Unfolding and Aggregation of Therapeutic Proteins

Summary

Protein drugs are the fastest growing class of drugs in the pharmaceutical industry. However, they can be plagued by long term stability issues, including aggregation and phase separation, that have yet to be fully understood. We have demonstrated an approach to characterize protein unfolding and aggregation and provide insight into destabilization mechanisms.

Description

A stable protein formulation is very important to the safety of mAb drugs. In particular, mAb drug solutions formulated at high concentrations can undergo various biophysical instabilities, such as aggregation, liquid-liquid phase separation, and can also have high viscosities. These undesired solution properties are primarily driven by attractive protein-protein interactions. In fact, preventing such interactions during long term storage is a major challenge faced by the biotech industry. Consequently, formulation condition is key to ensuring a robust and long-term shelf life of protein drug solutions.

A fundamental understanding of the impact of temperature, pH, viscosity, and addition of salts on the biophysical stability of mAbs could potentially help predict long term stability. Our group systematically studies the effects of salt (e.g., the Hofmeister series), temperature and pH on the unfolding and aggregation kinetics of a low pI mAb. This approach allows for a detailed, mechanistic investigation of salt effects across various charged states of the mAb while maintaining relevant formulations and physiological conditions.

We have studied the biophysical stability of a therapeutic protein sample using various optical methods [1]. Dependences of the thermal stability on pH, salt, and temperature provide insights into detailed mechanisms of domain unfolding and solvent interactions. This mechanistic study can help improve the shelf-life of protein drug products and understand protein aggregation-related disease mechanisms. We also monitored the aggregation kinetics by the solvent absorption compensation (SAC) IR spectroscopy. The newly developed SAC-IR spectroscopy

Figure 1. Isothermal aggregation kinetics of a low-pI mAb solution at 60 ºC monitored by light scattering [1]. (inset) Schematic diagram depicting possible mechanisms of the effects of chaotropic ions such as magnesium (Mg²⁺) and thiocyanate (SCN^–) on the physical stability of a slightly acidic mAb.

Figure 2. Time-resolved SAC-IR absorption spectra of insulin solutions at (a) 17.5 mg/mL and (b) 3.5 mg/mL concentrations at 74 °C and pH = 2 [2]. Each spectrum is baseline detrended by absorbances at 1480 and 1750 cm^-1. (c) and (d) Peak fitting of the amide I band with two Gaussian functions. (e) and (f) Areas of the two underlying peaks plotted as a function of time.

PUBLICATIONS

[1]  K. B. Rembert, J. Zhang, Y. J. Lee, Effects of Salts and Surface Charge on the Biophysical Stability of a low pI Monoclonal Antibody, J. Pharmaceut. Sci. Published online (2022). https://doi.org/10.1016/j.xphs.2022.11.010

[2] B. Chon, S. Xu, Y. J. Lee, Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions. Anal. Chem. 93, 2215 (2021). https://dx.doi.org/10.1021/acs.analchem.0c04091