Recombinant adeno-associated virus (rAAV) has been increasingly employed for in vivo gene therapy. To ensure the particle concentration meets specifications, strategies are necessary to minimize nonspecific adsorption of rAAVs onto solid surfaces during manufacturing and drug-product dispensing into vials. In this study, we first elucidated the physicochemical factors contributing to nonspecific adsorption of rAAVs by evaluating their adsorption on model surfaces with systematically controlled hydrophilicity and surface charge. Subsequently, we constructed a predictive model through multiple regression analysis of rAAV adsorption under various formulation conditions and the physicochemical parameters of both rAAV serotypes and investigated surfaces. The results revealed that both electrostatic and hydrophobic interactions were responsible for rAAV adsorption. Consequently, we designed a hydrophilic and near-neutral surface coating, which greatly suppressed the adsorption of multiple rAAV serotypes, even under the formulation conditions of marketed products, demonstrating the high versatility and predictive accuracy of the model. These findings validated the effectiveness of the surface modification strategy developed to ensure the stable manufacturing and quality control of rAAV products, offering a foundation for further material development toward clinical applications.

Recombinant adeno-associated virus (rAAV) manufacturing generates various particles in addition to full particles (FPs), posing a significant challenge for downstream purification. In this study, we applied anion-exchange chromatography (AEX) to resolve such particles of rAAV followed by the characterization of three fractions including an unidentified fraction, termed P3, that elutes at higher salt concentrations. Unexpectedly, density gradient ultracentrifugation (DGUC) clarified that P3 contained both empty particles (P3E) and full particles (P3F). No variation in genome packaging was observed between FPs and P3F, whereas zeta potential measurement showed that P3E and P3F exhibited increased negative surface charge compared with empty particles (EPs) and FPs in the earlier fractions (P1 and P2), respectively. Further characterizations using biolayer interferometry revealed the externalization of the negatively charged N-terminal region in viral protein 1 (VP1) of P3F in P3. In addition, P3F exhibited slightly elevated levels of deamidation, a critical quality attribute associated with vector potency, resulting in their reduced transduction efficiency. Notably, such P3F were not separated by DGUC solely. Collectively, AEX provides an effective strategy for isolating high-quality FPs by removing not only EPs but also structurally altered FPs with reduced potency.

Recombinant adeno-associated virus (rAAV) is widely used as a gene delivery vector. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is the gold standard for quantifying the ratio of full particles (FPs) to the sum of empty particles (EPs) and FPs (F/E ratio) of rAAVs. Here, we experimentally determined the molar extinction coefficients (ε) and mass extinction coefficients of highly purified FPs and EPs of AAV serotypes 2, 5, 6, 8, and 9 using SV-AUC with interference and multi-wavelength absorbance detection. At 230 nm, the difference in ε between EPs and FPs was the smallest, although the ε of FPs remained 1.2-fold higher than that of EPs. Expectedly, the differences in ε between FPs and EPs were almost identical across serotypes with the same genome length and increased linearly in a genome length-dependent manner, although both sets of ε differed across serotypes. Consequently, accurate quantification of F/E ratio requires the use of distinct ε values for EPs and FPs. The ε per base of single-stranded DNA was independent of serotype and genome length, allowing estimation of the ε of FPs from that of EPs. Coupling these ε values with SV-AUC enables the determination of absolute rAAV concentrations. This study provides practical guidance for accurate absorbance-based rAAV quantification.

Recombinant adeno-associated viruses (rAAVs) are a leading platform for in vivo gene therapy. However, limited information is available on the fluctuation of quality attributes (QAs), during manufacturing using the HEK293 suspension cell line system, which is suitable for small- to large-scale production using bioreactors. In this study, we evaluated 14 QAs and their variations, including the viral protein (VP) stoichiometric ratio and deamidation ratios of asparagine residues, of the products for AAV8 with enhanced green fluorescent protein as a gene of interest, which were produced employing essentially the same production procedures with slight modification in the upstream conditions. The VP stoichiometric ratios of the manufactured adeno-associated virus (AAV) vectors differed significantly among the products. Correlation analysis of the QAs revealed the attributes that could affect the transduction efficiency. Notably, the VP2 stoichiometric ratio was positively correlated with the transduction efficiency. The deamidation ratios of N57 and N94 in VP1 had a negative tendency to the transduction efficiency. Consistently, the accelerated stability testing showed a negative correlation of the time-dependent increase in the deamidation ratios of the two asparagine residues with the transduction efficiency. This study highlights the importance and implications of the examined QAs in the development of rAAVs.

