Hybrid Analysis of Mab Flexibility by Electron Microscopy and Scattering

Abstract

The biopharmaceutical industry is one of the most valuable business sectors of the US economy. In 2016, ten of the 15 top-selling drugs were protein biologics; and of these, five were monoclonal antibodies (mAbs). Biopharmaceuticals also represent at least 40% of the medicines in development today, and the global market for protein biologics in 2022 is projected to be over $200B. A key measurement gap in the biopharmaceutical industry is the inability of current methods to fully characterize the structure of highly flexible, protein-based drugs, such as mAbs. Crystallization is generally not successful due to their high flexibility; and even when successful, captures only a single conformational snapshot of the molecule. Similarly, the continuum of conformational states does not allow conventional multi-particle averaging in CryoEM. Using the NISTmAb (RM8671), a model drug-like monoclonal antibody reference material, we have developed hybrid methods to combine EM with small-angle x-ray and neutron scattering in order to build more complete models of antibody structure and flexibility in solution. In this methodology, Individual Particle Electron Tomography (IPET) is initially used to build a collection of conformational states of the antibody. These states can then be used to model scattering data under physiologically or pharmaceutically relevant solution conditions (e.g., different formulations). We have applied these methods to the NISTmAb reference material itself, as well as to soluble aggregates naturally present in small amounts in the product, and to a NISTmAb/antigen complex. Ultimately, the ability to measure the detailed conformational distribution of biotherapeutics may allow this behavior to be correlated with attributes of pharmaceutical significance, such as stability, efficacy, and immune tolerance. This understanding could allow for the more rational design of stabile and effective biopharmaceutical molecules and formulations.