Development and efficacy testing of a portable negative pressure enclosure for airborne infection containment.

OBJECTIVES: To overcome the shortage of personal protective equipment and airborne infection isolation rooms (AIIRs) in the COVID-19 pandemic, a collaborative team of research engineers and clinical physicians worked to build a novel negative pressure environment in the hopes of improving healthcare worker and patient safety. The team then sought to test the device's efficacy in generating and maintaining negative pressure. The goal proved prescient as the US Food and Drug Administration (FDA) later recommended that all barrier devices use negative pressure. METHODS: Initially, engineers observed simulations of various aerosol- and droplet-generating procedures using hospital beds and stretchers to determine the optimal working dimensions of the containment device. Several prototypes were made based on these dimensions which were combined with filters and various flow-generating devices. Then, the airflow generated and the pressure differential within the device during simulated patient care were measured, specifically assessing its ability to create a negative pressure environment consistent with standards published by the Centers for Disease Control and Prevention (CDC). RESULTS: The portable fans were unable to generate any airflow and were dropped from further testing. The vacuums tested were all able to generate a negative pressure environment with the magnitude of pressure differential increasing with the vacuum horsepower. Only the 3.5-horsepower Shop-Vac, however, generated a -3.0 pascal (Pa) pressure gradient, exceeding the CDC-recommended minimum of -2.5 Pa for AIIRs. CONCLUSION: A collaborative team of physicians and engineers demonstrated the efficacy of a prototype portable negative pressure environment, surpassing the negative pressure differential recommended by the CDC.

Design and in-vitro testing of a portable patient isolation chamber for bedside aerosol containment and filtration.

The emergence of the SARS-CoV-2 pandemic has imposed a multitude of complications on healthcare facilities. Healthcare professionals had to develop creative solutions to deal with resource shortages and isolation spaces when caring for COVID positive patients. Among many other solutions, facilities have utilized engineering strategies to mitigate the spread of viral contamination within the hospital environment. One of the standard solutions has been the use of whole room negative pressurization (WRNP) to turn a general patient room into an infection isolation space. However, this has not always been easy due to many limitations, such as direct access to the outdoors and the availability of WRNP units. In operating rooms where a patient is likely to go through aerosol-generating procedures, other solutions must be considered because most operating rooms use positive pressure ventilation to maintain sterility. The research team has designed, built, and tested a Covering for Operations during Viral Emergency Response (COVER), a low-cost, portable isolation chamber that fits over a patient's torso on a hospital bed to contain and remove the pathogenic agents at the source (i.e., patient's mouth and nose). This study tests the performance of the COVER system under various design and performance scenarios using particle tracing techniques and compares its efficiency with WRNP units. The results show that COVER can dramatically reduce the concentration of particles within the room, while WRNP is only effective in preventing the room-induced particles from migrating to adjacent spaces.

Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020.

The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis-targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA-binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre-existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid-liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams.

Latent Anti-nutrients and Unintentional Breeding Consequences in Australian Sorghum bicolor Varieties.

Modern feed quality sorghum grain has been bred to reduce anti-nutrients, most conspicuously condensed tannins, but its inclusion in the diets of monogastric animals can still result in variable performance that is only partially understood. Sorghum grain contains several negative intrinsic factors, including non-tannin phenolics and polyphenols, phytate, and kafirin protein, which may be responsible for these muted feed performances. To better understand the non-tannin phenolic and polyphenolic metabolites that may have negative effects on nutritional parameters, the chemical composition of sorghum grain polyphenol extracts from three commercial varieties (MR-Buster, Cracka, and Liberty) was determined through the use of an under-studied, alternative analytical approach involving Fourier-transform infrared (FT-IR) spectroscopy and direct ionization mass spectrometry. Supervised analyses and interrogation of the data contributing to variation resulted in the identification of a variety of metabolites, including established polyphenols, lignin-like anti-nutrients, and complex sugars, as well as high levels of fatty acids which could contribute to nutritional variation and underperformance in monogastrics. FT-IR and mass spectrometry could both discriminate among the different sorghum varieties indicating that FT-IR, rather than more sophisticated chromatographic and mass spectrometric methods, could be incorporated into quality control applications.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of-flight mass spectroscopy.

Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.

The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species.

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 µM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.

Resveratrol addition to Chinese hamster ovary cell culture media: The effect on cell growth, monoclonal antibody synthesis, and its chemical modification.

