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.     

Effects of Active‐Site Modification and Quaternary Structure on the Regioselectivity of Catechol‐O‐Methyltransferase

Catechol‐O‐methyltransferase (COMT), an important therapeutic target in the treatment of Parkinson's disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta‐ or para‐methylated catechols. X‐ray crystallography further revealed how the active‐site residues and quaternary structure govern regioselectivity. Finally, analogues of AdoMet are accepted by the regiocomplementary COMT mutants and can be used to prepare alkylated catechols, including ethyl vanillin.     

Searching for globally optimal functional forms for interatomic potentials using genetic programming with parallel tempering

Molecular dynamics and other molecular simulation methods rely on a potential energy function, based only on the relative coordinates of the atomic nuclei. Such a function, called a force field, approximately represents the electronic structure interactions of a condensed matter system. Developing such approximate functions and fitting their parameters remains an arduous, time-consuming process, relying on expert physical intuition. To address this problem, a functional programming methodology was developed that may enable automated discovery of entirely new force-field functional forms, while simultaneously fitting parameter values. The method uses a combination of genetic programming, Metropolis Monte Carlo importance sampling and parallel tempering, to efficiently search a large space of candidate functional forms and parameters. The methodology was tested using a nontrivial problem with a well-defined globally optimal solution: a small set of atomic configurations was generated and the energy of each configuration was calculated using the Lennard-Jones pair potential. Starting with a population of random functions, our fully automated, massively parallel implementation of the method reproducibly discovered the original Lennard-Jones pair potential by searching for several hours on 100 processors, sampling only a minuscule portion of the total search space. This result indicates that, with further improvement, the method may be suitable for unsupervised development of more accurate force fields with completely new functional forms.     

Aggregation, adsorption, and surface properties of multiply end-functionalized polystyrenes

The properties of polystyrene blends containing deuteriopolystyrene, multiply end-functionalized with C8F17 fluorocarbon groups, are strikingly analogous to those of surfactants in solution. These materials, denoted FxdPSy, where x is the number of fluorocarbon groups and y is the molecular weight of the dPS chain in kg/mol, were blended with unfunctionalized polystyrene, hPS. Nuclear reaction analysis experiments show that FxdPSy polymers adsorb spontaneously to solution and blend surfaces, resulting in a reduction in surface energy inferred from contact angle analysis. Aggregation of functionalized polymers in the bulk was found to be sensitive to FxdPSy structure and closely related to surface properties. At low concentrations, the functionalized polymers are freely dispersed in the hPS matrix, and in this range, the surface excess concentration grows sharply with increasing bulk concentration. At higher concentrations, surface excess concentrations and contact angles reach a plateau, small-angle neutron scattering data indicate small micellar aggregates of six to seven F2dPS10 polymer chains and much larger aggregates of F4dPS10. Whereas F2dPS10 aggregates are miscible with the hPS matrix, F4dPS10 forms a separate phase of multilamellar vesicles. Using neutron reflectometry (NR), we found that the extent of the adsorbed layer was approximately half the lamellar spacing of the multilamellar vesicles. NR data were fitted using an error function profile to describe the concentration profile of the adsorbed layer, and reasonable agreement was found with concentration profiles predicted by the SCFT model. The thermodynamic sticking energy of the fluorocarbon-functionalized polymer chains to the blend surface increases from 5.3kBT for x = 2 to 6.6kBT for x = 4 but appears to be somewhat dependent upon the blend concentration.     

The characterization of (+/-)-anti-benzo[a]pyrene diolepoxide-DNA adducts and (+/-)-anti-dibenzo[a,l]pyrene diolepoxide--DNA adducts in the same DNA sample using solid-matrix phosphorescence

Solid-matrix phosphorescence (SMP) spectra and lifetimes were used to characterize the (±)-anti-benzo[a]pyrene diolepoxide [(±)-anti-B[a]PDE] and (±)-anti-dibenzo[a,l]pyrene diolepoxide [(±)-anti-DB[a,l]PDE] bonded to the same sample of DNA. SMP spectra and lifetimes were also acquired for two samples of DNA that had only (±)-anti-B[a]PDE or (±)-anti-DB[a,l]PDE bonded to the individual samples of DNA. A detailed comparison of the SMP properties was made among the three samples of DNA. The SMP excitation spectra for the (±)-anti-B[a]PDE-DNA and the (±)-anti-DB[a,l]PDE-DNA adducts were very similar. However, the SMP emission spectra of the two DNA adduct systems were very dissimilar with a major emission band for the (±)-anti-B[a]PDE-DNA adducts appearing at 613 nm and for the (±)-anti-DB[a,l]PDE-DNA adducts a major band was at 558 nm. It was possible to selectively use SMP emission wavelengths and obtain a SMP excitation of spectrum of the (±)-anti-DB[a,l]PDE-DNA adducts in the dual adducted DNA sample without the (±)-anti-B[a]PDE-DNA adducts emitting SMP. In addition, it was shown that the SMP emission spectrum of the dual adducted DNA sample could be used to detect both adduct systems in the modified DNA sample. It was demonstrated that the SMP lifetimes could be effectively employed to characterize the dual adducted DNA sample. For example, the SMP decay curve for the (±)-anti-DB[a,l]PDE-DNA adducts could be acquired without any SMP emission from the (±)-anti-B[a]PDE-DNA adducts. Also, ln(SMP intensity) versus time plots were very useful in characterizing the dual adducted DNA sample.     

