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

Hydraulic characteristics of smart reactor for a nominal condition

SMART (System-integrated Modular Advanced ReacTor) is an integral-type reactor being developed, which has major components including core, pumps, steam generators and a pressurizer inside the reactor vessel. In order to analyze the various safety features of the reactor, the quantification for the flow and pressure distributions are very important. A test facility, named “SCOP”, was designed based on the conservation of Euler number which is a ratio of pressure drop to dynamic pressure with a sufficiently high Reynolds number. In order to preserve the flow distribution characteristics, the SCOP is linearly reduced with a scaling ratio of 1/5. For the present work, a total of 9 tests were performed for a nominal SMART flow condition. By using the test results, a statistical final flow distribution for the SMART reactor were presented. The current data could be applied for the validation of a CFD analysis method as well as reactor safety and system performance analysesPaper presented at the 9th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Malta, 16-18 July, 2012

Hydraulic characteristics of smart reactor for a nominal condition

Paper presented at the 9th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Malta, 16-18 July, 2012.SMART (System-integrated Modular Advanced ReacTor) is an integral-type reactor being developed, which has major components including core, pumps, steam generators and a pressurizer inside the reactor vessel. In order to analyze the various safety features of the reactor, the quantification for the flow and pressure distributions are very important. A test facility, named “SCOP”, was designed based on the conservation of Euler number which is a ratio of pressure drop to dynamic pressure with a sufficiently high Reynolds number. In order to preserve the flow distribution characteristics, the SCOP is linearly reduced with a scaling ratio of 1/5. For the present work, a total of 9 tests were performed for a nominal SMART flow condition. By using the test results, a statistical final flow distribution for the SMART reactor were presented. The current data could be applied for the validation of a CFD analysis method as well as reactor safety and system performance analyses.dc201

Electrodeposition of Co-Ni-MoxOy Powders: Part I. The Influence of Deposition Conditions on Powder Composition and Morphology

The Co-Ni-MoxOy powders were obtained electrochemically at a constant current density from ammonia electrolyte. Ni and Co were anomalously deposited, inducing Mo deposition, which cannot be deposited separately from aqueous solutions. The obtained Co-Ni-MoxOy powders were investigated by energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and scanning electon microscope (SEM) methods. Based on the obtained experimental results, it was concluded that the particle size of deposited powders is influenced by the chemical composition of the electrolyte and current density imposed. XRD results suggested that obtained powders were of amorphous structure, although a Co3Mo compound can be formed if certain experimental conditions are applied

Search for leptophobic Z ' bosons decaying into four-lepton final states in proton-proton collisions at root s=8 TeV

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Search for black holes and other new phenomena in high-multiplicity final states in proton-proton collisions at root s=13 TeV

Peer reviewe