Specific-Ion Effects on the Aggregation Mechanisms and Protein–Protein Interactions for Anti-streptavidin Immunoglobulin Gamma-1

Abstract

Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…The dramatic changes in aggregate growth behaviour observed for the 5R and 5K mutants reflect a change in growth pathways with increasing ionic strength, which has been previously observed for proteins such as a-chymotrypsinogen [51] and a handful of monoclonal antibodies [8,9,43,75]. With decreasing strength of electrostatic interactions, there is a transition from nucleation dominated growth (aggregates form but do not grow) to chain polymerization (aggregates only grow by addition of monomers or small building blocks) to aggregate-aggregate coalescence and finally to aggregate precipitation.…”

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

“…[24][25][26] In the current study, biophysical assays used to measure solution mediated properties were useful in predicting stability of G4-2, G1-2, and G1-7 at 40 C and emphasize the importance of maintaining colloidal stability in minimizing aggregation. However, these biophysical tools did not predict the stability for mAbs that degrade primarily through fragmentation.…”

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

“…This is in contrast to what has been found for IgG systems. 34,48,54 It also suggests that the Fab domains of IgG proteins carry most of the net charge in vitro, which is not what would be anticipated from theoretical calculations and amino acid sequence. 33 The effects of temperature on aggregation rates and mechanism (s) of the Fc1 were investigated by monitoring isothermal aggregation of the Fc1 over a broad temperature range, from 30 C to 77 C. The observed aggregation rate coefficient for monomer loss Figure 6.…”

“…However, the opposite result is observed. One hypothesis to account for this apparent discrepancy is that citrate ion binding 48 at pH 5 and 6 can compensate for the change in charge by binding to the glycans, mitigating the net charge of the O-glycosylated Fc1, compared to the Fc1 that is missing those O-glycans. However, a detailed characterization of all of the different glycosylated species was beyond the scope of this work.…”