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Background: No single pure enantiomeric pesticide residues was investigated and set regulations for tea quality safety and risk assessment. Objective: Due to lack of chiral pesticide analysis method and data, the Maximum residue limits (MRLs) about the chiral pesticides in tea was unknown. Method: An analytical method for the determination of chiral pesticide residues by QuEChERS combined chiral liquid chromatography quadruple/linear ion trap mass spectrometry (LC-MS/MS-Qtrap) was developed and applied to the analysis of various teas. Results: The mean recoveries for pesticides enantiomers ranged from 75.9% to 112.4%. Reproducibility represented by relative standard deviation percentage was 10% or less. Good linearity was achieved for all enantiomers with determination coefficients (r²) greater than 0.99. The detection of limit (CCa) and quantification of limit (CCb) were 0.2 ~1 mg/kg and 0.5~5 mg/kg, respectively. Conclusions: The method was suitable for monitoring the enantiomeric pesticide residues in various teas. Highlights: Enantioselective multiclass pesticide residues were determined in various teas by LC-MS/MS-Qtrap, additional Qtrap scan functions greatly enhance the performance of screening, confirmation, and identification of chiral pesticides. [ABSTRACT FROM AUTHOR]

The detection of partial discharge and analysis of the composition and content of sulfur hexafluoride SF6 gas components are important to evaluate the operating state and insulation level of gas-insulated switchgear (GIS) equipment. This paper reported a novel sensing material made of pure ZnO and NiO-decorated ZnO nanoflowers which were synthesized by a facile and environment friendly hydrothermal process for the detection of SF6 decomposition byproducts. X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) were used to characterize the structural and morphological properties of the prepared gas-sensitive materials. Planar-type chemical gas sensors were fabricated and their gas sensing performances toward the SF6 decomposition byproducts SO2, SO2F2, and SOF2 were systemically investigated. Interestingly, the sensing behaviors of the fabricated ZnO nanoflowers-based sensor to SO2, SO2F2, and SOF2 gases can be obviously enhanced in terms of lower optimal operating temperature, higher gas response and shorter response-recovery time by introducing NiO. Finally, a possible gas sensing mechanism for the formation of the p-n junctions between NiO and ZnO is proposed to explain the enhanced gas response. All results demonstrate a promising approach to fabricate high-performance gas sensors to detect SF6 decomposition byproducts. [ABSTRACT FROM AUTHOR]

The article describes a method for simultaneous extraction of coffee flavor compounds trigonelline, nicotinic acid and caffeine in Yunnan Arabica Coffee to be used for quality control (QC) of raw materials and monitoring of roasting condition. The method of extraction was based on microwave-assisted extraction (MAE) and separation of the three flavor compounds by high pressure liquid chromatography (HPLC) with two chromatographic columns in series.

new residue method for quantification of abamectin and ivermectin in vegetable is described in the article. The derivative process is devised that acylating chemical is firstly performed by N-methylimidazole (MI) and trifluoroacetic anhydride (TFAA), then which reacted with hydroxyl function of abamerctin to make fluorescence. The influence of triethylamin (TEA) is examined. Separation is resolute by a short column of 1.7 μm size and operated at high pressure values (10.000 psi). The optimal chromatographic condition and the highest sensitivity are achieved by acetonitrile: water (95: 5), 0.4 mL/min, 0.2 μL injector. The detection limits of abamectin and ivermectin are 1 μg/kg respectively. [ABSTRACT FROM AUTHOR]

Glycosylation is a common posttranslational modification and a key quality attribute of recombinant monoclonal antibodies. In particular, the level of core-fucosylation is critical to several Fe mediated biological functions. However, it is challenging to accurately determine the level of afucosylation due to its low level and possible distribution in multiple oligosaccharide species. Methods that can accurately determine the level of afucosylation were developed in the cunent study. Digestion of monoclonal antibodies with Endoglycosidases F2 and H, which cleave the glycosidic bond between the two primary GlcNAc residues, reduced complicated oligosaccharide population into two groups with either only GlcNAc or GlcNAc and the core-fucose residue. Analysis of the digested antibodies by LC-MS provided a quick estimate of the level of afucosylation. Alternatively, PNGaseF released oligosaccharides were labeled with 2-aminobenzamide, digested with Endoglycosidases F2 and H followed by normal phase HPLC with fluorescence detection. The results demonstrated that the latter method can provide an accurate quantitation of the level of afucosylation. [ABSTRACT FROM AUTHOR]

