Plasma-derived clotting factor VIII: heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors

In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma‐derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product‐specific variations from Emoclot^®, Alphanate^® and Haemate^® both in the presence and absence of reducing agents (dithiotreithol). All the plasma‐derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full‐length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate^® and 29 and 23% in Alphanate^® and Emoclot^®, respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B‐domain truncated forms of the FL protein along with biologically active digested‐A1 forms. Batch‐to‐batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter‐product comparisons yielded similar results for all the plasma‐derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma‐derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.     

Studies on the Selective Reduction of the Amide Link of Acyclic and Macrocyclic Amidoketals: Unexpected Cleavage and trans‐Acetalization with Red‐Al®

Selective reduction of the amide moiety of acyclic and macrocyclic amidoketals was studied in presence of various reagents (BH₃ · Me₂S, iBuAlH₂, Red‐Al^®, LiAlH₄). The best results were obtained with lithium aluminium hydride in the presence of triethylamine traces, whereas borane dimethyl sulfide gave rise to a partial ketal reduction of the acyclic compound and Red‐Al^® to a cleavage of the macrocyclic molecule accompanied by an unexpected trans‐acetalization.     

Utilization of Maltodextrin‐Based Enantioselective,Potentiometric Membrane Electrodes for the Enantioselective Assay of S‐Flurbiprofen

Abstract

Three enantioselective, potentiometric membrane electrodes (EPMEs) based on carbon paste impregnated with different maltodextrins [dextrose equivalent (DE) (DE 4.0–7.0 (I), 13–17 (II), 16.5–19.5 (III)], were proposed as chiral selectors for the assay of S‐flurbiprofen raw materials and from its pharmaceutical formulation, Froben 100^® tablets. The best response and enantioselectivity were obtained when maltodextrin with lower DE was used for the electrode design. The three EPMEs showed very low detection limits. The surfaces of the electrodes are easily renewable by simply polishing on an alumina paper.     

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE

2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.     

Fracture and failure behavior of partially consolidated ciscontinuous glass fiber mat‐reinforced polypropylene composites (Azdel SuperLite®)

The fracture and failure behavior of partially consolidated discontinuous glass fiber (GF) mat reinforced thermoplastic polypropylene (Azdel SuperLite^®) composite sheets of various densities (0.35, 0.5 and 0.7 g·cm⁻³) but with the same amount of GF (55 wt.%) were studied under static (in‐plane) and dynamic (out‐of‐plane perforation impact) conditions. The fracture toughness (K_Q) determined on single edge notched static tensile loaded (SEN‐T) specimens, increased with increasing density (or surface weight as the sheet thickness was constant, viz. 2 mm). Location of the acoustic emission (AE) and mapping the temperature rise during loading of the SEN‐T specimens via infrared thermography (IT) served to estimate the damage zone and trace the crack advance. Both techniques seem to be promising tools to determine the energy release rate directly. The unexpected high K_Q value was attributed to a combined effect of fiber nesting (achieved by the papermaking production technology) and high stress transfer GF length (owing to partial consolidation). This resulted in an efficient stress transfer and stress redistribution during damage zone development and growth. The resistance to perforation impact of the SuperLite^® sheets also increased with their density (surface weight).     

Investigation of commercial sorbents for the analysis of opioid peptides in human plasma by on‐line SPE‐CE

In this study, we investigated the performance of several commercial sorbents (Sep‐pack^® C18, ^tC18, C8 and ^tC2, Oasis^® HLB, Isolute^® ENV+, Strata^?‐X and Oasis^® MCX) for the determination of opioid peptides by solid‐phase extraction coupled on‐line to capillary electrophoresis (SPE‐CE). First, standard solutions were analyzed in order to achieve the lowest LOD and the best electrophoretic separations using UV detection. The best results were obtained using C18, C8 and ^tC2 sorbents, which were examined for the analysis of spiked human plasma samples. A double‐step sample clean‐up pretreatment, which consisted of precipitation with acetonitrile and filtration, was needed to prevent saturation of the on‐line SPE microcartridge. The filtration step was critical to obtain optimum analyte recovery and to clean up the sample matrix. A range of centrifugal filters and filtration conditions were tested and the recoveries of the sample pretreatment were evaluated by CE‐ESI‐MS. The LODs attained through SPE‐CE‐UV were approximately ten‐fold better with C18 than with C8 and ^tC2. The 0.1 μg/mL LODs achieved by C18‐SPE‐CE‐UV were further improved until we could detect 1 ng/mL concentrations of opioid peptides in plasma samples by C18‐SPE‐CE‐ESI‐MS, due to the outstanding selectivity of the MS detection.     

Enzyme‐Mediated Preparation of Chiral 1,3‐Dioxane Odorants

The enantiomerically enriched diastereoisomers of the chiral 1,3‐dioxane odorants Floropal^® ( 1 ) and Magnolan^® ( 2 ) were prepared by an enzyme‐mediated approach. Their olfactory properties were evaluated to investigate differences in the odor perception for the stereoisomers.     

