Concentrated Protein Solutions Research Articles

Selective-Integrative Technology for the Separation of Colostrum Into Components and the Possibilities of Obtaining Protein Substances From Different Sources

Background. Obtaining biologically active natural compounds involved in the regulation of metabolism is an important goal in biotechnology. Colostrum is a unique natural source of various biologically active compounds. However, the extremely high natural variability of colostrum composition does not meet the existing requirements for standardization in pharmaceutical preparations. Objective. To develop a method for separating colostrum into its basic components (lipids, casein, and protein fractions), thereby reducing the variability of whole colostrum composition, obtaining several target products, and demonstrating the possibility of acquiring new protein substances from different sources. Methods. Colostrum separation was carried out through centrifugation and membrane filtration. Plant proteins (sunflower) and milk proteins were used to obtain protein substances from different sources. The composition of proteins, carbohydrates, and nucleic acids was determined using mass spectrometry, centrifugation, and membrane filtration. Results. The proposed method for obtaining basic substances from colostrum significantly reduced the vari­ability in composition compared to whole colostrum. The efficiency of protein sedimentation in concentrated protein solutions by centrifugation and ultrafiltration was shown to depend on protein concentration. Additionally, the formation of non-specific protein aggregates in the centrifugal field allowed the extraction of protein substances from various natural sources, which is relevant for functional nutrition. Conclusions. The proposed selective-integrative technology for obtaining different substances from colostrum significantly reduces the high variability of whole colostrum composition. It increases the efficiency of component separation into lipid, casein fractions, low molecular weight protein fractions, and ultrafiltrate, while also enabling the acquisition of protein substances from diverse sources.

Diffracting crystals of an intrinsically disordered protein (IDP) AtPP16-1 grown in 1.3 V cm−1 DC field

DC electric field as weak as ∼1.3 V cm−1 induces crystal nucleation in very dilute protein solutions lacking precipitant. The basis of such growth is the microscopic model of interaction of protein dipoles with the Stark field, leading to glass-like amorphous aggregation and reconfiguration of the aggregates for crystal nucleation. This modest approach is very different from an earlier and rather ‘aggressive’ one in which electric field of ∼1 kV or orders of magnitude in excess is used to influence charge migration in a highly concentrated protein solution having precipitant confined to the crystallization drop. As an application of the precipitant-lacking ultralow protein method, the present work seeks the assistance of internally supplied 1.3 V cm−1 DC field to crystallize an intrinsically disordered protein (IDP) called AtPP16-1 in a 0.017 mg mL−1 solution. Crystallization is allowed in cuvette cells of spectrometers with online electric field, enabling measurement of real time changes in spectral features. The average crystal size increases with the time of passage of the electric field, from ∼0.042 at 10 min to 0.165 µm at the end of 300 min. The cubic crystals diffract electron and X-ray. Electron diffraction spot indexing yields lattice spacing dhkl ∼ 2.85 Å, consistent with 2.88 Å found from powder X-ray diffraction analysis. This level of lattice spacing will correspond to moderately resolved crystal structure of the IDP.

Modeling the Process of Ultrafiltration to Concentrate Protein Solutions by Neural Network Coding

Modeling the Process of Ultrafiltration to Concentrate Protein Solutions by Neural Network Coding

Flow Activation Energy of High-Concentration Monoclonal Antibody Solutions and Protein-Protein Interactions Influenced by NaCl and Sucrose.

The solution viscosity and protein-protein interactions (PPIs) as a function of temperature (4-40 °C) were measured at a series of protein concentrations for a monoclonal antibody (mAb) with different formulation conditions, which include NaCl and sucrose. The flow activation energy (Eη) was extracted from the temperature dependence of solution viscosity using the Arrhenius equation. PPIs were quantified via the protein diffusion interaction parameter (kD) measured by dynamic light scattering, together with the osmotic second virial coefficient and the structure factor obtained through small-angle X-ray scattering. Both viscosity and PPIs were found to vary with the formulation conditions. Adding NaCl introduces an attractive interaction but leads to a significant reduction in the viscosity. However, adding sucrose enhances an overall repulsive effect and leads to a slight decrease in viscosity. Thus, the averaged (attractive or repulsive) PPI information is not a good indicator of viscosity at high protein concentrations for the mAb studied here. Instead, a correlation based on the temperature dependence of viscosity (i.e., Eη) and the temperature sensitivity in PPIs was observed for this specific mAb. When kD is more sensitive to the temperature variation, it corresponds to a larger value of Eη and thus a higher viscosity in concentrated protein solutions. When kD is less sensitive to temperature change, it corresponds to a smaller value of Eη and thus a lower viscosity at high protein concentrations. Rather than the absolute value of PPIs at a given temperature, our results show that the temperature sensitivity of PPIs may be a more useful metric for predicting issues with high viscosity of concentrated solutions. In addition, we also demonstrate that caution is required in choosing a proper protein concentration range to extract kD. In some excipient conditions studied here, the appropriate protein concentration range needs to be less than 4 mg/mL, remarkably lower than the typical concentration range used in the literature.

