Stability Of Proteins In Solution Research Articles

알로에 베라 겔 가공부산물로서의 섬유질 분획의 성분 및 물리화학적 특성

The fibrous material fraction as a by-product from the commercial aloe vera gel processing was obtained and freeze dried. The physicochemical characteristics such as the proximate composition, crystalline/surface structures and several physical functionalities including the water holding capacity (WHC), swelling capacity (SW), oil holding capacity (OHC), emulsion/foam properties and viscosity properties of this powdered sample (100 mesh) were investigated and analyzed by comparison with commercial α-cellulose as a reference sample. The total dietary fiber content of powdered sample was very high as much as 87.5%, and the insoluble dietary and soluble dietary fiber content ratios were 77.6 and 22.4%, respectively. The FT-IR spectrum of powdered sample showed a typical polysaccharide property and exhibited a x-ray diffraction pattern for cellulose III and IV like structure. SW (8.24±0.15 mL/g), WHC(6.40±0.19 g water/g solid) and OHC(10.32±0.29 g oil/g solid) of freeze dried aloe cellulose were about 3.3, 1.4 and 2 times higher than those of commercial α-cellulose, respectively. Aloe cellulose (~2%, w/v) alone had no foam capacity while improved the foam stability of protein solution (1% albumin+0.5% CaCl2) by factor of 300%. Emulsion capacity of 2%(w/v) aloe cellulose was about 70% level of 0.5%(w/v) xanthan gum, but its emulsion stability was about 1.2 times higher than that of xanthan gum. Also, aloe cellulose containing CMC (carboxyl methyl cellulose) of 0.3%(w/v) showed a very good dispersity. Aloe cellulose dispersion of above 1%(w/v) exhibited higher pseudoplasticity and concentration dependence than those of α-cellulose dispersion, indicating the viscosity properties for new potential usage such as an excellent thickening agent.

High throughput thermostability screening of monoclonal antibody formulations

Differential scanning fluorimetry (DSF) was employed to increase the throughput of the thermostability screening of monoclonal antibody (mAb) formulations. The method consists of measuring the fluorescence intensity of a polarity sensitive probe at gradually increasing temperatures, and obtaining the transition temperature of exposure of the hydrophobic regions of proteins (Th). The change in fluorescence intensity was directly related to protein unfolding levels and temperatures. The results from thermostability measurements were compared with the data acquired using differential scanning calorimetry (DSC), and a good correlation between Th and the temperature of protein unfolding or melting (Tm) was observed. The method was applied to screen four mAb molecules in 84 different buffers. The studies revealed a good correlation of Th values with the known effects of pH and excipients on protein stability in solution. Specifically, the elevated aggregation levels induced by salt, low pH, and high protein concentrations could be successfully predicted by this thermal stability screening. This method is efficient, with high throughput capability, and could be widely applied in the biopharmaceutical industry for formulation and process development, and characterization. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1707–1720, 2010

Viscoelastic characterization of high concentration antibody formulations using quartz crystal microbalance with dissipation monitoring

With increasing protein concentrations, therapeutic protein formulations are increasingly demonstrating significant deviations from ideal dilute solution behavior due to protein–protein interactions. These interactions lead to unique biophysical challenges in the administration of biopharmaceuticals including high apparent viscosity and viscoelasticity as well as challenges in maintaining the physical stability of proteins in solution. Here, we describe a straightforward analytical method to calculate the complex modulus and viscosity of high concentration protein solutions from measurements made using quartz crystal microbalance with dissipation monitoring (QCM-D). Further, this methodology was used to investigate the dependence of the storage and loss moduli (G′ and G″, respectively) of a humanized monoclonal antibody solution on solution pH. Unlike recent reports, the effect of protein deposition onto the surface of the quartz sensor crystal was measured and explicitly accounted for during analysis when determining the solution's complex modulus. It was found that the ratio G″/G′ was significantly greater than unity for all solutions investigated, but demonstrated a distinct maximum at pH 5.5 indicating that the solution exhibited the greatest liquid-like behavior at this pH. In addition, measurements were made at higher frequencies, which were found to be more sensitive to the changes in pH than those made at lower frequencies. It was also found that the viscoelastic ratio was relatively insensitive to the frequency of measurement at lower pH, but showed greater dependence on frequency as pH increased. The characterization of the rheological properties of high concentration antibody solutions provides insight into protein–protein interactions, and the methodology presented here demonstrates a straightforward way to determine the viscoelastic properties using ultrasonic rheology without the drawbacks of numerical fitting. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3108–3116, 2009

The use of self‐interaction chromatography in stable formulation and crystallization of proteins

This paper reviews the basic principles of the recently developed self-interaction chromatography (SIC) technique with regard to protein solution stability and protein crystallization. It gives experimental protocols for both normal-scale and micro-scale SIC experiments and reviews recent developments and current applications of this novel technique in the biopharmaceutical area. This paper aims to be a benchmark in the further proliferation of this highly effective and fast technology for the rational design of stable aqueous formulations of therapeutic proteins and the determination of solution conditions favoring protein crystallization.

