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

An isothermal titration calorimetry study of phytate binding to lysozyme: A multisite electrostatic binding reaction

Isothermal titration calorimetry (ITC) was used to detect phytate binding to the protein lysozyme. This binding interaction was driven by electrostatic interaction between the positively charged protein and negatively charged phytate. When two phytate molecules bind to the protein, the charge on the protein is neutralised and no further binding occurs. The stoichiometry of binding provided evidence of phytate–lysozyme complex formation that was temperature dependent, being most extensive at lower temperatures. The initial stage of phytate binding to lysozyme was less exothermic than later injections and had a stoichiometry of 0.5 at 313 K, which was interpreted as phytate crosslinking two lysozyme molecules with corresponding water displacement. ITC could make a valuable in vitro assay to understanding binding interactions and complex formation that normally occur in the stomach of monogastric animals and the relevance of drinking water temperature on the extent of phytate–protein interaction. Interpretation of ITC data in terms of cooperativity is also discussed

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 13 C 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

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 [δC¯p/δp]T[δC¯p/δp]T 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 [δC¯p/δp]T[δC¯p/δp]T 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 [δC¯p/δp]T[δC¯p/δp]T are negative; this is indicative of the presence of isolated 2-propanol molecules surrounded by water molecules. Above 2.5 mol% 2-propanol [δC¯p/δp]T[δC¯p/δp]T 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

Multiresolution analysis of active region magnetic structure and its correlation with the Mt. Wilson classification and flaring activity

Two different multi-resolution analyses are used to decompose the structure of active region magnetic flux into concentrations of different size scales. Lines separating these opposite polarity regions of flux at each size scale are found. These lines are used as a mask on a map of the magnetic field gradient to sample the local gradient between opposite polarity regions of given scale sizes. It is shown that the maximum, average and standard deviation of the magnetic flux gradient for alpha, beta, beta-gamma and beta-gamma-delta active regions increase in the order listed, and that the order is maintained over all length-scales. This study demonstrates that, on average, the Mt. Wilson classification encodes the notion of activity over all length-scales in the active region, and not just those length-scales at which the strongest flux gradients are found. Further, it is also shown that the average gradients in the field, and the average length-scale at which they occur, also increase in the same order. Finally, there are significant differences in the gradient distribution, between flaring and non-flaring active regions, which are maintained over all length-scales. It is also shown that the average gradient content of active regions that have large flares (GOES class 'M' and above) is larger than that for active regions containing flares of all flare sizes; this difference is also maintained at all length-scales.Comment: Accepted for publication in Solar Physic

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

The tail of the maximum of smooth Gaussian fields on fractal sets

We study the probability distribution of the maximum $M_S$ of a smooth stationary Gaussian field defined on a fractal subset $S$ of $\R^n$. Our main result is the equivalent of the asymptotic behavior of the tail of the distribution $\P(M_S>u)$ as $u\rightarrow +\infty.$ The basic tool is Rice formula for the moments of the number of local maxima of a random field

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

Resveratrol addition to CHO 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 whilst 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 titre. 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

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