Development,validation, and implementation of capillary gel electrophoresis as a replacement for SDS‐PAGE for purity analysis of IgG2 mAbs

Capillary gel electrophoresis (CGE) methods with UV detection were developed for reduced and non‐reduced mAb analysis. These methods can be used to evaluate mAb purity, offering more reproducible quantitation compared with that of traditional SDS‐PAGE methods. These CGE methods have been utilized as platform technology for bioprocess development, formulation development, mAb characterization, drug substance/drug product release testing as well as a required methodology for stability testing. We have found these CGE methods to be applicable across a platform of mAbs in preclinical and clinical development, with the majority of mAbs requiring no modification to the method conditions. This methodology has been ICH validated and transferred to several supporting organizations. The data presented herein describes the development of CGE methodology, platform application to mAb purity analysis, ICH validation, reliability metrics, and considerations on technology enhancement for improved performance and throughput.     

Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies

A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary‐based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.     

Long-range electronic coupling of MM quadruple bonds (M = Mo or W) via a 2,6-azulenedicarboxylate bridge

The preparation of 2,6-azulenedicarboxylic acid (I) from its diester, 2-CO(2)(t)Bu-6-CO(2)-C(10)H(6) (II), is reported together with the crystal and molecular structure of the ester, II. From the reactions between the dicarboxylic acid I and the MM quadruply bonded complexes M(2)(O(2)C(t)Bu)(4), where M = Mo or W, the azulenedicarboxylate bridged complexes [M(2)(O(2)C(t)Bu)(3)](2)(mu-2,6-(CO(2))(2)-C(10)H(6)) have been isolated, III (M = Mo) and IV (M = W). The latter compounds provide examples of electronically coupled M(2) centers via a polar bridge. The compounds show intense electronic absorptions due to metal-to-bridge charge transfer. This occurs in the visible region of the spectrum for III (M = Mo) but in the near-IR for IV (M = W). One electron oxidation with Ag(+)PF(6)(-) in THF generates the radical cations III(+) and IV(+). By both UV-vis-NIR and EPR spectroscopy the molybdenum ion III(+) is shown to be valence trapped or Class II on the Robin and Day classification scheme. Electrochemical, UV-vis-NIR, and EPR spectroscopic data indicate that, in the tungsten complex ion IV(+), the single electron is delocalized over the two W(2) centers that are separated by a distance of ca. 13.6 A. Furthermore, from the hyperfine coupling to (183)W (I = (1)/(2)), the singly occupied highest molecular orbital is seen to be polarized toward one W(2) center in relationship to the other. Electronic structure calculations employing density functional theory indicate that the HOMO in compounds III and IV is an admixture of the two M(2) delta orbitals that is largely centered on the M(2) unit having proximity to the C(5) ring of the azulenedicarboxylate bridge. The energy of the highest occupied orbital of the bridge lies very close in energy to the M(2) delta orbitals. However, this orbital does not participate in electronic coupling by a hole transfer superexchange mechanism, and the electronic coupling in the radical cations of III and IV occurs by electron transfer through the bridge pi system.     

Interaction of mono- and diisocyanoazulenes with gold surfaces: first examples of self-assembled monolayer films involving azulenic scaffolds

The formation and properties of a new class of self-assembled monolayers (SAM) of aryl isocyanides and diisocyanides based on the nonbenzenoid azulenic framework have been investigated using FTIR spectroscopy and ellipsometry. Syntheses of several new members of the isocyanoazulene family, a recently established type of aryl isocyanides, are reported as well. The FTIR spectra for the isocyanoazulene derivatives absorbed on the gold surface indicate the terminal upright coordination of every isocyanoazulene molecule studied. In addition, the ellipsometric thicknesses have been measured and are consistent with those calculated for single monolayers of the isocyanides oriented along the surface normal. Unlike SAMs of some benzenoid aryl isocyanides, the nonbenzenoid isocyanoazule-based SAMs proved resistant to oxidation, oligomerization, and isomerization into the corresponding nitriles under ambient conditions, which is an important prerequisite to their future applications.     