Stability of Protein Pharmaceuticals: Recent Advances.

There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.'

Testing mixing rules for structural and dynamical quantities in multi-component crowded protein solutions.

Crowding effects significantly influence the phase behavior and the structural and dynamic properties of the concentrated protein mixtures present in the cytoplasm of cells or in the blood serum. This poses enormous difficulties for our theoretical understanding and our ability to predict the behavior of these systems. While the use of course grained colloid-inspired models allows us to reproduce the key physical solution properties of concentrated monodisperse solutions of individual proteins, we lack corresponding theories for complex polydisperse mixtures. Here, we test the applicability of simple mixing rules in order to predict solution properties of protein mixtures. We use binary mixtures of the well-characterized bovine eye lens proteins α and γB crystallin as model systems. Combining microrheology with static and dynamic scattering techniques and observations of the phase diagram for liquid-liquid phase separation, we show that reasonably accurate descriptions are possible for macroscopic and mesoscopic signatures, while information on the length scale of the individual protein size requires more information on cross-component interaction.

Protein Adsorption on Solid Surfaces: Data Mining, Database, Molecular Surface-Derived Properties, and Semiempirical Relationships.

Protein adsorption on solid surfaces is a process relevant to biological, medical, industrial, and environmental applications. Despite this wide interest and advancement in measurement techniques, the complexity of protein adsorption has frustrated its accurate prediction. To address this challenge, here, data regarding protein adsorption reported in the last four decades was collected, checked for completeness and correctness, organized, and archived in an upgraded, freely accessible Biomolecular Adsorption Database, which is equivalent to a large-scale, ad hoc, crowd-sourced multifactorial experiment. The shape and physicochemical properties of the proteins present in the database were quantified on their molecular surfaces using an in-house program (ProMS) operating as an add-on to the PyMol software. Machine learning-based analysis indicated that protein adsorption on hydrophobic and hydrophilic surfaces is modulated by different sets of operational, structural, and molecular surface-based physicochemical parameters. Separately, the adsorption data regarding four "benchmark" proteins, i.e., lysozyme, albumin, IgG, and fibrinogen, was processed by piecewise linear regression with the protein monolayer acting as breakpoint, using the linearization of the Langmuir isotherm formalism, resulting in semiempirical relationships predicting protein adsorption. These relationships, derived separately for hydrophilic and hydrophobic surfaces, described well the protein concentration on the surface as a function of the protein concentration in solution, adsorbing surface contact angle, ionic strength, pH, and temperature of the carrying fluid, and the difference between pH and the isoelectric point of the protein. When applying the semiempirical relationships derived for benchmark proteins to two other "test" proteins with known PDB structure, i.e., β-lactoglobulin and α-lactalbumin, the errors of this extrapolation were found to be in a linear relationship with the dissimilarity between the benchmark and the test proteins. The work presented here can be used for the estimation of operational parameters modulating protein adsorption for various applications such as diagnostic devices, pharmaceuticals, biomaterials, or the food industry.

Competitive sorption of low and high molecular weight proteins in model mixtures by Q HyperZ chromatographic medium. Part II: Mass transfer mechanisms

The kinetic uptake of Bovine Serum Albumin (BSA) and ferritin in mixtures by the composite anion exchanger Q HyperZ was studied. Transient adsorption experiments were performed to investigate the dominant resistance between external and internal mass transfer in simultaneous and sequential adsorption experiments. From the evolution of protein solution concentration with time, the external mass transfer coefficients determined were very low in simultaneous (7.1 10−7 and 1.1 10−7 m/s for BSA and ferritin respectively) and sequential (4.6 10−7 and 2.5 10−7 m/s for BSA and ferritin respectively) adsorption systems which is in agreement with the low agitation speed used. From the transient protein uptakes, the internal mass transfer was studied and the homogenous diffusion model applied provides a good predictability in single as well as in simultaneous and sequential adsorption experiments. The effective diffusion coefficients were determined in sequential (6.4 10−14 m2/s and 3.8 10−14 m2/s for BSA and ferritin respectively) and simultaneous (4.2 10−14 m2/s for both proteins) systems. In multi-component adsorption system, it was found that BSA was slowed down upon the presence of ferritin on mixture solutions contrarily to ferritin protein. This phenomenon, known as electrostatic coupling effect, indicates that diffusion of charged proteins in ion exchangers are not independent.

Inverted Transflection Spectroscopy of Live Cells Using Metallic Grating on Elevated Nanopillars.