Probability Distributions of Hydration Water Molecules around Polar Protein Atoms Obtained by a Database Analysis

Hydration structures on protein surfaces are visualized by high-resolution cryogenic X-ray crystallography. We calculated the probability distributions of 4,831,570 hydration water molecules found around the 4,214,227 polar atoms in main chains and hydrophilic side chains from the 17,984 crystal structures in the Protein Data Bank. The structures are refined using the diffraction data collected below 150 K and at resolutions of better than 2.2 A. The calculated distributions were nonrandom but condensed into a few clusters. The clusters were decomposed into the distance and angular distributions by viewing from the polar coordinate system. The major peaks in the clusters were almost located along the directions of the N-H and O-H bonds or the lone pairs of oxygen atoms. The Gaussian fitting method was applied for the distribution profiles to evaluate quantitatively the peak positions and the widths. The parameters characterizing the distributions apparently depended on the hydrogen-bond partners of water molecules and on the modes whether the water molecules acted as donors or acceptors of protons. This led to propose the different roles of NH(n) (n = 1, 3), OH, and CO groups in protein hydration and possible in protein-ligand and protein-protein interaction: While C horizontal lineO groups appear to control the H-bond distances, NH(n) groups likely limit the angular range of H-bonds. The OH groups have both characteristics. In addition, it was also demonstrated that polar protein atoms were arranged to satisfy the tetrahedral hydrogen-bond geometry of water molecules, suggesting essential roles of water molecules in the folding process and in the stabilization of protein structures. These probability distributions are probably one of fundamental data to better understand the roles of hydration water molecules in the folding process and the stability of proteins in solution.

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.

Characterization of Globular Protein Solutions by Dynamic Light Scattering, Electrophoretic Mobility, and Viscosity Measurements

In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.

Coarse-Grained Strategy for Modeling Protein Stability in Concentrated Solutions. III: Directional Protein Interactions

We extend our coarse-grained modeling strategy described in parts I and II of this investigation to account for nonuniform spatial distributions of hydrophobic residues on the solvent-exposed surfaces of native proteins. Within this framework, we explore how patchy surfaces can influence the solvent-mediated protein-protein interactions, and the unfolding and self-assembly behaviors of proteins in solution. In particular, we compare the equilibrium unfolding and self-assembly trends for three model proteins that share the same overall sequence hydrophobicity, but exhibit folded configurations with different solvent-exposed native-state surface morphologies. Our model provides new insights into how directional interactions can affect native-state protein stability in solution. We find that strongly-directional attractions between native molecules with patchy surfaces can help stabilize the folded conformation through the formation of self-assembled clusters. In contrast, native proteins with more uniform surfaces are destabilized by protein-protein attractions involving the denatured state. Finally, we discuss how the simulation results provide insights into the experimental solution behaviors of several proteins that display directional interactions in their native states.

Microcalorimetric studies of the effects on the interactions of human recombinant interferon-α2a

The applicability of the physical stability of protein solution monitored by isothermal titration calorimetry (ITC) was evaluated. The second virial coefficient, b 2, derived from the dilution enthalpies of protein solution measured by ITC under various experimental conditions was studied. The protein applied in this work is human recombinant interferon-α2a (hrIFN-α2a), which is a commercial drug applied for the treatment of virus-infected diseases. The results obtained were used to predict the possibility of hrIFN-α2a aggregation, and the prediction can be further confirmed by size-exclusion chromatography (SEC). Various factors affecting the stability of protein solution were investigated, for example, temperature, salts, surfactants, and mechanical stress. Specifically, the results show that the dilution enthalpy of hrIFN-α2a increased with increasing temperature and NaCl concentration, while b 2 decreased, indicating that the attraction between hrIFN-α2a molecules was enhanced under these conditions. On studying the effect of mechanical stress, the data obtained reveals that the introduction of centrifugal or vortex force strengthened the attractive forces between hrIFN-α2a molecules. These implications were supported by SEC data, demonstrating that the amount of aggregated hrIFN-α2a was increased. As a consequence, the methodologies presented in this investigation offer a possibility of monitoring the physical stability of protein solution at various stages of recovery, purification as well as the development of appropriate drug storage formulations.

Optical characterization of glutamate dehydrogenase monolayers chemisorbed on SiO2.

