Biophysical Signatures of Monoclonal Antibodies

Abstract

Monoclonal antibodies are the most common protein that is being developed by many companies as therapies against a wide range of diseases (Andreakos et al. 2002; Campbell and Marcus 2003; Trikha et al. 2002; Untch et al. 2003). With increasing interest in the use of monoclonal antibodies therapeutics, it is apparent that the combination of specificity and safety offered by these proteins will continue to drive the biotechnology industry forward in the coming years (Gelfand 2001; Brekke and Sandlie 2003). As is the case with any drug, a successful MAb formulation is dependant upon many factors, including understanding their biophysical properties to help describe their behavior and physical stability. While a wide variety of physical characterizations of monoclonal antibodies are routinely performed from basic research through their pharmaceutical development, little systematic information exists regarding the range of values commonly encountered during such studies. A new investigator is thus faced with some uncertainty concerning what might be considered “normal” values for the many different types of measurements. As will be shown below, despite the primary structure similarity of IgG molecules (with the exception of heavy chain subtype and hyper-variable regions, of course), generally, there is significant variation in the results of such studies. The primary purpose of this study is to establish the range of values obtained from a variety of biophysical measurements and not to examine in depth the origin or nature of such differences, although a brief discussion of these issues will be provided. Therefore, the identity of the 12 IgG molecules that were obtained from three of the leading producers of IgG monoclonal antibodies will not be provided. Rather, they are each moved into and maintained in identical buffer conditions (pH 7.4 PBS) and are identified by a simple letter designation. Spectroscopic, calorimetric, electrophoretic and solubility methods are employed for the analysis of MAbs and illustrate the diversity of their biophysical characteristics which pertain to a wide variety of pharmaceutically relevant structural, physical, and thermal stability parameters. Secondary structure is probed employing both FTIR and far-UV CD spectroscopy. Tertiary structure is examined using intrinsic fluorescence and high-resolution second derivative UV absorbance spectroscopy, while DLS is performed to gather information pertaining to protein hydrodynamic size. Other physical parameters of immediate pharmaceutical interest such as thermal unfolding temperatures, isoelectric point and heterogeneity and apparent solubility are obtained from DSC, capillary isoelectric focusing and PEG precipitation methods, respectively.