A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

Abstract

The early-stage assessment of the physical stability of new monoclonal antibodies in different formulations is often based on high-throughput techniques that suffer from various drawbacks. Accordingly, new approaches that facilitate the protein formulation development can be of high value to the industry. In this study, a dynamic light scattering plate reader is used to measure the aggregation (by means of the increase in the hydrodynamic radius [R]) of monoclonal antibody samples that were subject to incubation and subsequent dilution from different concentrations of a denaturing agent, that is, guanidine hydrochloride. The increase in the R of the protein samples is dependent not only on the denaturant concentration used but also on the buffer in which the incubation/dilution was performed. We also compare the aggregation after dilution from a denaturant with other high-throughput stability-indicating methods and find good agreement between the techniques. The proposed approach to probe the physical stability of monoclonal antibodies in different formulation conditions offers a unique combination of features-it is isothermal, probes both the resistance to denaturant-induced unfolding and the colloidal protein stability, it is entirely label-free, does not rely on complex data evaluation, and requires very short instrument measurement time on standard equipment.

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

“…We previously showed that assessing the aggregation during dilution-or dialysis-refolding from denaturants can be a valuable tool to select monoclonal antibody formulations with higher physical stability and suppressed aggregation during storage 30,37,44 . In an effort to standardize such experiments, we presented a ReFOLD assay that is based on microdialysis in deep multiwell plates 30 .…”

Evaluation of Two Novel Scale-Down Devices for Testing Monoclonal Antibody Aggregation During Large-Scale Freezing

“…An increase in hydrodynamic radius can be associated with the formation of aggregated particles. 36,37 The PDI allows to estimate the heterogeneity of this sample. For monodisperse samples a PDI ≤ 0.1 is expected.…”

Minimizing Oxidation of Freeze-Dried Monoclonal Antibodies in Polymeric Vials Using a Smart Packaging Approach

“…The bulk solutions of both mAbs were buffer exchanged to 20 mM (LMU1) or 10 mM (LMU2) histidine/histidine hydrochloride with pH 5.5 at 20 °C to 25 °C using Slide-A-Lyzer™ 10,000 molecular weight cut-off dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA). After extensive dialysis as described by Svilenov et al [ 28 ], the final buffers contained either 20 mM histidine and 0.04% ( w / v ) polysorbate 20 for LMU1 or 10 mM histidine and 0.05% ( w / v ) polysorbate 80 for LMU2. The concentration of both antibodies was measured with a Nanodrop 2000 UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).…”