This paper describes the formation of glutamate dehydrogenase monolayers on silicon dioxide, and their characterization by means of physical techniques, i.e., fluorescence spectroscopy and Fourier-transform infrared spectroscopy. Detailed investigations of the intrinsic stability of native proteins in solution were carried out to elucidate the occurrence of conformational changes induced by the immobilization procedure. The enzyme monolayers were deposited on SiO2 after preexposing silicon surfaces to 3-aminopropyltriethoxysilane and reacting the silylated surfaces with glutaric dialdehyde. The optical characterization demonstrates that the immobilization does not interfere with the fold pattern of the native enzyme. In addition, fluorescence spectroscopy, thermal denaturation, and quenching studies performed on the enzyme in solution well describe the folding and unfolding properties of glutamate dehydrogenase. The photophysical studies reported here are relevant for nanobioelectronics applications requiring protein immobilization on a chip.

Preparation of non-thrombogenic materials using 2-methacryloyloxyethyl phosphorylcholine

This review addresses the non-thrombogenic characteristics of copolymers based on 2-methacryloyloxyethyl phosphorylcholine (MPC), originally developed by Nakabayashi and colleagues. The hypothesis underlying these developments was that such materials would adsorb phospholipids from blood, yielding surfaces with good natural blood compatibility. Methacrylates were found to have excellent properties for this copolymerisation. The characteristics of the MPC copolymers relevant to the improved blood compatibility were minimisation of protein adsorption through an increase in the amount of free water in the MPC hydrogels, which prevents protein conformational change and increased protein stability in solution. Non-thrombogenicity has been evaluated by in vitro, ex vivo and in vivo procedures. Non-thrombogenic dialysis membranes and a durable glucose biosensor have been developed using this MPC copolymer.

Heat capacities of solid state proteins: implications for protein stability in solution

We show that experimentally measured heat capacities of six different proteins (lysozyme, myoglobin, chymotrypsinogen, lactoglobulin, ovalbumine, and ribonuclease A) in the solid state, in the temperature range from 260 to 420 K , can be well characterized as a sum of one Einstein mode, corresponding to the group vibrations, plus a constant, corresponding to the nearly excited skeletal vibrations. The relative root mean square deviations between experimental data and fitted data are less than 1%. We also show for lysozyme and ribonuclease A that the experimental values of the solid state heat capacities, corrected for intermolecular vibrational modes, deviate less than 15% relative experimental protein data in solution, where the hydration part of the latter data is excluded by a model compound evaluation (J. Mol. Biol. 224 (1992) 715). Thus, analyzing solid state proteins may give important information to energetics and stabilization of proteins in solution.

Refolding of the Escherichia coli expressed extracellular domain of alpha 7 nicotinic acetylcholine receptor.

Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand-binding sites. The ECD of the alpha 7 subunit of the homo-oligomeric alpha 7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle-type AChRs. The rat alpha 7 ECDs (amino-acid residues approximately 1-210) were recently expressed in Escherichia coli as fusion proteins with maltose-binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA 98, 3567-3570] and glutathione S-transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem. 276, 15810-15815]. However, these proteins exist in solution mostly as high-molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST-alpha 7-(1-208) protein in the presence of 0.1% SDS considerably decreases the formation of high-molecular mass aggregates. The C116S mutation in the alpha 7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for alpha-bungarotoxin (from Kd approximately 300 to 150 nm). Gel-permeation HPLC was used to isolate the monomeric form of the GST-alpha 7-(1-208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of beta structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the alpha 7 ECD in an oligomeric form.

Diffusion-controlled delivery of proteins from hydrogels and other hydrophilic systems.

The short in vivo half-life of many pharmaceutically active proteins necessitates the need for multiple administrations to produce a therapeutic response, emphasizing the applicability of controlled release formulations (Lee, 1992). The key variables that affect protein transport through hydrophilic polymers depend on the delivery mechanism and device properties, in a similar manner as in the case of lower-molecular-weight drug substances. However, in the case of proteins, the role of solute molecular size is much more dramatic in hindering the diffusion and release from hydrophilic polymers (am Ende, 1993). Another critical consideration in protein delivery from hydrogel systems is the potential for protein denaturation in the device. For diffusion-controlled delivery systems, where water is the main transporting medium, the protein solution stability governs the type of device. Extended releasing times can be achieved with reservoir systems (Fig. 1) for highly stable proteins (Langer, 1990). Alternatively, dehydrated delivery systems

Calorimetric studies of hydrophobic interactions of alkanols in concentrated aqueous solutions of glucose: Implications for the mechanism of protein stabilization by sugars