One of the basic structural features of human IgGi is the arrangement of the disulflde bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disuffide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgGi molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometty (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disuffide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disuffide bond of the two inter heavy chain disuffide bonds was more susceptible than the lower one. Furthermore, disullide bonds in the CR2 domain were the most susceptible to reduction. Disulfide bonds in VI, CL, Vii, and CH1 domains had shuilar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgGit and IgGl.% was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disuffide bonds was smaller in lgGlic than in IgG1L The intra chain disuffide bonds in the Fab region of IgGlic were also less susceptible than disulfide bonds in the Fab region of lgGl2. [ABSTRACT FROM AUTHOR]

Abstract Purpose  To determine the relative importance of direct hydrolysis and β-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH. Methods  A recombinant monoclonal antibody was incubated in buffers of various pH at 40°C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS). Results  Direct hydrolysis was accelerated by acidic and basic pH, while β-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5–9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain. Conclusions  Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4. [ABSTRACT FROM AUTHOR]

Artemisinin (an antimalaric compound) is isolated as the active compound of the medicinal plant Artemisia annua L. A simple, rapid, and high-efficient method of extraction is developed, in which it is extracted by matrix solid-phase dispersion (MSPD) and directly analyzed by post-column derivatization high-performance liquid chromatography (HPLC). The quantitation results from the MSPD method are compared with two conventional liquid solvent extraction processes, Soxhlet and ultrasonic wave by F-test, and the result indicates no significant difference. The production rate of Artemisinin during wild plant growth (tested over two years, acquired from Yunnan, provincial Yuan Yan country) is determined. The recovery range of determination is 88.1–91.2%. The relative standard deviation (RSD) is 4.55–6.43%. The limits of detection are 0.1 µg/mL, and the limits of quantitation are 0.5 µg/mL. [ABSTRACT FROM AUTHOR]

A coffee-certified reference material (CRM) for ochratoxin A, B, and C (OTA, OTB, and OTC) was created to improve the precision and reliability of these toxins. A collection of 100 bottles (each containing 10 g) of the CRM candidate for OTA, OTB, and OTC was produced by applying a mixed standard solution of OTA, OTB, and OTC to moderately roasted beans that subsequently ground and homogenized. Moreover, considering the intricate matrix interference in coffee, a rapid and sensitive method for OTA, OTB, and OTC in coffee was developed with minimal matrix effects. This approach employed liquid chromatography–tandem mass spectrometry and mixed purification materials (C18 and hydroxylated multi-walled carbon nanotubes (MWCNTs), OH-MWCNTs) for the analysis. Based on the standards GB/T 15000.3-2008/ISO Guide 35: 2017, the CRM demonstrated robust uniformity and stability when stored for 14 d at 4, 20, and 40 °C, and 12 months at −20 °C. Eight laboratories were employed to characterize the CRM. The certified values for OTA, OTB, and OTC in coffee were established to be 15.2 ± 0.4, 12.8 ± 0.4, and 3.4 ± 0.1 μg/kg (with a coverage factor k = 2, representing an approximate 95% confidence interval). [ABSTRACT FROM AUTHOR]

In inverse planning processes of radiotherapy, dose-volume histograms (DVH) have been commonly used as a measure of plan quality. The exact mathematical formulation of DVHs involves binary variables and is therefore intrinsically nonconvex. In view of the resulting computational intractability, existing treatment planning systems adopt convex surrogate models that involve many parameters. Manual parameter tweaking is then required to determine reasonable values for those parameters in order to generate plans that meet the target DVHs. However, conventional convex formulations for inverse planning entails non-trivial differences from an exact formulation and, therefore, provide only very limited control over the DVHs. In view of this limitation, this paper presents a new formulation that combines a novel folded concave penalty (FCP)-based constraint and a new kurtosis-based optimization criterion. The former presents a much sharper approximation to the exact DVH constraints than the existing counterparts, and the latter penalizes the occurrence of hot and cold spots in dose distributions. We investigated the effectiveness of the proposed planning formulations on three head-and-neck cases in comparison with conventional DVH-based formulations. Significant outperformance in terms of better distributed doses-at-volume was achieved through the proposed scheme. [ABSTRACT FROM AUTHOR]

