High throughput detection of antibody self-interaction by bio-layer interferometry

Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.     

Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen.

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.     

Enfuvirtide effects on human erythrocytes and lymphocytes functional properties.

Enfuvirtide (T-20) is the first inhibitor of human Immunodeficiency Virus type-1 (HIV-1) entrance on a target cell approved for clinical use. Recent studies indicated that its action mechanism involves the interaction with the membrane surface, increasing the concentration in the site of action. In the present study, the in vitro interaction between enfuvirtide and blood cells of healthy human donors, namely erythrocytes and lymphocytes, and the peptide effect on plasma and lymphocyte suspensions supernatant ions were evaluated, in order to better characterize the action of this peptide. Enfuvirtide causes a decrease in the concentration of hemoglobin and in the percentages of methemoglobin and carboxyhemoglobin, together with increased values of P50, pCO2, and [HCO3-], and significant decreases of pO2 and pH, in blood plasma. The supernatants of lymphocyte suspensions derived from blood incubated with enfuvirtide presented a decrease in pH and [HCO3-]. Fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), used to assess erythrocyte and lymphocyte membrane fluidity, did not yield enfuvirtide-induced changes (an effect could be expected due to peptide partition to lipid bilayers). Erythrocytes incubated with high enfuvirtide concentrations showed a significant decrease in osmotic fragility. As for erythrocyte deformability, enfuvirtide leads to increased elongation indexes for low shear stress values, whereas for high shear stress values it has the opposite effect. Despite the observed statistically significant variations in several parameters, these enfuvirtide-induced changes are not expected to lead to any detectable biomedical outcome for enfuvirtide-treated patients.     

Solution-Mediated Phase Transformation of Haloperidol Mesylate in the Presence of Sodium Lauryl Sulfate

Forming a salt is a common way to increase the solubility of a poorly soluble compound. However, the solubility enhancement gained by salt formation may be lost due to solution-mediated phase transformation (SMPT) during dissolution. The SMPT of a salt can occur due to a supersaturated solution near the dissolving surface caused by pH or other solution conditions. In addition to changes in pH, surfactants are also known to affect SMPT. In this study, SMPT of a highly soluble salt, haloperidol mesylate, at pH 7 in the presence of a commonly used surfactant, sodium lauryl sulfate (SLS), was investigated. Dissolution experiments were performed using a flow-through dissolution apparatus with solutions containing various concentrations of SLS. Compacts of haloperidol mesylate were observed during dissolution in the flow-through apparatus using a stereomicroscope. Raman microscopy was used to characterize solids. The dissolution of haloperidol mesylate was significantly influenced by the addition of sodium lauryl sulfate. In conditions where SMPT was expected, the addition of SLS at low concentrations (0.1–0.2 mM) reduced the dissolution of haloperidol mesylate. In solutions containing concentrations of SLS above the critical micelle concentration (CMC) (10–15 mM), the dissolution of haloperidol mesylate increased compared to below the CMC. The solids recovered from solubility experiments of haloperidol mesylate indicated that haloperidol free base precipitated at all concentrations of SLS. Above 5 mM of SLS, Raman microscopy suggested a new form, perhaps the estolate salt. The addition of surfactant in solids that undergo solution-mediated phase transformation can add complexity to the dissolution profiles and conversion.     

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein-protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column synthesis and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B(22), were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.     

Correlation of second virial coefficient with solubility for proteins in salt solutions

In this work, osmotic second virial coefficients (B(22)) were determined and correlated with the measured solubilities for the proteins, α-amylase, ovalbumin, and lysozyme. The B(22) values and solubilities were determined in similar solution conditions using two salts, sodium chloride and ammonium sulfate in an acidic pH range. An overall decrease in the solubility of the proteins (salting out) was observed at high concentrations of ammonium sulfate and sodium chloride solutions. However, for α-amylase, salting-in behavior was also observed in low concentration sodium chloride solutions. In ammonium sulfate solutions, the B(22) are small and close to zero below 2.4 M. As the ammonium sulfate concentrations were further increased, B(22) values decreased for all systems studied. The effect of sodium chloride on B(22) varies with concentration, solution pH, and the type of protein studied. Theoretical models show a reasonable fit to the experimental derived data of B(22) and solubility. B(22) is also directly proportional to the logarithm of the solubility values for individual proteins in salt solutions, so the log-linear empirical models developed in this work can also be used to rapidly predict solubility and B(22) values for given protein-salt systems.     

