Scan-Rate-Dependent Melting Transitions of Interleukin-1 Receptor (Type II):  Elucidation of Meaningful Thermodynamic and Kinetic Parameters of Aggregation Acquired from DSC Simulations

Abstract

The role of thermal unfolding as it pertains to thermodynamic properties of proteins and their stability has been the subject of study for more than 50 years. Moreover, exactly how the unfolding properties of a given protein system may influence the kinetics of aggregation has not been fully characterized. In the study of recombinant human Interleukin-1 receptor type II (rhuIL-1R(II)) aggregation, data obtained from size exclusion chromatography and differential scanning calorimetry (DSC) were used to model the thermodynamic and kinetic properties of irreversible denaturation. A break from linearity in the initial aggregation rates as a function of 1/T was observed in the vicinity of the melting transition temperature (T(m) approximately 53.5 degrees C), suggesting significant involvement of protein unfolding in the reaction pathway. A scan-rate dependence in the DSC experiment testifies to the nonequilibrium influences of the aggregation process. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from an irreversibly denatured process. The model was used to simulate how unfolding properties could be used to predict aggregation rates at different temperatures above and below the T(m) and to account for concentration dependence of reaction rates. The model was shown to uniquely identify the thermodynamic parameters DeltaC(P) (1.3 +/- 0.7 kcal/mol-K), DeltaH(m) (74.3 +/- 6.8 kcal/mol), and T(m) with reasonable variances.

Contribution of Variable Domains to the Stability of Humanized IgG1 Monoclonal Antibodies

“…There are few examples of quantitative analysis of thermograms with a model composed of a two-state reversible unfolding reaction followed by an irreversible process of aggregation. [31][32][33] We did not attempt to use that model because it is obvious that a two-state reversible equation followed by an irreversible step cannot describe the process of multi-domain unfolding of a monoclonal antibody.…”

Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

“…[94][95][96][97] It is well established that accurate values for unfolding thermodynamics in such cases cannot be directly obtained from DSC or similar measurements without additional analysis and assumptions. 57,96,98 Canonical signatures of nonnative aggregation during DSC or other thermal unfolding measurements include a significant downshift in peak position or inflection point with decreasing thermal scan rate and/or increasing c 0 .…”

“…[94][95][96][97] It is well established that accurate values for unfolding thermodynamics in such cases cannot be directly obtained from DSC or similar measurements without additional analysis and assumptions. 57,96,98 Canonical signatures of nonnative aggregation during DSC or other thermal unfolding measurements include a significant downshift in peak position or inflection point with decreasing thermal scan rate and/or increasing c 0 . 36,43,57,97 A pragmatic way to try to circumvent this problem for proteins that do not dissociate upon unfolding is to perform unfolding measurements at sufficiently low protein concentration to suppress aggregation rates on the time scales of the measurement ($10-10 2 min); 36,43 this follows naturally from the strong c 0 dependence of k obs for all but unfolding-limited kinetics.…”

“…[94][95][96][97] This is the same as the apparent melting temperature for irreversible DSC. For a two-state protein during thermal unfolding monitored spectroscopically, T m is effectively the temperature of the inflection point.…”

A thermodynamic analysis of the binding interaction between polysorbate 20 and 80 with human serum albumins and immunoglobulins: A contribution to understand colloidal protein stabilisation

“…However, for these therapeutics the doses per application are quite high reaching protein concentration up to 100 mg/ml. For such protein dosage forms, beyond chemical stability, physical and colloidal stability of the protein in an aqueous solution becomes of high relevance [3][4][5][6][7]. The main physical degradation encountered is protein aggregation, which reduces bioactivity and product quality [8,9].…”