P, Ballesteros MP, Torrado JJ, Serrano DR: Drug Stability: ICH versus Accelerated Predictive Stability Studies . Pharmaceutics 2022, 14 (11). Food, Drug Administration HHS: International Conference on Harmonisation; guidance on Q1A stability testing of new drug substances and products; availability. Notice . Fed Regist 2001, 66 (216):56332-56333. Food, Drug Administration HHS: International Conference on Harmonisation: guidance on Q1D bracketing and matrixing designs for stability testing of new drug substances and products; availability. Notice . Fed Regist 2003, 68 (11):2339-2340. Colomb-Delsuc M, Raim R, Fiedler C, Reuberger S, Lengler J, Nordstrom R, Ryner M, Folea IM, Kraus B, Hernandez Bort JA et al : Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM . PLoS One 2022, 17 (6):e0269139. Fiedler C, Lengler J, Gruber B, Scheindel M, Büngener C, Mittergradnegger D, Bendik M, Kraus B, Bort JAH: Characterizing the Biopotency of Truncated Transgene Variants in rAAV8 viral vectors: Essential Considerations for Gene Therapy Applications . 2024. Pistek M, Kahlig CI, Hackl M, Unterthurner S, Kraus B, Grabherr R, Grillari J, Hernandez Bort JA: Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications . Biotechnol J 2023, 18 (8):e2200513. Kahlig CI, Moser S, Micutkova L, Grillari J, Kraus B, Hernandez Bort JA: Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures . Biotechnol J 2024, 19 (5):e2300672. Thornburg CD: The benefits of gene therapy in people with haemophilia . J Viral Hepat 2024, 31 Suppl 1 :4-8. Hayashi Y, Sehara Y, Watano R, Ohba K, Takayanagi Y, Sakiyama Y, Muramatsu K, Mizukami H: Therapeutic Strategy for Fabry Disease by Intravenous Administration of Adeno-Associated Virus 9 in a Symptomatic Mouse Model . Hum Gene Ther 2024, 35 (5-6):192-201. Unnisa Z, Yoon JK, Schindler JW, Mason C, van Til NP: Gene Therapy Developments for Pompe Disease . Biomedicines 2022, 10 (2). Ramaswamy S, Kordower JH: Gene therapy for Huntington's disease . Neurobiol Dis 2012, 48 (2):243-254. Jennings TA: Lyophilization: introduction and basic principles : CRC press; 1999. Lengler J, Coulibaly S, Gruber B, Ilk R, Mayrhofer J, Scheiflinger F, Hoellriegl W, Falkner FG, Rottensteiner H: Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release . Mol Ther Methods Clin Dev 2020, 17 :581-588. Cossins J, Kozma I, Canzonetta C, Hawkins A, Beeson D, Sepulveda P, Dong Y: Dose escalation pre-clinical trial of novel DOK7-AAV in mouse model of DOK7 congenital myasthenia . bioRxiv 2024. Tong D, Zhang M, Yang Y, Xia H, Tong H, Zhang H, Zeng W, Liu M, Wu Y, Ma H et al : Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques . Protein Cell 2023, 14 (1):69-73. Kishimoto TK, Samulski RJ: Addressing high dose AAV toxicity - 'one and done' or 'slower and lower'? Expert Opin Biol Ther 2022, 22 (9):1067-1071. Gonzalez-Visiedo M, Li X, Munoz-Melero M, Kulis MD, Daniell H, Markusic DM: Single-dose AAV vector gene immunotherapy to treat food allergy . Mol Ther Methods Clin Dev 2022, 26 :309-322. Bennett A, Patel S, Mietzsch M, Jose A, Lins-Austin B, Jennifer CY, Bothner B, McKenna R, Agbandje-McKenna M: Thermal stability as a determinant of AAV serotype identity . Molecular Therapy-Methods & Clinical Development 2017, 6 :171-182. Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen H, Barungi T, Penaud-Budloo M et al : AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations . Mol Ther 2017, 25 (6):1375-1386. Lins-Austin B, Patel S, Mietzsch M, Brooke D, Bennett A, Venkatakrishnan B, Van Vliet K, Smith AN, Long JR, McKenna R et al : Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking . Viruses 2020, 12 (6). Lee JC, Timasheff SN: The stabilization of proteins by sucrose . J Biol Chem 1981, 256 (14):7193-7201. Pelliccia M, Andreozzi P, Paulose J, D'Alicarnasso M, Cagno V, Donalisio M, Civra A, Broeckel RM, Haese N, Jacob Silva P et al : Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months . Nat Commun 2016, 7 :13520. Ausar SF, Rexroad J, Frolov VG, Look JL, Konar N, Middaugh CR: Analysis of the thermal and pH stability of human respiratory syncytial virus . Mol Pharm 2005, 2 (6):491-499. Lengler J, Coulibaly S, Gruber B, Ilk R, Mayrhofer J, Scheiflinger F, Hoellriegl W, Falkner FG, Rottensteiner H: Development of an in vitro biopotency assay for an AAV8 hemophilia B gene therapy vector suitable for clinical product release . Molecular Therapy-Methods & Clinical Development 2020, 17 :581-588. Aronson SJ, Bakker RS, Moenis S, van Dijk R, Bortolussi G, Collaud F, Shi X, Duijst S, Ten Bloemendaal L, Ronzitti G: A quantitative in vitro potency assay for adeno-associated virus vectors encoding for the UGT1A1 transgene . Molecular Therapy Methods & Clinical Development 2020, 18 :250-258. Fiedler C, Fritscher E, Hasslacher M, Mittergradnegger D, Tabish T: Adeno-associated virus formulations . In . : Google Patents; 2023. Additional Declarations Competing interest reported. Johannes Lengler, Miruna Gavrila, Janina Brandis, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus and Juan A. Hernandez Bort are all employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owner of stock options. Juan A. Hernandez Bort conducted the research presented in this manuscript while employed at Baxalta Innovations GmbH, a part of Takeda companies. Between the completion of the research and the publication of this manuscript, he has transitioned to an additional affiliation with the University of Vienna, which is now listed as his correspondence address. 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Hernandez Bort","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYBACAwjFDMTnPz74wMDAA2YTqeWAseEMZC08hLUwmAmjqMKlxZy999mDnzus5eQbD6Qx27bdkTE4znyA4UcNg7w9Di2WPcfNDXvPpBszNhw49ji37RmPwWG2BMaeYwyGPbgcdiONTYK37XBiM8PBduPctsM8ks08Bgy8DQyMOLXcf8Ym+bftcH0bw2E2aUuwFv4PjH8bGOxx28LGJg20JYGH4RibNCNQCz8zMNCAtiTi1HImjU1ati0dGL5nmA17zoG0sBkcljkmkdxzAIeW48fYJN+2WcvLzzjD+OBH2WF7Nv7DDx++qbGxbW/AYQ0cSCAZCmRKEFIPBPwEDR0Fo2AUjIKRCgCk9FQfcujAsQAAAABJRU5ErkJggg==","orcid":"","institution":"Gene Therapy Process Development, Baxalta Innovations GmbH, a part of Takeda companies","correspondingAuthor":true,"prefix":"","firstName":"Juan","middleName":"A. Hernandez","lastName":"Bort","suffix":""}],"badges":[],"createdAt":"2024-07-04 09:18:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4685335/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4685335/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-79369-0","type":"published","date":"2024-11-12T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61389899,"identity":"8897b64b-9eef-4abc-9438-be0b07bd7447","added_by":"auto","created_at":"2024-07-30 07:48:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326933,"visible":true,"origin":"","legend":"\u003cp\u003eStability of AAV9-aGAL was challenged by A) freeze thaw cycles and B) change in pH of the matrix using two different transfection methods (TF). The potency was determined by conferred transgene activity in infected target cells and are reflected by relative biopotency units (BPU).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/9eb621c411e6184e905c091f.jpg"},{"id":61390520,"identity":"8c1d7bd6-d824-4f4a-8fbf-0ba742369aa5","added_by":"auto","created_at":"2024-07-30 07:56:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90911,"visible":true,"origin":"","legend":"\u003cp\u003eAddition of high concentration of sucrose protected AAV9 from thermal stress: Filled AAV9 capsids in a histidine buffered saline solution at pH=7 from project AAV9-aGAL and AAV9-GAA or filled AAV9-GAA capsids in tris buffered saline solution pH=7.4 with 50% sucrose were subjected to indicated temperature for 10 min at 1 mL in Eppendorf® tubes on a thermal shaker at 350rpm. Thus generated samples were subjected to: A) AAV9 ELISA for capsid concentration cp/mL and integrity, plots normalised to sample at +20°C, symbols represent average of n=3 technical replicates ± SD. B) ddPCR for vector genome concentration vg/mL and integrity, plots normalised to sample at +20°C, symbols represent average of n=2 technical replicates ± SD. C) size exclusion chromatography for % of aggregates, D) a relative biopotency assay.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/387de7d565da438b0ca04209.jpg"},{"id":61389902,"identity":"76050ac6-f6e9-432e-83cc-6ca173ba65c8","added_by":"auto","created_at":"2024-07-30 07:48:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":354194,"visible":true,"origin":"","legend":"\u003cp\u003eA short-term stability of the rAAV9-aGAL vector was performed at A) 5°C for 12 weeks (1≤n≤4), B) room temperature for 4 weeks (n=4) and C) 40°C for 1 week (n=4). The samples were analyzed for their vector genome titer and capsid titer, as well as for their biopotency at the indicated time points. D) A detailed analysis was performed at 37°C for 72 hours (n=4). The data reflect the biopotency as a function of time and are provided with a trend line.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/f3ba0e67b2e4e3859a12d168.jpg"},{"id":61389900,"identity":"aa3fb3bc-acfb-48eb-9fe2-6429dacfb51b","added_by":"auto","created_at":"2024-07-30 07:48:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":531930,"visible":true,"origin":"","legend":"\u003cp\u003eThe long-term stability of A) AAV8-FVIII (n=6) was observed for 36 months. For this purpose, the capsid titer (cp/ml) was determined by anti-AAV8 ELISA and the biopotency (BPU) was measured by transgene activity. B) Similarly, the AAV9-aGAL (n=2) was examined for 12 months including a determination of the genome titer (vg/ml). C) In addition, stability with lyophilized material is shown.