Title

The effect of solution environment on the conformational stability and aggregation of monoclonal antibody OKT-4

Date of Completion

January 2000

Keywords

Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

Monoclonal antibodies belong to a new generation of biotechnological products that are of growing interest for therapeutic applications in the treatment of organ transplantion, autoimmune diseases, malignancy etc. OKT-4 is an IgG2a antibody, specifically directed against T cells and binds to the CD4 antigen present on these cells. Presently it is in clinical trials for prevention of renal and other allograft rejections. The objectives of this research were to develop and/or utilize sensitive techniques to monitor changes in the conformation of OKT-4 and to investigate the solution stability and aggregation of OKT-4 under various conditions such as pH, ionic strength and temperature. ^ Fluorescence spectroscopy and thermal denaturation studies were used to monitor changes in the conformation of OKT-4. Our results showed the uniqueness of measuring the intrinsic fluorescence of tryptophan (Trp) in OKT-4 as a thermal switch to monitor changes in the tertiary structure and relate to the stability of OKT-4 in solution. Changes in the conformation and tertiary structure of OKT-4 upon heating and cooling (10°C → 85°C → 10°C) were monitored in the pH range 3.0 to 9.0. It was observed that the thermal denaturation of OKT-4 followed an irreversible pathway as observed from changes in the fluorescence intensity. With the decrease in pH, the transition temperature decreased significantly suggesting lower stability of OKT-4 in acidic environment. Changes in secondary structure were monitored by circular dichroism, which showed a decrease in the beta-sheet content with decrease in pH from 7.2 to 3.0 suggesting acid induced conformational change. Significant amount of secondary structural content is retained upon exposure to 60°C for 24 hours at pH 5.0, 7.2 and 9.0. ^ The effect of pH and temperature on aggregation kinetics of OKT-4 was investigated by size exclusion chromatography and light scattering. The sharp increase in the scattering intensity during thermal denaturation of OKT-4 indicated that large aggregates were formed upon unfolding. Laser light scattering studies showed the presence of dimer in the aggregation process. In addition to the physical denaturation, chemical denaturation was found to play a role in the thermal inactivation of OKT-4. ^