First homoleptic complexes of isocyanoferrocene

Interaction of aminoferrocene with a 65/35 mol % mixture of phenyl formate/phenol followed by subsequent dehydration of the resulting ferrocenylformamide with POCl3 produced a high yield of air- and thermally stable, peach-colored isocyanoferrocene (CNFc). Combining 6 equiv of CNFc with bis(naphthalene)chromium(0) afforded orange-red Cr(CNFc)6, the first homoleptic complex of CNFc. Successive one-electron oxidations of the latter with Ag+ quantitatively produced saddle-brown [Cr(CNFc)6]+ and forest-green [Cr(CNFc)6]2+. The compounds [Cr(CNFc)6]0,1+,2+ are air-, light-, and thermally robust and are remarkable due to the incorporation of seven potentially electroactive transition metal ions within a relatively compact ML6 motif. Their properties demonstrate that the pi-accepting potential of the ferrocenyl substituent, customarily compared to an alkyl group, may have been underestimated. Indeed, the donor/acceptor ratio of the CNFc ligand appears to be similar to those of aryl isocyanides but quite different from those of alkyl isocyanides. Spectroscopic (including multinuclear paramagnetic NMR), magnetic, structural, and electrochemical characteristics of the above substances are discussed. In addition, a DFT analysis of the Frontier molecular orbitals of CNFc is presented.     

Experimental calibration and field investigation of the oxygen isotopic fractionation between biogenic aragonite and water

Marine molluscs have long been recognised as potential records of palaeoclimate change using the patterns and differences in the stable isotopic composition of the carbonate shells. The aim of this study is to improve the robustness of this approach for aragonitic molluscs by completing the first experimental calibration of the fractionation between water and biogenic aragonite. Fractionation factors were calibrated by growing specimens of the freshwater mollusc Lymnaea peregra under controlled conditions of water temperature and isotopic composition. Fifteen populations of L. peregra were maintained at constant temperature and isotopic conditions for five months (at five different temperatures and using three different water compositions). Water samples and temperature measurements were taken regularly throughout the experiment. The temperature dependence of the fractionation factor, between 8 and 24 degrees C, is given by: 1000 ln alpha=16.74x(1000T(-1))-26.39 (T in Kelvin) and the relationship between temperature (T), delta(18)O(carb) and delta(18)O(wat) is given by: T=21.36-4.83xdelta(+ degrees )O(carb)-delta(+ degrees )O(wat) (T is in degrees C, delta(18)O(carb) is with respect to Vienna Pee Dee Belemnite (PDB), the International Atomic Energy Agency (IAEA) replacement standard for PDB, and delta(18)O(wat) is with respect to Vienna standard mean ocean water (VSMOW)) The outcome of the controlled experiment is compared with previous studies on synthetic, and biogenic, calcite and aragonite from field and laboratory investigations. These comparisons suggest that although a vital offset exists between the fractionation of isotopes in synthetic and biogenic aragonite for molluscs in general, there is no vital effect that is specific either to freshwater, or to individual, genera. Therefore, the calibrated relationship may be used for any freshwater or marine mollusc to derive palaeotemperatures providing the isotopic composition of the environmental water can be reliably constrained. Copyright 1999 John Wiley & Sons, Ltd.     

The 2,6-diisocyanoazulene motif: synthesis and efficient mono- and heterobimetallic complexation with controlled orientation of the azulenic dipole