The effect of the addition of resveratrol to cell culture media during the production of monoclonal antibodies was investigated. Treatments of Chinese hamster ovary (CHO) cells expressing immunoglobulin G (IgG) with 25 and 50 µM resveratrol showed that resveratrol was capable of slowing cell growth while almost doubling cell-specific productivity to 4.7 ± 0.6 pg IgG/cell·day, resulting in up to a 1.37-fold increase of the final IgG titer. A resveratrol concentration of 50 µM slowed the progression through the cell cycle temporarily by trapping cells in the S-phase. Cation exchange chromatography showed no significant difference in the composition of acidic or basic IgG species and size exclusion chromatography indicated no change in fragmentation or aggregation of the recombinant IgG in the treatment groups. Resveratrol could be used as a chemical additive to CHO media where it would enhance IgG productivity and provide a degree of protection against hydroxyl and superoxide free radicals, expanding the range of options for process improvement available to monoclonal antibody manufacturers.

Oxygen exposure during red wine fermentation modifies tannin reactivity with poly-l-proline.

Red wines injected with nitrogen or oxygen during fermentation were used to identify the effect of gas exposure on tannin structure and reactivity with poly-l-proline. Tannin was purified from wine after fermentation and after three years of bottle storage. Tannin from nitrogen-treated wine had a lower percentage of galloylation and were less pigmented than tannin from oxygen-exposed wine. Self-aggregation of tannin was measured by nanoparticle tracking analysis and a larger particle size was observed for the oxidized treatment. The interaction of tannin and poly-l-proline was measured by isothermal titration calorimetry, and involved more hydrogen bonding than hydrophobic interactions in the case of nitrogen-treated wine tannin. Conversely, oxidized tannin was more hydrophobic and the association with poly-l-proline was entropy-driven due to a change of solvation. The results show meaningful changes in the structure and reactivity of tannin as a result of oxygen exposure during fermentation, which may impact astringency perception.

Advances in liquid formulations of parenteral therapeutic proteins.

Liquid formulation of therapeutic proteins is a maturing technology. Demand for products that are easy to use in the clinic or that are amenable to self-administration make a ready to use liquid formulation desirable. Most modern liquid formulations have a simple composition; comprising a buffer, a tonicity modifier, a surfactant, sometimes a stabiliser, the therapeutic protein and water. Recent formulations of monoclonal antibodies often use histidine or acetate as the buffer, sucrose or trehalose as the tonicity modifier and polysorbate 20 or 80 as the surfactant with a pH of 5.7 +/- 0.4. The mechanisms for the behaviour of excipients is still debated by academics so formulation design is still a black art. Fortunately, a statistical approach like design of experiment is suitable for formulation development and has been successful when combined with accelerated stability experimentation. The development of prefilled syringes and pens has added low viscosity and shear resistance to the quality attributes for a successful formulation. To achieve patient compliance for self-administration, formulations that cause minimal pain and tissue damage is also desirable.

Screening Naturally Occurring Phenolic Antioxidants for Their Suitability as Additives to CHO Cell Culture Media Used to Produce Monoclonal Antibodies.

This study identified several antioxidants that could be used in Chinese hamster ovary (CHO)cell culture media and benefit monoclonal antibody production. The flavan-3-ols, catechin, epicatechin, epigallocatechin gallate and gallocatechin gallate all had no detrimental effect on cell viability at the concentrations tested, and they reduced the final viable cell count with a resulting rise in the cell specific productivity. The flavone, luteolin behave similarly to the flavan-3-ols. Resveratrol at 50 µM concentration resulted in the most pronounced reduction in viable cell density with minimal decrease in IgG synthesis and the largest increase in cell specific productivity. Low concentrations of α-tocopherol (35 µM) reduced viable cell density and raised cell specific productivity, but at higher concentration it had little additional effect. As high concentrations of α-tocopherol are not toxic to CHO cells, its addition as an anti-oxidant has great potential. Kaempferol up to 50 µM, curcumin up to 20 µM and piceid up to 100 µM showed little effect on growth or IgG synthesis and could be useful as antioxidants. Caffeic acid phenethyl ester was toxic to CHO cell and of no interest. Seven of the phenolic compounds tested are potential cell cycle inhibitors as well as having intrinsic antioxidant properties.

Analysis of Mesoscopic Structured 2-Propanol/Water Mixtures Using Pressure Perturbation Calorimetry and Molecular Dynamic Simulation.