Ultrasonic storage modulus as a novel parameter for analyzing protein-protein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements

The purpose of this work was to establish ultrasonic storage modulus (G') as a novel parameter for characterizing protein-protein interactions (PPI) in high concentration protein solutions. Using an indigenously developed ultrasonic shear rheometer, G' for 20-120 mg/ml solutions of a monoclonal antibody (IgG(2)), between pH 3.0 and 9.0 at 4 mM ionic strength, was measured at frequency of 10 MHz. Our understanding of ultrasonic rheology indicated decrease in repulsive and increase in attractive PPI with increasing solution pH. To confirm this behavior, dynamic (DLS) and static (SLS) light scattering measurements were conducted in dilute solutions. Due to technical limitations, light scattering measurements could not be conducted in concentrated solutions. Mutual-diffusion coefficient, measured by DLS, increased with IgG(2) concentration at pH 4.0 and this trend reversed as pH was increased to 9.0. Second virial coefficient, measured by SLS, decreased with increasing pH. These observations were consistent with the nature of PPI understood from G' measurements. Ultrasonic rheology, DLS, and SLS measurements were also conducted under conditions of increased ionic strength. The consistency between rheology and light scattering analysis under various solution conditions established the utility of ultrasonic G' measurements as a novel tool for analyzing PPI in high protein concentration systems.     

Role of surfactant and pH on dissolution properties of fenofibrate and glipizide—A technical note

Depending on the dose size and solubility characteristics of low solubility drugs, a meaningful and discriminatory power of dissolution rate testing can be demonstrated. Saturation solubility of fenofibrate and glipizide in different media were determined. Solubility of fenofibrate increased directly with SLS concentration. For a 54-mg fenofibrate tablet, SLS at 0.025 M level is required for a discriminative dissolution test, while for 160-mg tablet, dissolution condition and levels of SLS should be optimized; higher concentrations may be effective (ie, 0.052 M, ∼1.5%). A pH 6.8 phosphate buffer medium is appropriate for glipizide 10-mg tablet dissolution study, when formulation ingredients include excipients with surface activity (eg, HPMC).     

Screening of protein-ligand interactions by affinity chromatography

This paper examines affinity chromatography (AC) as an alternative tool for the determination of protein-ligand interactions for the particular case in which the ligand is the same protein. The methodology is less labor-intensive and more sample-efficient than traditional methods used to measure the second virial coefficient (B(22)), a parameter commonly used to evaluate protein-protein interactions. The chromatographic capacity factor (k') was studied for lysozyme and equine serum albumin for a wide range of experimental solution conditions such as crystallizing agent concentration, protein concentration and pH. Parallel experiments using AC to determine k' and static light scattering (SLS) to determine B(22) showed that the two parameters were highly correlated. Two different column volumes ( approximately 1 and approximately 0.1 mL) were tested and gave essentially the same values for k', showing the feasibility of miniaturization.     

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Conformational analysis of melittin in solution phase: vibrational circular dichroism study

The vibrational absorption and vibrational circular dichroism (VCD) spectra of melittin in D(2)O solutions at different pH values, different salt concentrations, or different 2,2,2-trifluoroethanol (TFE) concentrations are recorded in the amide I' (1850-1600 cm(-1)) region. Two models are used to simulate this peptide in different conditions, and a coupled oscillator program is used to obtain the calculated absorption and VCD spectra. This study indicates that melittin adopts a mixed structure in D(2)O solution at low pH, low salt concentration, or low TFE concentration. With an increase in pH, salt concentration, or TFE concentration, the structure changes to alpha-helix and further increases lead to aggregation. These results demonstrate the versatility of VCD in probing the conformations of peptides under different environmental perturbations.     

Oligomerization of the cytoplasmic fragment from the aspartate receptor of Escherichia coli.

