Glycan structures attached to the CH2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance ...is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.
A mass spectrometry-based method was developed to measure amino acid substitutions directly in proteins down to a level of 0.001%. When applied to recombinant proteins expressed in Escherichia coli, ...monoclonal antibodies expressed in mammalian cells, and human serum albumin purified from three human subjects, the method revealed a large number of amino acid misincorporations at levels of 0.001–0.1%. The detected misincorporations were not random but involved a single-base difference between the codons of the corresponding amino acids. The most frequent base differences included a change from G to A, corresponding to a G(mRNA)/U(tRNA) base pair mismatch during translation. We concluded that under balanced nutrients, G(mRNA)/U(tRNA) mismatches at any of the three codon positions and certain additional wobble position mismatches (C/U and/or U/U) are the main causes of amino acid misincorporations. The hypothesis was tested experimentally by monitoring the levels of misincorporation at several amino acid sites encoded by different codons, when a protein with the same amino acid sequence was expressed in E. coli using 13 different DNA sequences. The observed levels of misincorporation were different for different codons and agreed with the predicted levels. Other less frequent misincorporations may occur due to G(DNA)/U(mRNA) mismatch during transcription, mRNA editing, U(mRNA)/G(tRNA) mismatch during translation, and tRNA mischarging.
The evolution of Cenozoic continental climate signals from the Atlantic and Pacific side of Eurasia can be assessed for the first time by comparing climate records obtained for two mid-latitudinal ...regions. For the West, a detailed climate record over the past 45 Ma, based on palaeofloras from two Northern German Cenozoic basins (Mosbrugger et al., 2005) revealed major trends and shorter-term events throughout the Cenozoic Cooling, thus testifying the close correlation of continental and marine temperature evolution as derived from oxygen isotopes (Zachos et al., 2008). Using the same methodology, we analyze a total of 14 floral horizons originating from continental strata of Southern Primory'e (Russia) in order to study the evolution at the eastern side of the continent. The Primory'e record spans the middle Eocene to early Pleistocene. As the coeval record for the Atlantic side, it reflects major global signals of Cenozoic climate change such as the temperature decline throughout the late Eocene, coinciding with the growth of Antarctic Ice-sheets, warming during the Mid-Miocene Climatic Optimum, and step-wise cooling throughout the later Neogene. The comparison of both records reveals differing regional patterns. The considerable longitudinal temperature gradient, currently existing between both study areas, already began to evolve during the Aquitanian, and was very significant during the Mid-Miocene Climatic Optimum. The temperature offset between East and West is likely attributable to an effective North Atlantic Current, already operational from the late early Miocene onwards bringing about mild winters and low seasonality in Western Europe, while in Primory'e, seasonality steadily increased from the late Oligocene on. The strong late Pliocene decline of cold month mean temperatures recorded in Primory'e is supposed to coincide with the establishment of the Siberian High as semi-permanent structure of the Northern Hemisphere circulation pattern. When comparing the precipitation records obtained for both study areas, an unexpected co-variability at the longer-term (in the order of 5–20 Ma) is noted, pointing to continent-wide hydrological changes. The steady decline of mean annual precipitation in the Primory'e record, beginning in the Bartonian and culminating in the Aquitanian, coincides with an aridity increase reported from coeval Chinese inland localities of the mid-latitudes. The seasonality patterns of rainfall point to progressive intensification of the East Asian Summer Monsoon in Primory'e since the later Tortonian while the post-Zanclean decline of the precipitation of the dry season can be related to an increasing impact of the winter monsoon.
•We reconstruct a continental climate record over the last 45 Ma for Primory'e.•We compare the climatic evolution of the Atlantic and Pacific side of Eurasia.•The present-day longitudinal climate anomaly existed in the Neogene.•We study the evolution of the continental anomaly throughout the Cenozoic Cooling.•Maritime climates in the West point to an effective Neogene Palaeo-Gulf Stream.
A one-atom-layer compound made of one monolayer of Tl and one-third monolayer of Pb on a Si(111) surface having √3×√3 periodicity was found to exhibit a giant Rashba-type spin splitting of metallic ...surface-state bands together with two-dimensional superconducting transport properties. Temperature-dependent angle-resolved photoelectron spectroscopy revealed an enhanced electron-phonon coupling for one of the spin-split bands. In situ micro-four-point-probe conductivity measurements with and without magnetic field demonstrated that the (Tl, Pb)/Si(111) system transformed into the superconducting state at 2.25 K, followed by the Berezinskii-Kosterlitz-Thouless mechanism. The 2D Tl-Pb compound on Si(111) is believed to be the prototypical object for prospective studies of intriguing properties of the superconducting 2D system with lifted spin degeneracy, bearing in mind that its composition, atomic and electron band structures, and spin texture are already well established.
An approach to taking into account tensor correlations in the formation of charge-exchange giant spin-multipole resonances in medium-heavy magic parent nuclei is proposed within the particle–hole ...dispersive optical model based on the continuum version of the random-phase approximation. The approach is implemented via its application to describing strength functions for the Gamow–Teller resonance and giant spin-monopole resonances in the
Pb parent nucleus. The results of the calculations are compared with available experimental data.
Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. ...In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.
The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides ...should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r 2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples. Keywords: mass spectrometry • protein quantification • protein identification
The article presents the results of a study of the effect of thermal acid treatment of kaolinite on increasing its adsorption activity. Previous publications have presented the results of a study of ...the synthesis conditions and properties of the TiO
2
/kaolinite material—a filler of building materials with the function of passive degradation of household organic pollutants; the studies showed a significant increase in the adsorption–structural characteristics of the composite during synthesis compared to kaolinite and titanium dioxide as separate materials. The current article is devoted to study of changes in the sorption activity of potential carriers of titanium dioxide—kaolinite and montmorillonite—as a result of thermal acid treatment, which simulates the synthesis of a composite. A significant increase in the adsorption activity of kaolinite is obtained and the inefficiency of this treatment method for montmorillonite is shown.
PDF
