Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques.

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC-MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011-8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011-7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3-8% and 10-37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC-MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

Multi-layer mucilage of Plantago ovata seeds: Rheological differences arise from variations in arabinoxylan side chains.

Mucilages are hydrocolloid solutions produced by plants for a variety of functions, including the creation of a water-holding barrier around seeds. Here we report our discovery of the formation of three distinct mucilage layers around Plantago ovata seeds upon their hydration. Each layer is dominated by different arabinoxylans (AXs). These AXs are unusual because they are highly branched and contain ß-1,3-linked xylose in their side chains. We show that these AXs have similar monosaccharide and linkage composition, but vary in their polymer conformation. They also exhibit distinct rheological properties in aqueous solution, despite analytical techniques including NMR showing little difference between them. Using enzymatic hydrolysis and chaotropic solvents, we reveal that hydrogen bonding and side chain distribution are key factors underpinning the distinct rheological properties of these complex AXs.

Accountable care organizations--the promise, perils and pathway to value for plan sponsors.

If the potential of accountable care organizations (ACOs) is realized, they could significantly transform how health care is delivered and financed, bringing the promise of high-quality affordable health care within reach. This article explores the root causes that have handicapped the value in the health care delivery system historically and the critical requirements to overcome those issues. The authors describe how commercial and Medicare ACOs attempt to address those issues, the potential land mines in the transition to a new paradigm and the principles plan sponsors should consider in understanding and integrating ACOs into their health care benefits strategy.

Determining the mechanical properties of yeast cell walls.

The intrinsic cell wall mechanical properties of Baker's yeast (Saccharomyces cerevisiae) cells were determined. Force-deformation data from compression of individual cells up to failure were recorded, and these data were fitted by an analytical model to extract the elastic modulus of the cell wall and the initial stretch ratio of the cell. The cell wall was assumed to be homogeneous, isotropic, and incompressible. A linear elastic constitutive equation was assumed based on Hencky strains to accommodate the large stretches of the cell wall. Because of the high compression speed, water loss during compression could be assumed to be negligible. It was then possible to treat the initial stretch ratio and elastic modulus as adjustable parameters within the analytical model. As the experimental data fitted numerical simulations well up to the point of cell rupture, it was also possible to extract cell wall failure criteria. The mean cell wall properties for resuspended dried Baker's yeast were as follows: elastic modulus 185 ± 15 MPa, initial stretch ratio 1.039 ± 0.006, circumferential stress at failure 115 ± 5 MPa, circumferential strain at failure 0.46 ± 0.03, and strain energy per unit volume at failure 30 ± 3 MPa. Data on yeast cells obtained by this method and model should be useful in the design and optimization of cell disruption equipment for yeast cell processing.

Measuring the mechanical properties of single microbial cells.

Many cells are considered to be susceptible to mechanical forces or "shear" in bioprocessing, leading to undesirable cell breakage or adverse metabolic effects. However, cell breakage is the aim of some processing operations, in particular high-pressure homogenisation and other cell disruption methods. In either case, the exact mechanisms of damage or disruption are obscure. One reason for this is that the mechanical properties of the cells are generally unknown, which makes investigation or prediction of the damage difficult. There are several methods for measuring the mechanical properties of single microbial cells, and these are reviewed briefly. In the context of bioprocessing research, a powerful method of characterising the mechanical properties of single cells is compression testing using micromanipulation, supplemented by mathematical modelling of the cell behaviour in compression. The method and associated modelling are described, with results mainly from studies on yeast cells. Continuing difficulties in making a priori predictions of cell breakage in processing are identified. In future, compression testing by micromanipulation might also be used in conjunction with other single cell analytical techniques to study mechanisms controlling form, growth and division of cells and their consequential mechanical behaviour. It ought to be possible to relate cell wall mechanics to cell wall composition and structure, and eventually to underlying gene expression, allowing much greater understanding and control of the cell mechanical properties.