Streamlined Multi-Attribute Assessment of an Array of Clinical-Stage Antibodies: Relationship Between Degradation and Stability

Abstract

Clinical antibodies are an important class of drugs for the treatment of both chronic and acute diseases. Their manufacturability is subject to evaluation to ensure product quality and efficacy. One critical quality attribute is deamidation, a non-enzymatic process that is observed to occur during thermal stress, at low or high pH, or a combination thereof. Deamidation may induce antibody instability and lead to aggregation, which may pose immunogenicity concerns. The introduction of a negative charge via deamidation may impact the desired therapeutic function (i) within the complementarity-determining region, potentially causing loss of efficacy; or (ii) within the fragment crystallizable region, limiting the effector function involving antibody-dependent cellular cytotoxicity. Here we describe a transformative solution that allows for a comparative assessment of deamidation and its impact on stability and aggregation. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough platform technology is comprised of a quantum cascade laser microscope, a slide cell array that allows for flexibility in the design of experiments, and dedicated software. The enhanced spectral resolution is achieved using two-dimensional correlation, co-distribution, and two-trace two-dimensional correlation spectroscopies that reveal the molecular impact of deamidation. Eight re-engineered immunoglobulin G4 scaffold clinical antibodies under control and forced degradation conditions were evaluated for deamidation and aggregation. We determined the site of deamidation, the overall extent of deamidation, and where applicable, whether the deamidation event led to self-association or aggregation of the clinical antibody and the molecular events that led to the instability. The results were confirmed using orthogonal techniques for four of the samples.