A primary concern for protein-based pharmaceuticals is degradation under formulation conditions necessary for manufacturing, storage, and administration. Non-native aggregation is one common degradation route that may affect the quality and safety of protein therapeutics. Concerns about the formation of aggregates introduce a need to be able to understand, control, and predict aggregation rates, aggregate structure/morphology, and solubility at formulation conditions and temperatures of industrial interest. This dissertation addresses these challenges by characterizing aggregate structure, unfolding, association, and rates of formation for an IgG1 antibody, and providing a mechanistic analysis of aggregate formation as a function of solution conditions. Additionally, the results and analysis are used as a basis for a novel approach to quantitatively predict aggregation rates while minimizing time and protein material consumption.
Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5–5.5). A combination of intrinsic/extrinsic fluorescence, circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally-accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed non-native secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally- and chemically-induced aggregates showed similar ThT binding and secondary structure changes, and dissociated to 95% monomer in concentrated guanidine hydrochloride solutions. Interestingly, aggregates from chemical refolding did not grow significantly beyond dimers and trimers, while those created via thermal acceleration readily formed soluble aggregates of tetramers and larger as well as insoluble aggregates at high pH conditions.
Additional details of the pH-dependent aggregation were assessed via aggregation kinetics monitored by static laser light scattering and size-exclusion chromatography. Aggregation proceeded through a dimer nucleus for all conditions tested, based on direct observation (pH 3.5 and 4.5) and by the semi-quantitative agreement between theoretical expectations and the scaling of the characteristic time scale for nucleation (τn) with protein concentration (pH 4.5 and 5.5). Changing pH significantly altered the mechanism of aggregate growth, as well as the size and solubility of aggregates that formed. Aggregates at pH 3.5 grew primarily by monomer addition and remained small and soluble. Soluble aggregates at pH 4.5 grew first by chain polymerization, followed by condensation polymerization, leading ultimately to large insoluble particles. At pH 5.5, monomer loss resulted primarily in insoluble aggregate formation, with only small levels of soluble aggregate intermediates detected at early times. Qualitatively, the global aggregation behavior was consistent with reduction of charge-charge repulsions as a primary factor in promoting larger aggregates and aggregate phase separation.
Finally, the effects of adding three salts – NaCl, NaClO4 , and NaSCN – on the conformational stability, colloidal protein-protein interactions, and aggregation rates of an IgG1 antibody were examined at acidic solution conditions where soluble and insoluble aggregates form. Addition of the more chaotropic salts decreased monomer conformational stability, promoted attractive colloidal interactions, and increased aggregation rates at a given temperature. Observed rate coefficients of aggregation (kobs ) were determined from isothermal kinetic studies for each solution condition at several temperatures (T), corresponding to shelf lives spanning 4 to 5 orders of magnitude. kobs vs. T was predicted quantitatively at high T by a temperature-scanning approach. However, at lower temperatures non-Arrhenius behavior was observed and was captured by accounting for non-linear van’t Hoff contributions to monomer unfolding free energies. Overall, the results demonstrate a novel approach to predicting aggregation rates at time-scales of industrial interest, based on rational extrapolation of kobs from scanning experiments that inherently account for contributions from both colloidal and conformational stability, without the need to incorporate knowledge of colloidal interactions or unfolding parameters such as midpoint unfolding temperatures or unfolding enthalpies.