DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

Ionic Strength-Dependent Interactions and Dimensions of Adsorbed Zwitterionic Copolymers.

We report direct measurements of ionic strength-dependent interactions between different molecular weights of zwitterionic triblock copolymers adsorbed to hydrophobic colloids and surfaces. The zwitterionic copolymers investigated include phosphorylcholine [poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)] and sulfopropylbetaine [poly(3-( N-2-methacryloyloxyethyl- N, N-dimethyl)ammonatopropanesulfonate) (PMAPS)] end blocks separated by poly(propylene oxide) center blocks. The range of repulsion between adsorbed PMAPS copolymer layers increases with increasing NaCl from 0.01 to 3 M, and layer thicknesses range from ∼50 to 100% of the PMAPS block contour length. In contrast, repulsion between PMPC layers does not change for 0.01-3 M NaCl, and layers remain near full extension at their contour length. NaCl-dependent interactions and inferred layer dimensions correlate with hydrodynamic layer thickness and polymer second virial coefficients. These results suggest that the interaction range and layer thickness of adsorbed zwitterionic copolymers arise from a balance of intramolecular dipolar attraction and repulsion possibly mediated by water solvation. The balance between these competing effects and resulting ionic strength dependence is determined by specific zwitterionic moieties.

High throughput chromatography strategies for potential use in the formal process characterization of a monoclonal antibody.

High throughput experimental strategies are central to the rapid optimization of biologics purification processes. In this work, we extend common high throughput technologies towards the characterization of a multi-column chromatography process for a monoclonal antibody (mAb). Scale-down strategies were first evaluated by comparing breakthrough, retention, and performance (yields and clearance of aggregates and host cell protein) across miniature and lab scale columns. The process operating space was then evaluated using several integrated formats, with batch experimentation to define process testing ranges, miniature columns to evaluate the operating space, and comparison to traditional scale columns to establish scale-up correlations and verify the determined operating space. When compared to an independent characterization study at traditional lab column scale, the high throughput approach identified the same control parameters and similar process sensitivity. Importantly, the high throughput approach significantly decreased time and material needs while improving prediction robustness. Miniature columns and manufacturing scale centerpoint data comparisons support the validity of this approach, making the high throughput strategy an attractive and appropriate scale-down tool for the formal characterization of biotherapeutic processes in the future if regulatory acceptance of the miniature column data can be achieved. Biotechnol. Bioeng. 2016;113: 1273-1283. © 2015 Wiley Periodicals, Inc.

Synergistic Polymer-Surfactant-Complex Mediated Colloidal Interactions and Deposition.

Total internal reflection microscopy (TIRM) is used to directly, sensitively, and simultaneously measure colloidal interactions, dynamics, and deposition for a broad range of polymer-surfactant compositions. A deposition state diagram containing comprehensive information about particle interactions, trajectories, and deposition behavior is obtained for polymer-surfactant compositions covering four decades in both polymer and surfactant concentrations. Bulk polymer-surfactant phase behavior and surface properties are characterized to provide additional information to interpret mechanisms. Materials investigated include cationic acrylamide-acrylamidopropyltrimonium copolymer (AAC), sodium lauryl ether sulfate (SLES) surfactant, silica colloids, and glass microscope slides. Measured colloid-substrate interaction potentials and deposition behavior show nonmonotonic trends vs polymer-surfactant composition and appear to be synergistic in the sense that they are not easily explained as the superposition of single-component-mediated interactions. Broad findings show that at some compositions polymer-surfactant complexes mediate bridging and depletion attractions that promote colloidal deposition, whereas other compositions produce electrosteric repulsion that deters colloidal deposition. These findings illustrate mechanisms underlying colloid-surface interactions in polymer-surfactant mixtures, which are important to controlling selective colloidal deposition in multicomponent formulation applications.

A 3D-printed broadband millimeter wave absorber.

We present the design, manufacturing technique, and characterization of a 3D-printed broadband graded index millimeter wave absorber. The absorber is additively manufactured using a fused filament fabrication 3D printer out of a carbon-loaded high impact polystyrene filament and is designed using a space-filling curve to optimize manufacturability using the said process. The absorber's reflectivity is measured from 63 GHz to 115 GHz and from 140 GHz to 215 GHz and is compared to electromagnetic simulations. The intended application is for terminating stray light in cosmic microwave background telescopes, and the absorber has been shown to survive cryogenic thermal cycling.

Sulphur and carbon cycling in the subduction zone mélange.

Subduction zones impose an important control on the geochemical cycling between the surficial and internal reservoirs of the Earth. Sulphur and carbon are transferred into Earth's mantle by subduction of pelagic sediments and altered oceanic lithosphere. Release of oxidizing sulphate- and carbonate-bearing fluids modifies the redox state of the mantle and the chemical budget of subduction zones. Yet, the mechanisms of sulphur and carbon cycling within subduction zones are still unclear, in part because data are typically derived from arc volcanoes where fluid compositions are modified during transport through the mantle wedge. We determined the bulk rock elemental, and sulphur and carbon isotope compositions of exhumed ultramafic and metabasic rocks from Syros, Greece. Comparison of isotopic data with major and trace element compositions indicates seawater alteration and chemical exchange with sediment-derived fluids within the subduction zone channel. We show that small bodies of detached slab material are subject to metasomatic processes during exhumation, in contrast to large sequences of obducted ophiolitic sections that retain their seafloor alteration signatures. In particular, fluids circulating along the plate interface can cause sulphur mobilization during several stages of exhumation within high-pressure rocks. This takes place more pervasively in serpentinites compared to mafic rocks.

A practical strategy for using miniature chromatography columns in a standardized high-throughput workflow for purification development of monoclonal antibodies.

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high-throughput process development (HTPD) strategy implementing several high-throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high-throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high-throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high-throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance.