TY  - THES
AU  - TIAN, XINSHENG
TI  - Structural and biophysical characterization of humanized monoclonal antibodies by small angle X-ray scattering
PB  - University of Copenhagen
VL  - Dr.
M1  - PUBDB-2015-00713
SP  - -
PY  - 2014
N1  - No full text available.
N1  - University of Copenhagen, Diss., 2014
AB  - Monoclonal antibodies (mAbs) are the most popular macromolecular therapeutics and have been widely used for the treatment of a variety of diseases. To meet the therapeutic needs, the long-term storage stability and favourable clinical outcome of antibody candidates have to be carefully considered in biopharmaceutical product development. Several techniques such as dynamic light scattering (DLS) and size-exclusion chromatography (SEC) have been established to characterize the physicochemical properties of therapeutic proteins. However, due to the structual complexity of antibodies and various degaradation pathways, mutiple orthogonal analytical methods are often required to obtain complementary information on antibody stability and solution behaviors.In this PhD project, we introduce small-angle X-ray scattering (SAXS) into biophysical and structural analysis of humanized IgG1, IgG2 and IgG4 subclasses with identical light chains and  variable region of heavy chains. Firstly, several conventional analytical methods were employed to evaluate the physical and chemical stability of three IgGs. Then, their solution behaviors were investigated by SAXS in a broad range of formulation conditions, where we observed pH and excipient dependent as well as subclass-related antibody stability with respect to aggregation. SAXS analysis also revealed that the intermolecular respulsive forces of IgG1 contributed to its improved stability at low pH. Sucrose could dramatically influence such repulsive effects. In addition, the oligomeric states of mAbs could be identified based on SAXS data. Finally, we demonstrated that SAXS can be applied as a high-throughput method for preformulation development.Based on SAXS curves, the solution conformations were identical from pH 5.0 to pH 8.5 as well as between sucrose and NaCl containing formulations for each IgG subclass, while clearly different among the three IgG subclasses. The in-depth structural analyses were performed by using the advanced ensemble optimization method (EOM), and the structural ensembles reconstructed revealed significantly different flexibility of IgG subclasses. The subclass-related conformational preferences were further characterized through clustering the structural models based on their shape. We demonstrated that the distinct solution conformations of three IgG subclasses were predominantly determined by their hinge regions. The structural information obtained in this project provides new insights on rational design of therapeutic antibodies towards improved biophysical stability and tailored effector functions.
KW  - Dissertation (GND)
LB  - PUB:(DE-HGF)11
UR  - https://bib-pubdb1.desy.de/record/206199
ER  -