Comparative characteristics of immunoreagents based on aflatoxin B1 hemiacetals and sterigmatocystine

Aflatoxin B1 and sterigmatocystine hemiacetal derivatives were synthesized, and their conjugation to albumins and gelatin and also spectral and immunochemthe specificity and analytical properties of the antibodies produced by immunization with conjugated antigens. The possible mechanism of hemiacetal interaction with proteins is discussed. Based on immune reagents to sterigmatocystine hemiacetal, a test system was developed for determination of sterigmatocystine at the sensitivity of 0.1 ng/ml.     

Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan

A commercial laccase from Trametes versicolor was conjugated with biopolymer chitosan using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as the cross-linking agent. Laccase-chitosan conjugation strategies were tested using different molar ratios of glucosamine monomer/protein with different molar excess ratios of EDC relative to laccase. Immobilization techniques were developed to improve the stability against thermal and chemical denaturation, storage and reusability of this biocatalyst. The conjugation resulted in a solid biocatalyst with an apparent laccase activity of ±626 U/g, 12 and 60 folds higher in the conjugation efficiency of biocatalyst relative to the immobilized and free laccase activity respectively when compared with zero EDC/laccase ratio used in conjugation solution. The conjugated laccases formed successfully eliminated the emerging pollutant triclosan (TCS) from aqueous solutions, having a higher potential to transform TCS than free laccase. UPLC-QTOF results indicate the formation of TCS oligomers. Furthermore, they are the first evidence of direct dechlorination of TCS mediated by the oxidative action of laccases.     

Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.     

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin‐I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates

Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.     

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial ...     

Evaluation of ketone-oxime method for developing therapeutic on-demand cleavable immunoconjugates

The use of antibody molecules in immunoassay, molecular targeting, or detection techniques encompasses a broad variety of applications affecting nearly every field of medical science. In cancer therapy, monoclonal antibodies (mAb) have been used as vehicles to deliver radionuclides, toxins, or drugs to the target cancer cells. New conjugation methods are most needed to conjugate a wide variety of targeting small molecules and peptidomimatic compounds. Here, we exploited a keto-oxime method for conjugation of protease susceptible linkers to an antibody. This modified method involves two steps: (i) introduction of methyl ketone linkers (referred to as linker moiety) to the primary amines present in the antibody and (ii) conjugation of ketone linkers to aminoxy functional group present in the conjugated moiety (referred to as functional moiety). We have optimized this conjugation method and shown that approximately 10 functional moieties can be conjugated to antibody. Conjugation was verified by MALDI-TOF MS and Western blot analysis. The acidic pH conditions used in this method did not change the immune reactivity of the Ab. In addition, in vitro protease susceptibility assay was performed to validate this method for prodrug release assay as well as to remove excess radioimmune conjugates from circulation. This orthogonal method is compatible with peptides containing a thiol, amino, or carboxyl groups in the conjugation moiety.     

Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.     

Reagents for astatination of biomolecules. 6. An intact antibody conjugated with a maleimido-closo-decaborate(2-) reagent via sulfhydryl groups had considerably higher kidney concentrations than the same antibody conjugated with an isothiocyanato-closo-decaborate(2-) reagent via lysine amines

We are investigating the use of an (211)At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using (211)At-labeled anticanine CD45 mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27-0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted in prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with (123)I-labeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with (123)I-labeled CA12.10C12 conjugated with an amine-reactive phenylisothiocyanato-CHX-A″ derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and nonreducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g., 6 and 8), a new, easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with coadministered (125)I- and (211)At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.     

Substituted 2-iminothiolanes: reagents for the preparation of disulfide cross-linked conjugates with increased stability

Much attention has been focused recently on the stability of immunotoxin (antibody-toxin) conjugates linked by a disulfide bridge. Conflicting reports have appeared regarding the in vivo stability of such conjugates prepared with the two most commonly used cross-linking reagents, SPDP and 2-iminothiolane. We have developed (i) a series of reagents based on 2-iminothiolane substituted at the 4- and/or 5-positions (X2ITs) which, based on model studies with simple amines, should show enhanced disulfide stability when conjugated with antibodies or other proteins and (ii) a real-time method for monitoring the rate and extent of conjugation of these reagents with amino groups. Depending upon the substituent, the stability of model-activated disulfides relative to unsubstituted 2-iminothiolane was increased from 5- to 4000-fold as measured by glutathione-induced release of thionitrobenzoic acid. This family of cross-linking reagents should allow the construction of disulfide cross-linked toxin, drug, or enzyme conjugates with enhanced stability in vivo.     

Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention

Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated.     

