Optimization of Formulation Conditions for Broadly Neutralizing Antibodies (bnAb): Utilizing Isothermal Chemical Denaturation as a High-throughput Screening Method

Abstract

Broadly neutralizing antibodies (bnAb) were identified at the Vaccine Research Center (VRC) at NIH, towards the development of an anti-HIV vaccine. Examples of two such mAbs are CAP256 LS and N6 LS, targeting the V1V2 and CD4-binding sites, respectively, on the gp120 envelope glycoprotein. As part of mAb formulation development, it is critical to identify condition which can maintain the conformational and colloidal stability of the antibodies at 100mg/ml in frozen or liquid phase for two years. The factors which have the maximum impact on stability are formulation pH, ionic strength and stabilizers. High throughput methods to screen these three factors and identify optimal conditions are described here. The initial characterization of the mAbs using techniques including Differential Scanning Calorimetry (DSC), Dynamic Light Scattering (DLS), and Circular Dichroism (CD) indicated that they have very different physico-chemical properties. An assessment of intrinsic stability was conducted using Isothermal Chemical Denaturation (ICD) at a wide pH (4-8) and salt concentration range (0-200 mM). pH had the strongest effect on both CAP256LS and N6LS intrinsic stability. CAP256LS was most stable at a higher pH (>7) while N6LS stability improved above pH 6. Ionic strength had little effect on intrinsic stability for both mAbs. The stabilizing effects of various excipients on CAP256LS was screened using DLS, UV-Visible Spectroscopy, and Size Exclusion Chromatography (SEC). ICD was utilized to more rapidly screen the optimal concentration of these excipients. Balancing the results of the pH and ionic strength screens for both assays, optimal conditions were selected for both mAbs, and the high-throughput screening capacity of the ICD in conjunction with traditional stability-indicating assays helped identify optimal formulation conditions.