Water absorption of mid-infrared (MIR) radiation severely limits the options for vibrational spectroscopy of the analytes-including live biological cells-that must be probed in aqueous environments. While internal reflection elements, such as attenuated total reflection prisms and metasurfaces, partially overcome this limitation, such devices have their own limitations: ATR prisms are difficult to integrate with multiwell cell culture workflows, while metasurfaces suffer from a limited spectral range and small penetration depth into analytes. In this work, we introduce an alternative live cell biosensing platform based on metallic nanogratings fabricated on top of elevated dielectric pillars. For the MIR wavelengths that are significantly longer than the grating period, reflection-based spectroscopy enables broadband sensing of the analytes inside the trenches separating the dielectric pillars. Because the depth of the analyte twice-traversed by the MIR light excludes the highly absorbing thick water layer above the grating, we refer to the technique as inverted transflection spectroscopy (ITS). The analytic power of ITS is established by measuring a wide range of protein concentrations in solution, with the limit of detection in the single-digit mg mL-1. The ability of ITS to interrogate live cells that naturally wrap themselves around the grating is used to characterize their adhesion kinetic.

Mass spectrometric analysis of plant protein concentrates

The work is devoted to the study of the composition of protein concentrates from amaranth grains (Amaranthus hypochondriacus L.), Voronezh variety. Amaranth protein concentrates were obtained by the alkaline extraction of proteins, the neutralization of the solution, followed by ultrafiltration; by separating the starch fraction with amylolytic enzymes; alkaline extraction of proteins and their precipitation at pH 4.5. The conditions for protein extraction were selected, followed by gas chromatography-mass spectrometric analysis and their identification. It has been shown that proteins from amaranth grain were more efficiently extracted with a buffer containing urea at protein concentrations in solution of 1.7, 1.9, and 2.9 mg/cm3respectively, while the buffer with detergents was more effective for the extraction of low molecular weight proteins at protein concentrations in solution of 4.9, 2.9, and 9.0 mg/cm3 respectively. As a result of HPLC-MS/MS analysis followed by a search and identification in the UNIPROT database, it was established that the main protein of amaranth grain is 11S-globulin, which is a reserve protein of amaranth seeds. In amaranth concentrates, 14 unique proteins characteristic only for A.hipochondriacus L., as well as proteins that were not characteristic to this species, but were reliably identified. Based on the results of semi-quantitative analysis of the peptide profile of amaranth grain protein concentrates, a high frequency of occurrence of the main 11S-globulin proteins was established in all samples. The frequency of occurrence of other proteins in samples obtained by different methods differed significantly, which was associated with the peculiarities of protein isolation from amaranth grain. The obtained results can be used for the production of plant protein concentrates with a set protein composition.

Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

The dynamics of PEG‐coated nanoparticles in concentrated protein solutions up to the molecular crowding range

AbstractPolymer‐coated nanoparticles are widely studied in the context of nanomedicine and it is therefore of utmost importance to understand not only how their structure but also how their colloidal dynamics are affected by physiologically relevant conditions. A characteristic feature of the cytosol of cells is the very high concentration of proteins among other matrix components, often termed macromolecular crowding. Here, the structure and colloidal dynamics of poly(ethylene glycol) (PEG)‐coated gold nanoparticles in the presence of bovine serum albumin (BSA) concentrations ranging from 0 to 265 mg/mL are studied with X‐ray photon correlation spectroscopy. For protein–nanoparticle mixtures with high BSA concentrations, comparable to intracellular levels, a significant deviation of the apparent viscosity from expectations for pure BSA solutions is found. The findings strongly indicate that the nanoscopic viscous properties of the dense protein solutions are significantly affected by the nanoparticles. At these high concentrations, the colloidal stability of the samples depends on the molecular weight of the coating PEG–ligand, whereas at lower concentrations no differences are observed.

Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

In situ Fourier transform infrared-attenuated total reflection spectroscopy and modeling investigation of protein adsorption: Case of expanded bovine serum albumin on titanium dioxide anatase.

The purpose of this experimental and modeling research is to study the pH effect and to determine the surface coverage plus the adsorption constant (Ka) of bovine serum albumin (BSA) protein adsorbed on TiO2 anatase surface, respectively. In situ Fourier transform infrared-attenuated total reflection spectroscopy in a flow-through cell was used to study the BSA adsorption on porous TiO2 anatase films. The experiments were performed in water solution, under different pH values, at a concentration of 10-6 mol/l. Theoretically, we extended the two-state model, based on a system of coupled differential equations, by adding a desorption parameter Kd2, for unfolded state. The model was solved taking into account the adsorption (Ka), desorption (Kd1,2), transformation (Kf) coefficients, and the initial solution protein concentration (C0). The findings clearly illustrated that the solution pH drastically changed the behavior of BSA adsorption, whereas the mathematical analytical solutions allowed us to determine the native state (θ1), the unfolded state (θ2), and the full one (θ) surface coverages. Finally, a good application of the approximated model on the experimental work, expanded BSA adsorbed on TiO2 anatase at pH = 1.7, indicated a value of Ka = (408.36 ± 0.996) × 102 mol-1 l min-1.

