The microstructure of dense protein systems in biopharmaceutical applications
Date
2018
Authors
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Publisher
University of Delaware
Abstract
Dense protein systems are ubiquitous in biopharmaceutical processing but, while their microstructure plays a major role in the understanding of their structure-function relationships in biotherapeutic applications, microstructural characterization of these crowded, solid-like systems can be challenging. Here, we develop new methodologies and techniques to characterize the multiscale structure of dense protein systems, which are then used to understand fundamental problems in three general areas of the biopharmaceutical industry: separations, formulation, and drug delivery. ☐ In separations, we characterize the effect of the architecture of cellulose-based and traditional and dextran-modified agarose-based ion-exchange resins on the nanoscale distribution of a relatively small protein (lysozyme) and two larger proteins (lactoferrin and a monoclonal antibody) at different protein loadings. We show that different resins lead to distinct protein distributions on protein-size length scales which are smaller than those previously observed in situ. Based on the data we propose that entropic partitioning effects such as depletion forces may drive the observed protein crowding. Our observations of the nano-scale structure are fundamental in understanding the mechanism of protein partitioning in different classes of chromatographic materials, providing necessary information for designing resins with improved performance. ☐ In formulation, we demonstrate the use of confocal fluorescence microscopy with fluorescently-labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. Moreover, small-angle neutron scattering with a humidity control environment was used to characterize protein-scale microstructural changes in such solid-state formulations as they were humidified and dried in situ. The findings indicate that irreversible protein aggregates of stressed formulations do not form within the solid-state, but do emerge upon reconstitution of the formulation. After plasticization of the solid-state matrix by exposure to humidity, the formation of reversibly self-associating aggregates can be detected in situ. The characterization of the protein-scale microstructure in these solid-state formulations facilitates further efforts to understand the underlying mechanisms that promote long-term protein stability. ☐ In drug delivery, we developed a methodology to evaluate mathematical models for the prediction of sustained release from poly(lactic-co-glycolic acid)-based (PLGA) drug delivery systems. We show that a recently-developed, efficient stochastic optimization algorithm can be used not only to find global minima of such complex models robustly, but also to generate meta-data that allow quantitative evaluation of parameter sensitivity and correlation, which can be used for further model refinement and development. Furthermore, a predictive mathematical model was validated by (1) its use to design a desirable, zeroth-order release profile in injectable solvent depot release systems, and (2) the comparison between model predictions and experimental release data and microstructural observations for implantable solid rods. The novel observations for both experimental systems are essential for adequately describing the underlying drug-release mechanisms when designing predictive models such as the one evaluated here, and we directly illustrate how such a predictive model facilitates the development of sustained drug-release systems. ☐ In general, this dissertation highlights the broad range of phenomena that can influence dense protein systems, and emphasizes the value in bringing soft matter expertise to this field to better understand these systems. The tools and methods developed in this dissertation, including small-angle neutron scattering, confocal fluorescence microscopy, and mathematical modeling, will be invaluable in the study of the structure-function relationship of these and other dense protein systems throughout the biopharmaceutical field.