Optimization of capillary zone electrophoresis for charge heterogeneity testing of biopharmaceuticals using enhanced method development principles

CZE is a well‐established technique for charge heterogeneity testing of biopharmaceuticals. It is based on the differences between the ratios of net charge and hydrodynamic radius. In an extensive intercompany study, it was recently shown that CZE is very robust and can be easily implemented in labs that did not perform it before. However, individual characteristics of some examined proteins resulted in suboptimal resolution. Therefore, enhanced method development principles were applied here to investigate possibilities for further method optimization. For this purpose, a high number of different method parameters was evaluated with the aim to improve CZE separation. For the relevant parameters, design of experiments (DoE) models were generated and optimized in several ways for different sets of responses like resolution, peak width and number of peaks. In spite of product specific DoE optimization it was found that the resulting combination of optimized parameters did result in significant improvement of separation for 13 out of 16 different antibodies and other molecule formats. These results clearly demonstrate generic applicability of the optimized CZE method. Adaptation to individual molecular properties may sometimes still be required in order to achieve optimal separation but the set screws discussed in this study [mainly pH, identity of the polymer additive (HPC versus HPMC) and the concentrations of additives like acetonitrile, butanolamine and TETA] are expected to significantly reduce the effort for specific optimization.     

Synthesis of quinoline thioethers as novel small molecule enhancers of monoclonal antibody production in mammalian cell culture

A small library of novel quinoline derivatives containing different thioether substituents at position 4 have been synthesized and screened in murine hybridoma cell culture for their ability to enhance monoclonal antibody (mAb) production. From this set of compounds, four compounds have been discovered that enhanced immunoglobulin G (IgG) titer over negative control cultures due to an increase in specific productivity. These results demonstrate the utility of using organic synthesis and parallel ligand screening methods to discover novel cell culture additives that may be useful for increasing mAb yield in industrial biomanufacturing processes.     

Antibody Interactions with Ricinus communis Agglutinins Studied by Biolayer Interferometry

Two related agglutinins are present in the seeds of Ricinus communis (castor): ricin, a dichain ribosome-inactivating protein and Ricinus communis agglutinin-1, a much less toxic tetrameric hemagglutinin. The immunochemical analysis of these agglutinins is of special interest because ricin toxicity has resulted in both accidental and intentional poisonings, while it has also provided a potential cancer chemotherapeutic in the form of an immunoconjugate. We previously characterized a panel of monoclonal antibodies (mAbs) for the analysis of potential contamination with ricin in several food matrices. In this study, an optical sensing technique, biolayer interferometry (BLI), was used to study the binding of two mAbs to the agglutinins. MAbs were immobilized on sensors with amine-reactive, Fc-binding, and streptavidin-coated tips to study the interactions with the agglutinins and with ricin A- and B-chains in solution. The kinetically determined equilibrium dissociation constants generally agreed with the relative binding observed in ELISA, although binding was less predictable for the isolated ricin chains. BLI analysis of kinetic constants for mAb 1797 was not affected by nonfat milk (0.5% by volume). BLI provides a useful method to characterize the binding of antibodies, with the potential for immunodiagnostic applications in food matrices.     

有机质谱应用于抗体类药物结构解析的研究进展

抗体类药物因其靶向性,能够直接与目标特异结合,并且药物副作用小、毒性低,在临床上具有广阔的应用前景。质谱(MS)以其快速、高灵敏度和高分辨率成为抗体药物结构分析的重要手段,为药物的质量控制和安全性方面提供了强有力的技术支撑。该文针对快速发展的有机质谱技术在抗体类药物的氨基酸序列、高级结构以及修饰鉴定方面的应用进行了综述。     

一种新的混合斑精子分离方法

制备了精子表面膜抗原受精素β蛋白(Fertilinβ)的特异性抗体,通过抗原-抗体反应和抗体固相偶联技术,将抗体连接到琼脂糖球珠上,建立了混合斑精子分离纯化的新方法.首先,通过聚合酶链式反应(PCR)扩增编码人受精素β蛋白第341~373位氨基酸的基因序列,构建PGEX-4T-1/Fertilinβ原核表达质粒;其次,诱导表达GST-Fertilinβ融合蛋白,制备受精素β蛋白多克隆抗体,免疫荧光检测结果表明,受精素β蛋白定位于精子的头后部,并且阴道上皮细胞没有受精素β蛋白的表达;最后,将受精素β抗体与ProteinA琼脂糖球珠连接构建固相抗体系统,将精子吸附于表面,可从混合斑(精子与阴道上皮细胞混合液)中分离纯化精子.本文为性犯罪案件侦破提供了新的方法.     

磺胺类药物单链抗体的制备及其与磺胺噻唑相互作用研究

抗体与对应的小分子待测物之间的相互作用模式决定了免疫分析的特性。本研究以磺胺类药物杂交瘤细胞株4C7为起点,应用基因工程技术制备出单链抗体scFv4C7,采用间接竞争ELISA法对比其与母本单克隆抗体的识别特性,同时采用同源建模构建scFv4C7的三维立体结构,并与磺胺噻唑( STZ)进行分子对接。间接竞争ELISA结果显示scFv4C7保留了亲本单克隆抗体的识别特性,分子对接结果显示STZ深陷入抗体的重链和轻链形成的“口袋”中,STZ分子更靠近重链,且主要与抗原互补决定区CDR H3相互作用。本研究为制备识别谱更广、亲和力更高的磺胺类药物抗体提供了必要的结构信息。     

Interference‐free mass spectrometric detection of capillary isoelectric focused proteins,including charge variants of a model monoclonal antibody

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state‐of‐the‐art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE‐ESI‐MS applying a mechanical valve. During the CZE step, the remaining ESI‐interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart‐cut approach is presented expanding our previous single heart‐cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63–9.75 mg/L), covering a wide pI range (5–10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF‐CZE‐MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.     

河豚毒素的样品前处理与快速检测技术研究进展

河豚毒素是一种毒性极强的小分子、非蛋白神经毒剂,其性质相对稳定,缺乏特效解毒剂。其检测技术主要有生物检测法、理化检测法和免疫学检测法,其中免疫检测因快速、简便、灵敏而得到较为广泛的应用。该文综述了河豚毒素的样品前处理技术、免疫学快速检测技术及相关专利的研究进展情况,并对河豚毒素检测产品的开发进行了展望。     

Silicon nanowires as field-effect transducers for biosensor development: A review

The unique electronic properties and miniaturized dimensions of silicon nanowires (SiNWs) are attractive for label-free, real-time and sensitive detection of biomolecules. Sensors based on SiNWs operate as field effect transistors (FETs) and can be fabricated either by top–down or bottom–up approaches. Advances in fabrication methods have allowed for the control of physicochemical and electronic properties of SiNWs, providing opportunity for interfacing of SiNW-FET probes with intracellular environments. The Debye screening length is an important consideration that determines the performance and detection limits of SiNW-FET sensors, especially at physiologically relevant conditions of ionic strength (>100 mM). In this review, we discuss the construction and application of SiNW-FET sensors for detection of ions, nucleic acids and protein markers. Advantages and disadvantages of the top–down and bottom–up approaches for synthesis of SiNWs are discussed. An overview of various methods for surface functionalization of SiNWs for immobilization of selective chemistry is provided in the context of impact on the analytical performance of SiNW-FET sensors. In addition to in vitro examples, an overview of the progress of use of SiNW-FET sensors for ex vivo studies is also presented. This review concludes with a discussion of the future prospects of SiNW-FET sensors.