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

PURPOSE

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

METHODS

E 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 of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆H). 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.

RESULTS

E 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 of the CH unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆H and T of the CH transition. Moreover, the predictive parameters determined for the CH domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking E of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets.

CONCLUSION

E 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.