Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

Abstract

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).