Non-Arrhenius Protein Aggregation

Abstract

Abstract. Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

“…Finally, based on general principles of protein unfolding thermodynamics, DH un usually decreases greatly with decreasing temperature, and therefore diagrams such as Figure 7 are anticipated to show upward curvature for the non-Arrhenius portion of the diagram. 57 Returning to Figure 7, and with the above arguments in mind, the breakpoint occurring at the T m of the C H 2 domain argues that C H 2 unfolding is required for aggregation. That is qualitatively in keeping with prior results at much lower pH conditions for Fc1 fragments.…”

“…Finally, it is not currently possible to quantitatively predict, a priori, the curvature in the non-Arrhenius regimes for Figure 7 or analogous conditions for other proteins 34,57,58,59 without accurate determination of quantities such as the heat capacity of unfolding for the regime or domain that needs to unfold to aggregate. Unfortunately, unfolding in DSC is often not reversible and one can only qualitatively or semi-quantitatively estimate this key quantity.…”

“…Unfortunately, unfolding in DSC is often not reversible and one can only qualitatively or semi-quantitatively estimate this key quantity. 57,59 Therefore, predicting 55 and re-plotted here for comparison (breakpoints around 67 C, marked by the short dash-dot arrow). The short dash-dot lines are also only guides to the eyes.…”

Kinetic stability of membrane proteins

“…Linear Arrhenius plots indicate a common mechanism for thermal inactivation in the explored temperature range. On the contrary, non-linear Arrhenius plots can be found when aggregation becomes a primary cause of inactivation (Wang and Roberts 2013,) generally at the upper limit of the temperature ranges.…”

Commentary: Current Perspectives on the Aggregation of Protein Drugs

“…of Chemical and Biomolecular Engineering, University of Delaware) presented his group's recent work on the mechanisms of protein aggregation and origins of non-Arrhenius behavior (8,9). In this themed issue, he and Dr. Wei Wang (Pfizer BioTherapeutics) summarize these mechanistic insights and discuss the implications for accelerated stability testing (10).…”