We have observed that a 31-kDa cloned fragment from the Escherichia coli aspartate receptor exhibits a reversible monomer-oligomer reaction. The fragment, derived from the cytoplasmic region of the receptor (c-fragment), contains the signaling functions of the receptor. The wild-type and nine missense mutant fragments were analyzed. The latter were selected by the effect of the mutations on the signaling properties of the intact receptor, which induced either persistent smooth swimming or tumbling in bacteria [Mutoh, N., Oosawa, K., & Simon, M. I. (1986) J. Bacteriol. 167, 992-998]. In pH 7.0 buffer, the mutations caused five out of the six smooth mutant c-fragments to form oligomers, while neither the three tumble mutant nor wild-type fragments exhibited significant oligomer formation. At a lower pH (5.5), all of the fragments displayed some tendency to form oligomers. The equilibria between the monomer and the oligomers were monitored by gel permeation chromatography (GPC) which resolved two to three forms with apparent molecular weights between 110,000 and 270,000. The proportions of the different forms depended on concentration, indicating an association-dissociation reaction. Static light scattering (SLS) was used to demonstrate that the solution molecular mass of the wild-type c-fragment was 31 kDa and not 110 kDa as indicated by chromatography. One oligomer-forming c-fragment (S461L) eluted as the monomer and one other form, which was determined to be a dimer by SLS. The weight-average molecular weights, calculated from GPC data as a function of protein concentration, agreed well with the weight-average molecular weights obtained by SLS for this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-helix and beta-sheet conformations

Because poly-L-lysine (PLL) can exist in the alpha-helix or beta-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and alpha-helix or beta-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the beta-sheet conformation than for the alpha-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.     

Quantitative characterization of the concentration-dependent interaction between molecules of Dextran 70 in aqueous solution: Measurement and analysis in the context of thermodynamic and compressible sphere models

The static light scattering and sedimentation equilibrium of solutions of Dextran 70 were measured as functions of concentration up to 100 g/L in pH 7.4 phosphate-buffered saline at temperatures between 5 and 37 °C. The concentration dependence of scattering intensity and the apparent molar mass obtained from sedimentation equilibrium were found to be nearly independent of temperature over this range to within the uncertainty of measurement. Global analysis of the concentration dependence of both properties yielded a reliable estimate of the concentration-dependent thermodynamic activity coefficient, a quantitative measure of the free energy of self-interaction. The self-interaction between Dextran molecules is compared with that of a globular protein (BSA) and a highly crosslinked polymer of similar molar mass (Ficoll 70). The observed concentration dependence of the free energy of Dextran self-interaction may be quantitatively accounted for by a semi-empirical model in which the polymer molecule is represented by a compressible sphere.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : VI. THE ACTIVITY COEFFICIENTS OF THE IONS IN CERTAIN OXYHEMOGLOBIN SOLUTIONS.

1. The solvent action of a neutral salt upon a protein, oxyhemoglobin, has been found identical to the solvent action of a neutral salt upon a bi-bivalent or uni-quadrivalent compound. 2. The solubility of oxyhemoglobin in phosphate solutions of varying ionic strength has been defined by the equation: log See PDF for Equation in which µ is the ionic strength, and S ₀ is the solubility in the absence of salt. 3. The values of S ₀ have been calculated to be 12.2, 11.2, and 13.1 gm. per liter respectively at pH 6.4, 6.6, and 6.8. 4. The relatively great solubility of oxyhemoglobin in water has been ascribed to the strong affinity constants for acid and base of certain groups in oxyhemoglobin. 5. The small change in the solubility of oxyhemoglobin effected by neutral salts suggests that but few such groups are dissociated in oxyhemoglobin in the state in which it crystallizes near its isoelectric point. 6. Certain of the other properties of oxyhemoglobin, such as its low viscosity, are considered in the light of its molecular weight and its valence type.     

Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.     

Phase behavior of an intact monoclonal antibody

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.     

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

Optimizing the refolding conditions of self-assembling polypeptide nanoparticles that serve as repetitive antigen display systems

Nanoparticles show great promise as potent vaccine candidates since they are readily taken up by the antigen presenting cells of the immune system. The particle size and the density of the B cell epitopes on the surface of the particles greatly influences the strength of the humoral immune response. We have developed a novel type of nanoparticle composed of peptide building blocks (Raman et al., 2006) and have used such particles to design vaccines against malaria and SARS (Kaba et al., 2009; Pimentel et al., 2009). Here we investigate the biophysical properties and the refolding conditions of a prototype of these self-assembling polypeptide nanoparticles (SAPNs). SAPNs are formed from a peptide containing a pentameric and a trimeric coiled-coil domain. At near physiological conditions the peptide self-assembles into about 27 nm, roughly spherical SAPNs. The average size of the SAPNs increases with the salt concentration. The optimal pH for their formation is between 7.5 and 8.5, while aggregation occurs at lower and higher values. A glycerol concentration of about 5% v/v is required for the formation of SAPNs with regular spherical shapes. These studies will help to optimize the immunological properties of SAPNs.