Quantitative methods to elucidate and characterize various aggregation pathways of an IgG1 monoclonal antibody

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

Application of radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an IgG1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of Microflow Digital Imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size exclusion chromatography (SEC), Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions