3D Structure of Monoclonal Antibodies in Solution by Electron Density Topography

Cutting-edge mAb development aims to make more effective and sophisticated therapeutics by enhancing modalities. However, research and development processes are becoming increasingly complex and outcomes often do not match the intended design. The analysis of newly designed mAbs is becoming more challenging and requires a new method. Imagine being able to visualize a newly designed mAb within a few days of isolating it from the supernatant of the culture media, that is within in a similar time span as affinity and/or efficacy screening. This would allow you to engineer the molecule based on quick feedback from the experimentally determined molecular shape. For example, finding an X-shaped mAb in a bispecific constitution that is supposed to be Y-shaped, an unwanted pair of chains, or even fewer/shorter sugar chains might affect Fc activation. This technique would also allow direct epitope mapping and/or design effector control processes by monitoring multiple shapes and interactions among those components. To date, this examination of mAb shape at early stages has not happened because many structure analysis methods require fixing of the sample by cryocooling, crystallization, or immobilization; even if those are not required, one needs to prepare specific molecules to study, thus, not applicable to the mAb that was designed and produced. The process of structure determination is a study in and of itself. Here, we present the results of a novel technology to examine the molecular envelope in solution by determining the electron density of the molecule at low resolution without prior information, as if looking at the molecule in solution with a microscope. Using our technology, we determine the electron density of a flexible mAb in solution. The resultant envelopes are not at atomic resolution; nevertheless, there are enough details to obtain information on the above design requirements.