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/2812e22434f64d359236e2a8.jpg"},{"id":69284837,"identity":"1ca9c692-efd7-476b-9b2f-dee9b938417f","added_by":"auto","created_at":"2024-11-18 19:23:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3325990,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/83615658-b847-4e81-bd13-61f9a18cc615.pdf"},{"id":61389903,"identity":"45d33a78-bbd6-4a51-b876-12c43fc31313","added_by":"auto","created_at":"2024-07-30 07:48:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":185926,"visible":true,"origin":"","legend":"","description":"","filename":"LenglerJ2024SuppSR.docx","url":"https://assets-eu.researchsquare.com/files/rs-4685335/v1/6df7d1d8020333d56c976061.docx"}],"financialInterests":"Competing interest reported. Johannes Lengler, Miruna Gavrila, Janina Brandis, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus and Juan A. Hernandez Bort are all employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owner of stock options. \nJuan A. Hernandez Bort conducted the research presented in this manuscript while employed at Baxalta Innovations GmbH, a part of Takeda companies. Between the completion of the research and the publication of this manuscript, he has transitioned to an additional affiliation with the University of Vienna, which is now listed as his correspondence address.","formattedTitle":"Crucial Aspects for Maintaining rAAV Stability","fulltext":[{"header":"Background","content":"\u003cp\u003eGene therapy represents a groundbreaking approach in modern medicine, offering the potential to treat and cure a wide range of genetic disorders by introducing functional genes directly at their source. Central to the success of many gene therapy strategies are recombinant adeno-associated virus (rAAV) vectors, which are favored for their safety profile and efficacy in delivering genetic material into host cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. So far, several rAAV-based products have been commercialized: Glybera (rAAV1, 2012) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], Luxturna (rAAV2, 2017) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], Zolgensma (rAAV9, 2019) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], Hemgenix (rAAV5, 2021) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Roctavian (rAAV5, 2021) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], Elevidys (rAAVrh74, 2023) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, it is essential to continue improving knowledge about these vector systems. The clinical success of therapies utilizing rAAVs critically hinges on the stability of these vectors throughout the storage and handling processes. Ensuring rAAV stability is paramount, as it affects various facets ranging from dosing accuracy to the ultimate therapeutic outcomes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Product degradation, aggregation, and loss of efficacy during manufacturing, storage, and use are major concerns for rAAV therapies. Due to poor structural stability and suboptimal buffer conditions, many rAAV products must be stored frozen [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, maintaining the integrity of rAAV vectors across various environmental conditions during different stages of the workflow is crucial for maximizing their therapeutic potential and for guaranteeing consistent clinical benefits for patients [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcentrated rAAV stock solutions tend to aggregate of about 1x10\u003csup\u003e13\u003c/sup\u003e vector genomes (vg) per ml, resulting in losses during purification and inconsistencies in testing [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This aggregation may alter the bio-distribution or increase the immunogenicity of the vector when administered, highlighting the importance of robust storage studies. Although immunogenicity risks are typically lower with single doses, they remain significant for immunocompromised individuals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Higher concentrations lead to increased viscosity and aggregation, making it challenging to administer rAAVs to specific locations such as the central nervous system [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Common degradation pathways for rAAVs involve proteolysis and chemical alterations such as oxidation and deamidation. Viral vectors may unfold due to nonspecific binding to various surfaces during production and administration [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This degradation could directly impact the accuracy of the dosage that can be delivered to patients, which is crucial for achieving the desired therapeutic effect [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, improper storage could lead to degradation of the vectors, affecting their structural integrity and functional capacity.\u003c/p\u003e \u003cp\u003eBee JS et al. tested rAAV8 and rAAV9 subjected to five freeze-thaw cycles and various temperature conditions to assess their stability outside optimal storage. They revealed that formulations with low buffer concentrations or with 4% sucrose minimized DNA release from rAAV particles, indicating improved viral stability during these stress cycles [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, it is essential to address storage challenges to ensure that rAAV-based treatments are safe and effective, as variations in storage conditions, such as temperature fluctuations and exposure to light, can alter the potency of the rAAV vectors, thereby influencing their therapeutic effectiveness. Both, short-term and long-term storage studies are indispensable for the development and successful deployment of rAAV-based gene therapies. Short-term studies ensure that rAAVs are stable through the distribution and immediate post-distribution phase, whereas long-term studies guarantee that rAAVs retain their therapeutic properties throughout their intended usage period, aligning with regulatory, logistical, and clinical needs. Together, these studies build a comprehensive understanding of the product\u0026rsquo;s behavior over its entire lifecycle, from manufacture to administration [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the stability of rAAV9 vectors under various conditions to optimize their storage and handling. We assessed the impact of freeze-thaw cycles, pH exposure, and both, short-term and long-term storage at differing temperatures and conditions to understand how these factors influence rAAV9 biopotency. Additionally, we compared it to historical long-term stability data of the rAAV8 serotype. Although there are less data available for rAAV8. it confirmed observations obtained for rAAV9\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003erAAV Manufacturing Process\u003c/p\u003e \u003cp\u003eThe production and purification process of rAAV particles was adapted from methods outlined previously [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In brief, human embryonic kidney 293 (HEK293, #CRL-1573, ATCC) cells grown in FreeStyle\u0026trade; F17 media (ThermoFisher, NY, USA) were cultured in suspension under controlled conditions (+\u0026thinsp;37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e). rAAV8 and rAAV9 vectors were produced by triple transfection of plasmids harboring a helper containing Adenovirus 5 genes, the packaging genes rep2/cap8 or rep2/cap9, and the added transgene of interest. rAAV vectors incorporate the transgene of a correct DNA sequence version of either 1) coagulation factor IX [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which is deficient in hemophilia B disease, here termed as AAV8-FIX, or 2) the coagulation factor VIII, which is deficient in hemophilia A disease, here termed as AAV8-FVIII, or 3) the alpha-galactosidase (aGal) gene, which is deficient in Fabry disease, here termed as AAV9-aGAL [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], or 4) the alpha-glucosidase (GAA) gene, which is deficient in Pompe disease, termed here as rAAV9-GAA [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], or 5) the iduronate sulfatase (I2S) gene, which is deficient in Huntington\u0026rsquo;s disease, here termed as AAV9-I2S [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Transfection plasmids were complexed with polyethylenimine (PEI, Polysciences) according to the supplier's protocol. For rAAV8 serotypes, rAAVs released in the supernatant were harvested after five days post transfection. For rAAV9, three days post-transfection, cells were disrupted using an in-line disperser device to access the intracellular rAAVs. The suspension was diafiltrated and rAAVs were purified by ion exchange chromatography steps and ultracentrifugation as described elsewhere [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConditioning of rAAV Samples\u003c/p\u003e \u003cp\u003eThe following sections describe the conditions that were applied to the rAAV8 and rAAV9 to verify their durability, stability and efficacy. For this purpose, a series of freeze/thaw cycles, pH exposure screening, and short-term storage and long-term storage were performed.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFreeze/Thaw Cycles\u003c/h2\u003e \u003cp\u003e1 ml of rAAV9-aGAL vector was frozen at \u0026lt;-65\u0026deg;C and then thawed at room temperature (n\u0026thinsp;=\u0026thinsp;2). Subsequently, a 50 \u0026micro;l aliquot stored in the refrigerator for a maximum of 8 hours at approx. +5\u0026deg;C pending subsequent use. The remaining vector was refrozen. This cycle was repeated up to ten times. Afterwards, the functionality of the treated vector aliquots was determined via the biopotency assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003epH Exposure\u003c/h2\u003e \u003cp\u003erAAV9 samples were titrated with hydrochloric acid and adjusted to pH values of 8.5, 8.0, 6.0, 4.0 and 2.3. rAAV9-aGAL vectors derived from two different manufacturing processes underwent treatment. The vectors were then neutralized in cell culture medium and subsequently measured via biopotency assay.\u003c/p\u003e \u003cp\u003eShort-Term Storage of rAAV\u003c/p\u003e \u003cp\u003eVector material was stored and analyzed at four distinct temperatures for a duration of up to 12 weeks. For this purpose, AAV9-aGAL vectors were filtered at 0.22 \u0026micro;m (Thermo Fisher Scientific, Austria) to prevent microbial growth at higher temperatures, divided into aliquots, and maintained at the specific temperatures for the duration of the respective study period. To assess the effect of incubation at ambient temperature, +\u0026thinsp;40\u0026deg;C, and +\u0026thinsp;5\u0026deg;C, an aliquot was taken for each point in time and tested for capsid titers in anti-AAV9 ELISA, vector titers in droplet digital PCR (ddPCR) analysis, and functionality in biopotency assay. For incubations at +\u0026thinsp;37\u0026deg;C, vector material was taken from the ultra-low temperature freezer in a more close-meshed manner over 72 hours, according to the same sampling scheme, and analyzed for its efficacy in biopotency.