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
) and the zero-q limit static structure factor (S
), versus protein concentration (c
) from low to high c
. Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c
and to determine the protein interaction parameter (k
). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (H
), which accounts for changes in hydrodynamic PPI as a function of c
. 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
or B
as predictors of high viscosity.
Protein solution viscosity (η) as a function of temperature was measured at a series of protein concentrations under a range of formulation conditions for two monoclonal antibodies (MAbs) and a ...globular protein (aCgn). Based on theoretical arguments, a strong temperature dependence for protein–protein interactions (PPI) indicates highly anisotropic, short-ranged attractions that could lead to higher solution viscosities. The semi-empirical Ross-Minton model was used to determine the apparent intrinsic viscosity, shape, and “crowding” factors for each protein as a function of temperature and formulation conditions. The apparent intrinsic viscosity was independent of temperature for aCgn, while a slight decrease with increasing temperature was observed for the MAbs. The temperature dependence of solution viscosity was analyzed using the Andrade-Eyring equation to determine the effective activation energy of viscous flow (E a,η). While E a,η values were different for each protein, they were independent of formulation conditions for a given protein. PPI were quantified via the osmotic second virial coefficient (B 22) and the protein diffusion interaction parameter (k D) as a function of temperature under the same formulation conditions as the viscosity measurements. Net interactions ranged from strongly attractive to repulsive by changing formulation pH and ionic strength for each protein. Overall, larger activation energies for PPI corresponded to larger activation energies for η, and those were predictive of the highest η values at higher protein concentrations.
The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used ...to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.
When monoclonal antibodies are exposed to an air–water interface, they form aggregates, which negatively impacts their performance. Until now, the detection and characterization of interfacial ...aggregation have been difficult. Here, we exploit the mechanical response imparted by interfacial adsorption by measuring the interfacial shear rheology of a model antibody, anti-streptavidin immunoglobulin-1 (AS-IgG1), at the air–water interface. Strong viscoelastic layers of AS-IgG1 form when the protein is adsorbed from the bulk solution. Creep experiments correlate the compliance of the interfacial protein layer with the subphase solution pH and bulk concentration. These, along with oscillatory strain amplitude and frequency sweeps, show that the viscoelastic behavior of the adsorbed layers is that of a soft glass with interfacial shear moduli on the order of 10–3 Pa m. Shifting the creep compliance curves under different applied stresses forms master curves consistent with stress–time superposition of soft interfacial glasses. The interfacial rheology results are discussed in the context of the interface-mediated aggregation of AS-IgG1.
Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the ...relationship(s) among protein–protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein–protein interactions were quantified in terms of the protein–protein Kirkwood–Buff integral (G 22) determined from static light scattering and in terms of the protein effective charge (Z eff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein–protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Z eff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G 22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein–protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.
The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies ...was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding CH2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated CH2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of CH2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.
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 (Th, thermal stability) and the diffusion interaction parameter (kD, 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.
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.
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