Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

EmrE dimerization depends on membrane environment.

The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer-dimer equilibrium constant (KMD(2D)) of 0.002-0.009mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the KMD(2D) is only 0.8-0.95mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes KMD(2D) is 0.0005-0.0008mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.

Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products.

Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products.

Physicochemical Characterization of Sabin Inactivated Poliovirus Vaccine for Process Development.

Upscaling the production capacity of inactivated poliovirus vaccines (IPV) is urgently needed to eradicate polio worldwide. For the development of a robust manufacturing process for IPV, the impact of stresses on the properties of the poliovirus during manufacturing needs to be carefully evaluated. In this study, the physicochemical properties of Sabin poliovirus after low pH exposure were analyzed by asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4-MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS) and surface plasmon resonance (SPR). Low pH stress caused structural changes and aggregation of inactivated poliovirus virions, whereas degraded virion particles would not revert to native virions even after neutralization. Importantly, a complete loss of the D-antigenicity of IPV by low pH stress, followed by neutralization, was observed in SPR. These results suggest that the exposure of poliovirus particle to low pH stress would induce irreversible denaturation and aggregation of virus particles and lead to the loss of D-antigenicity; thus, low pH stress during the manufacturing of poliovirus vaccine should be minimized. The analytical methods above can be efficiently utilized in the development of high-integrity manufacturing processes and high-quality vaccines.

Comprehensive Size Distribution and Composition Analysis of Adeno-Associated Virus Vector by Multiwavelength Sedimentation Velocity Analytical Ultracentrifugation.

During the manufacturing of recombinant adeno-associated virus vectors, it is generally difficult to purify out vectors that lack nucleic acids (empty particles, EPs), contain incomplete nucleic acids (intermediate particles, IPs) or aggregates. These impurities may cause side effects and therefore it is essential to both quantify and reduce them; however, comprehensive identification of the size distribution and components of virus vectors have been lagging. We developed multiwavelength sedimentation velocity analytical ultracentrifugation to characterize EPs, full particles, IPs, and aggregates in adeno-associated virus vector samples. The wavelength-dependent ultraviolet (UV) absorption of capsid protein and encapsulated single-stranded DNA could be deduced from the multiwavelength detection followed by size distribution analysis and peak area integration. Subsequently, a spectral deconvolution analysis using the wavelength-dependent UV absorption data enabled the identification of the protein-nucleic acid ratio of all species. A comprehensive approach for quantifying the viral vector particles and related impurities was established.

Applying Two Orthogonal Methods to Assess Accuracy of Plasma Protein Binding Measurements for Highly Bound Compounds.

Significant advances have been made over the years to accurately measure plasma protein binding (PPB) of highly bound compounds. However, because of perceived uncertainty based on historical suboptimal methods and limitation of radiochemical purity of radiolabeled materials, current regulatory guidelines recommend using an arbitrary cutoff fraction unbound (fu) of 0.01 as the lower limit for drug-drug interaction (DDI) prediction. This can result in significant overprediction of DDI for highly bound compounds, unnecessary DDI clinical trials and more restrictive drug product labels. To build confidence in the accuracy of PPB measurement for highly bound compounds, 2 orthogonal methods, equilibrium dialysis and ultracentrifugation, are assessed in this study to measure PPB of 10 highly bound drugs (fu < 0.01). The results show that the 2 very different methods yield comparable fu values, generally within 2-fold of each other. The data suggest that PPB of highly bound compounds can be measured accurately using current state-of-art methods, and the experimental fu should be used for DDI prediction to provide a more realistic evaluation of DDI risk in the clinic.

Both Reversible Self-Association and Structural Changes Underpin Molecular Viscoelasticity of mAb Solutions.

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of "mAb1." These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein-protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions.

Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It.

Protein self-association or aggregation is a property of significant concern for biopharmaceutical products due to the potential ability of aggregates to cause adverse toxicological and immunological effects. Thus, during the development of a protein biopharmaceutical, it is important to detect and quantify the level and nature of aggregate species as early as possible in order to make well-informed decisions and to mitigate and control potential risks. Although a deeper understanding of the mechanism of aggregation (i.e., protein-protein interactions) is desirable, such detailed assessment is not always necessary from a biopharmaceutical process development point of view. In fact, the scope of characterization efforts is often focused on achieving a well-controlled process, which generates a product that reliably meets established acceptance criteria for safety and efficacy. In this brief note, we evaluated the utility of size-exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation in their simplest forms, to effectively reveal and confirm the presence of concentration-dependent reversible self-association (RSA) in a monoclonal antibody in the early stages of formulation development. Using these techniques, we also initiated preliminary work aimed at reducing the occurrence of this RSA behavior by varying the pH of the formulation buffer.