Adsorption and recovery issues of recombinant monoclonal antibodies in reversed‐phase liquid chromatography†

The poor recovery of large biomolecules is a well‐known issue in reversed‐phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state‐of‐the‐art reversed‐phase wide‐pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150 000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene‐bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42 000 and 443 000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.     

The pattern of methylcyclopentane and n-hexane reactions on Rh/Al2O3 in catalytic runs of various length

Methylcyclopentane and n-hexane were reacted on a Rh/alumina catalyst in a closed-loop reactor. The product composition was analyzed at different moments of the run. In spite of a slow self-deactivation, the distribution of primary products and the depth of hydrogenolysis showed hardly any dependence on the sampling time in the conversion range of 1.5% and 50%. This is discussed in terms of accumulation of hydrocarbonaceous residues and the low readsorption probability of primary products on rhodium surface.     

Interplay of Charge Transfer, Dimensionality, and Amide Hydrogen Bond Network Adaptability in TCNQF4 Complexes of EDO-TTF-CONH2 and EDT-TTF-CONH2

[EDO-TTF-CONH₂][TCNQF₄], triclinic system, space group P-1, a=8.2479(12) Å, b=12.282(2) Å, c=12.6842(18) Å, α=113.850(17)°, β=106.420(17)°, γ=90.284(19)°, V=1116.8(4) ų; and [EDT-TTF-CONH₂]₂[TCNQF₄], triclinic system, space group P-1, a=6.5858(9) Å, b=11.699(2) Å, c=12.2281(18) Å, α=104.000(19)°, β=93.611(17)°, γ=98.279(19)°, V=899.9(3) ų, whose π-donor molecules, (ethylenedioxo)-carbamoyltetrathiafulvalene and (ethylenedithio)-carbamoyltetrathiafulvalene, respectively, differ solely by the nature of the chalcogen atoms in their outer ethylene dichalcogeno bridge, yet form very different charge-transfer complexes with the same π-acceptor. [EDO-TTF-CONH₂^•+]₂ [TCNQF₄^•−]₂ is a diamagnetic insulating ionic salt with a three-dimensional rock-salt-type structure based on discrete dimers while in the semi-conducting mixed-valence complex, [EDT-TTF-CONH₂]₂^•+[TCNQF₄^•−], the mixed-valence dimers aggregate into infinite chains interspersed within parallel rows of non-interacting radical anions. It is shown how the robust and adaptable supramolecular amide hydrogen bond tweezers-like motifs common to the two solids simply comply to the 3-to-1 dimensionality reduction upon substitution of O for S.     

Benzyl and tert-butyl carbamate derivatives of 1,ω-amino acids as simple yet efficient gelators

We report the synthesis and a study of the gelation properties of a series of N-protected long-chain amino acids. Especially, benzyl and tert-butyl carbamate derivatives of 11-aminoundecanoic acid in their deprotonated form can gelate polar organic solvents and water at very low concentration (less than 5 mM). This is explained by the contribution of multiple forces—H-bond, van der Waals and ionic interactions—in the gel aggregate formation and stabilization, which is confirmed by the experimental data. Among the series of compounds investigated, only a dimer of 11-aminoundecanoic acid is capable of gelating toluene, which stems from the increased number of hydrogen bonding sites in the main aliphatic chain.     

Graphical Abstract: HCA 2/2003

The synthesis and antioxidant efficiencies of amphiphilic gallic acid derivatives are reported. To specify the impact of chemical structure on the antioxidant efficiency, several structural modifications of gallic acid were performed. The following structural features were chosen: i) introduction of hydrophobic or hydrophilic residues on the gallic acid and the type of their linkage, ii) the hydrophilic and/or lipophilic character of the whole molecule. The physico‐chemical studies of the different series prepared revealed that the antioxidant efficiency of this polyphenol depends clearly on the nature of the linkage with both hydrophilic and hydrophobic parts. A push‐pull effect is always necessary, and ester or amide bonds seem well adapted to increase the antioxidant efficiency. Second, under the oxidation conditions applied, it was observed that the hydrophilic and/or lipophilic character affects drastically the antioxidant activity of gallic acid derivatives. The results obtained are in accordance with the polar paradox, hydrophobic derivatives inhibit oxidation in an aqueous phase, whereas hydrophilic products are not efficient.     

The use of polyethyleneimine for fabrication of potentiometric cholinesterase biosensors

Potentiometric biosensors based on butyrylcholinesterase are developed by co-reticulation of the enzyme with glutaraldehyde on an electropolymerized polyethyleneimine film at the electrode surface. The BuChE-electrode was tested as biochemical sensor for detection of an organophosphorus pesticide, trichlorfon in liquid, the detection being based on the enzyme inhibition. The enzyme electrode showed a detection limit for trichlorfon below 10⁻⁷ M.     

Determination of chemical shift anisotropy tensors of carbonyl nuclei in proteins through cross-correlated relaxation in NMR.

The principal components and orientations of the chemical shift anisotropy (CSA) tensors of nearly all 13C carbonyl nuclei in a small protein have been determined in isotropic solution by a combination of three complementary cross-correlation measurements.     

Tandem copper-catalyzed enantioselective allylation-metathesis

Grignard reagents undergo enantioselective (up to 86% ee) copper-catalyzed S(N)2' substitution on achiral allylic chlorides. The reaction is wide in scope for both the Grignard reagent and the allylic substrate. The resulting terminal alkene could be submitted to intra- or intermolecular metathesis to afford new chiral synthons. The experimental conditions are compatible with a one-pot overall substitution-metathesis procedure without loss of enantioselectivity. [reaction: see text]     

Synthesis and Electrochemical Characterization of a New Electropolymerizable Hydrophilic Viologen Designed for Enzyme Wiring

A new viologen derivative functionalized by an electropolymerizable pyrrole group via a long hydrophilic spacer has been synthesized. This redox monomer has been electrochemically characterized both for its presence in organic and in aqueous media. Its electrooxidation in both solvents leads to the formation of a polymeric film exhibiting the regular electrochemical behaviour of the viologen groups. The electropolymerization process was applied to the immobilization of isocitrate dehydrogenase as an enzyme model. An electrical connection between the redox polymer and the immobilized enzyme molecules has been observed in the presence of oxoglutarate and CO₂.     

Docking macromolecules with flexible segments

We address a major obstacle to macromolecular docking algorithms by presenting a new method that takes into account the induced conformational adjustment of flexible loops situated at a protein/macromolecule interface. The method, MC2, is based on a multiple copy representation of the loops, coupled with a Monte Carlo conformational search of the relative position of the macromolecules and their side chain conformations. The selection of optimal loop conformations takes place during Monte Carlo cycling by the iterative adjustment of the weight of each copy. We describe here the parameterization of the method and trials on a protein-DNA complex of known 3-D structure, involving the Drosophila prd paired domain protein and its target oligonucleotide Wenqing, X. et al., Cell 1995, 80, 639. We demonstrate that our algorithm can correctly configure and position this protein, despite its relatively complex interactions with both grooves of DNA.