| dc.description.abstract | Characterization of biopharmaceuticals is necessary for ensuring their purity and structural integrity. Many classes of biopharmaceutical drugs are turbid solutions. Common optical characterization techniques such as absorbance and FTIR spectroscopy must be adapted for use with turbid solutions. The goal of this thesis is to develop and apply spectroscopic techniques for characterizing turbid solutions. In the first chapter we introduce the current techniques employed for characterization of biopharmaceuticals. Special attention is paid to spectroscopic techniques and possible pitfalls of their use with turbid solutions. In the second chapter we develop a new method for measuring absorbance spectra of turbid solutions. The chapters three through five we apply a variety of spectroscopic techniques to characterize three distinct turbid solutions: aggregated proteins, phase separated antibodies, and mRNA-cationic lipid nanoparticles. Chapter two describes a label-free, direct approach to measure protein concentrations in turbid solutions using a UV-Vis integrating cavity absorbance spectrometer. Protein-particle conjugates and mixtures have been investigated extensively for their diverse applications in biotechnology. However, general methods to measure protein concentration of protein-particle solutions are lacking. Typically, proteins in turbid solutions require separation or staining with another chromophore to quantitate their concentration. Three systems are used to test the ability to measure accurate protein concentrations: proteins adsorbed to Alhydrogel, proteins in solution with gold nanoparticles and proteins encapsulated within polymeric microspheres. Protein concentrations in each of the three protein-particle systems were successfully quantified using a calibration curve created from the absorbance at 280 nm. In chapter three we study the two main pathways of Polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis and oxidation. Polysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, while the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of non-degraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation. In chapter four we characterize three immunoglobulins which undergo liquid-liquid phase separation with the goal of better understanding how intermolecular protein-protein interactions affect protein conformation in the protein-rich phase. We performed extensive and direct physical characterization of both the protein-rich and protein-poor phases. In comparison, the protein-rich phases displayed blue-shifted tryptophan emission spectra and their amide I infrared absorbance spectra red shifted for all three immunoglobulins. We used Raman spectroscopy to observe disulfide bonds which were isomerized to higher energy strained conformations in the protein-rich phases. Changes in the structural organization of water molecules within the protein-rich phase for all three antibodies were observed, suggesting a role for water hydrogen-bonding in phase separation. Amide I and protein fluorescence spectra differences between the phases persisted at temperatures above the critical temperature, but ceased at the temperature where these proteins unfolded. Hydrogen exchange-mass spectrometry (HX-MS) analysis with two of the mAbs identified specific regions driving protein reversible self-association. For one of the antibodies, electrostatic interactions between two oppositely charged patches on the Fab region appeared to initiate protein self-interactions. For another antibody, a large hydrophobic patch on the IgG1 heavy chain variable domain facilitated protein self-interaction. We hypothesized that aggregation would occur at equivalent rates in both phases since the two phases have the same chemical potential. For two of the antibodies, the protein-poor phase aggregated more rapidly than the protein-rich phase, while the third antibody displayed equal rates in the two phases. In chapter five we characterize the effect of solution pH on mRNA–cationic lipid nanoparticles (mRNA–LNPs) structure and transfection efficiency. LNPs containing mRNA are able to deliver genetic material to cells for use as vaccines or protein replacement therapies. We compared the structural and colloidal properties of LNPs not containing mRNA and mRNA free in solution with mRNA–LNPs. We used a combination of biophysical techniques to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of FTIR spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA–LNPs transition from an inverse hexagonal phase at pH values below the pKa of the cationic lipid to a lamellar phase above the pKa. At higher temperatures the mRNA–LNPs also transition from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Circular dichroism was used to investigate changes to the mRNA. The mRNA within the LNP has more A form structure at pH’s below the pKa than above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA–LNPs. The particles were larger and more prone to aggregation below the pKa. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA–LNPs had the highest transfection efficiency and stability at pH values below the pKa. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hr transition) and the transition from lamellar to inverse hexagonal is irreversible. This suggests that the lamellar phase is less-stable and kinetically trapped. Our findings deepen our structural understanding of mRNA–LNPs and will aid in the development of liquid formulations. | |