Quartz Crystal Microbalance as a Predictive Tool for Drug-Material of Construction Interactions in Intravenous Protein Drug Administration

As a growing number of protein drug products are developed, formulation characterization is becoming important. An IgG drug product is tested at concentrations from 0.0001–0.1 mg/mL for adsorption behavior to polymer surfaces polyvinyl chloride (PVC) and polypropylene (PP) upon dilution in normal saline (NS) using quartz crystal microbalance with dissipation (QCM-D). The studies mimicked IgG antibody interaction during IV administration with polymeric surfaces within syringes, lines, and bags. Drug product was characterized with excipients, with focus on surfactant. Drug solutions were run over polymer-coated sensors to measure the adsorption behavior of the formulation with emphasis on the behavior of each of the formulation's components. Over 60 sensorgram data sets were correlated with assayed protein solution concentrations in mock NS-diluted infusions of drug product in the equivalent concentrations to QCM experiments to build a preliminary predictive model for determining fraction of drug and surfactant adsorbed and lost at the hydrophobic surface during administration. These results create a method for reliably and predictively estimating drug product adsorption behavior and protein drug dose loss on polymers at different protein drug concentrations.

Protein crystallization with DNA templates

Protein crystallization plays a significant role in three-dimensional structural analysis and protein purification. It is important to increase the crystallization efficiency, which is possible by adding heterogeneous templates in crystallization systems. DNA is biologically compatible and artificially designable polymer, which is easy to extract. In this study, single- and double-stranded DNA of precise sequences were designed and used as templates to promote protein crystallization of lysozyme and catalase. Influence of DNA, single-stranded DNA with 10, 20, 40 bases and double-stranded DNA with 10, 20, 40 base pairs, were investigated. The success rate of obtaining crystals of lysozyme and catalase in equal period was significantly improved with the addition of DNA comparing without templates added. Double-stranded DNA led to higher nucleation rate than that with single-stranded DNA. The promotion of nucleation was more obvious at low concentration of protein solution and with longer chain DNA templates. Crystal number and crystallization rate was enhanced with addition of long double-stranded DNA templates. All the results confirm that DNA is an effective polymer additive to enhance protein crystallization, especially for the application of the scarce protein crystallization.

Flow behavior of protein solutions in a lab-scale chromatographic system

A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening.

Effects of concentration by block freezing and vacuum evaporation on the physicochemical properties and digestibility of whey

ABSTRACT Whey is a source of protein of high nutritional value. In this work, the effects of block cryoconcentration assisted by centrifugation and vacuum evaporation on the physicochemical properties and in vitro digestibility of whey were determined. In vitro digestibility analysis was performed by evaluating the degrees of protein hydrolysis of the fresh, evaporated and cryoconcentrated whey protein samples from the third cycle. The results showed that the samples after the second and third cryoconcentration cycles had no significant differences in protein or lactose content. The in vitro digestibility analysis indicated that the protein obtained by cryoconcentration showed better gastrointestinal digestibility than that obtained by evaporation, with values of 68.2% and 55.4%, respectively. This study demonstrates that block cryoconcentration assisted by centrifugation is an efficient and novel technique that enables the preparation of concentrated protein solutions of high nutritional and biological value.

Aligned silver nanowires for plasmonically-enhanced fluorescence detection of photoactive proteins in wet and dry environment

We developed a method of aligning silver nanowires in a microchannel and fixing them to glass substrates via appropriate functionalization. The attachment of nanowires to the substrate is robust with no variation of their angles over minutes. Specific conjugation with photoactive proteins is observed using wide-field fluorescence imaging in real-time for highly concentrated protein solution, both in a microchannel and in a chip geometry. In the latter case we can detect the presence of the proteins in the dropcasted solution down to single proteins. The results point towards possible implementation of aligned silver nanowires as geometrically defined plasmonic fluorescence sensing platforms.

Probing Cage Relaxation in Concentrated Protein Solutions by X-Ray Photon Correlation Spectroscopy.

Diffusion of proteins on length scales of their size is crucial for understanding the machinery of living cells. X-ray photon correlation spectroscopy (XPCS) is currently the only way to access long-time collective diffusion on these length scales, but radiation damage so far limits the use in biological systems. We apply a new approach to use XPCS to measure cage relaxation in crowded α-crystallin solutions. This allows us to correct for radiation effects, obtain missing information on long time diffusion, and support the fundamental analogy between protein and colloid dynamical arrest.