\u003c/p\u003e \u003cp\u003eLong-Term Storage of rAAV\u003c/p\u003e \u003cp\u003eFor long term shelf life in the frozen liquid state, stability samples were stored in qualified, monitored storage areas at a temperature not exceeding \u0026minus;\u0026thinsp;60\u0026deg;C for a duration of up to 36 months. The initiation of the studies was defined by the set-down date of the samples in the stability chambers. The testing timepoints adhered to ICH guidance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], encompassing assessments at least every 3 months during the initial year, every 6 months in the subsequent year, and annually thereafter. At the designated testing timepoints, the samples were removed from the stability chambers, equilibrated at room temperature, aliquoted, and subjected to comprehensive analysis including physicochemical attributes and biopotency.\u003c/p\u003e \u003cp\u003eLyophilized study samples were maintained at +\u0026thinsp;2 to +\u0026thinsp;8\u0026deg;C (monitored and controlled) for 10 months and were reconstituted at defined intervals. Lyophilization was performed as stabilizing process in which the substance in the liquid formulation is first frozen and then the quantity of the solvent is reduced first by sublimation (primary drying) and then by desorption (secondary drying) to values that will no longer support biological growth or chemical reactions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. First freezing was conducted at -60\u0026deg;C to solidify the water content, followed by primary drying where a vacuum is introduced to reduce the pressure within the drying chamber, leading to direct sublimation of ice crystals from solid to vapor, bypassing the liquid phase. In this experiment, the shelf temperature was maintained at -55\u0026deg;C under a pressure of 1.6 Pa. Once most of the water has been removed, the sample underwent secondary drying to further remove any remaining bound water molecules. This involved raising the shelf temperature to a target of +\u0026thinsp;25\u0026deg;C and reducing the chamber pressure to 1.1 Pa. Finally, reconstitution of the lyophilized samples was performed at 0 M (months), 1 M, 2 M, 3 M, 6M, and 10 M testing timepoints by adding WFI (water for injection) to the dried material, which represented the volume prior lyophilization. The sample was then rehydrated under gentle agitation until the drug product was fully dissolved and subsequently subjected to stability testing.\u003c/p\u003e \u003cp\u003eAnalytical Methods\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDroplet Digital PCR\u003c/h2\u003e \u003cp\u003eFor vector genome quantification, a Bio-Rad based ddPCR method was used, applying the fully automated QX One System or semi-automated QX 200 AutoDG system. The key steps of this analytical method involved partitioning of the sample into as many as 20,000 oil droplets, allowing each droplet to function as an independent compartment for PCR reaction. Following PCR amplification using fluorescent-probe-based method, the fluorescence was determined by a droplet reader. Droplets containing the target sequence were identified through fluorescence and categorized as positive, while droplets lacking fluorescence were categorized as negative. Poisson statistical analysis of the counts of positive and negative droplets enabled the absolute quantification of the target sequence.Sample preparation: To remove extraneous DNA, rAAV samples were treated with 4U DNase I (2000 units/mL, New England Biolabs, Ipswitch, MA, USA) for 60 min at +\u0026thinsp;37\u0026deg;C. This reaction was stopped with 0.5M EDTA pH 8.0 (Art. No. E177, VWR Life Science, Austria). In order to enhance the efficiency of DNase I activity, samples from intermediate production steps were pre-diluted at a ratio of 1:500 in a dilution buffer containing 0.1% of 10% Pluronic F-68 (Art. No. 24040032, Poloxamer 188 Non-ionic Surfactant (100X), Thermo Fisher Scientific, Austria), 2 ng/mL Salmon Sperm DNA, sheared 10 mg/mL (Art. No. AM9680, Thermo Fisher Scientific, Austria) and 1x GeneAmp PCR Buffer (Art. No. 4379876, 10X PCR Buffer, Thermo Fisher Scientific, Austria). The dilution buffer described was essential for ensuring an even distribution of rAAV capsids in droplets. It was also utilized for further diluting the samples following DNase I treatment. The dilution factor was determined based on the sample concentration to achieve droplets containing capsids, as well as droplets without. For an expected sample concentration of 1.00E\u0026thinsp;+\u0026thinsp;13 vg/mL, the optimal total sample dilution, including all sample preparation steps prior to PCR, was determined to be 5.00E\u0026thinsp;+\u0026thinsp;07 vg/ml.\u003c/p\u003e \u003cp\u003eDroplet generation, ddPCR cycling and readout: Mastermix for ddPCR was prepared by using 2X ddPCR Supermix for Probes (no dUTP) (Art. No. 1863025, Bio-Rad, Austria), 900 nM forward and reverse primer each (Suppl. Table\u0026nbsp;1), 200 nM probe (Microsynth AG, Austria) and 2 \u0026micro;L of sample preparation in 20 \u0026micro;L total volume for each replicate. Droplets were generated automatically in the Bio-Rad Droplet Generator.\u003c/p\u003e \u003cp\u003eA three-step PCR was carried out with a reduced ramp rate of 2\u0026deg;C per second, initiating with a single cycle at a temperature of +\u0026thinsp;95\u0026deg;C for 10 minutes to facilitate the degradation of the capsids and enable DNA amplification. Subsequent PCR denaturation steps were performed at +\u0026thinsp;95\u0026deg;C for 30 seconds and PCR extension steps at +\u0026thinsp;72\u0026deg;C for 15 minutes. The first 5 PCR cycles were performed with an annealing temperature of +\u0026thinsp;65\u0026deg;C, followed by 42 cycles of +\u0026thinsp;60\u0026deg;C for 60 seconds respectively. The PCR was completed with one step at +\u0026thinsp;98\u0026deg;C for 10 minutes. Plate reading was performed according to Bio-Rad instructions. Vector genome concentration was calculated by the appropriate Bio-Rad software and the vector genome titer (vg) per milliliter (mL) of the rAAV sample was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eELISA of rAAV8 and rAAV9 Antigens\u003c/h2\u003e \u003cp\u003eThe quantification of rAAV8 capsids (cp) was conducted using an AAV-8 Titration ELISA Kit (Art. No. PRAAV8, Progen, Heidelberg, Germany) on a TECAN robotic system. Initially, microtiter strips were coated with a monoclonal antibody (ADK8), specifically targeting a conformational epitope on the assembled rAAV8 capsids. This coating facilitated the capture of rAAV8 particles. The detection of these captured rAAV8 particles was carried out in a two-step process. First, a biotin-conjugated monoclonal antibody, designed to specifically bind to the ADK8 antibody, was introduced to form an immune complex with the rAAV8 particles. Then, streptavidin peroxidase conjugates were added. These conjugates reacted with the biotin on the monoclonal antibody, forming a complex. For the detection an anti-AAV8 antibody (clone ADK8, Progen Germany) labelled with HRP (abcam HRP conjugation kit, ab102890 used as instructed by manufacturer) was added. To visualize the results, a peroxidase substrate solution was then applied. This addition initiated a chromogenic reaction, the intensity of which directly correlated with the quantity of rAAV8 particles present. Finally, The intensity was measured at a wavelength of 450, providing an estimate of the rAAV8 capsids concentration in the sample (cp/ml).\u003c/p\u003e \u003cp\u003eFor quantification of rAAV9 capsids, a microtiter plate was coated with anti-AAV9 (clone ADK9, Progen Germany) overnight in PBS pH 7.4 at +\u0026thinsp;4\u0026deg;C. After four washing steps with PBS\u0026thinsp;+\u0026thinsp;0.1% tween20, pH 7.4 (=\u0026thinsp;PBST), samples and a rAAV9 standard of defined concentration were incubated in the plate for 1 hour at +\u0026thinsp;37\u0026deg;C. Again, after four washing steps, the detection antibody anti-AAV9 (clone ADK9, Progen Germany) labelled with HRP (abcam HRP conjugation kit, ab102890 used as instructed by manufacturer) was added, and incubated under the same conditions. Finally, the plate was washed 5x with PBST and TMB (3,3\u0026rsquo;m5,5\u0026rsquo;-tetramethylbenzidine, ThermoFisher Scientific, Austria) solution was added. Color development was stopped after approximately 10 min using 0.25 M sulfuric acid. The plate was measured at 450 nm and corrected for 620 nm absorbance. Samples were quantified relative to a 4-parametric fit of the rAAV9 standard curve.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eIn Vitro\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBiopotency Assay\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe FIX in vitro biopotency assay was performed as described previously [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In brief, rAAV8-FIX vectors were quantified by ddPCR. Subsequently, the respective rAAV8 amount of each test item was used to infect the human liver cell line HepG2. During incubation, protein was expressed and released into the supernatant. In a second step, the activity of the FIX protein secreted into the supernatant was directly measured by a Rox Factor IX kit (Rossix, Moelndal, Sweden). The measurements of rAAV8-FIX samples are given as a percentage relative to a purified internal rAAV8-FIX vector standard material.\u003c/p\u003e \u003cp\u003eThe FVIII \u003cem\u003ein vitro\u003c/em\u003e biopotency assay was performed similarly as described above. Differing from the FIX assay, viral rAAV8-FVIII vector was used to infect HepG2 cells in the presence of 15 \u0026micro;g/mL VWF (von Willebrand factor), Takeda, Austria) and 7.5 \u0026micro;M EIPA (5-(N-Ethyl-N-isopropyl) amiloride, Sigma, Austria) supplemented in the cell supernatant to stabilize the expressed and secreted FVIII protein. Subsequently, the FVIII in the supernatant was used as cofactor for FX activation and its activity was measured in a Coatest\u0026reg; SP Factor VIII chromogenic assay (Chromogenix, Sweden).\u003c/p\u003e \u003cp\u003eSimilarly, potency assays were used for rAAV9 therapeutic Fabry- and Pompe-vectors. These assays determine the metabolic activity of the transgenes aGAL, and GAA, respectively. Again, the potency is expressed relative to a purified internal vector standard material.