An important yet challenging problem in LINAC-based rotational arc radiation therapy is the design of beam trajectory, which requires simultaneous consideration of delivery efficiency and final dose distribution. In this work, we propose a novel trajectory selection strategy by developing a Monte Carlo tree search (MCTS) algorithm during the beam trajectory selection process. To search through the vast number of possible trajectories, the MCTS algorithm was implemented. In this approach, a candidate trajectory is explored by starting from a leaf node and sequentially examining the next level of linked nodes with consideration of geometric and physical constraints. The maximum Upper Confidence Bounds for Trees, which is a function of average objective function value and the number of times the node under testing has been visited, was employed to intelligently select the trajectory. For each candidate trajectory, we run an inverse fluence map optimization with an infinity norm regularization. The ranking of the plan as measured by the corresponding objective function value was then fed back to update the statistics of the nodes on the trajectory. The method was evaluated with a chest wall and a brain case, and the results were compared with the coplanar and noncoplanar 4pi beam configurations. For both clinical cases, the MCTS method found effective and easy-to-deliver trajectories within an hour. As compared with the coplanar plans, it offers much better sparing of the OARs while maintaining the PTV coverage. The quality of the MCTS-generated plan is found to be comparable to the 4pi plans. Artificial intelligence based on MCTS is valuable to facilitate the design of beam trajectory and paves the way for future clinical use of non-coplanar treatment delivery. [ABSTRACT FROM AUTHOR]

-minimization-based sparse optimization was employed to solve the beam angle optimization (BAO) in intensity-modulated radiation therapy (IMRT) planning. The technique approximates the exact BAO formulation with efficiently computable convex surrogates, leading to plans that are inferior to those attainable with recently proposed gradient-based greedy schemes. In this paper, we alleviate/reduce the nontrivial inconsistencies between the -based formulations and the exact BAO model by proposing a new sparse optimization framework based on the most recent developments in group variable selection. We propose the incorporation of the group-folded concave penalty (gFCP) as a substitution to the -minimization framework. The new formulation is then solved by a variation of an existing gradient method. The performance of the proposed scheme is evaluated by both plan quality and the computational efficiency using three IMRT cases: a coplanar prostate case, a coplanar head-and-neck case, and a noncoplanar liver case. Involved in the evaluation are two alternative schemes: the -minimization approach and the gradient norm method (GNM). The gFCP-based scheme outperforms both counterpart approaches. In particular, gFCP generates better plans than those obtained using the -minimization for all three cases with a comparable computation time. As compared to the GNM, the gFCP improves both the plan quality and computational efficiency. The proposed gFCP-based scheme provides a promising framework for BAO and promises to improve both planning time and plan quality. [ABSTRACT FROM AUTHOR]

Traditional inverse planning relies on the use of weighting factors to balance the conflicting requirements of different structures. Manual trial-and-error determination of weighting factors has long been recognized as a time-consuming part of treatment planning. The purpose of this work is to develop an inverse planning framework that parameterizes the dosimetric tradeoff among the structures with physically meaningful quantities to simplify the search for clinically sensible plans. In this formalism, instead of using weighting factors, the permissible variation range of the prescription dose or dose volume histogram (DVH) of the involved structures are used to characterize the ‘importance’ of the structures. The inverse planning is then formulated into a convex feasibility problem, called the dosimetric variation-controlled model (DVCM), whose goal is to generate plans with dosimetric or DVH variations of the structures consistent with the pre-specified values. For simplicity, the dosimetric variation range for a structure is extracted from a library of previous cases which possess similar anatomy and prescription. A two-phase procedure (TPP) is designed to solve the model. The first phase identifies a physically feasible plan to satisfy the prescribed dosimetric variation, and the second phase automatically improves the plan in case there is room for further improvement. The proposed technique is applied to plan two prostate cases and two head-and-neck cases and the results are compared with those obtained using a conventional CVaR approach and with a moment-based optimization scheme. Our results show that the strategy is able to generate clinically sensible plans with little trial and error. In all cases, the TPP generates a very competitive plan as compared to those obtained using the alternative approaches. Particularly, in the planning of one of the head-and-neck cases, the TPP leads to a non-trivial improvement in the resultant dose distribution—the fractional volumes receiving a dose above 20 Gy for the spinal cord are reduced by more than 40% when compared to the alternative schemes, while maintaining the same PTV coverage. With physically more meaningful modeling of the inter-structural tradeoff, the reported technique enables us to substantially reduce the need for trial-and-error adjustment of the model parameters. The new formalism also opens new opportunities for incorporating prior knowledge to facilitate the treatment planning process. [ABSTRACT FROM AUTHOR]

