Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.     

Characterization of hyaluronic acid and synovial fluid in stagnation point elongational flow

Hyaluronic acid (HA) is an important biomacromolecule, which fulfils a number of vital physiological functions (especially in the joint synovial fluid) and also has consumer and pharmaceutical applications. HA solution properties have already been quite thoroughly characterized in response to steady shear flows but are less well understood in highly deforming extensional flows. In this study, flow‐induced birefringence measurements are made as a function of the strain rate in planar elongational flow at the stagnation point of a cross‐slot device using HA solutions of a range of molecular weights and at dilute concentrations. The results provide macromolecular relaxation times, molecular weight distributions and the extensional viscosities and Trouton ratios of the fluids. The HA relaxation time is found to vary as ^1.8, which is consistent with a partially solvated, expanded coil. An intrinsic Trouton ratio is defined, which varies as ². The measurement of birefringence with strain rate is shown to be highly sensitive to the molecular weight distribution and can resolve subtle changes due to macromolecular degradation and the presence of fracture products. Mechanical degradation experiments in the cross‐slots indicate midchain scission of HA macromolecules, strongly suggesting near full extension of the high‐molecular weight fraction in the stagnation point extensional flow field. Taken together the results suggest a possible method for analysis of the HA in synovial fluid, and this concept is tested using synovial fluid obtained from porcine tarsal joint. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 287–305, 2014.     

Optical Thromboelastography to evaluate whole blood coagulation

Measurement of blood viscoelasticity during clotting provides a direct metric of haemostatic conditions. Therefore, technologies that quantify blood viscoelasticity at the point‐of‐care are invaluable for diagnosing coagulopathies. We present a new approach, Optical Thromboelastography (OTEG) that measures the viscoelastic properties of coagulating blood by evaluating temporal laser speckle fluctuations, reflected from a few blood drops. During coagulation, platelet‐fibrin clot formation restricts the mean square displacements (MSD) of scatterers and decelerates speckle fluctuations. Cross‐correlation analysis of speckle frames provides the speckle intensity temporal autocorrelation, g₂(t), from which MSD is deduced and the viscoelastic modulus of blood is estimated. Our results demonstrate a close correspondence between blood viscoelasticity evaluated by OTEG and mechanical rheometry. Spatio‐temporal speckle analyses yield 2‐dimensional maps of clot viscoelasticity, enabling the identification of micro‐clot formation at distinct rates in normal and coagulopathic specimens. These findings confirm the unique capability of OTEG for the rapid evaluation of patients' coagulation status and highlight the potential for point‐of‐care use. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

The Rheology of Liquid Pentane Isomers by Non-Equilibrium Molecular Dynamics Simulations

Abstract

In this paper, non-equilibrium molecular dynamics (NEMD) simulations of planar Couette flow are reported for an expanded collapsed atom model for liquid pentane isomers at 273.15 K. The strain rate dependent viscosity for liquid pentane isomers exhibits shear-thinning and a linear dependence on γ^1/2. Newtonian viscosities for liquid pentane isomers obtained by a linear extrapolation to zero strain rate are: 0.256cP for normal pentane, 0.219cP for isopentane, and 0.168cP for neopentane. The strain rate dependent pressure difference and normal stress difference vary nearly linearly with the γ^3/2 law and the γ law, respectively, for all three liquid pentane isomers. The overall trend of the square of radius of gyration and end-to-end distance for normal pentane is a linear increase with strain rate. For isopentane, the trend hardly changes for the range of shear rate in this study. The alignment angle decreases with increasing strain rate and the alignment angle of the straight chain alkane is less than that of the branched chain alkane. The average percentage of C?C?C?C trans for normal pentane as a function of strain rate is in excellent correlation with the square of the radius of gyration and the average end-to-end distance. Applying the strain rate in the x-direction, the alignment angle is forced to decrease and the percentage of C?C?C?C trans increases with increasing strain rate.     

Increased diet viscosity by oat β-glucans decreases the passage rate of liquids in the stomach and affects digesta physicochemical properties in growing pigs

Rheological properties of digesta play a role in digesta passage kinetics through the gastrointestinal tract, in turn affecting nutrient absorption kinetics. Therefore, we studied the effects of diet viscosity on digesta passage and physicochemical properties in pigs. Twenty male growing pigs (35 kg body weight at the start) were assigned to one of five diets with increasing dietary concentrations of β-glucans (BG; from 0 % to 10 %), in exchange for maize starch. After a 17-day adaptation period, pigs were euthanised and the mean retention time (MRT) of digesta solids (TiO₂) and liquids (Cr-EDTA) in the stomach, and proximal and distal half of the small intestine was quantified. In the stomach, the MRT of liquids, but not of solids, increased when dietary BG level increased (6 min per % dietary BG, P = 0.008 and R² = 0.35). Concomitantly, stomach DM content (5 g/kg per % dietary BG, P < 0.001 and R² = 0.53) and apparent digesta viscosity (56 Pa × s at 1/s shear rate per % dietary BG, P = 0.003 and R² = 0.41) decreased. In the proximal half of the small intestine, no effects of dietary BG level were observed. In the distal half of the small intestine, water-binding capacity (WBC) of digesta increased (0.11 g/g digesta DM per % dietary BG, P = 0.028 and R² = 0.24) and starch digestibility decreased (0.3% per % dietary BG, P = 0.034 and R² = 0.23) when dietary BG level increased. In the colon, apparent digesta viscosity at 45/s shear rate increased (0.1 Pa × s per % dietary BG, P = 0.03 and R² = 0.24) in the proximal half of the colon, and digesta WBC increased (0.06 g/g digesta DM per % dietary BG, P = 0.024 and R² = 0.26) in the distal half of the colon when dietary BG level increased. To conclude, increasing dietary BG level caused the MRT of liquids, but not that of solids, to increase in the stomach, resulting in reduced separation of the solid and liquid digesta fractions. This caused dilution of the stomach content and reduction in digesta viscosity when dietary BG levels increased. Effects of dietary BG level on physicochemical properties in the proximal small intestine were absent and may have been due to a low DM content. The WBC of digesta in the distal small intestine and colon increased when dietary BG level increased, as did apparent digesta viscosity in the proximal colon. This likely reflects the concentration of BG in digesta when moving through the gastrointestinal tract.     

