Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies

A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary‐based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.     

Separation of post-translational modifications in monoclonal antibodies by exploiting subtle conformational changes under mildly acidic conditions

Chromatographic separation plays a key role in the identification, quantification, and characterization of protein variants. Here we describe separation of species containing two post-translational modifications (glycosylation and methionine oxidation) in the Fc fragment of a monoclonal antibody. The method is based on cation-exchange chromatography under mildly acidic conditions that destabilize mainly the CH2 domain. Our data suggest that the separation is not mediated by the chemical modification itself, but rather by subtle structural changes induced by the chemical modification in the domain-decoupled conformation that monoclonal antibodies adopt around pH 4. Compared to other procedures already described in the literature, this method demonstrates an improved separation and allows purification of species in the native fold for additional functional characterization. This approach of separation under conditions where the protein assumes an alternative conformation could find a more general utility for the separation of chemical modifications in proteins.     

pH-based cation exchange chromatography in the capture and elution of monoclonal antibodies

Cation exchange chromatography separates adsorbed proteins by controlling the salt concentration or the mobile phase pH. This study examines the pH‐based method for binding and elution of monoclonal antibodies (MAbs). Five different clones with isoelectric points from 6 to 9 were evaluated. We performed our studies using a new chromatography resin (Nuvia? S), which has high binding capacity. A three‐column process incorporating Nuvia S as a capture step was also demonstrated for the purification of MAb from tissue culture fluid. Chromatography performance of Nuvia S was demonstrated in a 50‐cycle study.     

Synthesis of protected peptides from the human IgG1 hinge region on PEG support using disulfide bond synthons and alkaline or enzymatic detachment

Peptides from the hinge region of human IgG1 are potential defined carriers of synthetic immunogens. Their synthesis was carried out on the soluble support—PEG-OMe 2000 using disulfide bond synthons. The loading capacity of the polymer was increased twofold by anchoring the lysine as a simple branching unit. The amino acid leucine was used as an enzymatically cleavable linker. Peptide detachment from the polymer was performed either by classical ester bond saponification or newly by peptide bond cleavage catalyzed by thermolysin in water.     

Conformations and vibrational properties of disulfide bridges: Potential energy distribution in the model system diethyl disulfide

Disulfide bridges are important structural elements in proteins. It is well-known that the position of the characteristic disulfide band at ca. 500 cm⁻¹ in the vibrational spectra varies with the conformation around the disulfide unit. In our computational study on the model system diethyl disulfide, both wavenumber and normal mode composition are analyzed simultaneously as a function of conformation. For the disulfide band, a negative correlation between the calculated vibrational wavenumber and the SS stretching contribution is detected. This trend in the normal mode composition provides an explanation for experimentally observed wavenumber shifts of the disulfide band.     

Emulsion stabilized with disulfide proteinoid and its stability in reducing condition

Reduction-responsive oil-in-water emulsion was prepared by stabilizing droplets using a disulfide proteinoid composed of Asp, Leu, and cystamine (Prot(ALC)). A disulfide-free proteinoid (Prot(AL)) was prepared as a control emulsifier. FTIR and ¹H NMR spectroscopy revealed that the disulfide bond was in Prot(ALC). The interfacial activity of Prot(ALC) in dithiothreitol (DTT) solution (10?mM) was significantly higher than in distilled water. The emulsion stabilized with Prot(ALC) had a few micrometers of droplets in diameter and it was more stable than the emulsion stabilized with Prot(AL) in phase separation. The emulsion with DTT was more stable than the emulsion without the reducing agent.     

Novel C-H activation and C-S formation reactions on disulfide and diselenide ligands in dinuclear ruthenium complexes

The C-S bond formation reactions of the transition metal sulfides with organic molecules are collected and reviewed to understand the reactivity of the sulfide ligands supported by the transition metals. As an example of the role of the sulfide, the C-H bond activation is focused and discussed.     

