452849 On the Small-Angle Scattering Signature of Monoclonal Antibodies

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Marco A. Blanco, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD; Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, Harold W. Hatch, Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, MD, Joseph E. Curtis, NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD and Vincent K. Shen, Chemical and Biochemical Reference Data Division, National Institute of Standards and Technology, Gaithersburg, MD

Among biotherapeutics, the high efficacy and specificity of monoclonal antibodies (mAbs) make them key in controlling different type of illnesses such as cancer, allergies, and autoimmune diseases. However, small changes in the protein sequence and formulating conditions may lead to physical instabilities such as protein phase separation and aggregation. These stability concerns constitute some of the main causes of impurities during biotherapeutic manufacturing, as well as may bring immunogenicity issues in patients. Understanding how different factors such as solution conditions, sequence identity, and protein concentration affect the behavior and stability of mAbs is critical for the rational design of new therapeutics. In that regard, small-angle scattering (SAS) experiments have been proven to be powerful methods to evaluate the stability of proteins over a wide range of length-scales (1–100nm) and formulation conditions. However, quantifying the behavior of proteins via SAS generally requires reliable theoretical models in order to relate scattering profiles at different length-scales to changes in protein structure, protein concentration, and solution conditions. In the case of mAbs, the use of such models results challenging due to their inherent anisotropy, as well as their high interdomain flexibility. In this work, a minimal, flexible molecular model was developed to evaluate the behavior of mAbs in solution and their corresponding SAS profiles, which coarse-grains antibodies based on the large domains (e.g., Fc and Fabs). Comparison against detailed models (e.g., fine-grain and atomistic representations) at dilute conditions showed that this minimal model effectively captures the main SAS signatures of mAbs. Unlike those detailed models, the simplicity of the working representation permits to evaluate the effect of high-concentration non-idealities such as molecular crowding on the behavior of mAbs. The results indicate there are significant structural changes as a consequence of high protein concentrations (>200mg/ml), where mAbs transition into closed, globular-like conformations. Finally, analysis of the effect of interdomain interactions provides a phenomenological phase boundary for different states that cause mAb instabilities, which include formation of micelles, gelation, and liquid-liquid phase separation.

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