Interaction of Liposomal Formulations of Meta-tetra(hydroxyphenyl)chlorin (Temoporfin) with Serum Proteins: Protein Binding and Liposome Destruction

mTHPC is a non polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy; however, neither was reported before. We studied drug binding to human serum proteins using size‐exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip^®) and PEGylated (Fospeg^®) liposomes does not affect equilibrium serum protein binding compared with solvent‐based mTHPC. At short incubation times the redistribution of mTHPC from Foslip^® and Fospeg^® proceeds by both drug release and liposomes destruction. At longer incubation times, the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared with conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long‐circulation times the pharmacokinetic behavior of Fospeg^® could be influenced by a combination of protein‐ and liposome‐bound drug. The study highlights the modes of interaction of photosensitizer‐loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in preclinical models, as well as the novel application of NTA to assess the destruction of liposomes.     

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit

DNA extracted from forensic samples can be degraded and also contain co‐extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpF?STR^® SGM Plus^® kit. The co‐amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX‐labelled primers. Co‐amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.     

Building an Air Stable and Lithium Deposition Regulable Garnet Interface from Moderate‐Temperature Conversion Chemistry

Garnet‐type electrolytes suffer from unstable chemistry against air exposure, which generates contaminants on electrolyte surface and accounts for poor interfacial contact with the Li metal. Thermal treatment of the garnet at >700 °C could remove the surface contaminants, yet it regenerates the contaminants in the air, and aggravates the Li dendrite issue as more electron‐conducting defective sites are exposed. In a departure from the removal approach, here we report a new surface chemistry that converts the contaminants into a fluorinated interface at moderate temperature <180 °C. The modified interface shows a high electron tunneling barrier and a low energy barrier for Li⁺ surface diffusion, so that it enables dendrite‐proof Li plating/stripping at a high critical current density of 1.4 mA cm^?2. Moreover, the modified interface exhibits high chemical and electrochemical stability against air exposure, which prevents regeneration of contaminants and keeps high critical current density of 1.1 mA cm^?2. The new chemistry presents a practical solution for realization of high‐energy solid‐state Li metal batteries.     

Odoriferous Cyclic Ethers via Co‐Halogenation Reaction: Facile Preparation of Nerol Oxide,Florol®, Florol® Methyl Ether,and Pityol® Methyl Ether

A series of odoriferous cyclic ethers, including nerol oxide ( 1 ), Florol^® ( 2 ), Florol methyl ether ( 3 ), and Pityol^® methyl ether ( 4b ), were prepared by a versatile synthetic protocol based on co‐halogenation with 1,3‐dibromo‐5,5‐dimethylhydantoin (= 1,3‐dibromo‐5,5‐dimethylimidazolidine‐2,4‐dione; DDH) as the key step. The methodology provides a facile access to important perfumery molecules from abundantly available monoterpene alcohols.     

A Study of the Reactivity of Secondary Phosphanes with Radical Sources: A New Dehydrocoupling Reaction

The reactions of secondary phosphanes with radical sources have been investigated. A stoichiometric dehydrocoupling of Ph₂PH with 1,1′‐azobis[cyclohexane‐1‐carbonitrile] (VAZO^® 88) affords tetraphenyldiphosphane in good yields, whilst reduction of the nitrosyl function was observed upon using 2,2,6,6‐tetramethylpiperidin‐1‐oxyl (TEMPO). Dialkylphosphane–borane adducts also undergo a dehydrocoupling reaction in the presence of VAZO^® 88 to form R₄P₂.     

Thickness dependence of thermally induced changes in surface and bulk properties of Nafion® nanofilms

Thermally induced changes in surface wettability, dewetting behavior, and proton transport of “self‐assembled” nanothin Nafion^® films (4–300 nm) on SiO₂ substrate is reported. Thermal annealing induces switching of the surface wettability of 55 nm and thinner films from hydrophilic to super‐hydrophobic. Thickness dependence of this behavior is observed with higher annealing temperature required for lower thickness films, indicating highly restrictive mobility of Nafion^® ionomer as film thickness decreases. Dewetting is only observed for 4‐nm thin film. Significant suppression in proton conductivity upon thermal annealing was noted. Similarly, two other bulk properties, water uptake and swelling, were found to decrease upon annealing. This work reports a systematic examination of the thickness dependence of thermally induced changes in both surface and bulk properties of ultra‐thin Nafion^®. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016 , 54, 1267–1277     

Stability of mycophenolate mofetil in polypropylene 5% dextrose infusion bags and chemical compatibility associated with the use of the Equashield® closed‐system transfer device