Terahertz time-domain and Raman spectroscopy of the sulfur-containing peptide dimers: Low-frequency markers of disulfide bridges

The terahertz time-domain and Raman spectra of sulfur-containing cystein-based peptides in the region of the low-frequency infrared vibrations have been measured at room temperature. The low-frequency bands that can be assigned to the S–S bridges are observed. The vibrational modes found in molecular crystalline materials should be described as phonon modes with strong coupling to the intra molecular vibrations.     

RhCl3-catalyzed disulfide exchange reaction using water solvent in homogeneous and heterogeneous systems

RhCl₃ catalyzed the alkylthio exchange reaction of hydrophilic disulfides in water under homogeneous conditions, and equilibrium was attained in several hours. The reaction was applied to the exchange of unprotected glutathione disulfide. The reaction of dimethyl disulfide and hydrophilic disulfides under heterogeneous conditions also proceeded effectively. The mechanism turned out to be dependent on the water solubility of the substrates: The reaction of bis(3-hydroxypropyl) disulfide took place in the dimethyl disulfide phase, whereas the reaction of bis(6-aminohexyl) disulfide dihydrochloride proceeded in the water phase.     

Synthesis of 2-arylbenzothiazoles via direct condensation between in situ generated 2-aminothiophenol from disulfide cleavage and carboxylic acids

In this work we describe a simple and efficient general methodology for 2-arylbenzothiazole preparation employing disulfides and carboxylic acids. The reaction is promoted by tributylphosphine that acts both in disulfide bond cleavage and as activating agent for coupling with carboxylic acids. The reaction scope was studied using bis(2-aminophenyl)disulfide and different carboxylic acids with donor/withdrawing substituents, which resulted in the desired 2-arylbenzothiazole with moderate to good yields. The method was tested with success in preparation of the amyloid probe 2-(4-aminophenyl)-6-methoxybenzothiazole that employed a substituted bis(2-aminophenyl)disulfide.     

Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins

Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content.     

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.      

Cutting‐edge mass spectrometry characterization of originator,biosimilar and biobetter antibodies

The approval process for antibody biosimilars relies primarily on comprehensive analytical data to establish comparability and high similarity with the originator. Mass spectrometry (MS) in combination with liquid chromatography (LC) and electrophoretic methods are the corner stone for comparability and biosimilarity evaluation. In this special feature we report head‐to‐head comparison of trastuzumab and cetuximab with corresponding biosimilar and biobetter candidates based on cutting‐edge mass spectrometry techniques such as native MS and ion‐mobility MS at different levels (top, middle and bottom). In addition, we discuss the advantages and the limitations of sample preparation and enzymatic digestion, middle‐up and ‐down strategies and the use of hydrogen/deuterium exchange followed by MS (HDX‐MS). Last but not least, emerging separation methods combined to MS such as capillary zone electrophoresis‐tandem MS (CESI‐MS/MS), electron transfer dissociation (ETD), top down‐sequencing (TDS) and high‐resolution MS (HR‐MS) that complete the panel of state‐of‐the‐art MS‐based options for comparability and biosimilarity evaluation are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Well-defined higher-molecular-weight polyacrylonitrile via RAFT technique in the presence of disulfide compounds as a source of chain transfer agent

Well-defined polyacrylonitrile with a higher number-average molecular weight () up to 200,000 and a lower polydispersity index (PDI, 1.7-2.0) was firstly obtained via reversible addition-fragmentation chain transfer (RAFT) process. This was achieved by selecting a stable, easy way to prepare disulfide compound intermediates including bis(thiobenzoyl) disulfide (BTBDS) and bis(thiophenylacetoyl) disulfide (BTPADS) to react with azobis(isobutyronitrile) to directly synthesize RAFT agents in situ. The polymerization of acrylonitrile (AN) displays the characteristics of controlled/living radical polymerization as evidenced by pseudo first-order kinetics of polymerization, linear evolution of molecular weight with increasing monomer conversion, and narrow PDIs. The polymerization rate and the efficiency for producing RAFT agent of BTPADS system are obviously higher than those of BTBDS system, whereas the control of the latter over the polymerization is superior to that of the former. ¹H NMR analysis has confirmed the dithioester chain-end functionality of the resultant polymer. The RAFT copolymerizations of AN and the comonomers including methyl acrylate, itaconic acid, methyl methacrylate, n-butyl acrylate, 2-hydroxyethyl acrylate, and acrylamide were also successfully carried out using the same polymerization system.     

