The stability of two purified monoclonal antibodies, MN12 and WT31, was investigated. The monoclonal antibodies were incubated for 32 days at different pH values (ranging from 3.0 to 10.0) at 4 and 37 degrees C. Various analytical methods were used to assess changes in physicochemical properties of the proteins. The monoclonal antibodies were more susceptible to degradation at 37 degrees C than at 4 degrees C. At low pH irreversible precipitation occurred. Decomposition of the proteins was enhanced at increasing pH values in the alkaline range. This was concluded from mouse IgG-specific and antigen-specific enzyme-linked immunosorbent assays, flow cytometry, analytical gel permeation chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and immunoblotting. No substantial change in the apparent affinity constant of MN12 was observed, as determined by an affinity enzyme-linked immunosorbent assay. Fluorescence spectra, fluorescence polarization values, and fluorescence quenching parameters of MN12 and WT31 were not substantially affected, indicating that no major irreversible conformational changes had occurred. It was concluded that each of the techniques used has only limited value for stability assessment of monoclonal antibodies and, hence, that the application of several analytical techniques is essential to gain insight into monoclonal antibody stability.