Expanding the toolbox for predictive parameters describing antibody stability considering thermodynamic and kinetic...

Introduction of the activation energy (E_a) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability.

E_a is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine E_a of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆H_vh). Using a set of 34 mAbs formulated in four different formulations, both the apparent thermodynamic and kinetic parameters together with apparent melting temperatures are correlated collectively with each other to storage stabilities to evaluate its predictive power with respect to long-term effects potentially reflected in shelf-life.

E_a allows for the discrimination of (i) different parent mAbs, (ii) different variants that originate from parent mAbs, and (iii) different formulations. Interestingly, we observed that the E_a of the CH₂ unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆H_vh and T_m of the CH₂ transition. Moreover, the predictive parameters determined for the CH₂ domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking E_a of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets.

E_a from IF unfolding transitions can be used in addition to other commonly used thermodynamic predictive parameters to discriminate and characterize thermal stability of different mAbs in different formulations. Hence, it shows great potential for antibody engineering and formulation scientists.