Characterization of the coupling of quantum dots and immunoglobulin antibodies

Water-soluble quantum dots (QDs) were used to label goat anti-human immunoglobulin antibodies (Abs), and the labeling process was characterized by column purification. The QDs obtained in organic solvent were modified with mercaptoacetic acid (MAA) and became water-soluble. These water-soluble QDs were linked to the antibodies using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The linking process was shown to be effective by ultra-filter centrifugation and column purification. After comparing the quantities of Abs and water-soluble QDs involved in the linking reaction via column purification, it was found that a molar Abs:QD ratio of >1.2 resulted in most of the water-soluble QDs becoming covalently linked to the Abs. The circular dichroism (CD) spectra of Abs and QD–Ab conjugates were very similar to each other, indicating that the secondary structure of Abs remained largely intact after the conjugation. Finally, antigen (Ag)–antibody (Ab) recognition reactions perfomed on the surface of a glass slide showed that the conjugate retained the activity of Abs. This work lends support to the idea of linking biomolecules to QDs, and thus should aid the application of QDs to the life sciences. Figure Firstly in this work, the conjugates of QDs-Ab were separated from EDC&NHS in the column of Sephadex G-100(left up). Then the bioactivity of QDs-Ab was analyzed in the immunoassay (right) and the immunofluorescent signals were detected (left bottom) finally     

A comparative study of LED-induced fluorescence and laser-induced fluorescence in SDS-CGE: application to the analysis of antibodies

LEDs present an alternative to lasers in LIF detection with CE, resulting in LED-induced fluorescence (LEDIF). LEDs are much less expensive, consume less energy and are more stable. In addition, LED light sources allow a greater range of wavelengths to better match the maximum wavelength for the fluorescence of the dye. Antibodies were largely studied in SDS capillary gel electrophoresis (SDS-CGE) and LIF detection with different dyes to label the proteins. In this work, our goal is to show that LEDs can advantageously replace lasers. We used 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA.SE), 3-(2-furoyl)-quinoline-2 carboxaldehyde (FQ), and naphthalene-2,3-dialdehyde (NDA) to label IgG and we compared the LIF sensitivity with that obtained from LEDIF. We measured that the LOD values of LEDIF are identical to that obtained with the wavelength equivalent laser, and for 5-TAMRA.SE analysis, LOD values are about six times better than when the classical 488 nm laser was used.     

特异性吗啡抗原和单克隆抗体的制备

吗啡分子的不同部位与蛋白偶联诱导出的抗体的专一性差异很大。为减少交叉反应,提高抗体分子对游离吗啡的特异性识别,选择吗啡分子N位进行修饰,合成了半抗原降吗啡,并设计和合成不同的连接臂,将半抗原用不同的连接臂与不同的载体蛋白共价结合分别制备了免疫抗原和筛选抗原,经细胞融合和筛选,成功地获得了5株可分汔抗吗啡单克隆抗体的细胞株：28H10,29D5,36G3,42D5,43C4。     

Production and characterization of monoclonal antibodies against urea derivatives

A panel of monoclonal antibodies was generated against the ureabased haptenN-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Crossreactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.     

Development and characterization of new rat monoclonal antibodies for procalcitonin

The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen-antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K (D)) of 3.42 x 10(-8) M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K (A) = 2.81-6.11 x 10(8) M(-1); K (D) = 1.64-3.56 x 10(-9) M) and have moderate dissociation rate constants (k (d) = 1.70-2.40 x 10(-3) s(-1)). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 microg L(-1). In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer(R)). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 microg L(-1) and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6-biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.