Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

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.     

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.     

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.     

水中硝基苯胺类化合物酶免疫化学分析技术研究

采用戊二醛法, 将4-硝基苯乙胺与牛血清蛋白(BSA)和卵清蛋白(OVA)共价偶联, 分别合成了免疫原4-硝基苯乙胺-BSA和包被原4-硝基苯乙胺-OVA, 经紫外分光光度计及飞行时间质谱扫描鉴定. 用合成的免疫抗原免疫新西兰大白兔, 并用合成的包被原进行间接竞争酶联免疫(ELISA)试验, 获得的抗血清效价达1∶32000. 方阵实验确定了包被抗原最佳浓度(0.5 mg/L)及抗血清最佳稀释度(1∶6000), 并建立了间接竞争ELISA方法. 工作曲线表明在1～1000 μg/L浓度范围内呈良好的线性关系, 该法IC₅₀值为(52.73±2.67) μg/L, 检测限为5.12 μg/L. 其它类似结构不干扰硝基苯胺的测定. 成功地建立了硝基苯胺类化合物的间接竞争酶免疫化学分析方法．     

Nanocatalytic resonance scattering spectral analysis

The resonance scattering spectral technique has been established using the synchronous scanning technique on spectrofluorometry.Because of its advantages of simplicity,rapidity and sensitivity,it has been widely applied to analyses of proteins,nucleic acids and inorganic ions.This paper summarizes the application of immunonanogold and aptamer modified nanogold(AptAu) catalytic resonance scattering spectral technique in combination with the work of our group,citing 53 references.     

稻瘟灵单克隆抗体的制备及评价

该文制备了农药稻瘟灵的单克隆抗体,并建立了稻瘟灵的酶联免疫吸附（ELISA）检测方法。在完全保留稻瘟灵结构的基础上从二硫杂环戊烷结构中衍生不同长度的活性手臂制备了2个半抗原,并分别与载体蛋白偶联合成免疫原与包被原。通过小鼠免疫、细胞融合、淘筛、腹水制备等步骤获得特异性识别稻瘟灵的单克隆抗体mAb-DWL。结果显示,基于mAb-DWL构建的间接竞争ELISA法的半抑制浓度（IC50）为55.2 ng/mL,线性范围为4.6～530.2 ng/mL,其与结构类似物的交叉反应可忽略不计。所建立的ELISA方法对蔬菜及粮食等样品的加标回收率为77.2%～116%,可用于实际样品的快速检测。     

Fe3O4/葡聚糖/抗体磁性纳米生物探针的制备和层析检测

在免疫检测中 ,经常利用一些具有特殊物理化学性质的标记物对抗体 (或抗原 )进行偶联标记 ,在抗体与抗原识别后 ,通过对标记物的定性和定量检测而达到对抗原 (或抗体 )检测的目的 .传统的免疫标记物包括放射性同位素 [1] 、酶 [2 ] 、胶体金 [3] 和有机荧光染料分子 [4 ] 等 .近年来 ,随着纳米技术的发展 ,半导体荧光纳米晶 [5,6 ] 和磁性纳米晶 [7] 在免疫检测方面受到了广泛关注 .磁性纳米晶性能稳定 ,较易制备 ,可与多种分子复合使粒子表面功能化 ,并且由于磁纳米晶具有超顺磁性 ,为样品的分离、富集和提纯提供了很大方便 .这些优点使它…