Concentrated Protein Solutions Research Articles

Conducting polymer nanowires for control of local protein concentration in solution

Interfacing devices with cells and tissues requires new nanoscale tools that are both flexible and electrically active. We demonstrate the use of PEDOT:PSS conducting polymer nanowires for the local control of protein concentration in water and biological media. We use fluorescence microscopy to compare the localization of serum albumin in response to electric fields generated by narrow (760 nm) and wide (1.5 µm) nanowires. We show that proteins in deionized water can be manipulated over a surprisingly large micron length scale and that this distance is a function of nanowire diameter. In addition, white noise can be introduced during the electrochemical synthesis of the nanowire to induce branches into the nanowire allowing a single device to control multiple nanowires. An analysis of growth speed and current density suggests that branching is due to the Mullins–Sekerka instability, ultimately controlled by the roughness of the nanowire surface. These small, flexible, conductive, and biologically compatible PEDOT:PSS nanowires provide a new tool for the electrical control of biological systems.

ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.

The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit.

Thermodynamic properties of amyloid fibrils in equilibrium

In this manuscript we use a two-dimensional coarse-grained model to study how amyloid fibrils grow towards an equilibrium state where they coexist with proteins dissolved in a solution. Free-energies to dissociate proteins from fibrils are estimated from the residual concentration of dissolved proteins. Consistent with experiments, the concentration of proteins in solution affects the growth rate of fibrils but not their equilibrium state. Also, studies of the temperature dependence of the equilibrium state can be used to estimate thermodynamic quantities, e.g., heat capacity and entropy.

In brief

PAGE 298 A method for identifying the tau protein involved in Alzheimer's disease has been developed by researchers in France. An acoustic wave system is used to differentiate the concentration of tau protein solutions (30-480 µM) by measuring the viscoelastic properties (G’ and G”); this provides information on the different states of polymerisation of the tau protein. PAGE 351 Researchers from China have developed the first effective data imaging method using airborne strip-mode synthetic aperture ladar (SAL). It is based on subaperture phase gradient autofocus (PGA) and normalised cross correlation (NCC). SAL works at a wavelength 104–5 times shorter than synthetic aperture radar (SAR) and so can be used at a finer resolution. Acoustic wave sensor used to detect tau protein involved in Alzheimer's disease. PAGE 308 A dual-mode IO driver and termination scheme is proposed, the device can efficiently trade-off between power, swing and impedance control. The hybrid transmitter design allows for low-speed data transmission under 0.5Gb/s and high-speed transmission up to 3.4 Gb/s. Korean researchers fabricated a prototype in 45nm CMOS process with a total chip area of 0.022 mm2. First effective airborne synthetic aperture ladar (SAL) data imaging method. PAGE 314 A kinaesthetically guided approach to tele-operation of anthropomorphic robot hands is presented in research from India. The computationally lean grasp generation procedure seeks to represent individual grasp poses in terms of an ordered set of contact points on a virtual sphere imagined within the grasp enclosure. Efficient dual-mode IO driver and termination scheme. PAGE 353 A reconfigurable phase converter that allows frequency-switching has been fabricated by researchers in China. The length of the slot in the ground plane can be altered using PIN-diodes, which permits switching at 0.9GHz and 1.6GHz. The simple structure allows for ordinary microwave integrated circuit fabrication. The approach circumvents the dimensional complexities of the hand kinesiology Switchable frequency (0.9GHz and 1.6GHz) phase inverter.

Intermediate scattering function for macromolecules in solutions probed by neutron spin echo.

The neutron-spin-echo method (NSE) is a powerful technique for studying internal dynamics of macromolecules in solutions because it can simultaneously probe length and time scales comparable to intramolecular density fluctuations of macromolecules. Recently, there has been increased, strong interest in studying protein internal motions using NSE. The coherent intermediate scattering function (ISF) measured by NSE depends on internal, rotational, and translational motions of macromolecules in solutions. It is thus critical, but highly nontrivial, to separate the internal motion from other motions in order to properly understand protein internal dynamics. Even though many experiments are performed at relatively high concentrations, current theories of calculating the ISF of concentrated protein solutions are either inaccurate or flawed by incorrect assumptions for realistic protein systems with anisotropic shapes. Here, a theoretical framework is developed to establish the quantitative relationship of different motions included in the ISF. This theory based on the dynamic decoupling approximation is applicable to a wide range of protein concentrations, including dilute cases. It is also, in general, useful for studying many other types of macromolecule systems studied by NSE.

Transmembrane Difference in Colloid Osmotic Pressure Affects the Lipid Membrane Fluidity of Liposomes Encapsulating a Concentrated Protein Solution.

