Mechanistic insights of protein aggregation at interfaces
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Monoclonal antibodies (MAbs) continue to be the largest selling class of therapeutic biologics. The success of MAb therapeutics is attributed to their potency, specificity, and ability to be engineered for different targets. A common obstacle during MAb development is non-native aggregation. While aggregation can decrease the final potency of the drug, aggregates present in the final drug formulation can potentially invoke an immune response that reduces drug efficacy and compromises patient safety. Therefore, the aggregation mechanisms and different aggregate species related to a therapeutic drug candidate must be tightly controlled. ☐ MAbs encounter a range of stresses during pharmaceutical development, manufacturing, transportation, and storage that can promote different aggregation mechanisms in bulk solution and at solid-liquid, liquid-liquid, and vapor-liquid interfaces. Aggregation in bulk solution has been studied extensively in a mechanistic context. In contrast, mechanistic understanding of surface-mediated aggregation remains largely putative, in part because of the limitations of techniques currently used to study surface-mediated aggregation. This thesis focuses on mechanistic insight into surface-mediated aggregation by introducing improved techniques for studying protein behavior at interfaces and using these techniques to systematically study protein behavior at interfaces under a range of conditions. ☐ Isothermal interfacial compression/dilation (IICD) cycles are advantageous for their ability to provide well-controlled and quantifiable turnover of air-water interfaces in a relatively high-throughput manner. This technique was used to evaluate the combined effects of temperature and compression/dilation of air-water interfaces for a model MAb in representative MAb formulations. Aggregation rates were quantified as a function of temperature and extent of IICD cycles using size exclusion chromatography, dynamic light scattering, and flow-imaging microscopy. The results indicated that competition exists between bulk- and surface-mediated aggregation mechanisms, and each pathway has a largely different temperature dependence that results in a crossover between the dominant aggregation mechanism as the sample temperature changes. Surface-mediated aggregation is also influenced by solution pH in a manner that correlates with electrostatic protein-protein interactions and does not mirror the pH dependence in bulk that instead trends with conformational stability. Polysorbate 20 reduces aggregation rates overall, but in some instances shifts the overall observed aggregation behavior towards bulk-mediated aggregation. ☐ To further improve the labor, time, and material needs of surface-mediated aggregation studies, microbubble aeration was introduced as a rapid, small-volume approach for evaluating protein aggregation via air-water interface exposure. Samples were aerated with microbubbles for short amounts of time (< 10 seconds), and the resulting particles were analyzed using backgrounded membrane imaging. The applicability of microbubble aeration was demonstrated by evaluating the surface-mediated aggregation propensity of two model MAbs and a globular protein as a function of pH and temperature. Temperature had a negligible effect under the rapid time scales of interface turnover of this technique, which indicates the technique can help to isolate some portions of the overall aggregation mechanism compared to longer time-scale experiments. Electrostatic colloidal interactions were more influential than conformational stability on surface-mediated aggregation rates, even when comparing different proteins. Polysorbate 20 substantially reduced aggregation for the MAbs but not aCgn, which exhibited rapid adsorption kinetics. ☐ In studies using IICD cycles and microbubble aeration, information about surface-mediated aggregation is primarily inferred through bulk solution measurements of monomer depletion and aggregation products after they putatively desorb from the interface. Interfacial rheology was introduced as a suitable technique for measuring the rheology of protein layers while still adsorbed to the interface. A Langmuir trough was used to improve the practicality of interfacial shear rheology measurements for MAb systems by accelerating meso-equilibration and reducing the preparation time for each experiment from several hours to approximately 30 minutes. Creep measurements and oscillatory strain and frequency sweep measurements revealed that an adsorbed MAb layer on the air-water interface resembles a soft glassy material. For each condition, creep compliance from different applied stresses could be horizontally shifted for superposition onto a master creep curve. The viscoelastic moduli, creep compliance, and superimposed master creep curves of the MAb layers were dependent on solution pH and bulk concentration in a manner that indicated that adsorbed MAbs form stronger interfacial films as the solution pH approaches the pI of the MAb, and at higher bulk concentrations. ☐ Lastly, to determine the influence of adsorption on surface-mediated aggregation, a microtensiometer was used to systematically measure the dynamic surface tension behavior of two model MAbs and a globular protein as a function of temperature, bulk concentration, and solution pH at air-water and oil-water interfaces. The meso-equilibrium surface pressure did not change between conditions at each type of interface, though the meso-equilibrium surface pressure of the air-water interface was significantly higher than that of the oil-water interface. While in the rest of the thesis, solution pH was a key parameter determining surface-mediated aggregation rates and the interfacial rheology of adsorbed protein layers, solution pH had no observable effect on dynamic surface tension at either interface. At the air-water interface, adsorption kinetics accelerated at higher temperatures and bulk concentrations. The three proteins exhibited distinguishable adsorption kinetics, but not in an order that reflected surface-mediated aggregation propensity measured using microbubble aeration. Trends relating to temperature, bulk concentration, and the identity of the molecule were indistinguishable at the oil-water interface and may have been hidden below the sensitivity limits of the instrument. A higher concentration of polysorbate 20 was required to reduce protein adsorption to oil-water interfaces than to air-water interfaces. ☐ Many of the results presented in this work have implications for how accelerated stability studies relating to surface-mediated aggregation should be designed during biopharmaceutical development. Overall, this dissertation illustrates the effect of solution pH, temperature, and bulk concentration on protein adsorption and aggregation behavior at interfaces and lays the foundation for future studies to uncover new mechanistic information relating to surface-mediated aggregation.
Description
Keywords
Monoclonal antibodies, Protein aggregates, Therapeutic biologics, Isothermal Interfacial Compression/Dilation