Boundary convection during velocity sedimentation in the Optima analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units used in conducting characterization work on adeno-associated virus (AAV) has shown evidence of sample convection. Further investigation reveals that this problem arises from the temperature control system design, which is prone to producing destabilizing temperature induced density gradients that can lead to density inversions. The observed convection impacts both the qualitative and quantitative data generated by the Optima. The problem is intermittent and variable in severity within a given Optima unit and between Optima units. This convection appears to be mainly associated with low rotor speeds and dilute samples in dilute solvents, such as AAV samples in formulation buffers containing relatively low concentrations of salts, sugars, etc. Under these conditions it is found that a sufficiently robust stabilizing density gradient is not always present during sedimentation, making the sample susceptible to convection by localized density inversions. Because SV-AUC is used as an analytical tool in making critical decisions in the development and quality control of biotherapeutics, it is imperative to alert users about this potential problem. In general special attention to data quality needs to be made by those researchers working with very large biopharmaceutical particles (e.g. gene therapy products that involve viral vectors or nanoparticles), where the conditions leading to convection are most likely to occur. It is important to note that the XL series analytical ultracentrifuges do not suffer from this problem, indicating that this problem is unique to the Optima. Attributes that reveal the presence of this problem and strategies for its elimination or minimization are provided.

Development of a submerged aquatic vegetation growth model in the Coupled Ocean–Atmosphere–Wave–Sediment Transport (COAWST v3.4) model

The coupled biophysical interactions between submerged aquatic vegetation (SAV), hydrodynamics (currents and waves), sediment dynamics, and nutrient cycling have long been of interest in estuarine environments. Recent observational studies have addressed feedbacks between SAV meadows and their role in modifying current velocity, sedimentation, and nutrient cycling. To represent these dynamic processes in a numerical model, the presence of SAV and its effect on hydrodynamics (currents and waves) and sediment dynamics was incorporated into the open-source Coupled Ocean–Atmosphere–Wave–Sediment Transport (COAWST) model. In this study, we extend the COAWST modeling framework to account for dynamic changes of SAV and associated epiphyte biomass. Modeled SAV biomass is represented as a function of temperature, light, and nutrient availability. The modeled SAV community exchanges nutrients, detritus, dissolved inorganic carbon, and dissolved oxygen with the water-column biogeochemistry model. The dynamic simulation of SAV biomass allows the plants to both respond to and cause changes in the water column and sediment bed properties, hydrodynamics, and sediment transport (i.e., a two-way feedback). We demonstrate the behavior of these modeled processes through application to an idealized domain and then apply the model to a eutrophic harbor where SAV dieback is a result of anthropogenic nitrate loading and eutrophication. These cases demonstrate an advance in the deterministic modeling of coupled biophysical processes and will further our understanding of future ecosystem change.

Electrostatic long-range interactions in macromolecules of flexible-chain linear polyelectrolytes with low charge density in aqueous solutions of different ionic strength

The methods of molecular hydrodynamics (translational diffusion, velocity sedimentation, viscometry) have been used to study copolymers of N‑methyl-N‑vinylacetamide and N‑methyl-N‑vinylamine hydrochloride with an average content of charged groups (4,4 0,2) mol.% in aqueous 0,2 M NaCl solution. Kuhn-Mark-Houwink-Sakurada scaling relations were obtained. Viscous flow was studied in the widest possible range of ionic strengths of aqueous solutions, from salt-free to 6 M NaCl. The data got were compared with those previously obtained for neutral poly-N‑methyl-N‑vinylacetamide. It was firstly shown experimentally that the character of the dependence of the intrinsic viscosity on the molecular weight of a copolymer of such composition in solutions of minimal ionic strength is typical for the chains exhibiting intrachain volume effects, i.e. electrostatic long-range interactions.

An optimised STAPUT method for the purification of mouse spermatocyte and spermatid populations

The purification of individual male germ cell populations is integral for the molecular and biochemical characterisation of specific spermatogenic phases. Although a number of more contemporary techniques have been developed, velocity sedimentation using the STAPUT method remains as a gold standard for this purpose. The gentle nature of the technique, wherein germ cell subpopulations are separated by sedimentation at unit gravity, results in the isolation of viable and high-purity cells. We provide an updated and simplified step-by-step version of the STAPUT protocol for the purification of mouse male germ cells. As per the original method, the protocol described herein allows for the purification of mouse spermatocyte and round spermatids, however it also allows for successful purification of elongating, and elongated spermatid populations, and is optimised for the preservation of cellular ultrastructure. This method yields sufficient numbers of high-purity cells from one adult mouse for RNA or protein extraction or for immunolocalisation studies.

Development of a Submerged Aquatic Vegetation Growth Model in a Coupled Wave-Current-Sediment-Transport Modeling System (COAWST v3.4)

The coupled biophysical interactions between submerged aquatic vegetation (SAV), hydrodynamics (currents and waves), sediment dynamics, and nutrient cycling have long been of interest in estuarine environments. Recent observational studies have addressed feedbacks between SAV meadows, current velocity, sedimentation, and nutrient cycling and suggest SAV are ecosystem engineers whose growth can be self-reinforcing. To represent these dynamic processes in a numerical model, the presence of SAV and its effect on hydrodynamics (currents and waves) and sediment dynamics was incorporated into the open source model COAWST. In this study, we extend the COAWST modelling framework to account for dynamic changes of SAV and associated epiphyte biomass. Modelled SAV biomass is represented as a function of temperature, light, and nutrient availability and exchanges nutrients, detritus, dissolved inorganic carbon, and dissolved oxygen with the water-column biogeochemistry model. The dynamic simulation of SAV biomass allows the plants to both respond to and cause changes in water column and sediment bed properties, hydrodynamics, and sediment transport (i.e., a two-way feedback). We demonstrate the behavior of these modelled processes through application to an idealized domain, then apply the model to a eutrophic harbour where SAV dieback is a result of anthropogenic nitrate loading and eutrophication. These cases demonstrate an advance in the deterministic modelling of coupled bio-physical processes and will further our understanding of future ecosystem change.

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. Figure Figure. Disclosures No relevant conflicts of interest to declare.