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In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments. . [Display omitted]. [ABSTRACT FROM AUTHOR]

In highly sensitive bioanalytical systems intended for the detection of protein biomarkers at low and ultra-low concentrations, the efficiency of capturing target biomolecules from the volume of the analyzed sample onto the sensitive surface of the detection system is a crucial factor. Herein, the application of excess electric charge for the enhancement of transport of target biomolecules towards the sensitive surface of a detection system is considered. In our experiments, we demonstrate that an uncompensated electric charge is induced in droplets of protein-free water owing to the separation of charge in a part of the Kelvin dropper either with or without the use of an external electric field. The distribution of an excess electric charge within a protein-free water droplet is calculated. It is proposed that the efficiency of protein capturing onto the sensitive surface correlates with the sign and the amount of charge induced per every single protein biomolecule. The effect described herein can allow one to make the protein capturing controllable, enhancing the protein capturing in the desired (though small) sensitive area of a detector. This can be very useful in novel systems intended for highly sensitive detection of proteins at ultra-low (≤10−15 M) concentrations. [ABSTRACT FROM AUTHOR]

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Fr€omter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, smallangle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation. [ABSTRACT FROM AUTHOR]

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities. [ABSTRACT FROM AUTHOR]

Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab’)2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.

A robust and applicable strategy of signal amplification for field effect transistor (FET) biosensor is of scientifically and technologically importance to detect wide range of target molecules, particularly for the molecules of low charge density, and to configure highly sensitive analysis. In this communication, we demonstrate a simple technique to modify the net charge of target protein based on the concept of protein conjugation for the detection of prion, which is infectious and transmissible protein in neurodegenerative disorders. The increases of prion net charge after chemical modification generate signal amplification and have remarkably enhanced the sensitivity of FET biosensor below than femtomolar (fM) level. Thus, we suggest that such strategy is promising technique for achieving a highly sensitive detection at very low concentration that is useful for early determination of protein biomarkers. [ABSTRACT FROM AUTHOR]

As interest in high-concentration protein formulations has increased, it has become apparent that routine, accurate protein charge measurements are necessary. There are several techniques for charge measurement, and a comparison of the methods is needed. The electrophoretic mobility, effective charge, and Debye- Hückel- Henry charge have been determined for bovine serum albumin, and human serum albumin. Three different electrophoretic methods were used to measure the electrophoretic mobility: capillary electrophoresis, electrophoretic light scattering, and membrane confined electrophoresis. In addition, the effective charge was measured directly using steady-state electrophoresis. Measurements made at different Na Cl concentrations, p H, and temperatures allow comparison with previous charge estimates based on electrophoresis, Donnan equilibrium, and pH titration. Similar charge estimates are obtained by all of the methods. The strengths and limitations of each technique are discussed, as are some general considerations about protein charge and charge determination. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2123-2131, 2015 [ABSTRACT FROM AUTHOR]

Purpose: To understand the apparent inconsistency between the dilute and high concentration viscosity behavior of bovine serum albumin (BSA). Method: Zeta potential and molecular charge on BSA were determined from Electrophoretic mobility measurements. Second virial coefficient ( B) and interaction parameter ( k) obtained from static and dynamic light scattering, respectively, quantified intermolecular interactions. Rheology studies characterized viscoelasticity at high concentration. The dipole moment was calculated using Takashima's approximation for proton fluctuations over charged residues. Results: The effective isoelectric point of BSA was pH 4.95. In dilute solutions (≤ 40 mg/ml), the viscosity was minimal at the pI; at high concentrations, pH 5.0 solutions were most viscous. B and k showed intermolecular attractions at pH 5.0; repulsions dominated at other pHs. The attractive interactions led to a high storage modulus (G′) at pH 5.0. Conclusion: In dilute solutions, the electroviscous effect due to net charge governs the viscosity behavior; at high concentrations, the solution viscosity cannot be justified based on a single parameter. The net interplay of all intermolecular forces dictates viscosity behavior, wherein intermolecular attraction leads to a higher solution viscosity. [ABSTRACT FROM AUTHOR]

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein-ion binding. Data for the pH offsets of an Fc-fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc-fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

The purpose of this work was to investigate the self-associating behavior observed in high concentration monoclonal antibody (MAb1) solutions at pH 6.0. Zeta potential measurements over the pH range (4.0–9.0) showed lower net charges present on the molecule at pH 6.0 and 7.0. The point of zero charge or crossover from positive to negative potential was at pH 6.7 and was different from the theoretical isoelectric point (pI) of 7.8. Interaction parameter (kD) from dynamic light scattering (DLS) studies indicated that attractive interactions of a similar magnitude span over a pH range of 5.0–8.0. Change in kD values with increase in ionic strength corroborated that the interactions were electrostatic in origin. Comparative rheology analysis of three different monoclonal antibodies at pH 6.0, using ultrasonic shear rheometer, showed a high solution storage modulus (G′) and its sharp increase in magnitude at high concentrations in MAb1. The shape of the rheology profile was indicative of strong attractive interactions between solute molecules. High specific attractive interactions may be attributed to charge–charge and charge–dipole interactions at the surface in the Fab regions of MAb1, and gives a reasonable explanation for the pH dependency of the rheological profile observed. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1152–1168, 2010 [ABSTRACT FROM AUTHOR]