Formation of thiol conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2

Albumin catalyzes the transformation of prostaglandin D2 to 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and to isomeric prostaglandin D2 compounds including delta 12(E)-prostaglandin D2. Both of these compounds are alpha,beta-unsaturated ketones, which should render them susceptible to nucleophilic addition. We therefore examined the ability of the compounds to form conjugates with thiols glutathione and cysteine. During incubation with excess glutathione, both 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2 formed a conjugate. Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 occurred very rapidly; approximately 70% was conjugated within 2 min. In contrast, conjugation of delta 12(E)-prostaglandin D2 with glutathione proceeded at a much slower rate; only 38% was conjugated at 60 min. The formation of both conjugates was enhanced by glutathione S-transferase. Conjugation of both compounds with cysteine was found to occur more rapidly than with glutathione. This effect was more pronounced with delta 12(E)-prostaglandin D2 in which 60% conjugated with cysteine within 2 min. These differences are likely attributed to greater steric hindrance for conjugation across the delta 12 double bond compared to that across the delta 9 bond. Analysis by fast atom bombardment mass spectrometry confirmed the formation of the glutathione conjugate of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2. Following prolonged incubation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with excess glutathione in the presence of glutathione S-transferase, a small quantity of a bis conjugate of this compound was also detected by mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-protein conjugation on a lectin matrix.

An efficient method of protein-protein conjugation yielding primarily monoconjugates is described. A glycoprotein enzyme, invertase, was ‘spaced-out’ on a succinyl concanavalin A sepharose matrix and reacted with 1% glutaraldehyde. The excess glutaraldehyde was washed out and a second, non-glycoprotein, enzyme, urease, was reacted with the ‘activated’ invertase. The column was washed till the washings were free of enzymatic activity. On elution with α-methyl glucoside both enzymes were detected in the eluate. Resolution on Sepharose 6B revealed that the eluted invertase was completely conjugated to urease. The molecular size of the conjugate suggested that it was a monoconjugate. The glutaraldehyde treated enzyme retained its immunological reactivity in the conjugate. This method of protein-protein conjugation is applicable if one of the two involved proteins is a glycoprotein.     

A novel one-pot de-blocking and conjugation reaction step leads to process intensification in the manufacture of PEGylated insulin IN-105

Bio-catalytic in vitro multistep reactions can be combined in a single step in one pot by optimizing multistep reactions under identical reaction condition. Using this analogy, the process of making PEGylated insulin, IN-105, was simplified. Instead of taking the purified active insulin bulk powder as the starting material for the conjugation step, an insulin process intermediate, partially purified insulin ester, was taken as starting material. Process intensification (PI) was established by performing a novel de-blocking (de-esterification) of the partially purified insulin ester and conjugation at B-29 Lys residue of B chain with a short-chain methoxy polyethylene glycol (mPEG) in a single-pot reactor. The chromatographic profile at the end of the reaction was found similar irrespective of whether both the reactions were performed sequentially or simultaneously. The conjugated product of interest, IN-105 (conjugation at LysB(29)), was purified from the heterogeneous mixture of conjugated products. The new manufacturing process was deduced to be more simplified and economical in making the insulin conjugates as several downstream purification steps could be circumvented. The physicochemical characteristics of IN-105 manufactured through this economic process was found to be indifferent from the product formed through the traditional process where the conjugation starting material was purified from bulk insulin.     

The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Alternatives for the intermediate recovery of plasmid DNA: Performance,economic viability and environmental impact

Robust cGMP manufacturing is required to produce high-quality plasmid DNA (pDNA). Three established techniques, isopropanol and ammonium sulfate (AS) precipitation (PP), tangential flow filtration (TFF) and aqueous two-phase systems (ATPS) with PEG600/AS, were tested as alternatives to recover pDNA from alkaline lysates. Yield and purity data were used to evaluate the economic and environmental impact of each option. Although pDNA yields =90% were always obtained, ATPS delivered the highest HPLC purity (59%), followed by PP (48%) and TFF (18%). However, the ability of ATPS to concentrate pDNA was very poor when compared with PP or TFF. Processes were also implemented by coupling TFF with ATPS or AS-PP. Process simulations indicate that all options require large amounts of water (100–200 tons/kg pDNA) and that the ATPS process uses large amounts of mass separating agents (65 tons/kg pDNA). Estimates indicate that operating costs of the ATPS process are 2.5-fold larger when compared with the PP and TFF processes. The most significant contributions to the costs in the PP, TFF and ATPS processes came from operators (59%), consumables (75%) and raw materials (84%), respectively. The ATPS process presented the highest environmental impact, whereas the impact of the TFF process was negligible.     

Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efficiently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Interestingly, UGT2B10 and UGT2B11, which are considered as orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, conjugated 12-HETE, 15-HETE, and 13-HODE. In summary, our data showed that several members of UGT1A and UGT2B families are capable of converting LA and AA metabolites into glucuronide derivatives, which is considered an irreversible step to inactivation and elimination of endogenous substances from the body.