Adsorption kinetics of deamidated antibody variants on macroporous and dextran-grafted cation exchangers. III. Microscopic studies

The kinetics of single and multicomponent adsorption of deamidated monoclonal antibody (mAb) charge variants is investigated using confocal laser scanning microscopy for two commercial cation exchangers, one with an open macroporous structure--UNOsphere S--and the other with charged dextran grafts--Capto S. Markedly different intraparticle concentration profiles are obtained, being very sharp for UNOsphere S, indicating pore diffusion control, but much more diffuse for Capto S, consistent with a solid or surface diffusion mechanism. For single-component adsorption, the mAb effective pore diffusivities for UNOsphere S are approximately D(e)=4.5×10(-8) and 8.3×10(-8) cm(2)/s at pH 5 and 7.5, respectively, while effective solid diffusivities for Capto S are D(s)=0.98×10(-9) and 5.0×10(-9) cm(2)/s at pH 5 and 7.5, respectively. Two-component adsorption at pH 7.5, where the deamidated variants are bound selectively also showed markedly different profiles for the two matrices. UNOsphere S showed distinct adsorption zones within the particles indicating that multicomponent transport occurs with continuous displacement of the more deamidated variant by the less deamidated one. Capto S, however, showed no spatial resolution of the variants within the particle during co-adsorption and very slow mass transfer during sequential adsorption suggesting that protein counter-diffusion is severely hindered in this material.     

Design of experiments as a valuable tool for biopharmaceutical analysis with (imaged) capillary isoelectric focusing

Capillary isoelectric focusing is indispensable for characterizing charge heterogeneity and isoelectric points of biopharmaceuticals. However, there are many influencing parameters and therefore method development is challenging. This study was performed to obtain an in‐depth understanding of the imaged CIEF methodology by applying a design of experiments approach. To describe the parameter's effects as objectively as possible, a polynomial regression model was derived for the most important responses. For this purpose, the reference monoclonal antibody suggested by the National Institute of Standards and Technology (NISTmAb) was used as test molecule. The total concentration and the mixing ratio of two types of carrier ampholytes and the added amounts of urea and l ‐arginine were selected as factors. The effects of these factors on 13 different responses such as resolution or pI values were investigated. In order to reduce the total number of experiments, a d ‐optimal design with 20 different parameter combinations and six replicates each was chosen. The most significant effects of the four factors were shown for the parameters related to separation efficiency and peak position. In addition, the extent of the factor's effect could be assessed. Depending on the selected factor combination, the pI value can differ up to approximately 0.15 pI units and the resolution value between main peak and adjacent basic peak can range from approximately 1.6 to 2.5, for example.     

基于成簇的规则间隔短回文重复序列的严重急性呼吸综合征冠状病毒2检测的最新进展北大核心CSCD

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)导致的新冠肺炎(COVID-19)迅速蔓延全球,给全球公共卫生系统带来了挑战。由于逆转录-定量聚合酶链反应(RT-qPCR)和抗原测试的普遍适用性和灵敏度较差,并且具有不同突变的SARS-CoV-2变体持续的出现,给疫情防控带来了更大的挑战,因此,高灵敏度、无需设备并且能够区分SARS-CoV-2变体的诊断方法亟须发展。基于成簇的规则间隔短回文重复序列(CRISPR)的诊断对设备要求低,具有可编程性、灵敏性和易用性,已经发展出多种核酸检测工具用于传染病的诊断,其在临床上具有巨大的应用潜力。文章聚焦于近期发表的基于CRISPR实现SARS-CoV-2检测和变体区分的最新技术,总结其特点并对其发展进行了展望。     

Analysis of intracellular and extracellular microcystin variants in sediments and pore waters by accelerated solvent extraction and high performance liquid chromatography-tandem mass spectrometry

The fate and persistence of microcystin cyanotoxins in aquatic ecosystems remains poorly understood in part due to the lack of analytical methods for microcystins in sediments. Existing methods have been limited to the extraction of a few extracellular microcystins of similar chemistry. We developed a single analytical method, consisting of accelerated solvent extraction, hydrophilic–lipophilic balance solid phase extraction, and reversed phase high performance liquid chromatography-tandem mass spectrometry, suitable for the extraction and quantitation of both intracellular and extracellular cyanotoxins in sediments as well as pore waters. Recoveries of nine microcystins, representing the chemical diversity of microcystins, and nodularin (a marine analogue) ranged between 75 and 98% with one, microcystin-RR (MC-RR), at 50%. Chromatographic separation of these analytes was achieved within 7.5 min and the method detection limits were between 1.1 and 2.5 ng g⁻¹ dry weight (dw). The robustness of the method was demonstrated on sediment cores collected from seven Canadian lakes of diverse geography and trophic states. Individual microcystin variants reached a maximum concentration of 829 ng g⁻¹ dw on sediment particles and 132 ng mL⁻¹ in pore waters and could be detected in sediments as deep as 41 cm (>100 years in age). MC-LR, -RR, and -LA were more often detected while MC-YR, -LY, -LF, and -LW were less common. The analytical method enabled us to estimate sediment-pore water distribution coefficients (K_d), MC-RR had the highest affinity for sediment particles (log K_d = 1.3) while MC-LA had the lowest affinity (log K_d = −0.4), partitioning mainly into pore waters. Our findings confirm that sediments serve as a reservoir for microcystins but suggest that some variants may diffuse into overlying water thereby constituting a new route of exposure following the dissipation of toxic blooms. The method is well suited to determine the fate and persistence of different microcystins in aquatic systems.     

Wave solutions for variants of the KdV–Burger and the K(n,n)–Burger equations by the generalized G′/G‐expansion method

An application of the ‐expansion method to search for exact solutions of nonlinear partial differential equations is analyzed. This method is used for variants of the Korteweg–de Vries–Burger and the K(n,n)–Burger equations. The generalized ‐expansion method was used to construct periodic wave and solitary wave solutions of nonlinear evolution equations. This method is developed for searching exact traveling wave solutions of nonlinear partial differential equations. It is shown that the generalized ‐expansion method, with the help of symbolic computation, provides a straightforward and powerful mathematical tool for solving nonlinear problems. Copyright © 2017 John Wiley & Sons, Ltd.     

New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database

The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a 'step-by-step' procedure, we have developed a 'single step' approach for the identification of Hb variants present in biological samples. This is based on the microHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin.     

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)₂ fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8–10.5 with PL 3–10 for variants of intact rituximab and of F(ab´)₂ fragments, respectively, whereas PL 6.7–7.7 in combination with PL 3–10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)₂. In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.     

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high‐resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis‐Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis‐Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε‐amino‐caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico‐chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5–15% and 15–30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.     

An estimate on the convergence of Baskakov-Bézier operators

In the present paper we estimate the rate of convergence on functions of bounded variation for the Bézier variant of the Baskakov operators Bn,α(f,x). Here we have studied the rate of convergence of Bn,α(f,x) for the case 0<α<1.