Developability Assessment of an Isolated CH2 Immunoglobulin Domain

The IgG C(H)2 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(H)2, C(H)2s, and m01s. The C(H)2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. C(H)2s 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(H)2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 degrees 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(H)2 and C(H)2s that are prone to self-association and establishing the differences in stability. Furthermore, C(H)2 helical segments, beta-sheets, beta-turns, and random coil regions were less stable than in C(H)2s and m01s because of the presence of the N-terminal 3(10)-helix and beta-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.

Automated summary

The IgG C(H)2 domain shows promise for new therapeutic entities due to its dual function as a biomarker and effector protein. A comprehensive developability assessment of three similar monoclonal antibody fragments revealed that m01s, with an engineered disulfide bridge, eliminated the self-association process and demonstrated the highest stability among the three proteins.