Electrostatically Mediated Attractive Self-Interactions and Reversible Self-Association of Fc-Fusion Proteins.

Attractive self-interactions and reversible self-association are implicated in many problematic solution behaviors for therapeutic proteins, such as irreversible aggregation, elevated viscosity, phase separation, and opalescence. Protein self-interactions and reversible oligomerization of two Fc-fusion proteins (monovalent and bivalent) and the corresponding fusion partner protein were characterized experimentally with static and dynamic light scattering as a function of pH (5 and 6.5) and ionic strength (10 mM to at least 300 mM). The fusion partner protein and monovalent Fc-fusion each displayed net attractive electrostatic self-interactions at pH 6.5 and net repulsive electrostatic self-interactions at pH 5. Solutions of the bivalent Fc-fusion contained higher molecular weight species that prevented quantification of typical interaction parameters (B22 and kD). All three of the proteins displayed reversible self-association at pH 6.5, where oligomers dissociated with increased ionic strength. Coarse-grained molecular simulations were used to model the self-interactions measured experimentally, assess net self-interactions for the bivalent Fc-fusion, and probe the specific electrostatic interactions between charged amino acids that were involved in attractive electrostatic self-interactions. Mayer-weighted pairwise electrostatic energies from the simulations suggested that attractive electrostatic self-interactions at pH 6.5 for the two Fc-fusion proteins were due to cross-domain interactions between the fusion partner domain(s) and the Fc domain.

A framework for 2-stage global sensitivity analysis of GastroPlus™ compartmental models.

Parameter sensitivity and uncertainty analysis for physiologically based pharmacokinetic (PBPK) models are becoming an important consideration for regulatory submissions, requiring further evaluation to establish the need for global sensitivity analysis. To demonstrate the benefits of an extensive analysis, global sensitivity was implemented for the GastroPlus™ model, a well-known commercially available platform, using four example drugs: acetaminophen, risperidone, atenolol, and furosemide. The capabilities of GastroPlus were expanded by developing an integrated framework to automate the GastroPlus graphical user interface with AutoIt and for execution of the sensitivity analysis in MATLAB®. Global sensitivity analysis was performed in two stages using the Morris method to screen over 50 parameters for significant factors followed by quantitative assessment of variability using Sobol's sensitivity analysis. The 2-staged approach significantly reduced computational cost for the larger model without sacrificing interpretation of model behavior, showing that the sensitivity results were well aligned with the biopharmaceutical classification system. Both methods detected nonlinearities and parameter interactions that would have otherwise been missed by local approaches. Future work includes further exploration of how the input domain influences the calculated global sensitivity measures as well as extending the framework to consider a whole-body PBPK model.

A New Miniature Respirable Sampler for In-mask Sampling: Part 2-Tests Performed Inside the Mask.

This paper is the second in a series of two describing the performance of a miniature and low-weight respirable sampler designed to fit inside filtering facepiece (FFP) and half-mask type respirators. The first paper described the design of the miniature sampler and evaluated the particle and collection performance of the miniature sampler. This paper assesses its comparability with the traditional inward leakage measurement technique, and its safe use. Simultaneous mass measurements of a respirable sodium chloride aerosol were taken inside a total inward leakage chamber by a miniature sampler and by sodium flame photometry. Direct side-by-side comparison of the two methods yielded excellent correlation (R 2 = 0.99), as did comparison when sampling from inside four different masks when worn by a breathing Sheffield dummy head. In addition, comparison tests were carried out using three models of FFP worn by human volunteers both with and without the miniature sampler, in order to test whether or not the presence of the miniature sampler negatively affected the protection offered. The difference between the tests carried out with the miniature sampler and without the miniature sampler was not statistically significant (P = 0.3). In all cases, the masks performed within their protection class, whether the miniature sampler was fitted or not. We therefore conclude that the miniature sampler does not significantly affect the protection offered by the masks. The miniature sampler may prove a viable option for in-mask measurements of respirable dust where low air concentrations of hazardous material are expected.

Simply scan--optical methods for elemental carbon measurement in diesel exhaust particulate.