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSize Exclusion Chromatography\u003c/h2\u003e \u003cp\u003erAAV9 samples were analyzed for aggregate formation by size exclusion chromatography (SEC). Analysis was performed on an Agilent 1260 HPLC system (Agilent, Waldbronn, Germany), consisting of a degasser, binary pump, autosampler, column oven coupled with a fluorescence detector, excitation at 280 nm and emission at 340 nm. For chromatographic separation an Agilent Bio SEC-5, 5 \u0026micro;m, 1000 \u0026Aring;, 7.8*300 mm (5190\u0026thinsp;\u0026minus;\u0026thinsp;2536) and a 1.47 mM KH\u003csub\u003e2\u003c/sub\u003ePO*2H\u003csub\u003e2\u003c/sub\u003eO, 2.68 mM KCl, 8.09 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 350 mM NaCl, 0.02% NaN\u003csub\u003e3\u003c/sub\u003e, pH 7.4 running buffer was used. All chemicals were purchased from Sigma Aldrich (Saint Louis, MO, USA). Samples were transferred to an Agilent 300 \u0026micro;L high recovery, amber and after the column was equilibrated at 1 mL/min with run buffer, 4E\u0026thinsp;+\u0026thinsp;11 rAAV9 capsids (based on ELISA) were injected. Aggregates were detected and integrated in a range of 5.5 to 8.5 min for signal intensity, and in the range of 8.5 to 10 min for monomer capsids. Aggregates % were calculated in relation to the total integrated area of aggregates plus monomer in each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Light Scattering (DLS)\u003c/h2\u003e \u003cp\u003eSample preparations were analyzed using the Wyatt PR III device (Wyatt Technology Corporation, Santa Barbara, CA, USA) and standard settings provided by the supplier. The rAAV particles were diafiltered and buffered in various buffers listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All samples were measured at a concentration of 1E\u0026thinsp;+\u0026thinsp;13 cp/mL, which corresponds to reported dosages in pre-clinical and clinical studies ranging from 1E\u0026thinsp;+\u0026thinsp;11 to 1E14 vg/ml [\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. With a theoretical molecular mass of 3729 kDa per empty capsid, the concentration of rAAV is approximately 0.064 mg/ml. This concentration was sufficient for DLS measurement due to the large size of rAAV capsids, compared to reference proteins such as BSA or lysozyme, which require higher minimum concentrations for detection. A data filter was employed to automatically exclude unsound measurements from the calculation of hydrodynamic radius (Rh).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eHydrodynamic ratio and thermal stability of rAAV8 and rAAV9 in various buffers.\u003c/b\u003e This table illustrates the impact of different buffer compositions on the hydrodynamic diameter (measured in nanometers, nm) of recombinant adeno-associated virus serotypes 8 and 9 (AAV8-FIX and rAAV9-I2S). The data highlights variations in particle size due to changes in the buffer environment, reflecting potential influences on stability and bioactivity relevant to gene therapy applications. The temperatures reflect the onset points where significant changes in the structural stability of the viral capsids are observed, indicating their thermal tolerance in different buffer environments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBuffer Description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003erAAV8 (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003erAAV9 (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003erAAV8 (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003erAAV9 (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10mM Histidine, 50mM glycine, 5% trehalose,0.005% Tween80, pH 7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10mM Histidine, 5mM sucrose, 0.005% Tween80, pH 7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50mM Histidine, 0.005% Tween80. pH 7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10mM Na\u003csub\u003e3\u003c/sub\u003eCitrate, 0.005% Tween80, pH 7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Glycine, 450mM NaCl, pH 5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Glycine, 150mM NaCl, pH 5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e67.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Glycine, 150mM NaCl, pH 2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;55.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;55.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Sodium Acetate, 150mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Sodium Acetate, 450mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Sodium Acetate, 1400mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM Glycine, 150mM, 150mM MgCl\u003csub\u003e2\u003c/sub\u003e, pH 3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80mM NaAcetate buffer, 0.05% Tween80, pH 6\u0026thinsp;\u0026minus;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;55.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1mM HCl, pH 3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e67.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mMTRIS, pH 8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mM TRIS, 100mM NaCl, pH 8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mM TRIS, 450mM NaCl, pH 8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mM TRIS, pH 10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mM TRIS, 100mM NaCl, pH 10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40mM TRIS, 450mM NaCl, pH 10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe samples were stored in either Eppendorf tubes or Falcon tubes. Prior to measurement, liquid samples were briefly vortexed to ensure even distribution, but further vortexing was avoided to prevent aggregation. Instead, samples were centrifuged at 10,000 rpm for 5 minutes to remove airborne contaminants gently, and then pipetted into well plate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eFactors Influencing rAAV9 stability\u003c/p\u003e \u003cp\u003eNumerous conditions require consideration when working with rAAV vectors. For example, freezing and thawing enable resource-saving handling, while pH adjustments and sucrose are additives that can contribute to the purification processes, provided that the viral titers and functionality are preserved. In this study, we focused on (i) the change in physical state, (ii) pH variations, (iii) sucrose, and (iv) other matrix compounds.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eChanges of the Aggregate State\u003c/h2\u003e \u003cp\u003eEvaluation of the impact of freeze/thaw cycles on the rAAV9 vector can simplify analysis and save materials, facilitating a comprehensive understanding of its stability. To simulate this scenario, AAV9-aGAL vectors underwent multiple freeze-thaw cycles, following assessment of their functionality via biopotency assays. It could be shown that up to ten freeze-thaw cycles have no detrimental effects on the effectiveness of the vector material. The biopotency of AAV9-aGAL remained consistently stable throughout this procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003epH Conditions\u003c/h2\u003e \u003cp\u003eCertain manufacturing process steps necessitate alterations in pH conditions. Consequently, it was imperative to quantify the extent to which pH irreversibly influenced the functionality of rAAV9 vectors. Therefore, pH titrations were performed utilizing rAAV9 and subsequently the biopotency was determined to track product quality. Across the range of pH 8.52 to pH 4.0, biopotency remained consistent. However, in a very acidic environment, biopotency abruptly dropped to less than one third of its original value (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consequently, stability of rAAV9-GAL was demonstrated to at least pH 4.0. While process steps involving lower pH values offered the possibility of more complete column cleaning, they may adversely impact the effectiveness of the rAAV9-GAL vector purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSucrose\u003c/h2\u003e \u003cp\u003eWe next sought to evaluate rAAV9 thermal stability. The Tm denoting the point at which half of the full rAAV9 capsids are denatured was found to be +\u0026thinsp;77.0\u0026deg;C in PBS using Differential Scanning Fluorimetry (DSF) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We thus subjected rAAV9 capsids of two different projects (rAAV9-aGAL and rAAV9-GAA) to selected temperatures, and then measured vector genome integrity (ddPCR, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), capsid integrity (ELISA, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), % of aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and biopotency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As expected, at +\u0026thinsp;80\u0026deg;C no biological activity, capsid or vector genome integrity could be detected. Already at +\u0026thinsp;50\u0026deg;C vector genome integrity and biopotency were affected, and a slight increase in aggregates was observed. We then used a fraction of AAVs from an ultracentrifugation process in 52% sucrose /TBS (tris buffered saline solution, pH\u0026thinsp;=\u0026thinsp;7.4), subjected it to the same heat treatment. The high sucrose concentration was protective as the AAV9 capsids did keep their protein and vector genome integrity as well as their biopotency. In addition, no detectable increase of aggregates was observed up to +\u0026thinsp;60\u0026deg;C. It was also striking to still detect intact capsid and vector genome at +\u0026thinsp;80\u0026deg;C with the addition of sucrose. To confirm this effect, we diluted the rAAV9 sample material of rAAV9-GAA 1:20 in either 50% sucrose/ or 50% sorbitol/ PBS (phosphate saline buffer a, pH\u0026thinsp;=\u0026thinsp;7.4). Again, the material maintained its capsid and vector genome integrity up to +\u0026thinsp;60\u0026deg;C with no detectable increase in aggregates (Suppl. Figure\u0026nbsp;1), However, no biopotency was recorded under these conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBuffer Compounds\u003c/h2\u003e \u003cp\u003eDLS was utilized to evaluate the impact of various buffers on rAAV9 stability. We analyzed how these buffers influenced the Rh and their effects on temperature onset, providing insights into how buffer composition affects the physical stability of AAV9-I2S under different temperature conditions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, historical data sets with rAAV8-FIX were included to enrich the analysis.\u003c/p\u003e \u003cp\u003eIn our analysis, rAAV9 displayed a significant increase in Rh in buffer B13, reaching a value of 44.8 nm compared to smaller sizes in contrast to other buffers. This could indicate that buffer B13 (low HCl) may cause substantial aggregation or conformational changes in rAAV9, making it appear much larger. Similarly, buffer B7 (Glycine buffer with medium ionic strength at low pH) increased rAAV9's radius to 33.6 nm. In contrast, rAAV8 showed less variability across buffers, with its smallest radius at 10.0 nm in buffer B12 (Sodium Acetate with high Tween80), a decrease from the typical 13 nm up to 14 nm range observed in other buffers.