Oxidation of methionine residues in protein pharmaceuticals

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004.Includes bibliographical references (p. 171-189).(cont.) of free methionine. Therefore, the environments surrounding different methionine sites in G-CSF mainly provide spatial restriction to the access to the solvent but do not affect oxidation in a specific manner, consistent with the good correlation between 2SWCN's and the rates of oxidation. A comprehensive picture of oxidation is thus developed. It allows an accurate prediction of protein oxidation, and provides a rationale for developing strategies to control oxidation, such as modulating protein conformation via adding excipients. This knowledge could aid in developing in a more rational manner solvent formulations that protect therapeutic proteins against oxidation.Oxidation of the amino acid methionine by peroxides in aqueous formulations of proteins is a critical issue in the development of therapeutic products. It must be controlled so that therapeutic proteins can maintain their activity. In addition, oxidized therapeutics are undesirable due to their possible immunogenetic effects. An understanding of the mechanism and the factors that influence the reactivity of different methionine sites toward oxidation is therefore important. In this thesis, computational methods are applied and developed to address these problems. First, a mechanism by which peroxides oxidize the sulfur atom of methionine is developed. The rate-limiting step was found to be the breaking of the O-O bond of H₂O₂ and the formation of the S-O bond during which significant charge separation is developed. The charge separation can be stabilized via specific interactions such as hydrogen bonding with surrounding water molecules. This "water-mediated" mechanism of oxidation is consistent with experimental data such as those on activation energies of oxidation and pH dependence of the rates of oxidation. Based on the "water-mediated" mechanism, a structural property, average 2-shell water coordination number (2SWCN), has been shown to correlate well to the rates of oxidation of different methionine groups in Granulocyte Colony-Stimulating Factor (G-CSF) and in a Human Parathyroid hormone fragment (hPTH(1-34)). Including the dynamics of protein and water molecules in an explicit manner was found to be important for such correlation. Via combined quantum mechanical and molecular mechanical free energy simulations, the activation free energies of the oxidation of methionine residues in G-CSF are found to be equivalent to the values for the oxidationby Jhih-Wei Chu.Ph.D

Understanding Oxidative Instability of Protein Pharmaceuticals

Mechanism of oxidation of methionine residues in protein pharmaceuticals by hydrogen peroxide was investigated via ab initio calculations. Specifically, two reactions, hydrogen transfer of hydrogen peroxide to form water oxide and the oxidation of dimethyl sulfide (DMS) by hydrogen peroxide to form dimethyl sulfoxide, were studied as models of these processes in general. Solvent effects are included both via including explicitly water molecules and via the polarizable continuum model. Specific interactions including hydrogen bonding with 2-3 water molecules can provide enough stabilization for the charge separation at the activation complex. The major reaction coordinates of the reaction are the breaking of the O-O bond of H₂O₂ and the formation of the S-O bond, the transfer of hydrogen to the distal oxygen of hydrogen peroxide occurring after the system has passed the transition state. Reaction barriers of the hydrogen transfer of H₂O₂ are in average of 10 kcal/mol or higher than the oxidation of DMS. Therefore, a two step oxidation mechanism in which the transfer of hydrogen atom occurs first to form water oxide and the transfer of oxygen to substrate occurs as the second step, is unlikely to be correct. Our proposed oxidation mechanism does not suggest pH dependence of oxidation rate within a moderate range around neutral pH (i.e. under conditions in which hydronium and hydroxide ions do not participate directly in the reaction), and it agrees with experimental observations over moderate pH values.Singapore-MIT Alliance (SMA

On the Oxidation of Methionine Residues during the Storage of Protein Pharmaceuticals in an Aqueous Formulation

This study addresses the fundamentals of an important degradation pathway of storing protein pharmaceuticals in an aqueous formulation, oxidation of methionine residues by peroxides. First, a mechanism by which methionine residues are oxidized is identified via ab initio calculations. The major difference of this new mechanism to previous ones is the role of solvent molecules in the oxidation process. Previously proposed mechanisms suggested that solvent molecules facilitate the transfer of hydrogen associated with the oxidation reaction, but the estimated activation energies and pH dependence of oxidation rates derived from this mechanism rates do not agree with experimental observations. In our proposed mechanism, however, water molecules stabilize the charge separation in the transition-state complex through specific interaction such as hydrogen bonding. This mechanism satisfies all experimental studies on the oxidation of organic sulfides by peroxides. A correct picture of instability mechanism is essential in developing stabilization strategies to design a robust formulation. Based on this mechanism, a structure/instability relationship is built to explain the oxidation rates of methionine residues in a protein molecule. Specifically, a structural property, two-shell water coordination number, is found to correlate semi-quantitatively to the rates of oxidation of methionine residues in G-CSF (granulocyte colony-stimulating factor) and hPTH (human parathoid hormone). We also show that a traditionally used structural property, solvent accessible area, can not provide such accurate correlation and that the dynamic motion of protein molecules and an explicit treatment of solvent molecules are essential to describe the rates of oxidation of methionine residues. Furthermore, the insight provided by the molecule-level understanding in developing a stabilizing formulation is discussed.Singapore-MIT Alliance (SMA

