Developability Assessment of an Isolated C_H2 Immunoglobulin Domain

Abstract

The IgG CH2 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: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s 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 CH2s, 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 CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, β-sheets, β-turns, and random coil regions were less stable than in CH2s 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.

The reduced form of the antibody CH2 domain

“…Among such small binders, the isolated CH2 domain, comprising residues 238–340 (Figure 1a), lacks the disulfide bridges present between domains in intact immunoglobulins and is considered a promising scaffold, having both neonatal Fc receptor and effector binding sites and reasonable pharmacokinetics 28,30–33 . Proof‐of‐concept for CH2 as a binder scaffold has been demonstrated, 34 and its aggregation propensity has been discussed and improved over the past few years 35–38 . However, most studies have been done in the context of fully oxidized CH2, with a disulfide‐bridge formation between C261 and C321, that has been purified in periplasmic conditions 39 or in eukaryotic cells with glycosylation 40 …”

“…28,[30][31][32][33] Proof-ofconcept for CH2 as a binder scaffold has been demonstrated, 34 and its aggregation propensity has been discussed and improved over the past few years. [35][36][37][38] However, most studies have been done in the context of fully oxidized CH2, with a disulfide-bridge formation between C261 and C321, that has been purified in periplasmic conditions 39 or in eukaryotic cells with glycosylation. 40 When Fv and Fab antibody fragments are expressed in a reducing, cytoplasmic environment, it is known that sufficient oxidization is needed to fold these proteins.…”