\u003c/p\u003e \u003cp\u003eUpon reviewing the temperature onset values for rAAV8 and rAAV9 in various buffers, it was observed that significant differences in stability existed. Buffer B10 (Sodium Acetate with high MgCl\u003csub\u003e2\u003c/sub\u003e) provided the highest stability for rAAV8, recording a temperature onset of +\u0026thinsp;68.8\u0026deg;C. In contrast, Buffer B7 presented the lowest stability for both, rAAV8 and rAAV9, with onset temperatures below +\u0026thinsp;55.0\u0026deg;C. For rAAV9, the most stable condition was found in Buffer B1 (histidine, glycine, trehalose, low Tween80), which achieved the highest onset temperature of +\u0026thinsp;73.2\u0026deg;C, indicating significant enhancement in the vector's thermal stability.\u003c/p\u003e \u003cp\u003eEffects of Short-Term Storage of rAAV9 at Different Temperatures\u003c/p\u003e \u003cp\u003eTwo insights are to be gained from short stability studies. First, is it feasible to utilize vector material at room temperature without complex handling, and can it also be stored in the refrigerator for a few days? Second, short-term storage above the freezing point allows rapid conclusions about the shelf life of the vector material, without a long-term stability study.\u003c/p\u003e \u003cp\u003eTherefore, the stability of rAAV9-aGAL vector material was investigated over days and weeks in a liquid state. Upon storage at room temperature, capsid and vector genomic titers were constant for 4 weeks. However, the biopotency decreased significantly and continuously over that period to half of its initial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). It is noteworthy that the decline in biopotency activity within the first week was small at just 8%, hence hardly less than the baseline activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, potency decreased much faster at the higher temperature of +\u0026thinsp;40\u0026deg;C and was already less than 10% of the initial activity after one week (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the capsid and vector genomic titers remained unchanged. Under both temperature conditions, room temperature and +\u0026thinsp;40\u0026deg;C, it is noticeable that the physical titers were proven to be stable, whereas the potency dropped. In contrast, shelf-life including biopotency was maintained over at least twelve weeks at +\u0026thinsp;5\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eA detailed potency assessment was performed at +\u0026thinsp;37\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). For this purpose, AAV9-aGAL vector was incubated for up to 72 hours. A continuous, approximately linear decrease in potency was observed, reaching half the efficacy at 70 hours. It should be noted that the activity decreased by only 3% or less within the initial eight hours. This showed that short-term stability was guaranteed for several hours, allowing uncooled handling.\u003c/p\u003e \u003cp\u003eLong-Term Stability of rAAV8 and rAAV9 Vectors\u003c/p\u003e \u003cp\u003eThe long-term stability of recombinant AAV materials is of utmost importance, due to the cost-intensive nature of production and the extended treatment periods, especially for rare diseases. In our longest study involving the recombinant AAV8-FVIII vector, the capsid titer was regularly determined by anti-AAV8 ELISA alongside with the vector potency. For this purpose, five different vector lots were placed on stability. There was a certain degree of variability in the individual determinations (Suppl. Figure\u0026nbsp;2). For the ELISA, this resulted in a relative standard deviation of 7\u0026ndash;12% for the respective time point. In case of the potency, the CV was significantly higher between 5% and 35% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Nevertheless, the mean values did not show a downward trend and thus showed physical and functional stability over 36 months.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe recombinant AAV9-aGAL vector also finds application in gene therapy programs. For this purpose, we could revert to stability data on the genome and capsid titer, as well as on biopotency over one year. None of these lot productions indicated a downward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Together, they show a notable level of both, physical and functional stability, for this serotype for at least one year.\u003c/p\u003e \u003cp\u003eUltra-low temperature freezers are not necessarily available in the clinical field and a material shipment of rAAV, as well as its ultra-low temperature storage, is always associated with increased costs. Therefore, AAV9-aGAL vector was lyophilized and tested for its stability at 4\u0026deg;C. The material for the capsid and ELISA titers was stable over the observation period of ten months (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Biopotency also remained stable. Lyophilized AAV8-FIX shelf-life confirmed this data (Suppl. Figure\u0026nbsp;2). In both analyses, however, a variable measurement of the starting point was observed. Nevertheless, lyophilization opened an opportunity to store rAAV vectors over a longer period. In summary, AAV8-FIX and rAAV9-aGAL could be stored in ultra-low temperature freezers for at least 12 months without losing its functionality. Its conversion into a lyophilizate enables bypassing the deep cooling.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of rAAVs in gene therapy requires their stability and storage, which can affect their efficacy. In this manuscript, it was shown that the manufacturing conditions, such as the pH value, temperature, and duration of storage, could be used within certain limits without suffering a major loss of effectiveness. Furthermore, rAAV was physically stable and its potency could be conserved for years at low temperatures.\u003c/p\u003e \u003cp\u003eWe observed that a very acidic pH caused an immense drop in rAAV9 efficacy, as measured by \u003cem\u003ein vitro\u003c/em\u003e biopotency. Such low pH values are commonly used in column elution, such as immunoaffinity chromatography. The melting temperatures of rAAV9, but also of other serotypes determined by DSF, also indicated pH-dependent destabilization, especially at low pH values [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In addition, a decrease in transduction efficiency was measured for the serotypes AAV1, AAV2, AAV5 and AAV8 (rAAV9 was not part of this study) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Taken together it is clearly beneficial to avoid very low pH exposure of rAAV. Once the freeze-thaw process was described to affect rAAV stability. In that study, it was shown that after ten freeze-thaw cycles, the AAV2 hydrodynamic particle radius swelled [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, in another study of Bee JS et al., rAAV8 and rAAV9 revealed minimal DNA release after several freeze-thaw cycles at low buffer concentrations or formulations including sucrose [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We did not observe any significant decrease in \u003cem\u003ein vitro\u003c/em\u003e biopotency for rAAV9 with similar matrix and treatment. At least for rAAV9, it appears that the vector can be reused after several freeze-thaw cycles without any loss of functionality.\u003c/p\u003e \u003cp\u003erAAV vectors are weakened in their potency by short-term exposure to high temperature. The higher the temperature, the more this accelerates the vector decrease in transduction efficiency and biologic effectiveness(Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By adding high concentrations of sucrose and sorbitol, we could show enhanced thermal stability of AAV vectors. The stabilizing effect of sucrose is well documented on proteins and viruses [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. An enhanced thermal stability could open an alternative process for the inactivation of unwanted enveloped viruses. Currently the AAV and similar manufacturing processes rely on solvent detergent for viral inactivation measures. It however needs to be shown that enveloped viruses like HIV and RSV, which usually denature at +\u0026thinsp;40 to +\u0026thinsp;60\u0026deg;C [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] are still inactivated at 60\u0026deg;C or less with a high concentrations of the protective agent present.\u003c/p\u003e \u003cp\u003eAs expected, temperature played a major role in the storage of rAAV. Our long-term storage of the rAAV8 and rAAV9 indicated that it can be stored for up to three years at temperatures lower than \u0026minus;\u0026thinsp;65\u0026deg;C. However, conducting such studies is expensive and time consuming, especially if they are required for each biologically different rAAV. For this reason, the physical and functional titers of the rAAV materials were also studied at higher temperatures. In this manner, we obtained a quick impression of its stability through accelerated conditions. We observed that rAAV is subject to a temperature-dependent decrease. It turned out that the physical titers remained quite stable up to 40\u0026deg;C for at least one week, although the biopotency decreased. Potential factors contributing to this phenomenon are deamidation and oxidation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Also, the vectors may unfold due to nonspecific binding to surfaces during storage [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This would explain why the capsid antigen determination and vector genome titer analysis did not yet show any effect, but the infectivity and ultimately the biopotency were substantially impaired. In a comparable study, similar observations on their infectivity were made for AAV1, AAV2, AAV5 and AAV8 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother aspect of long-term studies the preservation of biopotency over such periods of time. \u003cem\u003eIn vivo\u003c/em\u003e biopotency is usually subject to large fluctuations, as is \u003cem\u003ein vitro\u003c/em\u003e biopotency when measured absolutely, e.g. as FIX after AAV8-F9 infection of HepG2 cells [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A remarkable decrease in the variation of the \u003cem\u003ein vitro\u003c/em\u003e biopotency was achieved by normalizing it against an internal rAAV standard material. While a WHO or external rAAV control was not available, it raises the question whether the potency of the control decreases in a similar manner as observed in the stability test. Although a decline in efficacy cannot fully be ruled out, the standard vector material operated in a narrow range and did not show any drift of biopotency due to the rAAV control material. (Suppl. Figure\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eIn a previous work, we demonstrated that our lyophilization formulation for rAAV vectors enabled a cheaper way to easily store material in the refrigerator for long periods of time without major loss of efficacy [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Other studies go in the same direction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] offering opportunities for further exploration of the field, particularly concerning rAAV9. Short-term storage of rAAV9 in the liquid state in the refrigerator for days was shown to not be associated with titer decline and loss of efficacy. This observation is consistent with that of other serotypes, at least for physical parameters [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] (Benett et al., 2017, Thermal Stability.), and is a prerequisite for future clinical application.