A hemoglobin vesicle (Hb-V) is an artificial oxygen carrier encapsulating a highly concentrated hemoglobin solution (40 g/dL) in a liposome. The in vivo safety and efficacy of Hb-V suspension as a transfusion alternative and structural stability during storage have been studied extensively. Because the intraliposomal Hb aqueous solution can possess colloid osmotic pressure (COP, 200-300 Torr) that is much higher than that of blood plasma (20-25 Torr), a question arises as to whether the lipid membrane senses the transmembrane difference in COP. We examined the membrane microviscosity using a fluorescence polarization technique. To avoid the interference of red Hb on the fluorescence measurement, we used human serum albumin (HSA) as a substitute for Hb. Both HSA and Hb solutions show high COP depending on the concentration. Encapsulation of HSA solution (40 g/dL) in the liposome decreased the membrane microviscosity at a lower temperature (949 ± 8 cP → 607 ± 10 cP at 25 °C). The result indicates that the transmembrane osmotic stress induced by HSA encapsulation expands the liposome maximally with increasing spherical surface area, and the membrane fluidity is increased extremely. Even for such a condition, the lowest membrane microviscosity, 377 ± 10 cP at 60 °C, is much higher than that of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposome (40 ± 2 cP at 60 °C). Accordingly, Hb-V as well as HSA-V maintains a spherical structure and mechanical stability under transmembrane stress caused by high COP, as described in the literature.

Viscosity-Reducing Bulky-Salt Excipients Prevent Gelation of Protein, but Not Carbohydrate, Solutions.

The problem of gelation of concentrated protein solutions, which poses challenges for both downstream protein processing and liquid formulations of pharmaceutical proteins, is addressed herein by employing previously discovered viscosity-lowering bulky salts. Procainamide-HCl and the salt of camphor-10-sulfonic acid with L-arginine (CSA-Arg) greatly retard gelation upon heating and subsequent cooling of the model proteins gelatin and casein in water: Whereas in the absence of additives the proteins form aqueous gels within several hours at room temperature, procainamide-HCl for both proteins and also CSA-Arg for casein prevent gel formation for months under the same conditions. The inhibition of gelation by CSA-Arg stems exclusively from the CSA moiety: CSA-Na was as effective as CSA-Arg, while Arg-HCl was marginally or not effective. The tested bulky salts did not inhibit (and indeed accelerated) temperature-induced gel formation in aqueous solutions of all examined carbohydrates-starch, agarose, alginate, gellan gum, and carrageenan.

Role of Molecular Flexibility and Colloidal Descriptions of Proteins in Crowded Environments from Small-Angle Scattering.

Small-angle scattering is a powerful technique to study molecular conformation and interactions of proteins in solution and in amorphous solids. We have investigated the role of multiple protein configurations in the interaction parameters derived from small-angle scattering for proteins in concentrated solutions. In order to account for the wide configurational space sampled by proteins, we generate ensembles of atomistic structures for lysozyme and monoclonal antibodies, representing globular and flexible proteins, respectively. While recent work has argued that a colloidal approach is inadequate to model proteins, because of the large configurational space that they sample in solution, we find a range of length scales where colloidal models can be used to describe solution scattering data while simultaneously accounting for structural flexibility. We provide insights to determine the length scales where isotropic colloidal models can be used, and find smoothly varying sets of interaction parameters that encompass ensembles of structures. This approach may play an important role in the definition of long-range interactions in coarse-grained models of flexible proteins with experimental scattering constraints. Additionally, we apply the decoupling approximation to ensembles of lysozyme structures with atomistic detail and observe remarkably different results when using geometric solids, such as ellipsoids. The insights from this study provide guidelines for the analysis of small-angle scattering profiles of proteins in crowded environments.

Investigation of single-pass tangential flow filtration (SPTFF) as an inline concentration step for cell culture harvest

This work examined the use of single-pass tangential flow filtration (SPTFF) to concentrate cell culture harvest. Conventional tangential flow filtration (TFF) is routinely used in biotechnology and related industries to concentrate protein solutions and exchange them into the final formulation buffer. However, several pump passes and a recirculation skid are required to achieve the target concentration in conventional TFF. Thus, it is impractical to implement TFF for inline concentration of protein solutions during the manufacture of biologic products. New developments in the biotechnology industry have focused on using ultrafiltration in a single pump pass (also known as SPTFF) to concentrate protein solutions in a single pump pass as an inline concentration step. There is thus a potential utility for this technology to overcome manufacturing bottlenecks. While there is limited work in literature describing the use of SPTFF for concentration of in-process protein pools, no work has been reported on the use of SPTFF for concentration of cell culture harvest and the challenges faced in this situation. This work examines the utility of using a 30kDa ultrafiltration membrane in the SPTFF mode to concentrate cell culture harvest for six different biologic assets in order to determine its feasibility over a wide range of biologic cell culture harvest streams. Cell culture harvest provided unique challenges to SPTFF that were overcome using a synthetic adsorptive hybrid filter technology. In particular, the hydraulic conditions necessary to achieve a targeted concentration factor varied from lot-to-lot for the same molecule. This caused difficulty in implementing a given set of operating conditions for the same molecule because of process instability. To address these challenges, we used high capacity adsorptive filters that effectively reduced both particulates and soluble contaminants during primary recovery to improve the SPTFF performance. The adsorptive filter mainly removed process related impurities, specifically HCP and DNA that prematurely fouled the ultrafiltration membrane and changed the capacity of SPTFF. The adsorptive filter also eradicated cell-culture lot-to-lot performance variability with SPTFF by offering a relative standard deviation of <10%. The use of SPTFF by simultaneously coupling the clarification with the concentration was successfully demonstrated. This is the first work to report the use of SPTFF to concentrate cell culture harvest for six different biologic assets, overcoming challenges using adsorptive filtration, and demonstrate debottlenecking for manufacturing.

Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions

In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation.

A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles.

The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

Assessment for quantification of biopharmaceutical protein using a microvolume spectrometer on microfluidic slides

Spectrophotometric procedure is a traditional method for quantifying protein concentration in solution. The benefit of this method is simple and direct, not requiring standard materials, sample incubation, and exogenous chromophore addition. There are also some drawbacks, low detection limits and high amounts of protein needed. The recent advances in fiber optics and nano-technology enable to overcome those drawbacks by using microvolume sampling and extremely short pathlengths. In this study, the utility of the microvolume system with microfluidic carriers was assessed for concentration measurement of biopharmaceutical protein using absorbance at 280 nm and also compared to a traditional instrument. The results show a high correlation of estimation by Cohen’s d effect size and Pearson correlation coefficient between two different spectrometers. For UV spectral analysis, there are no statistically significant difference between those spectra from a microvolume spectrometer and a traditional one. For the extinction coefficient determination, two different spectrometers also give quite comparable and similar values. Thus a microvolume spectrometer can be used as an alternative over a traditional one to determine the concentration of biopharmaceutical protein. Moreover, the dynamic range for protein quantification by a microvolume spectrometer is over 10-fold higher than that of a traditional one.

Optimization of foaming properties of sludge protein solution by 60Co γ-ray/H2O2 using response surface methodology

Response surface methodology and Box-Behnken experimental design were used to model and optimize the operational parameters of foaming properties of the sludge protein solution by 60Co γ-ray/H2O2 treatment. The four variables involved in this research were the protein solution concentration, H2O2, pH and dose. In the range studied, statistical analysis of the results showed that selected variables had a significant effect on protein foaming properties. The optimized conditions contained: protein solution concentration 26.50% (v/v), H2O2 concentration 0.30% (v/v), pH value 9.0, and dose 4.81kGy. Under optimal conditions, the foamability and foam stability approached 23.3cm and 21.3cm, respectively. Regression analysis with R2 value of 0.9923 (foamability) and 0.9922 (foam stability) indicated a satisfactory correlation between the experimental data and predicted values (response). In addition, based on a feasibility analysis, the 60Co γ-ray/H2O2 method can improve odor and color of the protein foaming solution.

Prediction of Protein Aggregation in High Concentration Protein Solutions Utilizing Protein-Protein Interactions Determined by Low Volume Static Light Scattering

The development of highly concentrated protein formulations is more demanding than for conventional concentrations due to an elevated protein aggregation tendency. Predictive protein-protein interaction parameters, such as the second virial coefficient B22 or the interaction parameter kD, have already been used to predict aggregation tendency and optimize protein formulations. However, these parameters can only be determined in diluted solutions, up to 20 mg/mL. And their validity at high concentrations is currently controversially discussed. This work presents a μ-scale screening approach which has been adapted to early industrial project needs. The procedure is based on static light scattering to directly determine protein-protein interactions at concentrations up to 100 mg/mL. Three different therapeutic molecules were formulated, varying in pH, salt content, and addition of excipients (e.g., sugars, amino acids, polysorbates, or other macromolecules). Validity of the predicted aggregation tendency was confirmed by stability data of selected formulations. Based on the results obtained, the new prediction method is a promising screening tool for fast and easy formulation development of highly concentrated protein solutions, consuming only microliter of sample volumes.