Asparagine deamidation in the complementarity determining regions of recombinant monoclonal antibodies has been extensively studied and shown to have a negative impact on antigen binding. Those asparagine residues are typically exposed and thus have a higher tendency toward deamidation, depending also on local structure and environmental factors such as temperature and pH. Deamidation rates and products of a susceptible asparagine residue in the complementarity determining regions of a recombinant monoclonal antibody free in solution or in the antibody-antigen complex were studied. The results demonstrated that incubation of the antibody or its antigen complex generated a similar amount of aspartate. The expected amount of isoaspartate product was detected in free antibody, but it was completely lacking in the antibody-antigen complex. [ABSTRACT FROM AUTHOR]

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies. [ABSTRACT FROM AUTHOR]

A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydayl was first alkylated with 12C iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with 13C iodoacetic acid. Cysteine containing peptides that were modified by 13C iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by 12C iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with 12C or 13C. The percentage of free sulfhydryl was then calculated using the two mix series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies. [ABSTRACT FROM AUTHOR]

Each human IgG1 antibody contains a total of thirty-two cysteine residues. In theory, all of them are involved in disulfide bonds, and no free sulfhydryl should be detected. However, literature has suggested that the presence of low levels of free sulfhydryl groups is likely a common feature of recombinant and wild type IgG1 antibodies. Currently, there is little information correlating the presence of free sulfhydryl to specific cysteine residues. The presence of free sulfhydryl groups in five recombinant monoclonal antibodies and their locations were further investigated in the current study. Free sulfhydryl groups were first modified using 5-idoacetamidofluorescein (5-IAF), which was followed by reduction of disulfide bonds and alkylation with iodoacetic acid (IAA). This procedure allowed differentiation of free cysteine residues from cysteine residues that were involved in disulfide bonding. In addition, it allowed a sensitive fluorescence detection of peptides with free sulfhydryl groups. The locations of the free sulthydryl groups were determined using mass spectrometry after fraction collection. The results indicated that different antibodies had different levels of free sulfhydryl due to multiple unpaired cysteine residues commonly in the constant domains. Furthermore, free sulfhydryl due to unpaired cysteine residues in the variable domains varied for different antibodies. Interestingly, free sulfhydryl was rarely associated with cysteine residues that were involved in interchain disulfide bonds. [ABSTRACT FROM AUTHOR]

The inhibition of NaBr on the corrosion of cold rolled steel in 1.0 M phosphoric acid was studied by using weight loss method and polarization method. It was found that the adsorption of bromide ion can prevent steel from corrosion and the adsorption follows the Langmuir adsorption isotherm. Polarization studies showed that NaBr is a mixed-type inhibitor for steel corrosion in 1.0 M phosphoric acid. Thermodynamic parameters such as adsorption heat, adsorption entropy and adsorption free energy were obtained from the experimental data of temperature studies for the inhibition process at four temperatures ranging from 30 to 45°C. The kinetic data such as apparent activation energies and pre-exponential factors at different concentrations of the inhibitor were calculated, and the effects of the apparent activation energy and pre-exponential factor on the corrosion rate of cold rolled steel were discussed. The inhibitive action was satisfactorily explained by using thermodynamic and kinetic models. The results obtained from polarization experiment were in good agreement with those obtained from weight loss measurement. [ABSTRACT FROM AUTHOR]

A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab')2 fragment generated from IdeS digestion. LC-MS analysis of nonreduced tryptic peptides indicated trisulfide bonds are associated with the interchain disulfide bonds of both A isoform and A/B isoform and cysteinylation is associated only with the A isoform. A low percentage of trisulfide bonds was detected in between the light chain and heavy chain disulfide bond of the A and A/B forms. While the majority of trisulfide bonds and cysteinylation is associated with the hinge region peptide that involves the four closely spaced cysteine residues of the heavy chain. The locations of trisulfide bond and cysteinylation were determined using a combination of Edman sequencing and LC-MS. In the A isoform, the major site of the trisulfide bond and cysteinylation is between the first disulfide bond in the hinge region. In the A/B isoform, the trisulfide was also located in between the disulfide bond that is formed by the second pair of cysteine residues. [ABSTRACT FROM AUTHOR]

