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Non-native protein aggregation is a common concern for biopharmaceuticals. A given protein may aggregate through a variety of mechanisms that depend on solution and physico-chemical stress conditions. A thorough evaluation of aggregation behavior for a protein under all conditions of interest is necessary to ensure drug safety and efficacy. This work introduces a rapid, small-volume approach to evaluate protein aggregation propensity upon exposure to air-water interfaces (AWI). A microtensiometer apparatus is used to aerate a small volume of a protein solution with microbubbles for short periods of time (≤10 s). Sub-visible particles that form are captured and analyzed using backgrounded membrane imaging. This allows one to capture all particles in the solution while being sample sparing. The surface-mediated aggregation of two model monoclonal antibodies (MAbs) and a globular protein (aCgn) was tested as a function of pH and temperature. Temperature had a negligible effect under the rapid interface turnover time scales with this technique. Electrostatic protein-protein interactions, mediated by pH changes, were more influential for particle formation via AWI. Nonionic surfactants substantially reduced particle formation for all MAb solutions, but not aCgn. The results are contrasted with expectations when exposing samples to much larger air-water interfacial stress., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Non-native protein aggregation is a long-standing obstacle in the biopharmaceutical industry. Proteins can aggregate through different mechanisms, depending on the solution and stress conditions. Aggregation in bulk solution has been extensively studied in a mechanistic context and is known to be temperature dependent. Conversely, aggregation at interfaces has been commonly observed for liquid formulations but is less understood mechanistically. This work evaluates the combined effects of temperature and compression/dilation of air-water interfaces on aggregation rates and particle formation for anti-streptavidin immunoglobulin gamma-1. Aggregation rates are quantified via size-exclusion chromatography, dynamic light scattering, and microflow imaging as a function of temperature and extent of air-liquid interface compressions. Competition exists between bulk- and surface-mediated aggregation mechanisms. Each has a largely different temperature dependence that leads to a crossover between the dominant aggregation mechanisms as the sample temperature changes. Surface-mediated aggregation rates are pH dependent and correlate with electrostatic protein-protein interactions but do not mirror the pH dependence of bulk aggregation rates that instead follow trends for conformational stability. Mechanistic insights were informed by quiescent incubation of solutions before and after interface compressions. Detailed mechanistic conclusions require direct dynamic observation at the interface. Microbubble tensiometry is introduced as a promising tool for such measurements., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Protein-protein interactions (PPI) and solution viscosities were measured at low and high protein concentrations under a range of formulation conditions for 4 different monoclonal antibodies. Static light scattering was used to quantify the osmotic second virial coefficient (B 22 ) and the zero-q limit static structure factor (S q=0 ), versus protein concentration (c 2 ) from low to high c 2 . Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c 2 and to determine the protein interaction parameter (k D ). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (H q=0 ), which accounts for changes in hydrodynamic PPI as a function of c 2 . The net PPI ranged from strongly repulsive to attractive interactions, via changes in buffer pH, ionic strength, and choice of monoclonal antibodies. Multiple-particle tracking microrheology and capillary viscometery were used to measure monoclonal antibodies solution viscosities under the same solution conditions. In most cases, even large and qualitative changes in PPI did not result in significant changes in protein solution viscosity. This highlights the complex nature of PPI and how they influence protein solution viscosity and raises questions as to the validity of using experimental PPI metrics such as k D or B 22 as predictors of high viscosity., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Controlling and predicting unwanted degradation, such as non-native aggregation, is a long-standing challenge for mAbs and other protein-based products. mAb aggregation rates are typically sensitive to temperature, pH, and the addition of excipients. Quantitatively comparing temperature-dependent aggregation rates across multiple possible formulations is a challenge in product development. A parallel temperature initial rate method is used to efficiently and accurately determine initial rates for anti-streptavidin (AS) IgG1 aggregation as a function of pH, [NaCl], and in the presence of acetate versus citrate buffer. Parallel temperature initial rates are shown to agree with results from a traditional, isothermal method and permits direct comparison of the formulations across almost 3 orders of magnitude of aggregation rates. The apparent midpoint unfolding temperatures (through differential scanning calorimetry) and the effective activation energy values (Ea) are generally higher in acetate buffer compared with citrate buffer, which is consistent with preferential accumulation of citrate ions compared with acetate ions that was speculated in previous work (Barnett et al., J Phys Chem B, 2015). Static light scattering and Kirkwood-Buff analysis show that AS-IgG1 has stronger net repulsive protein-protein interactions in acetate compared with citrate buffer, also consistent with increased values of Ea. In an extreme case, aggregation of AS-IgG1 is effectively eliminated across all practical temperatures at pH 4 in 10 mM sodium acetate but proceeds readily in citrate buffer., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

In the present report, two formulation strategies, based on different aggregation models, were compared for their ability to quickly predict which excipients (cosolutes) would minimize the aggregation rate of an immunoglobulin G1 monoclonal antibody (mAb-1) stored for long term at refrigerated and room temperatures. The first formulation strategy assumed that a conformational change to an aggregation-prone intermediate state was necessary to initiate the association process and the second formulation strategy assumed that protein self-association was instead controlled by the solubility of the native state. The results of these studies indicate that the stabilizing effect of excipients formulated at isotonic concentrations is derived from their ability to solubilize the native state, not by the increase of protein conformational stability induced by their presence. The degree the excipients solvate the native state was determined from the apparent transfer free energy of the native state from water into each of the excipients. These values for mAb-1 and two additional therapeutic antibodies correlated well to their long-term 4°C and room temperature aggregation data and were calculated using only the literature values for the apparent transfer free energies of the amino acids into the various excipients and the three-dimensional models of the antibodies., (Copyright © 2012 Wiley Periodicals, Inc.)

Design of experiment and statistical analyses were applied to evaluate the effects of several formulation components on the thermal and colloidal stability for a series of monoclonal antibody (mAb) formulations. The high-throughput assessment of the protein stability was performed by measuring the temperature of hydrophobic exposure (T(h) , thermal stability) and the diffusion interaction parameter (k(D) , colloidal stability). To correlate the measured parameters with protein stability, the propensity to aggregate was tested by exposing the mAb samples to two types of stress: mechanical stress caused by shaking agitation and thermal stress. Mechanical stress led to increased formation of large particles, whereas temperature stress resulted in an increase in oligomers. The data obtained from the stress studies were used to determine the critical values for the stability parameters. The optimal formulation compositions were determined based on the statistical models and the predication tests. This approach of high-throughput thermal and colloidal stability screening can be applied to the characterization and prediction of protein formulation properties., (Copyright © 2011 Wiley-Liss, Inc.)

The purpose of this study was to demonstrate the utility of combining a design of experiment (DOE) approach with high-throughput formulation screening to identify the main factors affecting protein thermostability and solution viscosity. The optimization of buffer compositions was guided by statistical analysis of the data to obtain the targeted combination of low viscosity and high thermostability. Different monoclonal antibody (mAb) formulation variables were evaluated in the study to achieve optimization of two parameters: (i) thermostability characterized by temperature of hydrophobic exposure and (ii) viscosity. High-throughput measurements were employed to characterize both parameters. The significance of each factor and the two-way interactions between them was studied by multivariable regression analysis. An experimental design was used to estimate the significance of all factors, including interaction effects. The range of optimal buffer compositions that maximized thermostability and minimized viscosity of a mAb formulation was determined. The described high-throughput methods are well suited for characterization of multiple protein formulation compositions with minimized resources such as time and material. The DOE approach can be successfully applied to the screening of mAb formulations early in the development lifecycle., (Copyright © 2010 Wiley-Liss, Inc.)

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

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