Abstract
The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.
Highlights
The main variants generated from this system are hole-hole homodimers and, to a lesser extent, knob-knob homodimers, which are both product-related impurities and should be mostly removed through assembly and purification steps
The immunoglobulin-G molecules (IgGs) hole-hole homodimers produced as a side product from the assembly of the knob-into-hole bispecific antibody were examined by optimized negative-staining (OpNS) EM (Fig. 1A–D)[35,36,37,38]
The OpNS protocol was refined from conventional negative staining (NS) protocols by introducing three additional steps: (i) filtering the thawed staining solution (1% Uranyl formate, uranyl formate (UF)) using the smallest available filter (0.02 μm) to remove the precipitated particles immediately before usage, (ii) incubating and staining the EM grid within a black box to prevent the stain reagent from the light-exposure, and (iii) drying the specimen with nitrogen gas to prevent sample oxidation
Summary
The main variants generated from this system are hole-hole homodimers and, to a lesser extent, knob-knob homodimers, which are both product-related impurities and should be mostly removed through assembly and purification steps. Single particle cryo-EM has the capability for structure determination of protein at atomic resolution under near-native buffer conditions[30]. This method has its own limitations, such as difficulties in imaging small proteins (
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