Mechanistic studies on chemical instabilities of recombinant proteins
Author(s)Pan, Bin, Ph. D. Massachusetts Institute of Technology
Massachusetts Institute of Technology. Dept. of Chemical Engineering.
Daniel I. C. Wang and Bernhardt L. Trout.
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Protein molecules are being widely used as pharmaceuticals for treating diseases ranging from diabetes and haemophilia to various types of cancers due to their great potency and specificity. However, these macromolecules are intrinsically unstable in aqueous solutions, due to the existence of various physical and chemical degradation pathways. Degraded protein molecules have much reduced biological functions, and may also have adverse effects such as immunogenriicity or pharmacokinetic issues. Thus, understanding the underlying mechanisms of these degradation pathways is essential for rationally devising better ways to stabilize protein pharmaceuticals and extends their applicability. In this thesis, two important types of chemical degradation pathways, the oxidation of methionine residues and the hydrolysis of peptide bonds in monoclonal antibody molecules, are investigated from a mechanistic point of view. In the first half of the thesis, oxidation 'of methionine residues in a model protein G-CSF (Granulocyte-Colony Stimulating Factor) was studied to address the issue of how protein structure affects its reactivity. Comparative oxidation studies were performed where the kinetics of oxidation of methionine residues by hydrogen peroxide (H₂0₂) in G-CSF and corresponding chemically synthesized peptides thereof were measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on G-CSF to obtain the free energy of unfolding as a function of temperature.(cont.) A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range studied, 4°C to 45°C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One out of the four methionine residues, Met122, showed an activation energy significantly different than that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Phenomenological modeling trying to understand the temperature dependence of rate constants was pursued. Finally, we show that the data obtained from accelerated oxidation can be used in conjunction with our models to get predictions about the long-term shelf-life oxidation comparable with experimental results. In the latter half of this thesis, three approaches in a hierarchical order were taken in order to explain the higher rate of un-catalyzed hydrolysis of peptide bonds only in the hinge region of antibody molecules. First, ab initio molecular dynamic simulations were performed to understand the reaction mechanism of the hydrolysis of peptide bonds. The system solvated in explicit water molecules was modeled quantum mechanically and dynamic transition trajectories of the chemical reaction were computed at ambient conditions.(cont.) Since no unique pathway can be used to describe the reaction process due to fluctuations at finite temperature, path sampling technique was applied to obtain an ensemble of trajectories. A statistical tool, likelihood maximization, was used to extract physically important degrees of freedom by screening a large number of reaction coordinate models. The same approach was applied to the hydrolytic reactions under both acidic and neutral pH conditions, which are the most relevant to the formulation of antibody molecules. In both cases, changes in local bonding pattern close to the reaction center, as well as the solvent network, showed importance in determining the reaction dynamics of the hydrolysis of the peptide bond. Then classical molecular dynamic simulations were performed to study the dynamics of a free hinge fragment and -the hinge fragments in the antibody molecule. Important structural and dynamic differences between the two situations were revealed, especially the observation that the free hinge fragment takes on configurations much less frequently accessed by the hinge fragment when situated inside the antibody molecule. In the third approach, a coarse-grained reaction rate model was proposed in order to explain the experimentally observed higher rate of hydrolysis of peptide bonds. A hypothesis involving a mechano-chemical mechanism was motivated by the essential constraining effect of Fab and Fc domains on the hinge region in the antibody molecule revealed in the second approach.(cont.) Combining the information obtained from the previous two approaches, force was calculated along the reaction coordinate direction that was determined and verified previously. This information was integrated into a reaction rate model in order to compute the reaction rate constants. The computational results show that the mechano-chemical mechanism can yield reasonable rate constants comparable with available experimental data.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Chemical Engineering.; Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology