Application Notes

Getting the full picture: predicting the aggregation propensity of mAbs using chemical and thermal denaturation on a single, fully automated platform

Issue link: https://resources.nanotempertech.com/i/1050515

Contents of this Issue

Navigation

Page 1 of 9

2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Introduction Biologics belong to the fastest growing group of drugs, and an ever growing number of complex molecules such as mAbs, antibody-drug conjugates (ADCs) and biosimilars demands novel, predictive analytical methods to streamline the development process. An important aspect during drug development is the long- term stability of the medicinal product [3, 10]. The most critical parameter is the formation of protein particles, especially at high protein concentrations. Thereby, protein particles can serve as nucleation seeds and might promote protein aggregation. Consequently, particle formation does not only reduce the amount of active native-like protein, but can also cause unwanted effects which render the drug ineffective, or even harmful to patients [2, 9]. In order to predict the aggregation propensity of biologics, the assessment of their relative thermal stability and the measurement of ∆G° of unfolding are becoming invaluable tools during formulation development and protein engineering [11]. The principle behind the chemical denaturation approach is the following: By increasing the concentration of a chaotropic salt, here guanidine hydrochloride (GuaHCl), protein conformation equilibrium is shi ed from a folded to a partially unfolded up to a completely unfolded state. The fraction of unfolded protein at each measured GuaHCl concentration can be determined, e.g. by monitoring changes in the F350/F330 fluorescence ratio. From this GuaHCl concentration dependence of unfolding, ∆G° can be calculated (Figure 1). Importantly, based on the Gibbs-Helmholtz law and van´t Hoff equation (∆G° = - RTlnK), ∆G° can also be used to very precisely determine the fraction of unfolded protein, which can then be used to calculate back the fraction of unfolded protein at [D] = 0 (Table 1). Figure 1: Chemical denaturation curve of a mAb, analyzed by detecting changes in the fluorescence emission ratio using the Prometheus NT.Plex. The fit was performed using a three-state unfolding model.

Articles in this issue

view archives of Application Notes - Getting the full picture: predicting the aggregation propensity of mAbs using chemical and thermal denaturation on a single, fully automated platform