Exploiting space‐group symmetry in fragment‐based molecular crystal calculations

Recent developments in fragment‐based methods make it increasingly feasible to use high‐level ab initio electronic structure techniques to molecular crystals. Such studies remain computationally demanding, however. Here, we describe a straightforward algorithm for exploiting space‐group symmetry in fragment‐based methods which often provides computational speed‐ups of several fold or more. This algorithm does not require a priori specification of the space group or symmetry operators. Rather, the symmetrically equivalent fragments are identified automatically by aligning the individual fragments along their principle axes of inertia and testing for equivalence with other fragments. The symmetry operators relating equivalent fragments can then be worked out easily. Implementation of this algorithm for computing energies, nuclear gradients with respect to both atomic coordinates and lattice parameters, and the nuclear hessian is described. © 2014 Wiley Periodicals, Inc.     

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.     

Point‐of‐Care Amperometric Testing of Diabetic Marker (HbA1c) Using Specific Electroactive Antibodies

Specific immune detection of glycated hemoglobin is still a great challenge owing to the small epitopic difference between Hemoglobin (Hb) and HbA1c. We report a new electrochemical immunoassay format for point of care testing of HbA1c. A conducting self‐assembled monolayer of mercaptophenyl boronic acid (MPBA) was used as a capture layer for binding of glycated proteins and ferrocene tagged anti‐HbA1c antibody (FcAb) as a tracer molecule on a gold screen printed electrode. Validation of the new HbA1c assay was carried out using 6 clinical samples with known HPLC values and a correlation coefficient of 98 % was observed.     

Exploring Weak Ligand–Protein Interactions by Long‐Lived NMR States: Improved Contrast in Fragment‐Based Drug Screening

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long‐lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N‐terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K_D. This property makes the LLS method particularly attractive for the initial steps of fragment‐based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.     

Voltammetric Label‐free Immunosensors for the Diagnosis of Cystic Echinococcosis

Cystic echinococcosis (CE) or hydatid disease is a parasitic infection caused by Echinococcus granulosus. Early serodiagnosis and continuous monitoring of the disease is very important for medical treatment. Here, we report the detecting of both echinococcus antigen and antibody for the diagnosis of hydatid disease using square wave voltammetry (SWV)‐based immunosensors. The gold electrodes were functionalized using cysteamine/phenylene diisothiocyanate linkers and used for the immunosensors fabrication. The hydatid antigen and antibody immunosensors were constructed by the immobilization of either purified rabbit polyclonal antibody or recombinant antigen B (AgB), respectively on the functionalized gold electrodes surfaces. The detection in both cases was achieved by following the change in the SWV reduction peak current of the ferro/ferricyanide redox couple upon antibody or antigen binding. These immunosensors enabled the detection of echinococcus antigen and antibody within a concentration range of 1 pg.mL^?1 to 1 μg.mL^?1 with detection limits of 0.4 pg.mL^?1 and 0.3 pg.mL^?1, respectively. A preliminary application of the developed immunosensor was performed in spiked serum sample showing good recovery percentages ranging from 102 to 110 % for both hydatid antibody and antigen detection. This easy‐to‐use, sensitive, and low cost quantitative method holds great promise for the early diagnosis of hydatid disease and thus, better managements and treatment outcomes.     

Development of supercritical fluid chromatography coupled with mass spectrometry method for characterization of a nonionic surfactant and comparison with liquid chromatography coupled with mass spectrometry method

The supercritical fluid chromatography coupled with mass spectrometry (SFC‐MS) method and liquid chromatography coupled with mass spectrometry (LC‐MS) method were developed for the separation and characterization of poly (ethylene oxide) methyl glucose sesquistearate (PEO‐Glu‐sesquistearate). The products of PEO‐Glu‐sesquistearate are composed of complex oligomers. The relationship between molecular structure of these oligomers and chromatographic retention behavior in both SFC and LC were discussed and compared. As compared with LC, hydrophobic moieties of compounds favor the fast elution in SFC. The different series can be better separated by LC, while the homologues compounds in same series can be better separated by SFC, and SFC‐MS provided more comprehensive structural information. Different series such as PEO‐distearate, PEO‐stearate, PEO, PEO‐Glu‐tetrastearate, PEO‐Glu‐tristearate, PEO‐Glu‐distearate, PEO‐Glu‐stearate, and PEO‐Glu were identified by MS/MS.     

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

N‐glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co‐existing glycosylation variants. However, quantification of N‐glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O‐glycosylation. Herein, we describe a simple strategy to reveal actual N‐glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the “hexosylation bias”. We show that actual N‐glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N‐glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.     

Evaluation of hydrophobic‐interaction chromatography resins for purification of antibody‐drug conjugates using a mimetic model with adjustable hydrophobicity

Antibody drug conjugates are cytotoxic pharmaceuticals, designed to destroy malignant cells. A cytotoxic molecule is attached to an antibody that binds specific to a cancer‐cell surface. Given the high toxicity of the drugs, strict safety standards have to be kept. For this reason, an antibody drug conjugates model was developed with fluorescein 5‐isothiocyanate as the nontoxic payload surrogate. Due to the similar hydrophobicity, this model is used to establish a suitable purification process and characterization method for antibody drug conjugates. Because of the pH dependent solubility of fluorescein, the hydrophobicity of conjugates can be modulated by the pH value. Based on the complex heterogeneity and hydrophobicity of the conjugates a chromatographic purification is challenging. Hydrophobic interaction chromatography is used for analytical as well as for preparative separation. Because of the increased hydrophobicity of the conjugates compared to native antibody, hydrophobic interaction chromatography often suffer from resolution and recovery problems. Conjugates were separated differing on the number of payloads attached to the antibody. For this matter, the drug–antibody ratio is determined and used as a quantitative term. The conjugates are purified at high recoveries and resolution by step gradients using suitable resins, allowing the separation of the target drug–antibody ratio.     

钨球对高硬度钢斜侵彻效应

为研究高硬度钢板抗不同着角钨球的侵彻性能及破坏模式,通过弹道枪进行了?8 mm、?11 mm钨合金球形破片以0°、20°、40°着角撞击厚度为6 mm、8 mm的高硬度钢板试验,得到了极限贯穿速度v₅₀;分析了钨球轴向径向变形及靶板失效模式与撞击速度的关系,发现高硬度钢板失效模式主要为压缩开坑破坏和沿厚度方向剪切破坏。采用有限元方法对试验进行了模拟,验证了数值模型及参数的合理性,并运用数值模拟方法研究了撞击着角对靶板吸能模式影响,结合试验数据,修正已有极限贯穿速度计算公式。结果表明:随侵彻着角增大,极限贯穿速度提高,且着角越大,极限贯穿速度增长越快;随着角增大,靶板吸能模式逐渐由压缩开坑向剪切冲塞过渡,且着角大于50°时,剪切冲塞耗能将超过压缩开坑耗能;修正后极限贯穿速度计算公式适用范围更广、精度更高,具有较好工程应用价值。