This article describes a performance assessment of three optical methods, a Magee Scientific OT21 Transmissometer, a Hach-Lange Microcolor II difference gloss meter, and a combination of an office scanner with Adobe Photoshop software. The optical methods measure filter staining as a proxy for elemental carbon in diesel exhaust particulate (DEP) exposure assessment and the suitability of each as a replacement for the existing Bosch meter optical method. Filters loaded with DEP were produced from air in a non-coal mine and the exhaust gases from a mobile crane. These were measured with each apparatus and then by combustion to obtain a reference elemental carbon value. The results from each apparatus were then plotted against both the Bosch number and reference elemental carbon values. The equations of the best fit lines for these plots were derived, and these gave functions for elemental carbon and Bosch number from the output of each new optical method. For each optical method, the range of DEP loadings which can be measured has been determined, and conversion equations for elemental carbon and Bosch number have been obtained. All three optical methods studied will effectively quantify blackness as a measure of elemental carbon. Of these the Magee Scientific OT21 transmissometer has the best performance. The Microcolor II and scanner/photoshop methods will in addition allow conversion to Bosch number which may be useful if historical Bosch data are available and functions for this are described. The scanner/photoshop method demonstrates a technique to obtain measurements of DEP exposure without the need to purchase specialized instrumentation.

Toward a Suite of Coarse-Grained Models for Molecular Simulation of Monoclonal Antibodies and Therapeutic Proteins.

A series of coarse-grained models for molecular simulation of proteins are considered, with emphasis on the application of predicting protein-protein self-interactions for monoclonal antibodies (MAbs). As an illustrative example and for quantitative comparison, the models are used to predict osmotic virial coefficients over a broad range of attractive and repulsive self-interactions and solution conditions for a series of MAbs where the second osmotic virial coefficient has been experimentally determined in prior work. The models are compared based on how well they can predict experimental behavior, their computational burdens, and scalability. An intermediate-resolution model is also introduced that can capture specific electrostatic interactions with improved efficiency and similar or improved accuracy when compared to the previously published models. Guidance is included for the selection of coarse-grained models more generally for capturing a balance of electrostatic, steric, and short-ranged nonelectrostatic interactions for proteins from low to high concentrations.

A Physiologically-Based Pharmacokinetic Model for Targeting Calcitriol-Conjugated Quantum Dots to Inflammatory Breast Cancer Cells.

Quantum dots (QDs) conjugated with 1,25 dihydroxyvitamin D3 (calcitriol) and Mucin-1 (MUC-1) antibodies (SM3) have been found to target inflammatory breast cancer (IBC) tumors and reduce proliferation, migration, and differentiation of these tumors in mice. A physiologically-based pharmacokinetic model has been constructed and optimized to match experimental data for multiple QDs: control QDs, QDs conjugated with calcitriol, and QDs conjugated with both calcitriol and SM3 MUC1 antibodies. The model predicts continuous QD concentration for key tissues in mice distinguished by IBC stage (healthy, early-stage, and late-stage). Experimental and clinical efforts in QD treatment of IBC can be augmented by in silico simulations that predict the short-term and long-term behavior of QD treatment regimens.

Analytical protocol for the sensitive determination of mannitol, sorbitol and glucose containing powders in pharmaceutical workplaces by ion chromatography using a pulsed amperometric detector.

Workers in the pharmaceutical industry can potentially be exposed to airborne dusts and powders that can contain potent active pharmaceutical ingredients (API). Occupational hygienists and health and safety professionals need to assess and ultimately minimise such inhalation and dermal exposure risks. Containment of dusts at source is the first line of defence but the performance of such technologies needs to be verified, for which purpose the good practice guide: assessing the particulate containment performance of pharmaceutical equipment, produced by the International Society for Pharmaceutical Engineering (ISPE), is a widely used reference document. This guide recommends the use of surrogate powders that can be used to challenge the performance of such containment systems. Materials such as lactose and mannitol are recommended as their physical properties (adhesion, compactability, dustiness, flow characteristics and particle sizes) mimic those of API-containing materials typically handled. Furthermore they are safe materials to use, are available in high purity and can be procured at a reasonable cost. The aim of this work was to develop and validate a sensitive ion-chromatography based analytical procedure for the determination of surrogate powders collected on filter samples so as to meet analytical requirements set out in this ISPE guide.