Synthesis and reactivity of a dipyrrinatolithium complex

The synthesis and characterization of the first dipyrrinato-alkali-metal complex is reported herein. The novel reactivity of this lithium complex is demonstrated in the preparation, isolation, and characterization of a heteroleptic zinc(II) complex in high yield.     

Structure and properties of Gd3Ge4: the orthorhombic RE3Ge4 structures revisited (RE = Y, Tb-Tm)

The new binary compound Gd(3)Ge(4) has been synthesized and its structure has been determined from single-crystal X-ray diffraction. Gd(3)Ge(4) crystallizes in the orthorhombic space group Cmcm (No. 63) with unit cell parameters a = 4.0953(11) A, b = 10.735(3) A, c = 14.335(4) A, and Z = 4. Its structure can be described as corrugated layers of germanium atoms with gadolinium atoms enclosed between them. The bonding arrangement in Gd(3)Ge(4) can also be derived from that of the known compound GdGe (CrB type) through cleavage of the (infinity)(1)[Ge(2)] zigzag chains in GdGe and a subsequent insertion of an extra germanium atom between the resulting triangular fragments. Formally, these characteristics represent isotypism with the Er(3)Ge(4) type (Pearson's oC28). However, re-examination of the crystallography in the whole RE(3)Ge(4) series (RE = Y, Tb-Tm) revealed discrepancies and called into question the accuracy of the originally determined structures. This necessitated a new rationalization of the bonding, which is provided in the context of a comparative discussion concerning both the original and revised structure models, along with an analysis of the trends across the series. The temperature dependence of the magnetic susceptibility of Gd(3)Ge(4) shows that it is paramagnetic at room temperature and undergoes antiferromagnetic ordering below 29 K. Magnetization, resistivity, and calorimetry data for several other members of the RE(3)Ge(4) family are presented as well.     

Toward actinide molecular magnetic materials: coordination polymers of U(IV) and the organic acceptors TCNQ and TCNE

Molecular materials of transition metal ions and organic acceptors of the general formula M(TCNX)2 M=V, Mn, Fe, Co, Ni; X=Q=7,7,8,8-tetracyano-p-quinodimethane, E=tetracyanoethylene, are known to exhibit magnetic ordering resulting from magnetic interactions between the 3d metal orbitals of the paramagnetic metal ion and pi* orbitals of the organic radical spin centers. The reaction of THF solutions of UI3(THF)4 with neutral TCNQ and TCNE instantaneously produce insoluble coordination polymers that can be assigned a formula of [U(TCNX)2I2(THF)2]n on the basis of elemental analysis. Similar reactions between (K-18-crown-6)(TCNX) reagents and UI3(THF)4 produce materials with slightly different phase compositions that are structurally distinct from the neutral TCNQ and TCNE reaction products, as judged by infrared spectroscopy. In all cases the magnetic response of these materials is consistent with the presence of the U(IV) rather than U(III) ion. Voltammetric data obtained for all the synthetic precursors are consistent with the U(IV)/TCNX radical anion formulation. None of the materials undergo magnetic ordering, presumably due to the singlet magnetic ground state that results from the U(IV) ion in a low-symmetry ligand field.     

Infrared spectra of the Li+-(H2)n (n=1-3) cation complexes

The Li+-(H2)n n=1-3 complexes are investigated through infrared spectra recorded in the H-H stretch region (3980-4120 cm-1) and through ab initio calculations at the MP2/aug-cc-pVQZ level. The rotationally resolved H-H stretch band of Li+-H2 is centered at 4053.4 cm-1 [a -108 cm-1 shift from the Q1(0) transition of H2]. The spectrum exhibits rotational substructure consistent with the complex possessing a T-shaped equilibrium geometry, with the Li+ ion attached to a slightly perturbed H2 molecule. Around 100 rovibrational transitions belonging to parallel Ka=0-0, 1-1, 2-2, and 3-3 subbands are observed. The Ka=0-0 and 1-1 transitions are fitted by a Watson A-reduced Hamiltonian yielding effective molecular parameters. The vibrationally averaged intermolecular separation in the ground vibrational state is estimated as 2.056 A increasing by 0.004 A when the H2 subunit is vibrationally excited. The spectroscopic data are compared to results from rovibrational calculations using recent three dimensional Li+-H2 potential energy surfaces [Martinazzo et al., J. Chem. Phys. 119, 11241 (2003); Kraemer and Spirko, Chem. Phys. 330, 190 (2006)]. The H-H stretch band of Li+-(H2)2, which is centered at 4055.5 cm-1 also exhibits resolved rovibrational structure. The spectroscopic data along with ab initio calculations support a H2-Li+-H2 geometry, in which the two H2 molecules are disposed on opposite sides of the central Li+ ion. The two equivalent Li+...H2 bonds have approximately the same length as the intermolecular bond in Li+-H2. The Li+-(H2)3 cluster is predicted to possess a trigonal structure in which a central Li+ ion is surrounded by three equivalent H2 molecules. Its infrared spectrum features a broad unresolved band centered at 4060 cm-1.