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis manuscript explored approaches for working with rAAV without jeopardizing its stability. Particular attention was directed towards the vector genome or capsid titers, as well as the vector biopotency, which was the primary focus of the analysis. We show that physical stability is not necessarily in line with the preservation of full efficacy of rAAV viral vectors. Besides that, rAAV had a shelf life of years at temperatures below \u0026minus;\u0026thinsp;65\u0026deg;C foryears.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"650\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eDefinition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003e6FAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003e6-Carboxyfluorescein, fluorescent dye\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eaGAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eAlpha-galactosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eAUC \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eAnalytical Ultracentrifugation.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eBPU \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eBiopotency Unit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003ecp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eCapsid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eddPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eDroplet digital Polymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eDLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eDynamic Light Scattering\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eDSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eDifferential Scanning Fluorimetry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003edUTP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eDeoxyuridine triphosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eEIPA \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003e5-(N-Ethyl-N-isopropyl) Amiloride.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eELISA \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eEnzyme-Linked Immunosorbent Assay.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eFabry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eThe correct gene used in this work for treating Fabry\u0026apos;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eFIX \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eFactor IX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eFVIII \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eFactor VIII\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eGAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eAlpha-acidic glucosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHEK293 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eHuman Embryonic Kidney 293 cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHemA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eFVIII\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHemB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eFIX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHepG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eHumane Hepatocyte Cell Line\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eHorseradish Peroxidase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eHuntington\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eThe correct gene used in this work for treating Huntington\u0026apos;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eMonths\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eMGBNFQ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eMinor groove binder non fluorescent quencher\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003ePhosphate-Buffered Saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003ePompe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eThe correct gene used in this work for treating Pompe\u0026rsquo;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003erAAV \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eRecombinant Adeno-Associated Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003erAAV8 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eRecombinant Adeno-Associated Virus Serotype 8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003erAAV9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eRecombinant Adeno-Associated Virus Serotype 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003ePBST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003ePhosphate-Buffered Saline with Tween\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eROX FIX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eRox Factor IX (a specific reagent)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eTMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003e3,3\u0026rsquo;,5,5\u0026rsquo; Tetramethylbenzidine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eVIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eVIC phosphoramidite, fluorescent dye\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003evg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eVector Genome\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.615384615384617%\" valign=\"bottom\"\u003e\n \u003cp\u003eVWF \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.38461538461539%\" valign=\"bottom\"\u003e\n \u003cp\u003eVon Willebrand Factor.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors of this manuscript consent to its publication in this journal\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eJohannes Lengler, Miruna Gavrila, Janina Brandis, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus and Juan A. Hernandez Bort are all employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owner of stock options.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJuan A. Hernandez Bort conducted the research presented in this manuscript while employed at Baxalta Innovations GmbH, a part of Takeda companies. Between the completion of the research and the publication of this manuscript, he has transitioned to an additional affiliation with the University of Vienna, which is now listed as his correspondence address.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe presented work was funded by Baxalta Innovations GmbH, a part of Takeda companies.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization JL, MG, JB, KP, SU, FF, BK \u0026amp; JAHB; Methodology: JL, MG, KP, FD, SU, FF; Visualization: JL,MG, JB,KP,FF \u0026amp; JAHB; Writing \u0026ndash; Original Draft Preparation: JL, JB, KP, FF, JAHB; Writing \u0026ndash; Review \u0026amp; Editing: All co-authors\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors would like to express their gratitude to former project collaborators: Falko G. Falkner, Hanspeter Rottensteiner, Josef Mayrhofer, Eva Fritscher and Theresa Pfisterer\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; information\u003c/h2\u003e\n\u003cp\u003eAll authors are affiliated with Baxalta Innovations GmbH, a part of Takeda companies:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eDepartment Gene Therapy Process Development, Uferstra\u0026szlig;e 15, 2304, Orth an der Donau, Austria\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eJohannes Lengler, Miruna Gavrila, Kristina Palavra, Felix Dieringer, Sabine Unterthurner, Felix Fuchsberger, Barbara Kraus, and Juan A. Hernandez Bort\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eDepartment Drug Product Development Europe, Industriestrasse 72, A-1221, Vienna, Austria\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eJanina Brandis\u003c/p\u003e\n\u003cp\u003eIn addition, Juan A. Hernandez Bort is affiliated to the University of Vienna, Austria\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSrivastava A: \u003cstrong\u003eRationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy\u003c/strong\u003e. \u003cem\u003eMol Ther Nucleic Acids \u003c/em\u003e2023, \u003cstrong\u003e32\u003c/strong\u003e:949-959.\u003c/li\u003e\n\u003cli\u003eMiller N: \u003cstrong\u003eGlybera and the future of gene therapy in the European Union\u003c/strong\u003e. \u003cem\u003eNat Rev Drug Discov \u003c/em\u003e2012, \u003cstrong\u003e11\u003c/strong\u003e(5):419.\u003c/li\u003e\n\u003cli\u003eYla-Herttuala S: \u003cstrong\u003eEndgame: glybera finally recommended for approval as the first gene therapy drug in the European union\u003c/strong\u003e. \u003cem\u003eMol Ther \u003c/em\u003e2012, \u003cstrong\u003e20\u003c/strong\u003e(10):1831-1832.\u003c/li\u003e\n\u003cli\u003eKeeler AM, Flotte TR: \u003cstrong\u003eRecombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?\u003c/strong\u003e \u003cem\u003eAnnu Rev Virol \u003c/em\u003e2019, \u003cstrong\u003e6\u003c/strong\u003e(1):601-621.\u003c/li\u003e\n\u003cli\u003eUrquhart L: \u003cstrong\u003eFDA new drug approvals in Q2 2019\u003c/strong\u003e. \u003cem\u003eNat Rev Drug Discov \u003c/em\u003e2019, \u003cstrong\u003e18\u003c/strong\u003e(8):575.\u003c/li\u003e\n\u003cli\u003eMullard A: \u003cstrong\u003e2021 FDA approvals\u003c/strong\u003e. \u003cem\u003eNat Rev Drug Discov \u003c/em\u003e2022, \u003cstrong\u003e21\u003c/strong\u003e(2):83-88.\u003c/li\u003e\n\u003cli\u003ePhilippidis A: \u003cstrong\u003eBioMarin\u0026apos;s ROCTAVIAN Wins Food and Drug Administration Approval As First Gene Therapy for Severe Hemophilia A\u003c/strong\u003e. \u003cem\u003eHum Gene Ther \u003c/em\u003e2023, \u003cstrong\u003e34\u003c/strong\u003e(15-16):665-668.