Date of Award
Croinne S. Lengsfeld
Aggregation, Cavitation, Nebulization, Protein
Therapeutic proteins represent an essential piece of a health management plan for diseases such as diabetes, cancer, hemophilia, Crohn's Disease, and myocardial infarction. These proteins, however, must be maintained in their correct, biologically active conformation throughout processing, transportation, and delivery. This requirement poses serious engineering challenges because of a protein's susceptibility to thermodynamic instabilities resulting from the weak bonds driving the tertiary structure of the molecule. A particularly problematic type of protein degradation is aggregation. Administration of aggregated proteins, a particularly problematic degradation form, can have dire consequences, including blocking a patient's responsiveness to therapy, inducing immunogenicity, and even anaphylactic shock and death. Normal shipping and delivery methodologies are suspected of causing protein aggregation after the normal quality control process has been completed. This work investigates the effect of acoustic cavitation on protein aggregation as a function of impurity level, gas-liquid surface to value ratios, protein concentration, solution viscosity, density, surface tension, and nebulization time. A 0.2M and pH of 4.2 Glycine buffer solution was utilized with IVIg protein at 0.5, 1.0, 5.0, and 10.0 mg/ml and 20ºC. Protein aggregates were characterized using Microflow imaging and NanoSight tracking analysis. Transient cavitation and formation of radicals was monitored using classical iodine assays. Higher protein aggregation is observed in solutions that initially contain greater amounts of impurities or have a larger contact area with the gas interface. Aggregate production in hyper clean solutions, with no gas-liquid interface, initially increases with protein concentration, but eventually decreases at high concentrations.
In contrast, aggregation rates in hyper clean solution with a gas-liquid interface continue to fall with increasing protein concentration. The size of the particulate in these two conditions suggests different degradation pathways. The small sizes when a gas interface is available are likely a result of the large area over which the process takes place. The effect of concentration is actually an effect of diffusion or availability for proteins at the surface. The large sizes found in conditions with no gas interface suggest a much more concentrated process consistent with an intense energy release at a single location. Moreover, monitoring of the formation of I3- from iodine as a function of nebulization time shows increasing production or radicals. All this supports the hypothesis that ultrasonic pressure waves in protein solutions cause transient cavitation which upon bubble implosion release hydroxyl radicals that can attack the protein in solution. In this circumstance, a rise in viscosity at higher protein concentration inhibits cavitation by elevating the lowest pressure region based on a specified pressure drop.
Giarratano, John Angelo, "Protein Aggregation through Acoustic Cavitation" (2012). Electronic Theses and Dissertations. 240.
Recieved from ProQuest
John Angelo Giarratano