Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE‐ESI‐MS/MS system

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic‐labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope‐labeled lysine or arginine. The heavy‐labeled peptides were spiked in the HCP digests at known concentrations. After CZE‐ESI‐MS/MS analysis, the peaks of native and isotopic‐labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic‐labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R² values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7–30 ppt level in the purified HCP samples.     

A charge iteration-corrected extended-Hückel study of the electronic and spectroscopic properties of conjugated heterocycles

A computationally inexpensive modified extended-Hückel method using coordinates from STO-6G(d,p) geometry optimizations is described and shown to accurately simulate the spectra of conjugated heterocycles. The origins of the natural optical activities of blue indophenines and diheptylindophenines, and several related brown, red, and colorless compounds, were investigated with this method to show how it can be used to examine the changes in spectra and electronic structure that occur with substituent additions and changes. Diheptylindophenines were found to be blue because of absorptions from electronic transitions between spatially congruent π and π* molecular orbitals delocalized across carbon, nitrogen, oxygen, and sulfur atomic orbitals. The effects of conjugated thiophene moieties replacing the isatin moiety β-carbonyls move these absorptions to the blue relative to their locations in n-heptylisatin, and the diheptyl groups move these absorptions slightly to the red relative to indophenines.     

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies

We have evaluated CZE‐ESI‐MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5‐point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE‐MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ～70‐min separation window (～90‐min total analysis duration) and ～300‐peak capacity. We also analyzed the sample using ultra‐performance LC‐MS/MS. CZE‐MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low‐level spiked proteins. Both methods detected all proteins spiked at ～100 ppm level with respect to the antibody.     

Search for approaches to improving the calculation accuracy of the protein—ligand binding energy by docking

The following reasons limiting the accuracy of calculations of the protein—ligand binding energy by the molecular docking programs are considered: the limited facilities of algorithms of finding a global minimum on a complicated multi-dimensional protein—ligand energy surface, restrictions on the degrees of freedom of a protein—ligand system including docking into a rigid protein, inadequacy of the existing force fields, a lack of taking into account the solvent or too rough allowance for the solvent in the docking procedure, a lack of the local energy optimization in the docking process, an inaccuracy of the construction of models of a target protein and a ligand, simplification of the calculation method of the Gibbs free energy of a molecular system, and limited computer resources for docking of one ligand. A new approach to the development of the new generation of docking programs is proposed. The approach allows one to remove step-by-step the existing simplifications and to increase considerably the accuracy of the whole docking process, including the calculation accuracy of the protein—ligand binding energy. The results of the study are presented and demonstrate the computational feasibility of the assigned docking problem.     

Comparing normal modes across different models and scales: Hessian reduction versus coarse‐graining

Dimension reduction is often necessary when attempting to reach longer length and time scales in molecular simulations. It is realized by constraining degrees of freedom or by coarse‐graining the system. When evaluating the accuracy of a dimensional reduction, there is a practical challenge: the models yield vectors with different lengths, making a comparison by calculating their dot product impossible. This article investigates mapping procedures for normal mode analysis. We first review a horizontal mapping procedure for the reduced Hessian techniques, which projects out degrees of freedom. We then design a vertical mapping procedure for the “implosion” of the all‐atom (AA) Hessian to a coarse‐grained scale that is based upon vibrational subsystem analysis. This latter method derives both effective force constants and an effective kinetic tensor. Next, a series of metrics is presented for comparison across different scales, where special attention is given to proper mass‐weighting. The dimension‐dependent metrics, which require prior mapping for proper evaluation, are frequencies, overlap of normal mode vectors, probability similarity, Hessian similarity, collectivity of modes, and thermal fluctuations. The dimension‐independent metrics are shape derivatives, elastic modulus, vibrational free energy differences, heat capacity, and projection on a predefined basis set. The power of these metrics to distinguish between reasonable and unreasonable models is tested on a toy alpha helix system and a globular protein; both are represented at several scales: the AA scale, a Gō‐like model, a canonical elastic network model, and a network model with intentionally unphysical force constants. Published 2012 Wiley Periodicals, Inc.     

Geometric limits of knot complements, II: graphs determined by their complements

We prove that there are compact submanifolds of the 3-sphere whose interiors are not homeomorphic to any geometric limit of hyperbolic knot complements.     

In Situ Formation of Gold Nanoparticles within a Polymer Particle and Their Catalytic Activities in Various Chemical Reactions

Composite materials consisting of nanoscale gold particles and protective polymer shells were designed and tested as catalysts in various chemical reactions. Initially, the systematic incorporation of multiple gold nanoparticles into a poly(N-isopropylacrylamide) particle was achieved by an in situ method under light irradiation. The degree of gold nanoparticle loading, along with the structural and morphological properties, was examined as a function of the amount of initial gold ions and reducing agent. As these gold nanoparticles were physically-embedded within the polymer particle in the absence of strong interfacial interactions between the gold nanoparticles and polymer matrix, the readily-accessible surface of the gold nanoparticles with a highly increased stability allowed for their use as recyclable catalysts in oxidation, reduction, and coupling reactions. Overall, the ability to integrate catalytically-active metal nanoparticles within polymer particles in situ allows for designing novel composite materials for multi-purpose catalytic systems.     

Synthesis,Structural, and Optical Characterization of a New Europium(II) Tin Selenide,Eu8(Sn4Se14)(Se3)2 with a (Sn4Se14)12– Building Block

A new phase in europium‐tin‐chalcogenide chemistry has been prepared using the reactive flux method: Eu₈(Sn₄Se₁₄)(Se₃)₂. The compound crystallizes in the orthorhombic space group P2₁2₁2 with cell parameters a = 11.990(2) Å, b = 16.425(4) Å, c = 8.543(1) Å, and Z = 2. Eu₈(Sn₄Se₁₄)(Se₃)₂ is a three dimensional structure with Eu^II cations linked together with an unusual (Sn₄Se₁₄)^12– anionic unit and (Se₃)^2– chains. UV‐VIS‐NIR band‐gap analysis shows that these black metallic crystals are likely semiconductors with an optical band‐gap of 1.07 eV.     

Double-walled pyr topology networks from a novel fluoride-bridged heptanuclear metal cluster

Two isostructural metal–organic materials, Tripp-1-M (Tripp = 2,4,6-tris(4-pyridyl)pyridine; M = Co, Ni), that exhibit binodal 3,6-connected pyr network topology have been prepared and characterized. Tripp-1-M are based upon a novel M₇F₁₂ ²⁺ cluster that possesses 12 connection points but, because of double cross-linking by 3-connected Tripp ligands, it functions as a 6-connected supermolecular building block (SBB).     

PHOTOPHYSICAL BEHAVI0RS OF OLIGOMER BASED ON 1, 1′-BINAPHTHOL WITH 3, 3′-ACETYLENE SPACER*

The photophysical behaviors of the oligomer based on 1,1'-binaphthol with3,3'-acetylene spacer were investigated. The oligomer molecule has a naphthyl-acetylene-naphthyl effective conjugation segment. The atropic of the 1,1'-binaphthyl moiety led totwisted and rigid main chain in the oligomer. With the changes of the external environ-ment such as solvents used, solvent viscosity and ambient temperature, the wavelengthsof absorption and the intensities of fluorescence and absorption are changed slightly, butthe fluorescent intensity and quantum yield can be influenced. The luminescent behav-iors of the oligomer exhibit twisted intramolecular charge transfer characteristics, whichcould have a potential application in wavelength-stable light emitting material adaptableto ambient temperature and the solvents used in wide range.