Highly concentrated monoclonal antibody solutions: Direct analysis of physical structure and thermal stability

Abstract

Virtually all current analytical methods employed in the development of highly concentrated monoclonal antibody (MAb) formulations require dilution of the sample before acquiring data. Thus, there is an unmet need for methods to study proteins directly at high concentration, since extrapolation of stability indicating parameters obtained from dilute studies may not be representative of the high concentration solution. Only slight or no modifications of biophysical methods including fluorescence, UV absorbance, circular dichroism, and FTIR (ATR and transmittance) spectroscopies as well as differential scanning calorimetry (DSC) are described here that permit the direct study of immunoglobulins (and other proteins) at high concentrations. Although FTIR spectra show differences that are dependant upon sampling geometry, other spectroscopic data from two different recombinant MAbs suggests that structure of each antibody exists in a physically similar state in the concentration range of 0.1–190 mg/mL in 40 mM pH 6 citrate–phosphate buffer. Upon thermally stressing these proteins, spectroscopic techniques that probe tertiary structure demonstrate a decrease in the apparent thermal melting temperature of ∼5–20°C of both proteins with increasing concentration. In contrast, DSC thermograms and CD thermal experiments suggest a minor degree of stabilization (∼2°C) for both antibodies although protein association could be responsible for these observations. Empirical phase diagrams produced from spectroscopic data also suggest (1) the existence of similar structural states at low temperatures independent of concentration and (2) a decrease in the temperature at which phase changes are observed with increasing concentration. The decrease in structural stability observed in these studies is probably the result of aggregation or self-association of the recombinant MAbs upon heating in crowded solutions and not due to a decrease in the intrinsic structural stability of the MAbs. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci