Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics

Abstract

Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an “IgG1-like” single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.

Bispecific Antibodies: From Research to Clinical Application

“…The Fab arm of the IgG4 antibody can be dynamically recombined and exchanged in vivo to formed BsAbs, in a process referred to as named Fab-arm exchange (FAE) (38)(39)(40). Controlled Fab-arm exchange (cFAE) is the core technology of the Duobody platform.…”

The unique properties of IgG4 and its roles in health and disease

“…Comparing IgG4 with IgG1, two mutations in the latter are required to enable Fab-arm exchange: a P228S mutation in the hinge region allowing for the disulfide bonds that normally connect the heavy chains to be easily broken, and a K409R mutation in the carboxy-terminal domains that results in weaker non-covalent interactions between the heavy chains 48 . Conversely, therapeutic IgG4 monoclonal antibodies often contain a S228P mutation to prevent Fab-arm exchange in vivo 49 .…”