Recombinant adeno-associated virus (rAAV) has attracted attention as a gene therapy vector. Monitoring the percentage of full particles (FPs) to the sum of empty particles (EPs) and FPs (F/E ratio) is required to optimize the rAAV production conditions; however, there is a lack of analytical methods to identify FPs and EPs and quantify the F/E ratio of rAAV without purification. Here, we established a direct analysis method for identifying FPs and EPs and quantifying the F/E ratio and genomic titer of unpurified rAAV in crude cell lysate and conditioned medium by mass photometry (MP). MP can detect the events of both molecules that bind to the glass surface and molecules that unbind from the glass surface. Few unbinding molecules were detected in the cell lysate and conditioned medium, but unbinding particles were as prevalent as binding particles in rAAV. By analyzing the unbinding side of the histogram, the F/E ratio of rAAV in the cell lysate was directly quantified with accuracy comparable to that of purified rAAV, which showed there was no interference from impurities. The genomic titer of rAAV in cell lysate was also estimated using particle counts of the unbinding side. This method can successfully determine the F/E ratio and estimate genomic titers of rAAV in crude cell lysate and conditioned medium during the manufacturing process. Direct quantification by MP is a convenient, rapid, and accurate method for quantifying unpurified rAAV and will be useful for improving rAAV production processes, for example, by screening manufacturing conditions.

The role of viral protein (VP) 1 and VP2, which comprise the recombinant adeno-associated virus (rAAV) capsid, in heat-induced genome release was investigated using rAAV serotype 8 (rAAV8) samples with a high VP1/VP2 to VP3 ratio, a low VP1/VP2 to VP3 ratio, and VP3 only. The thermal unfolding of the VP1 N-termini was closely monitored by nano-differential scanning fluorimetry with an onset temperature (Tonset1) of ∼55°C and a melting temperature of ∼60°C (which was below the onset temperature of capsid disassembly [Tonset2] >70°C), which is related to genome release upon heating. The folded VP1 N-termini prevented release of the full-length genome at temperatures below 60°C, whereas unfolding of the VP1 N-termini facilitated genome release above 60°C. Above Tonset1 and below Tonset2, most rAAV8 particles remained as monomeric particles in three states: capsids encapsidating their single-stranded DNA (ssDNA), capsids that had fully released their genome, and capsids that had fully ejected the genome while tethering the genome on the capsid surface as evidenced by large frictional ratios in analytical ultracentrifugation. The ratio of VP1 and/or VP2 to total VPs had little effect on the extent of genome release. These findings provide new insights into heat-induced genome release from rAAV at the molecular level.

Physical stresses such as agitation induce protein aggregation, which causes adverse effects on the immune system of patients, leading to challenges in drug development. Aggregation induced by physical stresses can be minimized by formulation optimization. In this study, 120 combinations of 10 therapeutic proteins and 12 different formulations (4 pH conditions and 3 salt concentrations) were prepared. Subsequently, the agitation-induced aggregation propensity of each protein was investigated by evaluating its monomer recovery (%) using size exclusion chromatography. Hierarchical clustering was applied to categorize each protein according to its aggregation propensity, resulting in two groups of proteins: group A and B. The aggregation propensity of proteins in group A was insensitive to changes in formulation conditions because conformational, colloidal, and interfacial stabilities were minimally affected by changes in the pH and salt concentration and a compensation mechanism existed between conformational and colloidal stabilities. Thus, proteins in group A can be formulated with a relatively high degree of freedom. In contrast, the aggregation propensity of proteins in group B was sensitive to changes in formulation conditions. Multiple regression analysis of the physicochemical parameters and monomer recovery of proteins in group B clarified that changes in conformational stability in response to changes in formulations primarily contributed to the sensitivity of the monomer recovery to changes in formulation conditions. For all antibodies, there was a positive correlation between the monomer recovery after agitation and that after quiescent storage at 40 °C for 1 month, suggesting that a stable formulation can be obtained without the quiescent testing. Therefore, a proposed formulation optimization strategy based on the agitation-induced monomer recovery can improve the efficiency of formulating selected therapeutic proteins. This strategic approach is expected to accelerate the development of therapeutic proteins while reflecting the importance of aggregation factors and quiescent stability in the optimization of therapeutic protein formulations.

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/ MS revealed that rAAV6 was O-glycosylated with the mucintype glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galb1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

Improving protein stability is important for industrial applications, and one promising method for achieving this is backbone circularization. As connector length affects stability, predicting and elucidating a more stable connector length is necessary for development of the backbone circularization method. However, the relationship between connector length and protein stability has not been completely elucidated. Here, we determined the most stable connector length for granulocyte colony‐stimulating factor by changing one residue at a time to produce connector length variants and then measuring their thermal stability. Analysis of the local structures obtained from the predicted structures of the circularized variants revealed that an approach using helix length, dihedral backbone angle, and number of unbonded hydrogen bond donors and acceptors is suitable for identifying connector lengths with higher stability.

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM.

On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill's Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.