Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis: Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

ABSTRACTA crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development. © 2014 The Authors. Journal of Pharmaceutical Sciences published by Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1701-1710, 201

In-depth analysis of subclass-specific conformational preferences of IgG antibodies

IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy

AbstractWe have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% α-helix, 28% extended structures, 12% β-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112–452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2–110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein