A methodological approach for the thermal stability and stress exposure studies of a model antibody.

The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials. In the developed method, the anti-HRP antibody is covalently immobilized onto a solid surface. After the direct recognition of its antigen HRP, the signal is proportional to the number of antibody active binding sites. The method was successfully utilized to accurately evaluate the anti-HRP antibody melting temperature (Tm was 73.5 ±â€¯0.2 °C). The method is a rapid and reliable tool with minimal cost for studying the anti-HRP antibody stability to solvent stress, freeze-thawing cycles, and freeze-drying process. The obtained information may be useful for routine analysis or in the development of antibody-based materials. This can be also proposed as an easy way to control antibody freeze-drying process.

Evaluation of capillary zone electrophoresis for the quality control of complex biologic samples: Application to snake venoms.

Snake venoms constitute a very promising resource for the development of new medicines. They are mainly composed of very complex peptide and protein mixtures, which composition may vary significantly from batch to batch. This latter consideration is a challenge for routine quality control (QC) in the pharmaceutical industry. In this paper, we report the use of capillary zone electrophoresis for the development of an analytical fingerprint methodology to assess the quality of snake venoms. The analytical fingerprint concept is being widely used for the QC of herbal drugs but rarely for venoms QC so far. CZE was chosen for its intrinsic efficiency in the separation of protein and peptide mixtures. The analytical fingerprint methodology was first developed and evaluated for a particular snake venom, Lachesis muta. Optimal analysis conditions required the use of PDADMAC capillary coating to avoid protein and peptide adsorption. Same analytical conditions were then applied to other snake venom species. Different electrophoretic profiles were obtained for each venom. Excellent repeatability and intermediate precision was observed for each batch. Analysis of different batches of the same species revealed inherent qualitative and quantitative composition variations of the venoms between individuals.

Optimization of a nano-enzymatic reactor for on-line tryptic digestion of polypeptide conjugates by capillary electrophoresis.

This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.

A rapid and reversible colorimetric assay for the characterization of aminated solid surfaces.

The covalent immobilization of synthetic or natural macromolecular compounds containing amino groups onto polystyrene (PS) solid surfaces is of great interest in diagnostic applications. A sensitive assay allowing the determination of reactive end groups is therefore a powerful tool for predicting the performance of the active surface. Recently, we reported the use of the Coomassie brilliant blue (CBB) colorimetric reagent to quantify protonated groups (N(+)) in linear and dendritic structures in solution (Coussot et al., Polym Int 58(5):511-518, 2009). In this work, a simple method using CBB dye for the characterization of PS aminated solid surfaces is developed. The proposed amino density estimation by colorimetric assay (ADECA) method is based on the reversible complexation of the dye with the N(+) groups on solid surfaces. The assay measures the released dye thanks to the use of a unique sodium carbonate-methanol buffer. Thereby, for the first time, the same surface can be used for characterization and for further coupling applications. A surface density of four N(+) groups per square nanometer can be measured in PS microwell format, the whole characterization being done within 30 min. Performances of this new colorimetric-based method are detailed. The ADECA method is further demonstrated to be useful for the characterization of aminated polypropylene and glass materials with various sizes and shapes.

Dendrigraft poly-L-lysine: a non-immunogenic synthetic carrier for antibody production.

An easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens.

α-Glucosidase Inhibitory Activity of Tannat Grape Phenolic Extracts in Relation to Their Ripening Stages.

The present study aimed to screen grape extracts as novel α-glucosidase inhibitors to prevent type-2 diabetes and hyperglycemia. The total polyphenol content (TPC) was measured by Folin-Ciocalteu assay and the stilbene, anthocyanin and flavan-3-ol compounds were measured by Ultra High-Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). The α-glucosidase inhibitory of seed and skin Tannat grape extracts at four ripening stages were investigated. The highest TPC values were measured in seeds at the "veraison stage" (65.29 ± 5.33 g of Gallic Acid Equivalent (GAE) per kilogram of Fresh Weight (FW)). This was in accordance with the high flavan-3-ol contents measured for these two extracts (43.22 ± 2.59 and 45.45 ± 6.48 g/kg of seeds FW, respectively). The skin and seed extracts at the first stage of ripening exerted strong α-glucosidase inhibition, exceeding 95% (p < 0.05). A high linear correlation (R = 0.723, p ≤ 0.05) was observed between flavan-3-ol contents and the α-glucosidase inhibitory activity. The stilbene contents and this activity were moderately to strongly anti-correlated (R = -0.828, p ≤ 0.05 for trans-resveratrol). The enzyme kinetic studies revealed a mixed type of inhibition. This study brings promising results for the therapeutic potential of seed and skin Tannat grape extracts as a functional food product with anti-diabetic activity.

In-line tryptic digestion of therapeutic molecules by capillary electrophoresis with temperature control.

The main purpose of the present work is to provide a fully integrated temperature control of in-line tryptic digestion in order to facilitate the quality control of polypeptidic therapeutic compounds. The in-line enzymatic reaction was performed in 100 mM bicarbonate ammonium whereas a mixture of citric acid/ε-amino caproic acid (pH 5.0 and I 75 mM) was used as a background electrolyte (BGE). After the injection of all reactants (substrate, enzyme, proteolysis buffer), a BGE plug was injected to push all reactants until a position where the capillary is thermostated. Then, the enzymatic reaction was initiated during 15 min of incubation time and finally, a voltage was applied to separate the generated proteolysis products. The methodology was developped regarding the effects of the BGE plug length and pressure on the reactants plug mixing and on the advanced of the tryptic digestion. Successful temperature control of in-line proteolysis with excellent repeatability was obtained in optimal cleavage and separation conditions.

Photochemistry on the Space Station-Antibody Resistance to Space Conditions after Exposure Outside the International Space Station.

Antibody-based analytical instruments are under development to detect signatures of life on planetary bodies. Antibodies are molecular recognition reagents able to detect their target at sub-nanomolar concentrations, with high affinity and specificity. Studying antibody binding performances under space conditions is mandatory to convince space agencies of the adequacy of this promising tool for planetary exploration. To complement previous ground-based experiments on antibody resistance to simulated irradiation, we evaluate in this paper the effects of antibody exposure to real space conditions during the EXPOSE-R2 mission outside the International Space Station. The absorbed dose of ionizing radiation recorded during the 588 days of this mission (220 mGy) corresponded to the absorbed dose expected during a mission to Mars. Moreover, samples faced, at the same time as irradiation, thermal cycles, launch constraints, and long-term storage. A model biochip was used in this study with antibodies in freeze-dried form and under two formats: free or covalently grafted to a solid surface. We found that antibody-binding performances were not significantly affected by cosmic radiation, and more than 40% of the exposed antibody, independent of its format, was still functional during all this experiment. We conclude that antibody-based instruments are well suited for in situ analysis on planetary bodies.

Photochemistry on the Space Station-Aptamer Resistance to Space Conditions: Particles Exposure from Irradiation Facilities and Real Exposure Outside the International Space Station.

Some microarray-based instruments that use bioaffinity receptors such as antibodies or aptamers are under development to detect signatures of past or present life on planetary bodies. Studying the resistance of such instruments against space constraints and cosmic rays in particular is a prerequisite. We used several ground-based facilities to study the resistance of aptamers to various types of particles (protons, electrons, neutrons, and carbon ions) at different energies and fluences. We also tested the resistance of aptamers during the EXPOSE-R2 mission outside the International Space Station (ISS). The accumulated dose measured after the 588 days of this mission (220 mGy) corresponds to the accumulated dose that can be expected during a mission to Mars. We found that the recognition ability of fluorescently labeled aptamers was not significantly affected during short-term exposure experiments taking into account only one type of radiation at a time. However, we demonstrated that the same fluorescent dye was significantly affected by temperature variations (-21°C to +58°C) and storage throughout the entirety of the ISS experiment (60% of signal loss). This induced a large variability of aptamer signal in our analysis. However, we found that >50% of aptamers were still functional after the whole EXPOSE-R2 mission. We conclude that aptamer-based instruments are well suited for in situ analysis on planetary bodies, but the detection step requires additional investigations.

Phenyl phosphotriester derivatives of AZT: variations upon the SATE moiety.

Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'-dideoxythymidine) are described herein. These pronucleotides are characterized by the presence of polar functions on the SATE biolabile phosphate protections. Whereas derivatives incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moieties confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, the monohydroxylated SATE derivative of AZT 2, is able to cross a Caco-2 cell monolayer mainly in intact form, probing that further development is warranted as a possible HIV-pronucleotide candidate.

A gold standard method for the evaluation of antibody-based materials functionality: Approach to forced degradation studies.

The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 1012 to 2.98 × 1012 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses.

Integrated microreactor for enzymatic reaction automation: An easy step toward the quality control of monoclonal antibodies.

The main purpose of the present work is to provide a fully integrated miniaturized electrophoretic methodology in order to facilitate the quality control of monoclonal antibodies (mAbs). This methodology called D-PES, which stands for Diffusion-mediated Proteolysis combined with an Electrophoretic Separation, permits to perform subsequently mAb tryptic digestion and electrophoresis separation of proteolysis products in an automated manner. Tryptic digestion conditions were optimized regarding the influence of enzyme concentration and incubation time in order to achieve similar enzymatic digestion efficiency to that obtained with the classical methodology (off-line). Then, the optimization of electrophoretic separation conditions concerning the nature of background electrolyte (BGE), ionic strength and pH was realized. Successful and repeatable electrophoretic profiles of three mAbs digests (Trastuzumab, Infliximab and Tocilizumab), comparable to the off-line digestion profiles, were obtained demonstrating the feasibility and robustness of the proposed methodology. In summary, the use of the proposed and optimized in-line approach opens a new, fast and easy way for the quality control of mAbs.

Separation of 3'-azido-2',3'-dideoxythymidine pronucleotide diastereoisomers in biological samples by CZE with cyclodextrin addition.

The possibility of using capillary electrophoresis as an alternative technique to HPLC for the separation of pronucleotide diastereoisomers of AZT was investigated. In the pH range 6.2-7.2 where the analytes are stable, a chiral additive, carboxymethyl-beta-CD, was found appropriate to enable the separation of the uncharged diastereoisomers. An experimental design strategy was used to study the influence of several parameters (CD and phosphate buffer concentration, methanol content of the electrolyte, injected volume, capillary length, electric field and separation temperature) on the separation and find suitable analytical conditions for monitoring the prodrugs in cell extracts. The diastereoisomers of the three tBuSATE phenylphosphotriester derivatives of AZT studied could be fully resolved within short analysis time (less than 10 min). Method validation results showed satisfactory results for linearity, accuracy and repeatability.

S-acyl-2-thioethyl aryl phosphotriester derivatives of AZT: synthesis, antiviral activity, and stability study.

The synthesis, antiviral activity, and stability study of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing modified l-tyrosinyl residues are reported. These compounds were obtained via phosphoramidite (P(III)) chemistry from the appropriate aryl precursors. All the derivatives were evaluated for their in vitro anti-HIV activity, and they appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments, with EC(50) values between the micro- and nanomolar range, especially in thymidine kinase deficient (TK(-)) cells, showing their ability to act as mononucleotide prodrugs. The proposed decomposition process of these mixed mononucleoside aryl phosphotriesters successively involves an esterase and a phosphodiesterase hydrolysis.

Antibody-based surfaces: rapid characterization using two complementary colorimetric assays.

Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A(2)HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.