\u003c/li\u003e\n\u003cli\u003eHoy SM: \u003cstrong\u003eDelandistrogene Moxeparvovec: First Approval\u003c/strong\u003e. \u003cem\u003eDrugs \u003c/em\u003e2023, \u003cstrong\u003e83\u003c/strong\u003e(14):1323-1329.\u003c/li\u003e\n\u003cli\u003eAu HKE, Isalan M, Mielcarek M: \u003cstrong\u003eGene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings\u003c/strong\u003e. \u003cem\u003eFront Med (Lausanne) \u003c/em\u003e2021, \u003cstrong\u003e8\u003c/strong\u003e:809118.\u003c/li\u003e\n\u003cli\u003eCroyle MA, Cheng X, Wilson JM: \u003cstrong\u003eDevelopment of formulations that enhance physical stability of viral vectors for gene therapy\u003c/strong\u003e. \u003cem\u003eGene Ther \u003c/em\u003e2001, \u003cstrong\u003e8\u003c/strong\u003e(17):1281-1290.\u003c/li\u003e\n\u003cli\u003eGrossen P, Skaripa Koukelli I, van Haasteren J, A HEM, Durr C: \u003cstrong\u003eThe ice age - A review on formulation of Adeno-associated virus therapeutics\u003c/strong\u003e. \u003cem\u003eEur J Pharm Biopharm \u003c/em\u003e2023, \u003cstrong\u003e190\u003c/strong\u003e:1-23.\u003c/li\u003e\n\u003cli\u003eZhi L, Chen Y, Lai KN, Wert J, Li S, Wang X, Tang XC, Shameem M, Liu D: \u003cstrong\u003eLyophilization as an effective tool to develop AAV8 gene therapy products for refrigerated storage\u003c/strong\u003e. \u003cem\u003eInt J Pharm \u003c/em\u003e2023, \u003cstrong\u003e648\u003c/strong\u003e:123564.\u003c/li\u003e\n\u003cli\u003eRieser R, Menzen T, Biel M, Michalakis S, Winter G: \u003cstrong\u003eSystematic Studies on Stabilization of AAV Vector Formulations by Lyophilization\u003c/strong\u003e. \u003cem\u003eJ Pharm Sci \u003c/em\u003e2022, \u003cstrong\u003e111\u003c/strong\u003e(8):2288-2298.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eErratum to \u0026quot;Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors\u0026quot; [Journal of Pharmaceutical Sciences 110 (2021) 2609-2624]\u003c/strong\u003e. \u003cem\u003eJ Pharm Sci \u003c/em\u003e2021, \u003cstrong\u003e110\u003c/strong\u003e(9):3324.\u003c/li\u003e\n\u003cli\u003eSrivastava A, Mallela KMG, Deorkar N, Brophy G: \u003cstrong\u003eManufacturing Challenges and Rational Formulation Development for AAV Viral Vectors\u003c/strong\u003e. \u003cem\u003eJ Pharm Sci \u003c/em\u003e2021, \u003cstrong\u003e110\u003c/strong\u003e(7):2609-2624.\u003c/li\u003e\n\u003cli\u003eFu Q, Polanco A, Lee YS, Yoon S: \u003cstrong\u003eCritical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing\u003c/strong\u003e. \u003cem\u003eBiotechnol Bioeng \u003c/em\u003e2023, \u003cstrong\u003e120\u003c/strong\u003e(9):2601-2621.\u003c/li\u003e\n\u003cli\u003eSnyder RO, Audit M, Francis JD: \u003cstrong\u003erAAV vector product characterization and stability studies\u003c/strong\u003e. \u003cem\u003eMethods Mol Biol \u003c/em\u003e2011, \u003cstrong\u003e807\u003c/strong\u003e:405-428.\u003c/li\u003e\n\u003cli\u003eCashen P, McLaughlin, K.: \u003cstrong\u003eOverview of Current Downstream Processing for Modern Viral Vectors\u003c/strong\u003e. In:\u003cem\u003e Bioprocess and Analytics Development for Virus-based Advanced Therapeutics and Medicinal Products (ATMPs).\u003c/em\u003e Edited by Gautam S, Chiramel, A.I., Pach, R.: Springer, Cham; 2023.\u003c/li\u003e\n\u003cli\u003eXie Q, Hare J, Turnigan J, Chapman MS: \u003cstrong\u003eLarge-scale production, purification and crystallization of wild-type adeno-associated virus-2\u003c/strong\u003e. \u003cem\u003eJ Virol Methods \u003c/em\u003e2004, \u003cstrong\u003e122\u003c/strong\u003e(1):17-27.\u003c/li\u003e\n\u003cli\u003eWright JF, Qu G, Tang C, Sommer JM: \u003cstrong\u003eRecombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector\u003c/strong\u003e. \u003cem\u003eCurr Opin Drug Discov Devel \u003c/em\u003e2003, \u003cstrong\u003e6\u003c/strong\u003e(2):174-178.\u003c/li\u003e\n\u003cli\u003eWright JF, Le T, Prado J, Bahr-Davidson J, Smith PH, Zhen Z, Sommer JM, Pierce GF, Qu G: \u003cstrong\u003eIdentification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation\u003c/strong\u003e. \u003cem\u003eMol Ther \u003c/em\u003e2005, \u003cstrong\u003e12\u003c/strong\u003e(1):171-178.\u003c/li\u003e\n\u003cli\u003eWright JF: \u003cstrong\u003eProduct-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment\u003c/strong\u003e. \u003cem\u003eBiomedicines \u003c/em\u003e2014, \u003cstrong\u003e2\u003c/strong\u003e(1):80-97.\u003c/li\u003e\n\u003cli\u003eRodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM: \u003cstrong\u003ePharmaceutical Development of AAV-Based Gene Therapy Products for the Eye\u003c/strong\u003e. \u003cem\u003ePharm Res \u003c/em\u003e2018, \u003cstrong\u003e36\u003c/strong\u003e(2):29.\u003c/li\u003e\n\u003cli\u003ePuhl DL, D\u0026apos;Amato AR, Gilbert RJ: \u003cstrong\u003eChallenges of gene delivery to the central nervous system and the growing use of biomaterial vectors\u003c/strong\u003e. \u003cem\u003eBrain Res Bull \u003c/em\u003e2019, \u003cstrong\u003e150\u003c/strong\u003e:216-230.\u003c/li\u003e\n\u003cli\u003eHocquemiller M, Giersch L, Audrain M, Parker S, Cartier N: \u003cstrong\u003eAdeno-Associated Virus-Based Gene Therapy for CNS Diseases\u003c/strong\u003e. \u003cem\u003eHum Gene Ther \u003c/em\u003e2016, \u003cstrong\u003e27\u003c/strong\u003e(7):478-496.\u003c/li\u003e\n\u003cli\u003eZhou K, Han J, Wang Y, Zhang Y, Zhu C: \u003cstrong\u003eRoutes of administration for adeno-associated viruses carrying gene therapies for brain diseases\u003c/strong\u003e. \u003cem\u003eFront Mol Neurosci \u003c/em\u003e2022, \u003cstrong\u003e15\u003c/strong\u003e:988914.\u003c/li\u003e\n\u003cli\u003eJia L, Sun Y: \u003cstrong\u003eProtein asparagine deamidation prediction based on structures with machine learning methods\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2017, \u003cstrong\u003e12\u003c/strong\u003e(7):e0181347.\u003c/li\u003e\n\u003cli\u003eZhou Y, Wang Y: \u003cstrong\u003eDirect deamidation analysis of intact adeno-associated virus serotype 9 capsid proteins using reversed-phase liquid chromatography\u003c/strong\u003e. \u003cem\u003eAnal Biochem \u003c/em\u003e2023, \u003cstrong\u003e668\u003c/strong\u003e:115099.\u003c/li\u003e\n\u003cli\u003eGiles AR, Sims JJ, Turner KB, Govindasamy L, Alvira MR, Lock M, Wilson JM: \u003cstrong\u003eDeamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function\u003c/strong\u003e. \u003cem\u003eMol Ther \u003c/em\u003e2018, \u003cstrong\u003e26\u003c/strong\u003e(12):2848-2862.\u003c/li\u003e\n\u003cli\u003eXiang YS, Hao GG: \u003cstrong\u003eBiophysical characterization of adeno-associated virus capsid through the viral transduction life cycle\u003c/strong\u003e. \u003cem\u003eJ Genet Eng Biotechnol \u003c/em\u003e2023, \u003cstrong\u003e21\u003c/strong\u003e(1):62.\u003c/li\u003e\n\u003cli\u003eFrancois A, Bouzelha M, Lecomte E, Broucque F, Penaud-Budloo M, Adjali O, Moullier P, Blouin V, Ayuso E: \u003cstrong\u003eAccurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls\u003c/strong\u003e. \u003cem\u003eMol Ther Methods Clin Dev \u003c/em\u003e2018, \u003cstrong\u003e10\u003c/strong\u003e:223-236.\u003c/li\u003e\n\u003cli\u003eBee JS, Zhang YZ, Phillippi MK, Finkner S, Mezghebe T, Webber K, Cheung WD, Marshall T: \u003cstrong\u003eImpact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9\u003c/strong\u003e. \u003cem\u003eJ Pharm Sci \u003c/em\u003e2022, \u003cstrong\u003e111\u003c/strong\u003e(5):1346-1353.\u003c/li\u003e\n\u003cli\u003eWilliams HE, Bright J, Roddy E, Poulton A, Cosgrove SD, Turner F, Harrison P, Brookes A, MacDougall E, Abbott A\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA comparison of drug substance predicted chemical stability with ICH compliant stability studies\u003c/strong\u003e. \u003cem\u003eDrug Dev Ind Pharm \u003c/em\u003e2019, \u003cstrong\u003e45\u003c/strong\u003e(3):379-386.\u003c/li\u003e\n\u003cli\u003eGonzalez-Gonzalez O, Ramirez IO, Ramirez BI, O\u0026apos;Connell P, Ballesteros MP, Torrado JJ, Serrano DR: \u003cstrong\u003eDrug Stability: ICH versus Accelerated Predictive Stability Studies\u003c/strong\u003e. \u003cem\u003ePharmaceutics \u003c/em\u003e2022, \u003cstrong\u003e14\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eFood, Drug Administration HHS: \u003cstrong\u003eInternational Conference on Harmonisation; guidance on Q1A stability testing of new drug substances and products; availability. Notice\u003c/strong\u003e. \u003cem\u003eFed Regist \u003c/em\u003e2001, \u003cstrong\u003e66\u003c/strong\u003e(216):56332-56333.\u003c/li\u003e\n\u003cli\u003eFood, Drug Administration HHS: \u003cstrong\u003eInternational Conference on Harmonisation: guidance on Q1D bracketing and matrixing designs for stability testing of new drug substances and products; availability. Notice\u003c/strong\u003e. \u003cem\u003eFed Regist \u003c/em\u003e2003, \u003cstrong\u003e68\u003c/strong\u003e(11):2339-2340.\u003c/li\u003e\n\u003cli\u003eColomb-Delsuc M, Raim R, Fiedler C, Reuberger S, Lengler J, Nordstrom R, Ryner M, Folea IM, Kraus B, Hernandez Bort JA\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAssessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2022, \u003cstrong\u003e17\u003c/strong\u003e(6):e0269139.\u003c/li\u003e\n\u003cli\u003eFiedler C, Lengler J, Gruber B, Scheindel M, B\u0026uuml;ngener C, Mittergradnegger D, Bendik M, Kraus B, Bort JAH: \u003cstrong\u003eCharacterizing the Biopotency of Truncated Transgene Variants in rAAV8 viral vectors: Essential Considerations for Gene Therapy Applications\u003c/strong\u003e. 2024.\u003c/li\u003e\n\u003cli\u003ePistek M, Kahlig CI, Hackl M, Unterthurner S, Kraus B, Grabherr R, Grillari J, Hernandez Bort JA: \u003cstrong\u003eComprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications\u003c/strong\u003e. \u003cem\u003eBiotechnol J \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(8):e2200513.\u003c/li\u003e\n\u003cli\u003eKahlig CI, Moser S, Micutkova L, Grillari J, Kraus B, Hernandez Bort JA: \u003cstrong\u003eEnhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures\u003c/strong\u003e. \u003cem\u003eBiotechnol J \u003c/em\u003e2024, \u003cstrong\u003e19\u003c/strong\u003e(5):e2300672.\u003c/li\u003e\n\u003cli\u003eThornburg CD: \u003cstrong\u003eThe benefits of gene therapy in people with haemophilia\u003c/strong\u003e. \u003cem\u003eJ Viral Hepat \u003c/em\u003e2024, \u003cstrong\u003e31 Suppl 1\u003c/strong\u003e:4-8.