Quantitative methods to elucidate and characterize various aggregation pathways of an IgG1 monoclonal antibody
Issue Date
2016-05-31Author
Kalonia, Cavan Kumar
Publisher
University of Kansas
Format
227 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Pharmaceutical Chemistry
Rights
Copyright held by the author.
Metadata
Show full item recordAbstract
The term protein aggregation is used in pharmaceutical biotechnology literature to describe a collection of different mechanisms that produce non-native, net irreversibly associated protein molecules. The presence of protein aggregates in therapeutic protein drugs is problematic because certain protein aggregates may induce an anti-drug immune response in patients. The work described in this Ph.D. thesis is focused on developing quantitative tools and theoretical models to better characterize and understand protein aggregation as a function of formulation conditions (solution pH, ionic strength, excipients, etc.) and external stresses (e.g., temperature, agitation, light, etc.). The methods developed here are then applied to develop a comprehensive understanding of the aggregation pathways of an IgG1 monoclonal antibody (mAb) under various conditions. Depending upon the protein itself, the formulation composition and the processing stress, protein aggregation may produce heterogeneous distribution of aggregates with different sizes and morphological properties. This work presents a novel data visualization method to monitor aggregate size, morphology, and concentration as a function of formulation variables such as solution composition, type of external stress, and stress duration. Such data visualization methods can be useful for isolating the effect of single variables. Another complementary method to determine the mass of subvisible particles (large aggregates with an equivalent spherical diameter of ~1-100 μm) was also developed in this work to better relate protein subvisible particle numbers to sample stability. Such calculation methods are often necessary because particle mass is often too low to be measured experimentally. When combined, these two methodologies serve as a set of powerful tools to better analyze protein aggregation because stressed samples with high particle numbers and small masses suggest that aggregate nucleation (monomers forming aggregate seeds) mechanisms are dominant. On the other hand, stressed protein samples with low particle numbers and large masses may point to aggregate growth mechanisms being dominant. Application of the data visualization and mass calculation methods to better characterize mAb aggregation led to a striking experimental observation that certain formulations could stabilize the IgG1 mAb against elevated temperatures but destabilize it against mechanical agitation. This, among other observations, led to the hypothesis that elevated temperatures promoted aggregation of this mAb in the bulk solution while mechanical agitation induced mAb particle formation at the air-solution interface. To better understand how formulation affected temperature induced aggregation of this mAb, kinetic models describing nucleation and growth processes were fit to mAb aggregation data collected at several incubation temperatures (25, 40 and 57 °C). The results of this study suggested that partially unfolded intermediates are an important driver of both aggregate nucleation and growth. Protein-protein interactions, on the other hand, only appeared to affect aggregate growth for this mAb. To further elucidate how mechanical agitation of mAb solutions could induce protein particle formation, controlled compression-expansion cycles were applied to the air water interface of mAb solutions. In this study, in collaboration with the Dhar laboratory, slow compression-expansion rates were found to induce aggregate formation at the air-solution interface. Faster compression-expansion rates appeared to disrupt the air-solution interface releasing protein particles into the bulk mAb solution. In addition, formulation composition was observed to affect the tendency of this IgG1 mAb to aggregate at the air-solution interface.
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