Survey of chemical unfolding complexity as a unique stability assessment assay for monoclonal antibodies.

Abstract

Seventy-two intentionally sequence-diverse antibody variable regions were selected, expressed as IgG1 antibodies, and evaluated by chemical unfolding to survey the complexities of denaturant induced unfolding behavior. A two-transition fit well described the curves and uncovered a wide range of sensitivities to denaturant. Four general types of unfolding curves were observed: balanced traces (each transition responsible for half of the total unfolding curve), low-unfolding traces (first transition is a majority of the unfolding curve), high-unfolding traces (the second transition is the majority of the unfolding curve), and coincident traces (the two transitions are found close to each other, approximating a single transition). The complexity of the data from this survey indicates that focusing on the first inflection point or fitting a single transition model is likely an over-simplistic method for measuring stability by the chemical unfolding assay. Additionally, other conformational assays, such as thermal and low pH unfolding, showed no correlation with the chemical unfolding results, indicating that each of these assays provide alternate information on the different pathways of antibody conformational stability. These results provide a basis for beginning to better connect unfolding behavior to other physical phenotypic behaviors and production process behaviors.