Date of Award

Spring 2009

Project Type


Program or Major

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy

First Advisor

Tom Laue


This study was conducted to address the aggregation/opalescence relationship of an IgG 1 therapeutic antibody under a variety of conditions. The opalescence characteristics of three antibodies, Mab 1, 2 and 3, were examined as a function of salt dependence, protein concentration and temperature, using a variety of biophysical techniques. The high molecular weight species were initially identified using batch static and dynamic light scattering and separated by asymmetric flow field flow fractionation. When the salt dependence was examined, any structural changes were characterized using differential scanning calorimetry, circular dichroism and fluorescence, while the association state and temperature dependence was measured by viscometry. The resulting non-ideality was determined using multi-angle light scattering over a range of NaCl and protein concentration, and then used to assess the nature of the noncovalent association. In addition, the valence of the antibody therapeutics was directly measured using capillary electrophoresis and assessed as a predictive tool of non-ideal behavior.

The antibody that exhibited the highest degree of opalescence, and therefore was the main focus of the study, was Mab 1, with Mab 3 exhibiting a lower degree of opalescence and Mab 2 acting as a control. The results suggest that the opalescence phenomenon of Mab 1 was due to a salt induced non-mass-action driven self-association which is mainly entropic in origin and is driven predominantly by NaCl. The intermolecular interactions appear to be facilitated by enhanced flexibility of Fc region and the independent Fab folding domain on Mab 1. The opalescence phenomenon also appears to be a relatively small fraction of discrete HMW material which is freely reversible and results in a liquid-liquid or liquid-solid phase transition which is either clear or cloudy depending on A2. Finally, it was determined that there is a discrepancy between the measured and calculated valence of the IgG1 antibodies in the study and this may be used as a predictive tool of non-ideal behavior. Together, this information was used to propose a model of the opalescence phenomenon of Mab 1 based on an electrostatic interaction involving mainly the CDR region on the Fab.