Towards a greater mechanistic understanding of reversible protein-protein interactions and irreversible aggregation of IgG1 monoclonal antibodies

Abstract

Immunoglobulin G1 monoclonal antibodies (IgG1-mAbs) are one of the most important and fastest growing class of biotherapeutic agents. These mAbs are being used to treat a wide range of medical conditions such as cancer, macular degeneration, autoimmune diseases, rheumatoid arthritis, etc. Antibodies are dynamic molecules and their dynamic nature governs their physiological and biological functions. Antibodies are protein based drugs and are prone to both physical and chemical degradation in vitro. The biggest concerns related to IgGs mAb stability is their propensity to form protein aggregates (at low and high protein concentrations) and transient protein-protein interactions at high protein concentrations leading to dramatic increase in solution viscosity. Predicting protein stability and mapping protein interactions at high protein concentrations remains some of the most desirable long term goals of protein formulation development. This dissertation’s second chapter focused on exploring the utility of hydrogen exchange mass spectrometry (HX-MS) in predicting stability profile of an IgG1 mAb, mAb-4, upon addition of various destabilizing phenolic antimicrobial preservatives (APs). The trends in mAb-4’s physical stability measurements using differential scanning calorimetry and extrinsic fluorescence spectroscopy (thermal stability) and size exclusion chromatography (aggregation propensity) showed correlations with significant increases in local flexibility of the aggregation hot-spot peptide segment in the CH2 domain of mAb-J (HC 237-254) upon addition of APs. Most hydrophobic AP (m-cresol) caused the greatest decrease in physical stability of mAb-4 and also the biggest increase in the local flexibility of CH2 domain peptide hot-spot of the antibody. Global deuterium uptake by mAb-4 was also highest in the presence of m-cresol, followed by phenol, phenoxyethanol and benzyl alcohol. In the third and fourth chapter, the effect of different solution and environmental variables on viscoelastic behavior and propensity of concentration-dependent self-association of two IgG1 mAbs was tested. One of the IgG1 mAbs, mAb-C showed a primarily hydrophobic interaction driven protein association behavior, however, the other IgG1 mAb, mAb-J, showed a distinct association mechanism where the antibody monomers associated through electrostatic attractive interactions. A novel HX-MS methodology was developed to map protein-protein interaction interfaces of transient intermolecular antibody associations at high protein concentrations. Antibody solutions of low (non-associating) and high protein concentrations (associating) were lyophilized and reconstituted directly in D2O solutions to initiate HX process directly at target concentrations. Protein interface of mAb-C reversible self-association was confined in the relatively hydrophobic VH and VL domains of the antibody that spanned its CDR2H and CDR2L loops, indicating towards a Fab-Fab interaction drive association event. HX-MS also revealed distant dynamic coupling effects of mAb-C association in the form of significant increases in local flexibility of certain peptide segments in the VH and CH2 domains of the antibody. HX-MS analysis of mAb-J reversible self-association revealed one of the interfaces in the VH and VL domain (positively charged, spanning CDR3H and CDR2L) and other interface in the CH3 domain of the antibody (negatively charged). Hence, HX-MS can provide a robust solution to protein physical stability prediction for rational design of protein formulation strategies and high-resolution protein-protein interaction interface mapping at high protein concentrations for engineering mutant antibody molecules with superior physiochemical properties and enhanced stability.