Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.     

聚合物流动行为的光散射表征

简要地介绍了光散射原理及目前光散射技术在聚合物流变学的中的应用，归纳并分析了前人的一些主要工作成果。对于一些至今难以解释的实验现象提出了自己的一些认识，并对仍未有人涉足的振动力场下聚合物复杂流动光散射表征提出了新的设想。     

溴虫腈的质谱特征与核磁共振波谱分析

报道了溴虫腈的质谱特征与核磁共振波谱分析结果。在GC/MS上得到的EI质谱,分子离子峰很弱,基峰为m/z59;以甲醇/水作流动相和负离子模式下,在LC/MS上得到的CI质谱,未见分子离子峰,基峰为m/z349;对所有1H和13C NMR数据进行了鉴定。     

酪蛋白溶解与酶解行为的动态光散射

采用动态光散射技术,以磷酸盐为缓冲液,研究了温度、离子强度和酶解行为对酪蛋白胶束平均流体力学半径(Rh)的影响规律. 结果表明,Rh值在升温过程中发生不可逆减小;随着离子强度增大,Rh值先减小再增大;酪蛋白在胰蛋白酶作用下,溶液光散射强度对应于分子量大小先急剧下降,再逐渐趋于平缓,而Rh值的变化规律与之相反;并推测了酪蛋白胶束酶解过程的结构变化模型,即由原始致密的球状体逐渐舒展为松散而规则的毛束状蛋白肽链.     

Application of Dynamic and Static Light Scattering for Size and Shape Characterization of Small Extracellular Nanoparticles in Plasma and Ascites of Ovarian Cancer Patients

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (R_h) and the radius of gyration (R_g) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. R_h and R_g of the predominant ENP population of OC patients were in the range 20–30 nm (diameter 40–60 nm). In thawed samples, larger particles (R_h mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The R_h and R_g of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1–2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.     

光散射技术及其在高分子溶液研究中的应用

从重均分子量,均方旋转半径及平均扩散系数等高分子溶液中较为重要的几个物理量入手介绍了近十几年来光散射技术在高分子溶液领域的应用。同时,对静态光散射和动态光散射的几个重要参数以及其较为重要的公式也做了介绍。最后,还综述了光散射的发展简史、基本原理、有关实验的技术方法等内容。     

氯化银纳米粒子散射光谱的应用研究

研究了氯化银纳米粒子的散射光谱及其应用,与透射光谱相比,用散射光谱法测定微量银离子,具有更好的线性关系、更宽的线性范围和更高的灵敏度。     

Assessment of Isoprostanes in Human Plasma: Technical Considerations and the Use of Mass Spectrometry

Oxygenated lipid mediators released from non‐enzymatic peroxidation of polyunsaturated fatty acids (PUFA) are known to have functional roles in humans. Notably, among these lipid mediators, isoprostanes molecules are robust biomarkers of oxidative stress but those from n‐3 PUFA are also bioactive molecules. In order to identify and assess the isoprostanes, the use of mass spectrometry (MS) for analysis is preferable and has been used for over two decades. Gas chromatography (GC) is commonly coupled to the MS to separate the derivatized isoprostanes of interest in biological samples. In order to increase the accuracy of the analytical performance, GC–MS/MS was also applied. Lately, MS or MS/MS has been coupled with high‐performance liquid chromatography to assess multiple isoprostane molecules in a single biological sample without derivatization process. However, there are limitations for the use of LC–MS/MS in the measurement of plasma isoprostanes, which will be discussed in this review.     

High Pressure Synthesis and Characterization of Acrylonitrile Based Copolymers in Aqueous Free Radical Polymerization Using NMR Spectroscopy

Abstract

The influence of reaction pressure on rate of polymerization and intrinsic viscosity in acrylonitrile (AN), methyl acrylate (MA) and itaconic acid (IA) ternary suspension polymerization system has been studied using free radical forming potassium persulphate and sodium metabisulphite water soluble redox initiator pair. The resulting polymers synthesized at various reaction pressures were characterized for tacticity and composition by NMR and other spectroscopic (FT-IR. ESCA) techniques. These polymers were also subjected to differential scanning calorimetric (DSC) analysis and significant change in heat of cyclization were observed with the change in reaction pressure.     

