Blech Michaela, Melien Richard, Tschammer Nuska, Presser Beate, Hinderberger Dariush, Garidel Patrick
Boehringer Ingelheim Pharma GmbH & Co. KG, Innovation Unit, Pharmaceutical Development Biologicals, 88397, Biberach an der Riss, Germany.
Institute of Chemistry, Martin-Luther-Universität Halle-Wittenberg, von-Danckelmann-Platz 4, 06120, Halle (Saale), Germany.
Pharm Res. 2021 Dec;38(12):2065-2089. doi: 10.1007/s11095-021-03120-x. Epub 2021 Dec 13.
Introduction of the activation energy (E) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability.
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.
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.
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.
引入活化能（E）作为动力学参数，以描述和区分单克隆抗体（mAb）的稳定性。
E由内在荧光（IF）解折叠热谱图得出。基于阿伦尼乌斯积分建立了一个表观不可逆三态拟合模型，以确定各个解折叠转变的E。将这些活化能与范特霍夫焓（∆H）的热力学参数进行比较。使用一组以四种不同配方配制的34种单克隆抗体，将表观热力学和动力学参数以及表观解链温度与储存稳定性进行集体关联，以评估其对可能反映在保质期内的长期影响的预测能力。
E能够区分（i）不同的亲本单克隆抗体，（ii）源自亲本单克隆抗体的不同变体，以及（iii）不同的配方。有趣的是，我们观察到，与CH转变的∆H和T相比，在加速和应激条件下储存后，CH解折叠转变的E与单体和聚集体含量的相关性最强。此外，CH结构域确定的预测参数与单体和聚集体含量的相关性通常比Fab结构域确定的参数更强。通过对Fab结构域的E进行排序进行的定性评估与各个单克隆抗体亚组储存稳定性中的单体含量显示出良好的一致性。
除其他常用的热力学预测参数外，IF解折叠转变的E可用于区分和表征不同配方中不同单克隆抗体的热稳定性。因此，它在抗体工程和配方科学家方面显示出巨大潜力。