Stability studies are necessary in healthcare settings as they facilitate fast, cost‐effective and efficient work related to batch manufacturing and availability of supplies. We studied the stability of 1–10 mg/mL mycophenolate mofetil (MMF) in polypropylene 5% dextrose infusion bags prepared from Cellcept^® and with a generic brand name (Micofenolato de Mofetilo Accord) at different storage temperatures. To ensure chemical compatibility during preparation, we also tested MMF sorption to the Equashield^® closed‐system drug transfer device used in this step. For this, a validated stability‐indicating high‐performance liquid chromatography method was developed for the quantification and identification of MMF in the infusion bags. The analytical selectivity of the assay was determined by subjecting an MMF sample to extreme values of pH, oxidative stress and heat conditions to force degradation. Protected from light, 1–10 mg/mL MMF in infusion polypropylene bags prepared from reconstituted Cellcept^® 500 mg or Accord 500 mg in 5% dextrose was stable for at least 35 days when stored at 2–8°C or between ?15 and ?25°C, and for 14 days when stored at 25°C. MMF loss owing to chemical sorption to the Equashield^® closed‐system drug transfer device set was negligible.     

Gas‐transport properties in crosslinked polymers. I. Aliphatic polyurethane–acrylate‐based adhesives

The gas‐transport properties of one of a family of well‐known adhesives, Loctite 350^®, were studied. Permeability, solubility, and diffusivity coefficients, together with the activation energies of diffusion and permeation and the solution enthalpy, were determined from 20 to 40 °C for oxygen, nitrogen, carbon dioxide, and methane. Loctite 350^® showed relatively high permselectivity and permeability for the gas pairs O₂/N₂ and CO₂/CH₄, especially for the former. The possibility of preparing very thin layers on various kinds of supports from these photocurable polymers makes them promising materials for gas‐separation devices. © 2001 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 786–795, 2001     

Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3

Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4–4.5 h. The parallel quantification was possible in a concentration range of 9.6?×?10⁵–1.4?×?10¹⁰ genomic units (GU)/mL for bacteriophage MS2, 1.4?×?10⁵–3.7?×?10⁸ GU/mL for bacteriophage ΦX174, and 6.5?×?10³–1.2?×?10⁵ for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6?×?10⁵ GU/mL for MS2, 5.3?×?10³ GU/mL for ΦX174, and 1.5?×?10² GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.     

Synthesis of Oligo(thienylfuran)s with Thiophene Rings at Both Ends and Their Structural,Electronic, and Field‐Effect Properties

Oligo(thienylfuran)s with thiophene rings at both ends ( SOSOSOS , DE‐SOSOS , DH‐SOSOS , DE‐SOSOSOS , and DH‐SOSOSOS ; S and O denote thiophene and furan rings, respectively, DE and DH denote diethyl‐ and dihexyl‐substituted, respectively) were newly synthesized by repetitive Stille coupling reactions. The UV/Vis maximum absorptions of the oligomers, SO , SOSO , SOSOS , SOSOSO , and SOSOSOS , exhibited a clear bathochromic shift with increasing number of heterocycles. The value of the oxidation peak potential (E_pa¹) determined by cyclic voltammetry decreased with an increase in the number of heterocycles by 0.06–0.08 V per heterocycle. The crystal‐packing structures of DE‐SOSOS and DH‐SOSOS determined by X‐ray crystallography have a herringbone motif and are denser than the reported structures of pentacene and α‐sexithiophene. The morphologies of thin films prepared by vacuum deposition and spin coating were investigated by atomic force microscopy and X‐ray diffraction. Among these films, those of DE‐SOSOS and DH‐SOSOS exhibited highly ordered arrangements. The devices based on vacuum‐deposited and spin‐coated films of DE‐SOSOS and DH‐SOSOS displayed the highest FET mobilities of 10^?2–10^?3 cm² V^?1 s^?1 among the oligomers reported in this study.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

Extraction, thermal cleanup, and GC/MS quantification with disposable solid extractants: application to hydrophobic analytes in aqueous surfactant solutions

Fragranced consumer products are generally formulated together with surfactants. In application, these products are often highly diluted with water. Analyzing trace amounts of fragrance ingredients in such mixtures is challenging and usually requires either time‐consuming sample cleanup or extensive cleaning of the trapping device to avoid memory effect and cross‐contamination between samples. To overcome these limitations, a new disposable extraction device has been developed to be used in combination with a thermodesorption–GC–MS unit. Made of PDMS foam cylinders, it efficiently extracts trace amounts of hydrophobic compounds from complex aqueous solutions and provides an online sample cleanup, thanks to the controlled desorption temperature, which allows retaining the low volatile constituents of the matrix within the absorptive foam. Combined with a stable isotope dilution assay, accurate quantifications of Cetalox^®, Muscenone?, Helvetolide^®, Polysantol^®, Dartanol, and Myrrhone^® from aqueous solution containing surfactant and from water from the aeration tank of a sewage plant were successfully conducted. LOQ varied between 1 and 25 ppb (20% confidence interval, α=0.1).     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.