Scalable Fabrication of Electrospun Nanofibrous Membranes Functionalized with Citric Acid for High-Performance Protein Adsorption

Fabricating protein adsorbents with high adsorption capacity and appreciable throughput is extremely important and highly desired for the separation and purification of protein products in the biomedical and pharmaceutical industries, yet still remains a great challenge. Herein, we demonstrate the synthesis of a novel protein adsorbent by in situ functionalizing eletrospun ethylene-vinyl alcohol (EVOH) nanofibrous membranes (NFM) with critic acid (CCA). Taking advantage of the merits of large specific surface area, highly tortuous open-porous structure, abundant active carboxyl groups introduced by CCA, superior chemical stability, and robust mechanical strength, the obtained CCA-grafted EVOH NFM (EVOH-CCA NFM) present an excellent integrated protein (take lysozyme as the model protein) adsorption performance with a high capacity of 284 mg g(-1), short equilibrium time of 6 h, ease of elution, and good reusability. Meanwhile, the adsorption performance of EVOH-CCA NFM can be optimized by regulating buffer pH, ionic strength, and initial concentration of protein solutions. More importantly, a dynamic binding efficiency of 250 mg g(-1) can be achieved driven solely by the gravity of protein solution, which matches well with the demands of the high yield and energy conservation in the actual protein purification process. Furthermore, the resultant EVOH-CCA NFM also possess unique selectivity for positively charged proteins which was confirmed by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Significantly, the successful synthesis of such intriguing and economic EVOH-CCA NFM may provide a promising candidate for the next generation of protein adsorbents for rapid, massive, and cost-effective separation and purification of proteins.

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

ToF-SIMS and XPS Characterization of Protein Films Adsorbed onto Bare and Sodium Styrenesulfonate-Grafted Gold Substrates.

The adsorption of single-component bovine serum albumin (BSA), bovine fibrinogen (Fgn), and bovine immunoglobulin G (IgG) films as well as multicomponent bovine plasma films onto bare and sodium styrenesulfonate (NaSS)-grafted gold substrates was characterized. The adsorption isotherms, measured via X-ray photoelectron spectroscopy, showed that at low solution concentrations all three single-component proteins adsorb with higher affinity onto gold surfaces compared to NaSS surfaces. However, at higher concentrations, NaSS surfaces adsorb the same or more total protein than gold surfaces. This may be because proteins that adsorb onto NaSS undergo structural rearrangements, resulting in a larger fraction of irreversibly adsorbed species over time. Still, with the possible exception of BSA adsorbed onto gold, neither surface appeared to have saturated at the highest protein solution concentration studied. Principal component (PC) analysis of amino acid mass fragments from time-of-flight secondary ion mass spectra distinguished between the same protein adsorbed onto NaSS and gold surfaces, suggesting that proteins adsorb differently on NaSS and gold surfaces. Explored further using peak ratios for buried/surface amino acids for each protein, we found that proteins denature more on NaSS surfaces than on gold surfaces. Also, using peak ratios for asymmetrically distributed amino acids, potential structural differences were postulated for BSA and IgG adsorbed onto NaSS and gold surfaces. PC modeling, used to track changes in plasma adsorption with time, suggests that plasma films on NaSS and Au surfaces become more Fgn-like with increasing adsorption time. However, the PC models included only three proteins, where plasma is composed of hundreds of proteins. Therefore, while both gold and NaSS appear to adsorb more Fgn with time, further study is required to confirm that this is representative of the final state of the plasma films.

Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it.

Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.

Studying solutions at high shear rates: a dedicated microfluidics setup

The development of a dedicated small-angle X-ray scattering setup for the investigation of complex fluids at different controlled shear conditions is reported. The setup utilizes a microfluidics chip with a narrowing channel. As a consequence, a shear gradient is generated within the channel and the effect of shear rate on structure and interactions is mapped spatially. In a first experiment small-angle X-ray scattering is utilized to investigate highly concentrated protein solutions up to a shear rate of 300000 s(-1). These data demonstrate that equilibrium clusters of lysozyme are destabilized at high shear rates.

Electrokinetic Properties of Lubricin Antiadhesive Coatings in Microfluidic Systems

Lubricin is a glycoprotein found in articular joints which has long been recognized as being an important biological boundary lubricant molecule and, more recently, an impressive antiadhesive that readily self-assembles into a well ordered, polymer brush layer on virtually any substrate. The lubricin molecule possesses an overabundance of anionic charge, a property that is atypical among antiadhesive molecules, that enables its use as a coating for applications involving electrokinetic processes such as electrophoresis and electroosmosis. Coating the surfaces of silica and polymeric microfluidic devices with self-assembled lubricin coatings affords a unique combination of excellent fouling resistance and high charge density that enables notoriously "sticky" biomolecules such as proteins to be used and controlled electrokinetically in the device without complications arising from nonspecific adsorption. Using capillary electrophoresis, we characterized the stability, uniformity, and electrokinetic properties of lubricin coatings applied to silica and PTFE capillaries over a range of run buffer pHs and when exposed to concentrated solutions of protein. In addition, we demonstrate the effectiveness of lubricin as a coating to minimize nonspecific protein adsorption in an electrokinetically controlled polydimethylsiloxane/silica microfluidic device.