TY - JOUR

T1 - Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis

T2 - Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

AU - Tian, Xinsheng

AU - Langkilde, Annette Eva

AU - Thorolfsson, Matthias

AU - Rasmussen, Hanne B

AU - Vestergaard, Bente

N1 - © 2014 The Authors. Journal of Pharmaceutical Sciences published by Wiley Periodicals, Inc. and the American Pharmacists Association.

PY - 2014/6

Y1 - 2014/6

N2 - A 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 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

AB - A 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 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

U2 - 10.1002/jps.23964

DO - 10.1002/jps.23964

M3 - Journal article

C2 - 24700358

VL - 103

SP - 1701

EP - 1710

JO - Journal of Pharmaceutical Sciences

JF - Journal of Pharmaceutical Sciences

SN - 0022-3549

IS - 6

ER -