Abstract: Understanding of electrophoretic mobility and zeta potential is an important issue in charge characteristics of proteins for proteomics research. By exploring the electrostatic and electrokinetic principles, we presented a framework that would allow rigorous interpretations of surface zeta potential and effective charge for highly charged conditions holding the full range of buffer pH. Together with an analytic expression by considering the Henry''s formula with Debye–Hückel ansatz, the zeta potential at the protein surface was evaluated from an integral expression for the electrophoretic mobility of a spherical particle on the basis of nonlinear Poisson–Boltzmann equation. Subsequently, the effective protein charge was evaluated from the corresponding relationships between potential distribution and surface charge, with accounting for the presence of an electric double layer. Applying the microchip electrophoresis, the experimental results of mobility data were obtained for model proteins with bovine serum albumin and ovalbumin in different pH of buffer solution. To illustrate the usefulness of our considerations, experimental data of electrophoretic mobility available in the literature are also included. It is evident that the results by linear correlations are identified to overestimate the zeta potential, while they underestimate the effective charge. The discrepancy between linear and nonlinear correlations is trivial for the zeta potential less than 25.69mV, however, it fairly increases with increasing the absolute value of zeta potential. [Copyright &y& Elsevier]

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders that may occur in plasma cell dyscrasias with dysproteinemia. In systemic AL, the deposits are fibrillar and patchy in distribution, within and amongst different organs, whereas in NALCDD, the deposits are granular and diffusely distributed in systemic basement membranes, suggesting different mechanisms of aggregation and deposition. Previous evidence, that charge differences between the light chains in AL and NALCDD might account for their different phenotypes, prompted the present study, which compared the isoelectric points (pIs) of AL and NALCDD protein deposits extracted from human tissues. The pI profiles (5·2–8·8) of polypeptides in AL deposits were heterogenous in four cases, with a spread of both anionic and cationic isoforms; in contrast, in three of NALCDD the pI profiles (8·2–8·8) were homogeneous and restricted in the cationic range. These in vivo findings in human disease, together with other reported in vitro and in vivo experimental data, suggest that the fibrillar deposits in AL may form by electrostatic interaction between oppositely charged polypeptides, whereas the granular deposits in NALCDD form by the binding of cationic polypeptides to anionic proteoglycans sites in basement membranes. [ABSTRACT FROM AUTHOR]

Abstract: We derive a simple formula for the free solution electrophoretic mobility of protein by including both molecular shape and charge distribution effects. The molecular shape of protein is described by a deformed sphere model, while the charge distribution is represented in terms of net charge, charge dipole, and charge quadrupole. The deformed sphere model approximates the radial coordinate of the protein surface as a simple quadratic equation based on the atomic coordinate data. Charge dipole does not affect the mobility of protein. Combined with the quadratic coefficients of the surface equation, charge quadrupole affects the mobility. When the charge quadrupole contribution is negligible, the mobility equation simplifies to the Henry equation in which the sphere radius is replaced with the hydrodynamic radius of protein. The deformed sphere model predicts correctly the hydrodynamic radius of protein from the atomic coordinate data. The hydrodynamic radius is not the radius of sphere of equal volume but the effective radius that correlates with the translational diffusivity of protein. To illustrate the utility of our mobility equation we study the electrophoresis of lysozyme and compare our results with previously published works. [Copyright &y& Elsevier]

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a “charge ladder” approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein. [Copyright &y& Elsevier]

The electrophoretic mobility of a macro-ion is affected in a complex manner by a variety of forces that arise from the applied field. Coupling of the macro-ion and small-ion flows gives rise to non-conserved forces that are greater than those expected from ordinary hydrodynamic considerations. It is difficult to separate the steady-state hydrodynamic and electrodynamic contributions to the macro-ion mobility. Membrane-confined electrophoresis (MCE), a free solution technique, provides an experimental means by which to gain insight into these contributions. In this work we used MCE steady-state electrophoresis (SSE) of a series of T4 lysozyme charge mutants to investigate these effects and to examine the existing theoretical descriptions. These experiments isolate the effects of charge on electrophoretic mobility and permit a unique test of theories by Debye–Hu¨ckel–Henry, Booth and Allison. Our results show that for wild type (WT) T4, where divergence is expected to be greatest, the predicted results are within 15, 8 and 1%, respectively, of experimental SSE results. Parallel experiments using another free-solution technique, capillary electrophoresis, were in good agreement with MCE results. The theoretical predictions were within 20, 13 and 5% of CE mobilities for WT. Boundary element modeling by Allison and co-workers, using continuum hydrodynamics based on detailed structural information, provides predictions in excellent agreement with experimental results at ionic strengths of 0.11 M. [Copyright &y& Elsevier]

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein interactions, using a Debye­Hückel-based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface-charge/E° relationships. This approach shows that a number of anions, most of which are of biological relevance, namely Cl¯, HPO42-, HCO3¯, NO42-, SO42-, ClO4¯, citrate3- and oxalate2-, bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities. The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence-related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. continue [ABSTRACT FROM AUTHOR]

Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab')2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)'2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab')2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab')2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided. [ABSTRACT FROM AUTHOR]

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)