Structural Characterization of Frustrated Lewis Pairs and Their Reaction Products Using Modern Solid-State NMR Spectroscopy Techniques

Frustrated Lewis pair (FLP) chemistry has provided a new strategy for small-molecule binding and/or catalytic activation. The most prominent FLPs are based on intramolecular phosphane borane adducts, the catalytic properties of which can be tailored over wide ranges of reactivity and selectivity. Advanced solid-state NMR spectroscopic techniques, together with DFT calculations, can provide new structural insights in these systems. This review illustrates the utility of ³¹P and ¹¹B NMR chemical shifts, ¹¹B electric field gradient tensors, and ³¹P ¹¹B indirect and direct dipole dipole interactions for characterizing intramolecular borane phosphane FLPs. We demonstrate the potential of this method to 1) quantify the extent of boron phosphorus bonding interactions (and hence, the “degree of frustration”); 2) reveal specific structural details (i.e., boron phosphorus distances and other local geometric aspects) related to their catalytic activities; and (3) characterize products of FLP reactions with regard to molecular structure, stereochemistry, and aggregation properties in terms of internuclear distances, bonding connectivities, and orientational parameters.     

Characterization of Structure and Dynamics of the Guanidine-II Riboswitch from Escherichia coli by NMR Spectroscopy and Small-Angle X-ray Scattering (SAXS)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg²⁺ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg²⁺ and ligand binding.     

Mass Spectrometry,Ion Mobility Separation and Molecular Modelling: A Powerful Combination for the Structural Characterisation of Substituted Cyclodextrins Mixtures

When working on the synthesis of substituted cyclodextrins (CDs), the main challenge remains the analysis of the reaction media content. Our objective in this study was to fully characterise a complex isomers mixture of Lipidyl-βCDs (LipβCD) obtained with a degree of substitution 1 (DS = 1) from a one-step synthesis pathway. The benefit of tandem mass spectrometry (MS/MS) and ion mobility separation hyphenated with mass spectrometry (IM-MS) was investigated. The MS/MS fragment ion‘s relative intensities were analysed by principal component analysis (PCA) to discriminate isomers. The arrival time distribution (ATD) of each isomer was recorded using a travelling wave ion mobility (TWIM) cell allowing the determination of their respective experimental collision cross section (CCS_exp). The comparison with the predicted theoretical CCS (CCS_th) obtained from theoretical calculations propose a regioisomer assignment according to the βCD hydroxyl position (2, 3, or 6) involved in the reaction. These results were validated by extensive NMR structural analyses of pure isomers combined with molecular dynamics simulations. This innovative approach seems to be a promising tool to elucidate complex isomer mixtures such as substituted cyclodextrin derivatives.     

Characterization of spatial inhomogeneities and dynamic properties of random cross-linked polystyrene networks by dynamic light scattering

The network inhomogeneity and the cooperative motion of the network chains of random cross-linked poly(styrene-co-maleic anhydride) gels were investigated by dynamic light scattering. Measurements were performed for gels in the preparation state as well as in the swelling equilibrium. Network inhomogeneities and cooperative motion were analyzed at varying the cross-linker concentration and the polymer volume fraction. While the cross-linker concentration has only a minor influence on the inhomogeneity and the diffusion constant D_coop, the polymer volume fraction clearly influences both measured properties. The concentration dependence of D_coop can be well described by a power law, as known for semi-dilute polymer solutions. In the preparation state the networks appear homogeneous, exhibiting dynamic contributions to the scattering intensity of 70-90%. Swollen to equilibrium stage, significant heterogeneities emerge, reducing the dynamic contributions to 10-20%.     

