Rational downstream development for adeno-associated virus full/empty capsid separation - A streamlined methodology based on high-throughput screening and mechanistic modeling.

Recombinant adeno-associated virus (AAV) has emerged as one of the most promising systems for therapeutic gene delivery and has demonstrated clinical success in a wide range of genetic disorders. However, manufacturing of high-quality AAV in large amounts still remains a challenge. A significant difficulty for downstream processing is the need to remove empty capsids that are generated in all currently utilized expression systems and that represent product-related impurities that adversely affect safety and efficacy of AAV vectors. Empty and full capsids exhibit only subtle differences in surface charge and size, making chromatography-based separations highly challenging. Here, we present a rapid methodology for the systematic process development of the crucial AAV full/empty capsid separation on ion-exchange media based on high-throughput screening and mechanistic modeling. Two of the most commonly employed serotypes, AAV8 and AAV9, are used as case studies. First, high-throughput studies in filter-plate format are performed that allow the rapid and comprehensive study of binding and elution behavior of AAV on different resins, using different buffer systems, pH, salt conditions, and solution additives. Small amounts of separated empty and full AAV capsids are generated by iodixanol gradient centrifugation that allow studying the binding and elution behavior of the two vector species separately in miniaturized format. Process conditions that result in maximum differences in elution behavior between empty and full capsids are then transferred to benchtop chromatography systems that are used to generate calibration data for the estimation of steric mass-action isotherm and mass transport parameters for process simulation. The resulting column models are employed for in-silico process development that serves to enhance understanding of separation constraints and to identify optimized conditions for the removal of empty particles. Finally, optimized separation conditions are verified experimentally. The methodology presented in this work provides a systematic framework that affords mechanistic understanding of the crucial empty/full capsid separation and accelerates the development of a scalable AAV downstream process.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Analytical band centrifugation for the separation and quantification of empty and full AAV particles.

Analytical band centrifugation (ABC) was first developed for the separation of macromolecules in centrifugation cells ~60 years ago. Since its development, ABC has been predominantly utilized to study macromolecular interactions or chemical reactions between two solutions in situ upon mixing. In this current study, we evaluated ABC separations on modern analytical ultracentrifugation (AUC) instruments for therapeutic adeno-associated viruses (AAVs). ABC provided sufficient separation between the genome-containing full AAV particle and the empty AAV capsid, which need to be controlled during the manufacturing process. Because ABC produces a physical separation, no complex algorithm or sophisticated software is needed to process the experimental raw data. ABC profiles, dubbed "centrifugrams", can be analyzed with a similar approach as typically used for electrophoretic separations to produce relative percent area. Sedimentation coefficients (s) of analytes can also be determined from ABC. The relative area percent and s value obtained in ABC experiments were shown to be consistent with those determined by conventional sedimentation velocity AUC (SV-AUC). Additionally, the separation and quantification by ABC were found to be reproducible and did not appear to be sensitive to experimental variations of initial rotor temperature or cell misalignment. The robustness of the separation, ease of data processing, and universal applicability for analysis of different AAV serotypes make ABC a promising technique for routine analysis of empty and full AAV particle composition in therapeutic products.

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate.

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from -80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at -20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.

Studies of the aggregation of RNase Sa.

Thirty-eight mutants of RNase Sa (ribonuclease from Streptomyces aureofaciens) were examined for their structure, thermal sensitivity, and tendency to aggregate. Although a biphasic correlation was seen between the effect of temperature on structure and the free energy of transfer changes in many of the mutants, little correlation was seen between the time at which aggregation is initiated or the rate of aggregation and the thermal sensitivity of the mutants. It is hypothesized that the nature of contacts between protein molecules in the associated (aggregated) phase rather than structural changes dominates the aggregation process for these series of mutants.