Developability Assessment of an Isolated C_H2 Immunoglobulin Domain

The IgG C_H2 domain continues to hold promise for the development of new therapeutic entities because of its bifunctional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody (mAb) fragments: C_H2, C_H2s, and m01s. The C_H2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. C_H2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to C_H2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 °C for all three proteins in an array. Co-distribution and two-dimensional infrared correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within C_H2 and C_H2s that are prone to self-association and establishing the differences in stability. Furthermore, C_H2 helical segments, β-sheets, β-turns, and random coil regions were less stable than in C_H2s and m01s because of the presence of the N-terminal 3₁₀-helix and β-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.