Dynamic light scattering and viscoelasticity of semidilute polystyrene/cyclohexane solutions

In the present work, dynamic light scattering (DLS) measurements from polystyrene in cyclohexane semidilute solutions were carried out at 60 °C with scattering angles varied from 30° to 120°. The correlation functions were analyzed with the double KWW function and the CONTIN program. The amplitude of the fast mode A_f of the field correlation function g¹(t) was used to calculate the longitudinal stress modulus M_o using Wang’s theory. A comparison between M_o calculated from DLS data and the shear stress modulus G obtained from mechanical measurement was made.     

高效液相色谱-串联质谱法测定粮谷中十二环吗啉和十三吗啉残留量

建立了高效液相色谱-串联质谱法(HPLC-MS/MS)测定粮谷中十二环吗啉和十三吗啉残留量的分析方法。样品经1%甲酸-乙腈超声提取、石墨化炭黑/氨基复合固相萃取柱净化、HPLC-MS/MS多反应监测模式测定、外标法定量。结果表明,十二环吗啉和十三吗啉在2.0~200.0μg/L范围内线性关系良好,相关系数均大于0.999。本方法对十二环吗啉和十三吗啉的定量限分别为2.0和5.0μg/kg,样品基质中两种目标物在3个添加水平的平均回收率为78.0%~96.0%,相对标准偏差(RSD)为3.0%~8.2%。该方法简单、灵敏、准确、可靠,可用于粮谷中十二环吗啉和十三吗啉残留的分析。     

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.     

Quantitative Analysis of TAG in Oils Using Lithium Cationization and Direct‐Infusion ESI Tandem Mass Spectrometry

This study was undertaken to determine whether triple‐stage mass spectrometry (MS³) could be employed to obtain quantitative and regioisomeric data from complex oil samples without the need for a chromatographic step in the analysis protocol. Lithium‐7 trifluoroacetate and electrospray ionization were used to form lithium adducts of the triacylglycerols (TAG) in a fish oil sample. The first‐generation precursor ion was the lithium‐TAG adduct, the second‐generation precursor ion was formed by loss of a neutral acid side chain in the first fragmentation. The ions used for analysis were formed in the second fragmentation by loss of the lactones of the acid side chains remaining after the first fragmentation. This analysis scheme provided quantitative and regioisomeric data without interference from TAG in the sample other than TAG with the same acyl carbon number, one more double bond, and two acyl side chains in common with the analyte. Even in this case a majority of the interferences could be estimated and compensated. Analysis of synthetic samples containing the fish oil matrix indicated that both absolute and relative quantitative data could be obtained with average errors of approximately 5 %. The method proved well suited to routine analyses of complex oil samples.     

固相膜萃取-气相色谱/离子阱质谱法测定水中17种多环芳烃

利用固相萃取膜富集,气相色谱-离子阱质谱联用法测定水中17种多环芳烃。通过实验表明,该方法操作简便,灵敏度高,精密度好,采用气质联用技术,内标法测定,定性、定量准确。用该方法测定实际样品,平均回收率为80%～113%,17种多环芳烃的方法检出限为0.07～0.23μg/L。     

Dynamic light scattering studies on the aggregation behavior of tributyl phosphate and straight chain dialkyl amides during thorium extraction

Dynamic light scattering (DLS) studies were carried out to investigate the aggregation behavior of 1.1 M solutions of tributyl phosphate (TBP), N,N-dihexyl octanamide (DHOA) and of N,N-dihexyl decanamide (DHDA) in n-dodecane equilibrated with varying concentrations of nitric acid (0.1–6 M) and of Th (10–200 g/L). There was a gradual increase in thorium extraction with increased aqueous phase acidities. A significant enhancement in the aggregate sizes was observed with increasing concentration of thorium in the organic phase. The effect of 1-octanol as phase modifier was also investigated on the aggregation behavior of extracted species for TBP system.