Stabilization of Therapeutic Proteins

We present results of molecular simulations, quantum mechanical calculations, and experimental data aimed towards the rational design of solvent formulations. In particular, we have found that the rate limitation of oxidation of methionine groups is determined by the breaking of O-O bonds in hydrogen peroxide, not by the rate of acidic catalysis as previously thought. We have used this understanding to design molecular level parameters which are correlated to experimental data. Rate data has been determined both for G-CSF and for hPTH(1-34).Singapore-MIT Alliance (SMA

Molecular computations for reactions and phase transitions: applications to protein stabilization, hydrates and catalysis

In this work we have made significant contributions in three different areas of interest: therapeutic protein stabilization, thermodynamics of natural gas clathrate-hydrates, and zeolite catalysis. In all three fields, using our various computational techniques, we have been able to elucidate phenomena that are difficult or impossible to explain experimentally. More specifically, in mixed solvent systems for proteins we developed a statistical-mechanical method to model the thermodynamic effects of additives in molecular-level detail. It was the first method demonstrated to have truly predictive (no adjustable parameters) capability for real protein systems. We also describe a novel mechanism that slows protein association reactions, called the “gap effect.” We developed a comprehensive picture of methioine oxidation by hydrogen peroxide that allows for accurate prediction of protein oxidation and provides a rationale for developing strategies to control oxidation. The method of solvent accessible area (SAA) was shown not to correlate well with oxidation rates. A new property, averaged two-shell water coordination number (2SWCN) was identified and shown to correlate well with oxidation rates. Reference parameters for the van der Waals Platteeuw model of clathrate-hydrates were found for structure I and structure II. These reference parameters are independent of the potential form (unlike the commonly used parameters) and have been validated by calculating phase behavior and structural transitions for mixed hydrate systems. These calculations are validated with experimental data for both structures and for systems that undergo transitions from one structure to another. This is the first method of calculating hydrate thermodynamics to demonstrate predictive capability for phase equilibria, structural changes, and occupancy in pure and mixed hydrate systems. We have computed a new mechanism for the methanol coupling reaction to form ethanol and water in the zeolite chabazite. The mechanism at 400°C proceeds via stable intermediates of water, methane, and protonated formaldehyde.Singapore-MIT Alliance (SMA

SIMD Everywhere Optimization from ARM NEON to RISC-V Vector Extensions

Many libraries, such as OpenCV, FFmpeg, XNNPACK, and Eigen, utilize Arm or x86 SIMD Intrinsics to optimize programs for performance. With the emergence of RISC-V Vector Extensions (RVV), there is a need to migrate these performance legacy codes for RVV. Currently, the migration of NEON code to RVV code requires manual rewriting, which is a time-consuming and error-prone process. In this work, we use the open source tool, "SIMD Everywhere" (SIMDe), to automate the migration. Our primary task is to enhance SIMDe to enable the conversion of ARM NEON Intrinsics types and functions to their corresponding RVV Intrinsics types and functions. For type conversion, we devise strategies to convert Neon Intrinsics types to RVV Intrinsics by considering the vector length agnostic (vla) architectures. With function conversions, we analyze commonly used conversion methods in SIMDe and develop customized conversions for each function based on the results of RVV code generations. In our experiments with Google XNNPACK library, our enhanced SIMDe achieves speedup ranging from 1.51x to 5.13x compared to the original SIMDe, which does not utilize customized RVV implementations for the conversions

Ferroelectric Control of the Conduction at the LaAlO 3 /SrTiO 3 Hetero-interface

Abstract The LaAlO 3 /SrTiO 3 (LAO/STO) interface serves as a model system in which a highly mobile quasi-twodimensional electron gas (2DEG) forms between two band insulator

CHARMM at 45: Enhancements in Accessibility, Functionality, and Speed

Since its inception nearly a half century ago, CHARMM has been playing a central role in computational biochemistry and biophysics. Commensurate with the developments in experimental research and advances in computer hardware, the range of methods and applicability of CHARMM have also grown. This review summarizes major developments that occurred after 2009 when the last review of CHARMM was published. They include the following: new faster simulation engines, accessible user interfaces for convenient workflows, and a vast array of simulation and analysis methods that encompass quantum mechanical, atomistic, and coarse-grained levels, as well as extensive coverage of force fields. In addition to providing the current snapshot of the CHARMM development, this review may serve as a starting point for exploring relevant theories and computational methods for tackling contemporary and emerging problems in biomolecular systems. CHARMM is freely available for academic and nonprofit research at https://academiccharmm.org/program