In this paper we demonstrate the application of pressure perturbation calorimetry (PPC) to the characterization of 2-propanol/water mixtures. PPC of different 2-propanol/water mixtures provides two useful measurements: (i) the change in heat (ΔQ); and (ii) the [Formula: see text] value. The results demonstrate that the ΔQ values of the mixtures deviate from that expected for a random mixture, with a maximum at ~20-25 mol% 2-propanol. This coincides with the concentration at which molecular dynamics (MD) simulations show a maximum deviation from random distribution, and also the point at which alcohol-alcohol hydrogen bonds become dominant over alcohol-water hydrogen bonds. Furthermore, the [Formula: see text] value showed transitions at 2.5 mol% 2-propanol and at approximately 14 mol% 2-propanol. Below 2.5 mol% 2-propanol the values of [Formula: see text] are negative; this is indicative of the presence of isolated 2-propanol molecules surrounded by water molecules. Above 2.5 mol% 2-propanol [Formula: see text] rises, reaching a maximum at ~14 mol% corresponding to a point where mixed alcohol-water networks are thought to dominate. The values and trends identified by PPC show excellent agreement not only with those obtained from MD simulations but also with results in the literature derived using viscometry, THz spectroscopy, NMR and neutron diffraction.

Magnetic-Silk Core-Shell Nanoparticles as Potential Carriers for Targeted Delivery of Curcumin into Human Breast Cancer Cells.

Curcumin is a promising anticancer drug but its applications in cancer therapy are limited due to its poor solubility, short half-life, and low bioavailability. In this article, we present a curcumin loaded magnetic silk fibroin core-shell nanoparticle system for sustained release of curcumin into breast cancer cells. Curcumin loaded magnetic silk fibroin core-shell nanoparticles were fabricated by a simple salting-out method using sodium phosphate with magnetic nanoparticles. The size, zeta potential, encapsulation/loading efficiency, and curcumin release rate were controlled and optimized by regulating silk fibroin concentration, pH value of the phosphate solution, and curcumin usage. Curcumin loaded magnetic silk fibroin core-shell nanoparticles showed enhanced cytotoxicity and higher cellular uptake in the human Caucasian breast adenocarcinoma cell line (MDA-MB-231cells) evidenced by MTT and cellular uptake assays. In addition, silk fibroin nanoparticles and magnetic silk fibroin nanoparticles without curcumin loaded were used as controls. The particles prepared using sodium phosphate showed significantly smaller diameter (90-350 nm) compared with those prepared using potassium phosphate, which possess a diameter range of 500-1200 nm. These smaller particles are superior for biomedical applications since such a size range is particularly desired for cell internalization. In addition, the magnetic cores inside the particles provide the possibility of using an external magnet for cancer targeting.

Control of Globular Protein Thermal Stability in Aqueous Formulations by the Positively Charged Amino Acid Excipients.

The positively charged amino acids are commonly used excipients in biopharmaceutical formulations for stabilization of therapeutic proteins, yet the mechanisms for their modulation of protein stability are poorly understood. In this study, both lysine and histidine are shown to affect the thermal stability of myoglobin, bovine serum albumin, and lysozyme through a combination of mechanisms governed by their respective functional side chains and glycine, similar to arginine. This study provides evidence that at low concentrations, lysine and histidine interact with proteins by a combination of (1) direct electrostatic interactions with negatively charged side chains, (2) possible binding to high-affinity hydrophobic binding sites, and (3) glycine-mediated weak interactions with peptide backbone and polar side chains. At high concentrations, lysine and histidine act via (4) glycine-mediated competition for water between the unfolding protein and the excipient and (5) sidechain-mediated interaction with apolar regions exposed during unfolding (histidine). Lysine and histidine are useful for biopharmaceutical formulations as they were less destabilizing of the protein structures tested than arginine at concentrations above 100 mM.

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015.

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x-ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion-π and π-π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd.

Formulations for microprojection/microneedle vaccine delivery: Structure, strength and release profiles.

To develop novel methods for vaccine delivery, the skin is viewed as a high potential target, due to the abundance of immune cells that reside therein. One method, the use of dissolving microneedle technologies, has the potential to achieve this, with a range of formulations now being employed. Within this paper we assemble a range of methods (including FT-FIR using synchrotron radiation, nanoindentation and skin delivery assays) to systematically examine the effect of key bulking agents/excipients - sugars/polyols - on the material form, structure, strength, failure properties, diffusion and dissolution for dissolving microdevices. We investigated concentrations of mannitol, sucrose, trehalose and sorbitol from 1:1 to 30:1 with carboxymethylcellulose (CMC), although mannitol did not form our micro-structures so was discounted early in the study. The other formulations showed a variety of crystalline (sorbitol) and amorphous (sucrose, trehalose) structures, when investigated using Fourier transform far infra-red (FT-FIR) with synchrotron radiation. The crystalline structures had a higher elastic modulus than the amorphous formulations (8-12GPa compared to 0.05-11GPa), with sorbitol formulations showing a bimodal distribution of results including both amorphous and crystalline behaviour. In skin, diffusion properties were similar among all formulations with dissolution occurring within 5s for our small projection array structures (~100µm in length). Overall, slight variations in formulation can significantly change the ability of our projections to perform their required function, making the choice of bulking/vaccine stabilising agents of great importance for these devices.

Molecular Mechanism for the Hofmeister Effect Derived from NMR and DSC Measurements on Barnase.

The effects of sodium thiocyanate, sodium chloride, and sodium sulfate on the ribonuclease barnase were studied using differential scanning calorimetry (DSC) and NMR. Both measurements reveal specific and saturable binding at low anion concentrations (up to 250 mM), which produces localized conformational and energetic effects that are unrelated to the Hofmeister series. The binding of sulfate slows intramolecular motions, as revealed by peak broadening in 13C heteronuclear single quantum coherence spectroscopy. None of the anions shows significant binding to hydrophobic groups. Above 250 mM, the DSC results are consistent with the expected Hofmeister effects in that the chaotropic anion thiocyanate destabilizes barnase. In this higher concentration range, the anions have approximately linear effects on protein NMR chemical shifts, with no evidence for direct interaction of the anions with the protein surface. We conclude that the effects of the anions on barnase are mediated by solvent interactions. The results are not consistent with the predictions of the preferential interaction, preferential hydration, and excluded volume models commonly used to describe Hofmeister effects. Instead, they suggest that the Hofmeister anion effects on both stability and solubility of barnase are due to the way in which the protein interacts with water molecules, and in particular with water dipoles, which are more ordered around sulfate anions and less ordered around thiocyanate anions.

The complexity of condensed tannin binding to bovine serum albumin--An isothermal titration calorimetry study.

Isothermal titration calorimetry was applied to study the binding of purified proanthocyanidin oligomers to bovine serum albumin (BSA). The molecular weight of the proanthocyanidin oligomer had a major impact on its binding to BSA. The calculated change in enthalpy (ΔH) and association constant (Ka) became greater as the oligomer size increased then plateaued at the heptameric oligomer. These results support a model for precipitation of proteins by proanthocyanidin where increased oligomer size enhanced the opportunity for cross linkages between proteins ultimately forming sediment-able complexes. The authors suggest tannin binding to proteins is opportunistic and involves multiple sites, each with a different Ka and ΔH of binding. The ΔH of binding comprises both an endothermic hydrophobic interaction and exothermic hydrogen bond component. This suggests the calculated entropy value (ΔS) for tannin-protein interactions is subject to a systematic error and should be interpreted with caution.

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy.

Terahertz time-domain spectroscopy (THz-TDS) has been shown to detect overlapping extended hydration layers around proteins. Here, we used THz-TDS to detect modulation of the extended hydration layer around monoclonal antibodies (mAbs) by the introduction of commonly used excipients. Proline and sucrose altered the hydration layer around a mAb (mAb1), which was observed as a negative shift in the plateau in absorbance above ~100 mg/mL mAb1 (~70,000 water molecules per mAb); arginine had no effect. At lower concentrations of ~10 mg/mL mAb1 (~700,000 water molecules per mAb) proline and arginine modulated the hydration layer, which was observed as a negative shift in the relative absorbance, whereas sucrose had no effect. The changes in the extended hydration layer were not translated to shifts in the thermal stability or protein:protein interaction parameter. The hydration layer of a second mAb (mAb2) was further shown to be modulated by more complex formulations composed of two or more excipients; although the differences in terahertz absorbance were not predictive of viscosity or long-term stability. THz-TDS promises to be a useful tool for understanding a protein's interaction with excipients in solution and the challenge will be to determine how to apply this knowledge to protein formulation.

A study of the relationship between water and anions of the Hofmeister series using pressure perturbation calorimetry.

Pressure perturbation calorimetry (PPC) was used to study the relationship between water and sodium salts with a range of different anions. At temperatures around 25 °C the heat on pressurisation (ΔQ) from 1 to 5 bar was negative for all solutions relative to pure water. The raw data showed that as the temperature rose, the gradient was positive relative to pure water and the transition temperature where ΔQ was zero was related to anion surface charge density and was more pronounced for the low-charge density anions. A three component model was developed comprising bulk water, the hydration layer and the solute to calculate the molar expansivity of the hydration layer around the ions in solution. The calculated molar expansivities of water in the hydration layer around the ions were consistently less than pure water. ΔQ at different disodium hydrogen phosphate concentrations showed that the change in molar enthalpy relative to pure water was not linear even as it approached infinite dilution suggesting that while hydration layers can be allocated to the water around ions this does not rule out interactions between water and ions extending beyond the immediate hydration layer.