Role of erythrocyte deformability during capillary wetting

Deformability of erythrocyte was found to fundamentally alter the wetting dynamics of red blood cell (RBC) suspensions during their invasion into capillaries. Normal RBC suspensions failed to penetrate more than 1 cm into a glass capillary when the capillary radius was smaller than a critical value that is dependent on the erythrocyte concentration (about 50 microm for whole blood). In contrast, suspensions of rigidified RBCs, after cross-linking with different concentrations of glutaraldehyde or incubating with 100 ng/mL of an endotoxin, could penetrate any capillary larger than the erythrocyte dimension. The effect of RBC deformability on penetration was attributed to the enhanced shear-induced migration of normal deformable RBCs toward the capillary centreline, which imparted a higher average velocity to the RBCs than the average plasma velocity. As a result, the erythrocytes advanced into the capillary faster than the wetting meniscus, packing behind it to form a concentrated slug. This tightly packed slug had a high hydrodynamic resistance that could arrest the penetrating flow of concentrated suspensions into the small capillaries.     

Unexpected high pressure effects on the structural properties of condensed whey protein systems

We show that application of high hydrostatic pressure (600 MPa for 15 min) on condensed whey protein (WP) systems (e.g., 80% w/w solids content) results in unexpected structure–function behavior when compared with conventional thermal treatment. Unraveling the relaxation properties in first‐order thermodynamic transitions, the manifestation of glass transition phenomena and the preservation of native conformation in condensed preparations were recorded using small‐deformation dynamic oscillation in shear, modulated differential scanning calorimetry, and infrared spectroscopy. Informed temperature application results in the formation of continuous networks at the denaturation temperature, which undergo vitrification at subzero temperatures. In contrast, high‐pressure‐treated WPs resist physicochemical denaturation, hence preserving the native conformation of secondary and tertiary structures. This was rationalized on the basis of a critical concentration threshold where transfer of water molecules to nonpolar residues in the protein interior is minimized because of low moisture content and restricted molecular mobility. The physical state and morphology of these high‐solid preparations were further examined by the combined framework of reduced variables and Williams, Landel, and Ferry equation/free volume theory. Theoretical treatment of experimental observations unveils the dynamic range of the mechanical manifestation of the glass transition region in samples subjected to heat or pressure. In addition to preserving native conformation, WPs subjected to high pressure form glassy systems at parity with the structural functionality of the thermally treated counterparts. © 2012 Wiley Periodicals, Inc. Biopolymers 97:963–973, 2012.     

Effects of Pulsed Electric Field on the Viscoelastic Properties of Potato Tissue

We have investigated whether transient permeabilization caused by the application of pulsed electric field would give rise to transient changes in the potato tissue viscoelastic properties. Potato tissue was subjected to nominal field strengths (E) ranging from 30 to 500 V/cm, with a single rectangular pulse of 10⁻⁵, 10⁻⁴, or 10⁻³ s. The changes on the viscoelastic properties of potato tissue during pulsed electric fields (PEF) were monitored through small amplitude oscillatory dynamic rheological measurements. The elastic (G′) and viscous moduli (G″) were measured every 30 s after the delivery of the pulse and the loss tangent change (tan-δ) was calculated. The results were correlated with measurements of changes on electrical resistance during the delivery of the pulse. Results show a drastic increase of tan-δ in the first 30 s after the application of the pulse, followed by a decrease 1 min after pulsation. This response is strongly influenced by pulsing conditions and is independent of the total permeabilization achieved by the pulse. Our results, supported by similar measurements on osmotically dehydrated control samples, clearly show that PEF causes a rapid change of the viscoelastic properties of the tissue that could be attributed to a partial loss in turgor pressure. This would be an expected consequence of electroporation. The recovery of tan-δ to values similar to those before pulsation strongly suggests recovery of cell membrane properties and turgor, pointing at reversible permeabilization of the cells. A slight increase of stiffness traduced by a negative change of tan-δ after application of certain PEF conditions may also give an indication of events occurring on cell wall structure due to stress responses. This study set the basis for further investigations on the complex cell stress physiology involving both cell membrane functional properties and cell wall structure that would influence tissue physical properties upon PEF application.     

Current views on pectin substances

This review concerns pectin substances, the most complex class of plant polysaccharides. For the most part, the data reported after 1998 are presented; the references to earlier works are made only in the historical aspect. New data on the structure of pectin substances, their physiological activity, their role in plants, and their valuable physical properties are surveyed.