Calorimetric measurements were carried out at 298 K on concentrated aqueous solutions of glucose, ranging from 0 to 5 mol kg −1, containing alkan-1-ols, alkane-1,2-diols and alkane-α,ω-diols from C 2 to C 7. The purpose of this study is to obtain more information about the influence of glucose on hydrophobic hydration and interactions. The pair-wise interaction coefficients of the virial expansion of the excess enthalpies were evaluated, and the results rationalized according to the preferential configuration model. At increasing glucose concentration, alkanols and 1,2-diols maintain almost unaltered the coefficients they have in water, while α,ω-diols show a smooth decrease only at 3 mol kg −1 glucose. Urea and glycine have been studied in concentrated glucose, too. Their coefficients indicate that glucose is able to reduce hydrophilic interactions. Then, the invariance of the coefficients for alkanols means that both hydrophilic and hydrophobic interactions are attenuated in the presence of high concentrations of the cosolvent. The data show that the nature of the hydration cosphere of the alkyl chain does not change, so that the same interaction mechanism operates in water and in concentrated glucose solutions. The results obtained are compared with those found for diols in concentrated solutions of urea and ethanol: some comments are made on the possible mode of action of these substances on the stability of proteins in solution.

Probing hydration and the stability of protein solutions — a colloid science approach

The stability of protein solutions is a topic of much theoretical and practical interest. The theory of Derjaguin, Landau, Verweey and Overbeek (DLVO theory) has long been used by colloid scientists and chemical engineers to define conditions leading to stability of dispersion, but has found very little application by protein chemists. We apply DLVO theory to demonstrate that protein solution stability appears to be closely related to the presence of the hydration layer, rather than to significant inter-molecular net repulsive force. A novel but simple formalism is then developed which indicates, via computation of a newly defined parameter (‘site relaxation time’), that estimated ‘dwell times’ for water molecules on a protein surface are of a magnitude adequate to account for such a role.

Approaches for increasing the solution stability of proteins

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.

Effect of salt concentration on the pKa of acidic residues in lysozyme.

We determined the pKa values of acidic residues in hen lysozyme by comparing the pH dependency of stability between wild type and mutant lysozymes in which a negative charge is eliminated. In the comparison of the stability between wild type and a mutant lysozyme, the difference in pH titration curve between them could be expressed as a two-state process involving protonation of a single acidic residue. The results strongly indicated that the Aune and Tanford theory of protein denaturation [Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4579-4585] is applicable to protein stability in solution. On the other hand, the pKa values of acidic residues in the presence of low (5 mM) or high (400 mM) salt concentration were determined by means of two-dimensional NMR. We found that the pKa values obtained from the pH dependency of stability were close to those from the NMR experiment under the high salt condition. Moreover, by comparing pKa values at high salt and low salt concentrations, we could evaluate the dependency of two electrostatic interactions (salt bridge and charge-helix dipole interaction) on salt concentration.

Modes of stabilization of a protein by organic solutes during desiccation

We have investigated the role that stabilization of proteins in solution by organic solutes plays in stabilizing labile enzymes during air-drying. Phosphofructokinase (PFK) was dehydrated to various water contents and rehydrated, and the resulting enzyme activity was then measured. When 60% of the water was removed, 30% of the initial enzyme activity was lost. This loss in activity was clearly due to changes in solution properties during dehydration, since it occurred in excess water. No activity was measurable following dehydration to only 3% of the initial water content. This final loss in activity was attributed to dehydration of the protein. Inactivation at any stage of air-drying was irreversible. In the presence of 100 m M trehalose, by contrast, there was no loss of enzyme activity induced at any stage of the progressive desiccation. Similar results were seen with 100 m M sucrose or maltose or with 200 m M glucose, but the degree of protection was less. Addition of 0.6 m M ZnSO 4 to glucose/PFK solutions enhanced the protection provided by the sugar. Glycerol, proline, and trimethylamine N-oxide protected PFK until up to 90% of the initial water was removed, but not against complete dehydration. Sugars and the other organic solutes inhibited pH-induced dissociation of PFK, indicating that these solutes have a more generalized capacity to stabilize this protein in solution. We conclude that the capacity of sugars to stabilize labile enzymes in solution is a prerequisite for the maintenance of catalytic activity when water is removed by air-drying, but that this effect is not in itself sufficient to stabilize fully dried proteins. We suggest that the capacity of sugars to preserve dried PFK may be dependent on the binding of the sugar to the protein during the final stages of dehydration.

Stability of glycolytic enzymes of human erythrocytes.

The stability of various glycolytic enzymes of human erythrocytes has been studied by the mechanical shaking method. The rate of denaturation apparently followed first order kinetics. The t1/2, the shaking time required to denature 50% of the original activity, for glucose-6-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase was less than 1 min; that for hexokinase, 6-phosphogluconate dehydrogenase, and monophosphoglyceromutase was between 2 and 13 min; that for all the other enzymes was more than 30 min. Since the t1/2 value for each enzyme is highly reproducible if the shaking conditions are kept constant, these parameters may be used as an indicator of protein stability in solution. The mechanical denaturation method may also be used to remove unstable components from a mixture of proteins with different stabilities.