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution. [ABSTRACT FROM AUTHOR]

Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed dioroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures. [ABSTRACT FROM AUTHOR]

Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. β-Galactosidase and β-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after β-galactosidase and β-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics. [ABSTRACT FROM AUTHOR]

Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increases of molecular weights of 72 and 54 Da, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals. [ABSTRACT FROM AUTHOR]

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in 18O water. When using H218O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the 18O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two 18O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of 18O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using 18O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of 18O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is a complex polygenic disease. Although recent genome-wide association (GWA) studies have identified multiple susceptibility loci for lung cancer, most of these variants have not been validated in a Chinese population. In this study, we investigated whether a genetic risk score combining multiple. Methods: Five single-nucleotide polymorphisms (SNPs) identified in previous GWA or large cohort studies were genotyped in 5068 Chinese case-control subjects. The genetic risk score (GRS) based on these SNPs was estimated by two approaches: a simple risk alleles count (cGRS) and a weighted (wGRS) method. The area under the receiver operating characteristic (ROC) curve (AUC) in combination with the bootstrap resampling method was used to assess the predictive performance of the genetic risk score for lung cancer. Results: Four independent SNPs (rs2736100, rs402710, rs4488809 and rs4083914), were found to be associated with a risk of lung cancer. The wGRS based on these four SNPs was a better predictor than cGRS. Using a liability threshold model, we estimated that these four SNPs accounted for only 4.02% of genetic variance in lung cancer. Smoking history contributed significantly to lung cancer (P < 0.001) risk [AUC = 0.619 (0.603-0.634)], and incorporated with wGRS gave an AUC value of 0.639 (0.621-0.652) after adjustment for over-fitting. This model shows promise for assessing lung cancer risk in a Chinese population. Conclusion: Our results indicate that although genetic variants related to lung cancer only added moderate discriminatory accuracy, it still improved the predictive ability of the assessment model in Chinese population. [ABSTRACT FROM AUTHOR]

Asparagine (Asn) deamidation is a major source of antibody instability and micro heterogeneity. For this reason, it is critical to accurately characterize both the levels and the sites of Asn deamidation in therapeutic antibodies. Asn deamidation is normally quantified by analyzing antibodies at the peptide level by liquid chromatography-mass spectrometry. This requires denaturation, reduction, alkylation, and enzyme digestion of the antibody prior to analysis. These steps in sample preparation may directly contribute to the total levels of Asn deamidation detected. Therefore, to obtain accurate levels and sites of Asn deamidation, it is important to determine if any deamidation occurred during the sample preparation steps. However, this could be challenging because deamidation that occurred prior to and during sample preparation resulted in peptides with the same retention times and the same molecular weight increase of 1 Da. Sample preparation was carried out in 18O-water in the current study to differentiate between the two events of Asn deamidation. Using this method, deamidation that occurred during sample preparation resulted in a molecular weight increase of 3 Da instead of 1 Da. This molecular weight difference was readily detected by inspection of the isotopic peak cluster of the peptides containing the deamidation products, isoAsp and Asp residues. It enabled discrimination of deamidation that was due to analytical artifacts and thus determination of the level of deamidation that was present in the samples. [ABSTRACT FROM AUTHOR]

The synergistic effect of the mixture of hexadecyl trimethyl ammonium bromide (HTAB) and various concentrations of NaBr on the corrosion of cold rolled steel in 0.5 M sulfuric acid has been investigated using weight loss method and polarization method. The results reveal that the corrosion of cold rolled steel has been efficiently inhibited by HTAB in the presence of NaBr in sulfuric acid, and inhibition efficiency increases with increasing concentration of NaBr at the same temperature. A synergistic effect has been observed for HTAB with NaBr at every experimental temperature. The polarization curves show that the complex of HTAB and NaBr is a mixed-type inhibitor. The experimental results suggest that the presence of bromide ion in the solution stabilizes the adsorption of HTAB molecules on the metal surface and improves the inhibition efficiency of HTAB. Langmuir adsorption isotherm equation was obeyed at all the temperatures studied. The synergism parameters have been calculated and discussed. The results obtained from weight loss method and polarization method are in good agreement. [ABSTRACT FROM AUTHOR]