\u003c/li\u003e\n\u003cli\u003eHayashi Y, Sehara Y, Watano R, Ohba K, Takayanagi Y, Sakiyama Y, Muramatsu K, Mizukami H: \u003cstrong\u003eTherapeutic Strategy for Fabry Disease by Intravenous Administration of Adeno-Associated Virus 9 in a Symptomatic Mouse Model\u003c/strong\u003e. \u003cem\u003eHum Gene Ther \u003c/em\u003e2024, \u003cstrong\u003e35\u003c/strong\u003e(5-6):192-201.\u003c/li\u003e\n\u003cli\u003eUnnisa Z, Yoon JK, Schindler JW, Mason C, van Til NP: \u003cstrong\u003eGene Therapy Developments for Pompe Disease\u003c/strong\u003e. \u003cem\u003eBiomedicines \u003c/em\u003e2022, \u003cstrong\u003e10\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eRamaswamy S, Kordower JH: \u003cstrong\u003eGene therapy for Huntington\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eNeurobiol Dis \u003c/em\u003e2012, \u003cstrong\u003e48\u003c/strong\u003e(2):243-254.\u003c/li\u003e\n\u003cli\u003eJennings TA: \u003cstrong\u003eLyophilization: introduction and basic principles\u003c/strong\u003e: CRC press; 1999.\u003c/li\u003e\n\u003cli\u003eLengler J, Coulibaly S, Gruber B, Ilk R, Mayrhofer J, Scheiflinger F, Hoellriegl W, Falkner FG, Rottensteiner H: \u003cstrong\u003eDevelopment of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release\u003c/strong\u003e. \u003cem\u003eMol Ther Methods Clin Dev \u003c/em\u003e2020, \u003cstrong\u003e17\u003c/strong\u003e:581-588.\u003c/li\u003e\n\u003cli\u003eCossins J, Kozma I, Canzonetta C, Hawkins A, Beeson D, Sepulveda P, Dong Y: \u003cstrong\u003eDose escalation pre-clinical trial of novel DOK7-AAV in mouse model of DOK7 congenital myasthenia\u003c/strong\u003e. \u003cem\u003ebioRxiv \u003c/em\u003e2024.\u003c/li\u003e\n\u003cli\u003eTong D, Zhang M, Yang Y, Xia H, Tong H, Zhang H, Zeng W, Liu M, Wu Y, Ma H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSingle-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques\u003c/strong\u003e. \u003cem\u003eProtein Cell \u003c/em\u003e2023, \u003cstrong\u003e14\u003c/strong\u003e(1):69-73.\u003c/li\u003e\n\u003cli\u003eKishimoto TK, Samulski RJ: \u003cstrong\u003eAddressing high dose AAV toxicity - \u0026apos;one and done\u0026apos; or \u0026apos;slower and lower\u0026apos;?\u003c/strong\u003e \u003cem\u003eExpert Opin Biol Ther \u003c/em\u003e2022, \u003cstrong\u003e22\u003c/strong\u003e(9):1067-1071.\u003c/li\u003e\n\u003cli\u003eGonzalez-Visiedo M, Li X, Munoz-Melero M, Kulis MD, Daniell H, Markusic DM: \u003cstrong\u003eSingle-dose AAV vector gene immunotherapy to treat food allergy\u003c/strong\u003e. \u003cem\u003eMol Ther Methods Clin Dev \u003c/em\u003e2022, \u003cstrong\u003e26\u003c/strong\u003e:309-322.\u003c/li\u003e\n\u003cli\u003eBennett A, Patel S, Mietzsch M, Jose A, Lins-Austin B, Jennifer CY, Bothner B, McKenna R, Agbandje-McKenna M: \u003cstrong\u003eThermal stability as a determinant of AAV serotype identity\u003c/strong\u003e. \u003cem\u003eMolecular Therapy-Methods \u0026amp; Clinical Development \u003c/em\u003e2017, \u003cstrong\u003e6\u003c/strong\u003e:171-182.\u003c/li\u003e\n\u003cli\u003ePacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen H, Barungi T, Penaud-Budloo M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations\u003c/strong\u003e. \u003cem\u003eMol Ther \u003c/em\u003e2017, \u003cstrong\u003e25\u003c/strong\u003e(6):1375-1386.\u003c/li\u003e\n\u003cli\u003eLins-Austin B, Patel S, Mietzsch M, Brooke D, Bennett A, Venkatakrishnan B, Van Vliet K, Smith AN, Long JR, McKenna R\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAdeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking\u003c/strong\u003e. \u003cem\u003eViruses \u003c/em\u003e2020, \u003cstrong\u003e12\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eLee JC, Timasheff SN: \u003cstrong\u003eThe stabilization of proteins by sucrose\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e1981, \u003cstrong\u003e256\u003c/strong\u003e(14):7193-7201.\u003c/li\u003e\n\u003cli\u003ePelliccia M, Andreozzi P, Paulose J, D\u0026apos;Alicarnasso M, Cagno V, Donalisio M, Civra A, Broeckel RM, Haese N, Jacob Silva P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAdditives for vaccine storage to improve thermal stability of adenoviruses from hours to months\u003c/strong\u003e. \u003cem\u003eNat Commun \u003c/em\u003e2016, \u003cstrong\u003e7\u003c/strong\u003e:13520.\u003c/li\u003e\n\u003cli\u003eAusar SF, Rexroad J, Frolov VG, Look JL, Konar N, Middaugh CR: \u003cstrong\u003eAnalysis of the thermal and pH stability of human respiratory syncytial virus\u003c/strong\u003e. \u003cem\u003eMol Pharm \u003c/em\u003e2005, \u003cstrong\u003e2\u003c/strong\u003e(6):491-499.\u003c/li\u003e\n\u003cli\u003eLengler J, Coulibaly S, Gruber B, Ilk R, Mayrhofer J, Scheiflinger F, Hoellriegl W, Falkner FG, Rottensteiner H: \u003cstrong\u003eDevelopment of an in vitro biopotency assay for an AAV8 hemophilia B gene therapy vector suitable for clinical product release\u003c/strong\u003e. \u003cem\u003eMolecular Therapy-Methods \u0026amp; Clinical Development \u003c/em\u003e2020, \u003cstrong\u003e17\u003c/strong\u003e:581-588.\u003c/li\u003e\n\u003cli\u003eAronson SJ, Bakker RS, Moenis S, van Dijk R, Bortolussi G, Collaud F, Shi X, Duijst S, Ten Bloemendaal L, Ronzitti G: \u003cstrong\u003eA quantitative in vitro potency assay for adeno-associated virus vectors encoding for the UGT1A1 transgene\u003c/strong\u003e. \u003cem\u003eMolecular Therapy Methods \u0026amp; Clinical Development \u003c/em\u003e2020, \u003cstrong\u003e18\u003c/strong\u003e:250-258.\u003c/li\u003e\n\u003cli\u003eFiedler C, Fritscher E, Hasslacher M, Mittergradnegger D, Tabish T: \u003cstrong\u003eAdeno-associated virus formulations\u003c/strong\u003e. In\u003cem\u003e.\u003c/em\u003e: Google Patents; 2023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gene therapy, rAAV8, rAAV9, stability, biopotency","lastPublishedDoi":"10.21203/rs.3.rs-4685335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4685335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe storage of rAAV vectors for gene therapy applications is critical for ensuring a constant product quality and defined amount of medication at the time of administration. Therefore, we determined the influence of different storage conditions on the physicochemical and biological properties of rAAV8 and rAAV9 preparations. Particular attention was paid to short-term storage, which plays a crucial role in both the manufacturing process and in clinical applications. Additionally, we addressed the question, of viability of rAAV8 and rAAV9 when subjected to very low-temperature storage conditions (\u0026lt; -65\u0026deg;C) or lyophilization. To determine the impact on rAAV vectors, various analyses were used, including the quantification of capsid and genome titers, as well as biopotency assessments, which are pivotal determinants in characterizing vector behavior and efficacy.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur data showed that freeze/thaw cycles hardly affected the functionality of rAAV9-aGAL vectors. In contrast, prolonged storage at room temperature for several days, resulted in a discernible decrease in biopotency despite consistent capsid and genome titers. When the storage temperature was further increased, the rAAV8-aGAL decay accelerated. For example, a short-term exposure of +\u0026thinsp;40\u0026deg;C and more, led to a reduction in the physical viral titer and to an even faster decline in efficacy determined by biopotency. However, the addition of sucrose and sorbitol to the rAAV9-aGAL and rAAV9-GAA preparations reduced the temperature sensitivity of rAAV and improved its stability. Furthermore, exposure of rAAV9-aGAL to highly acidic conditions (pH 2.5) dramatically reduced its biopotency by 70% or more. Most interestingly, a long-term storage of rAAV9-aGAL and rAAV8-FVIII vectors over 12 months and 36 months, respectively, demonstrated exceptional stability at storage temperatures below \u0026minus;\u0026thinsp;65\u0026deg;C. Also lyophilization conserved functionality for at least 10 months.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur data showed how to maintain rAAV biopotency levels over the time without substantial loss. Storage at very low temperatures (\u0026lt; -65\u0026deg;C) preserved its effectiveness over years. Overall, pH and temperature conditions during the manufacturing process, storage and clinical application are worth considering. Consistency in the rAAV capsid titer determination did not necessarily indicate the preservation of biopotency. In conclusion, our approach determined several options for maximizing rAAV stability.\u003c/p\u003e","manuscriptTitle":"Crucial Aspects for Maintaining rAAV Stability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 07:48:53","doi":"10.21203/rs.3.rs-4685335/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-11T05:53:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T02:15:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-28T03:39:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-26T21:30:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T08:35:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207397887215317451827915972127815181285","date":"2024-07-18T14:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85729380123229906881306368512357706986","date":"2024-07-18T01:35:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138291820717616224061101988535461411475","date":"2024-07-15T17:09:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11919927573858755735216137713970839477","date":"2024-07-13T14:49:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-13T14:43:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-13T14:38:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-07T13:42:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-05T06:35:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-04T09:17:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9cbbb8d7-8220-4860-b89d-6d96eff17418","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T19:16:38+00:00","versionOfRecord":{"articleIdentity":"rs-4685335","link":"https://doi.org/10.1038/s41598-024-79369-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-12 15:57:59","publishedOnDateReadable":"November 12th, 2024"},"versionCreatedAt":"2024-07-30 07:48:53","video":"","vorDoi":"10.1038/s41598-024-79369-0","vorDoiUrl":"https://doi.org/10.1038/s41598-024-79369-0","workflowStages":[]},"version":"v1","identity":"rs-4685335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4685335","identity":"rs-4685335","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}