AUC Sedimentation Velocity Studies of Therapeutic Proteins in Serum

Abstract

The preclinical characterization of therapeutic proteins seeks to determine the stability, state of aggregation, and interaction with other macromolecules in serum. Sedimentation velocity is one of the experimental methods required to determine and understand these factors. Our goal is to develop analysis methods for sedimentation velocity data acquired using absorbance, multi-wavelength and the Aviv-FDS AUC. The Aviv-FDS utilizes a single fluorescently labeled sedimenting species against a concentrated and heterogeneous background of serum proteins which results in the phenomenon known as the Johnston-Ogston (J-O) effect. The J-O effect describes the hydrodynamic and thermodynamic nonideal sedimentation of a single component as a function of high concentrations of other components in solution. Thus, the aim of our current work is to study the sedimentation of a fluorescently labeled therapeutic antibody in the presence of high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, (∼35-40 mg/ml) and γ-globulins, IgG, (∼10-15 mg/ml). To study and understand the behavior of therapeutic antibodies in the presence of HSA, human IgG, and other serum components, tracer experiments are done pairwise as a function of HSA, IgG and therapeutic protein concentration. A plot of 1/s vs concentration reveals thermodynamic nonideality or a slowing of the sedimentation rate due to itself or another component (s = s0/(1 + Ksc)). This generates a 3x3 matrix of data that describes self- and cross-term hydrodynamic nonideality Ks. The goal is to develop a preclinical method for quantitative hydrodynamic analysis of therapeutic proteins in crowded environments like serum. (Supported by Boehringer-Ingelheim.)