Advanced analyses of kinetic stabilities of iggs modified by mutations and glycosylation

Abstract

The stability of Immunoglobulin G (IgG) affects production, storage and usability, especially in the clinic. The complex thermal and isothermal transitions of IgGs, especially their irreversibilities, pose a challenge to the proper determination of parameters describing their thermodynamic and kinetic stability. Here, we present a reliable mathematical model to study the irreversible thermal denaturations of antibody variants. The model was applied to two unrelated IgGs and their variants with stabilizing mutations as well as corresponding non-glycosylated forms of IgGs and Fab fragments. Thermal denaturations of IgGs were analyzed with three transitions, one reversible transition corresponding to C H 2 domain unfolding followed by two consecutive irreversible transitions corresponding to Fab and C H 3 domains, respectively. The parameters obtained allowed us to examine the effects of these mutations on the stabilities of individual domains within the full-length IgG. We found that the kinetic stability of the individual Fab fragment is significantly lowered within the IgG context, possibly because of intramolecular aggregation upon heating, while the stabilizing mutations have an especially beneficial effect. Thermal denaturations of nonglycosylated variants of IgG consist of more than three transitions and could not be analyzed by our model. However, isothermal denaturations demonstrated that the lack of glycosylation affects the stability of all and not just of the C H 2 domain, suggesting that the partially unfolded domains may interact with each other during unfolding. Investigating thermal denaturation of IgGs according to our model provides a valuable tool for detecting subtle changes in thermodynamic and/or kinetic stabilities of individual domains.

Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering

“…For the analysis of the kinetic stability of IgG with our previously derived mathematical model we chose the well‐characterized antibody IgG6B3, a human IgG1, that contains a heavy chain of subclass V H 6 and a light chain of the V λ 3 family …”

“…The well‐characterized IgG6B3 binds myoglobin and had been selected from the Human Combinatorial Antibody Library (HuCAL) . Protein was produced from a stable HEK293 clone as described previously as well as from transiently transfected FreeStyle 293‐F cells (Thermo Fisher Scientific) according to the protocol for HEK cell lines summarized by Hacker et al Subsequently, IgGs were affinity‐purified from cleared supernatants on Protein A columns (GE Healthcare Life Sciences). Eluted fractions were pooled and dialyzed against PBS, pH 7.4, (Thermo Fisher Scientific) before proteins were used for subsequent analyses.…”

“…The experimental data of the excess heat capacity of IgG obtained from DSC were fitted by our recently derived equation which reflects the model of thermal denaturation of IgG consisting of three consecutive transitions (one reversible step followed by two irreversible transitions): where N is the native state, U and D are intermediate states of thermal denaturation and F is the final (denatured) state of the IgG; K is the equilibrium constant of the first transition, k 2 and k 3 are the rate constants of the corresponding irreversible reactions. The excess heat capacity, being the parameter measured in the DSC experiments, is expressed by the general equation satisfying the model: where Δ H 1 , Δ H 2 , and Δ H 3 are the molar enthalpy changes for the first, second and third steps, respectively.…”

“…Hence, the long‐term stability of therapeutic candidates in the pharmaceutical development of new biological drugs is the focus of intensive studies regarding both their conformational and colloidal stabilities. Thus, understanding factors determining IgG stability or predicting it at suitable storage temperatures is a critical goal in biopharmaceutical studies.…”

“…Recently, we have developed a kinetic model addressing the complex thermal denaturation of IgGs that consists of three consecutive steps: one reversible transition (corresponding to C H 2 domain unfolding) followed by two irreversible transitions, corresponding to the unfolding of the Fab and C H 3 domains, respectively . This model enabled us to determine parameters quantitatively describing each step of the IgG thermal denaturation, such as the equilibrium constant of the first transitions and the rate constants of the irreversible steps at any given temperature.…”