Synthesis of the remarkably air- and thermally stable 2,6-diisocyano-1,3-diethoxycarbonylazulene linker from 2-amino-1,3-diethoxycarbonylazulene in 57% cumulative yield was developed. Incorporation of the ester "arms" in the design of this first diisocyanoazulene bridge permitted fully controlled stepwise installation and complexation of its isocyano junction groups. The -CO(2)Et arms in 2,6-diformamido-1,3-diethoxycarbonylazulene effectively suppress the rate of dehydration of its 2-NHCHO end relative to that of the 6-NHCHO end leading to practically exclusive formation of 6-isocyano-2-formamido-1,3-diethoxycarbonylazulene upon treatment of the above diformamide with an equimolar amount of POCl(3). This crystallographically characterized 6-isocyano-2-formamidoazulene derivative was employed to access mono- and heterobimetallic complexes of the 2,6-diisocyanoazulene scaffold with controlled orientation of the azulenic dipole. A complete series of monometallic, homobimetallic, and isomeric heterobimetallic ([M] = M(CO)(5), M = Cr and/or W) complexes of the 2,6-diisocyanoazulene motif was isolated and studied by a variety of techniques, including X-ray crystallography. The metal-to-bridge charge transfer in mono- and dinuclear adducts of 2,6-diisocyanoazulene, the assignment of which was corroborated by time-dependent density functional theory calculations, occurs at a dramatically lower energy as compared to the analogous systems featuring the 1,4-diisocyanobenzene scaffold. Moreover, the metal-to-diisocyanide charge transfer exhibits a substantially greater red shift upon binucleation of the mononuclear [M(CO)(5)] adducts of the nonbenzenoid 2,6-diisocyanoazulene linker versus the 1,4-diisocyanobenzene bridge.     

Effect of three-body forces on the lattice dynamics of noble metals

A simple method to generate an effective electron-ion interaction pseudopotential from the energy wave number characteristic obtained by first principles calculations has been suggested. This effective potential has been used, in third order perturbation, to study the effect of three-body forces on the lattice dynamics of noble metals. It is found that three-body forces, in these metals, do play an important role. The inclusion of such three-body forces appreciably improves the agreement between the experimental and theoretical phonon dispersion curves.     

First isocyanoazulene and its homoleptic complexes

An efficient synthesis of remarkably stable 6-isocyanoazulene (CN6Az), the first nonbenzenoid organic isocyanide, is described. Its superb pi-accepting potential as a ligand was demonstrated through cyclic voltammetry studies on the binary complexes [Cr(CN6Az)6]0/+. The paramagnetic shift patterns for [Cr(CN6Az)6]+, the only azulenic pi-system studied by paramagnetic NMR, suggest that Cr(dpi) --> CN6Az(ppi*) electron delocalization involves both rings of the azulenyl substituents in [Cr(CN6Az)6]+. This conclusion is supported by density functional theory calculations on the complex [Cr(CN6Az)6][BF4].     

Multipoint anchoring of the [2.2.2.2]metacyclophane motif to a gold surface via self-assembly: coordination chemistry of a cyclic tetraisocyanide revisited

A one-pot transformation of bis(2-isocyano-3-methylphenyl)ethane affords gram quantities of 8,16,24,32-tetraisocyano[2.2.2.2]metacyclophane ( 3). The solid state structure of 3 is remarkably close to the lowest energy conformation found on the potential energy landscape for 3 by DFT. In solution, the structure of metacyclophane 3 is mobile but can be locked in a rectangular gauche- anti- gauche- anti conformation by coordination of the isocyanide substituents to the [W(CO) 5] units to give [M] 4(mu 4-eta (1):eta (1):eta (1):eta (1)- 3) ( 5). The tetranuclear [M] 4(mu 4-eta (1):eta (1):eta (1):eta (1)- 3) motif featured in crystallographically characterized 5 may be present in several insoluble complexes of 3 previously described as mononuclear eta (4) species. A self-assembled monolayer of metacyclophane 3 is formed upon exposing a solution of 3 to the gold(111) surface with no precautions to exclude air or light. The monolayer nature of the film was confirmed by optical ellipsometry. The isocyanide stretching band for 3 shifts from 2119 cm (-1) in solution to 2175 cm (-1) upon chemisorption to metallic gold. The FTIR spectrum of the film indicates interaction of 3 with the gold surface via all four of its isocyanide anchors. No gold-facilitated oxidation of the -NC junctions was detected under ambient conditions. The energy cost associated with accessing the conformations of 3 suitable for mu 4-eta (1):eta (1):eta (1):eta (1) interaction of the molecule with the Au(111) surface is under 8 kcal/mol, a value that can be easily offset by formation of a gold-isocyanide bond. Two different mu 4-eta (1):eta (1):eta (1):eta (1) coordination arrangements of 3 with respect to gold atoms on the (111) face of the fcc Au lattice are suggested.