Search

Abstract Background Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo. Results In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium. Conclusion The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications.

Monoclonal antibodies (mAbs) are of crucial interest for therapeutic purposes, particularly in vaccination and immunization, and in the treatment of life-threatening diseases. However, their downstream processing from the complex cell culture media in which they are produced still requires multiple steps, making mAbs extremely high-cost products. Therefore, the development of cost-effective, sustainable and biocompatible purification strategies for mAbs is in high demand to decrease the associated economic, environmental and health burdens. Herein, novel aqueous biphasic systems (ABS) composed of glycine–betaine analogue ionic liquids (AGB-ILs) and K2HPO4/KH2PO4 at pH 7.0, the respective three-phase partitioning (TPP) systems, and hybrid processes combined with ultrafiltration were investigated and compared in terms of performance as alternative strategies for the purification and recovery of anti-human interleukin-8 (anti-IL-8) mAbs, which are specific therapeutics in the treatment of inflammatory diseases, from Chinese Hamster Ovary (CHO) cell culture supernatants. With the studied ABS, mAbs preferentially partition to the IL-rich phase, with recovery yields up to 100% and purification factors up to 1.6. The best systems were optimized in what concerns the IL concentration, allowing to take advantage of IL-based three-phase partitioning approaches where a precipitate enriched in mAbs is obtained at the ABS interface, yielding 41.0% of IgG with a purification factor of 2.7 (purity of 60.9%). Hybrid processes combining the two previous techniques and an ultrafiltration step were finally applied, allowing the recovery of mAbs from the different fractions in an appropriate buffer solution for further biopharmaceutical formulations, while allowing the simultaneous IL removal and reuse. The best results were obtained with the hybrid process combining TPP and ultrafiltration, allowing to obtain mAbs with a purity higher than 60%. The recyclability of the IL was additionally demonstrated, revealing no losses in the purification and recovery performance of these systems for mAbs. The biological activity of anti-IL-8 mAbs is maintained after the several purification and recovery steps, indicating that the novel ABS, three-phase partitioning and hybrid processes comprising AGB-ILs are promising and sustainable strategies in mAbs downstream processing. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Monoclonal antibodies are becoming a leading class of biopharmaceuticals but to increase their accessibility by the general population, new production processes must be developed in particular for the downstream processing. RESULTS: In this work, an alternative and innovative affinity chromatographic method using quaternary amine matrices is proposed. Separation is driven by the dual affinity ligand LYTAG‐Z, composed of a choline binding polypeptide (LYTAG) and the synthetic antibody binding Z domain. A two‐elution method was developed for the purification of mAbs and the performance of different anion exchangers containing quaternary amines that act as choline analogues – CIMmultus Q, Q Sepharose and gPore Q – were tested and compared, with both CIMmultus Q and Q Sepharose allowing a recovery of more than 94% of mAbs from a CHO cell supernatant with a purity greater than 95%. An integrated platform combining an initial affinity extraction step for the clarification and capture of mAbs and a subsequent chromatographic separation using Q‐matrices for the polishing of mAbs is also proposed. LYTAG‐Z triggers the extraction of 94.7 ± 1.7% mAbs to the PEG‐rich phase, as opposed to 26.9 ± 0.6% in the absence of the ligand, using 7% PEG 3350 and 6% dextran 500 k. Further purification using Q Sepharose allowed a mAb recovery of 95.3 ± 1.4% with a purity level of 91.4 ± 13.0%. CONCLUSION: An integrated platform based on two purification steps – affinity extraction and affinity chromatography – results in an overall process yield of 90%, allowing the processing of mAbs directly from a non‐clarified CHO cell culture. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL−1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL−1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications. [ABSTRACT FROM AUTHOR]

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains. [ABSTRACT FROM AUTHOR]

This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (p DNA)-containing Escherichia coli lysates by phenyl-boronic ( PB) acid chromatography using columns packed with 7.6 and 15.2 cm3 of controlled porous glass beads ( CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm3/min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of p DNA was recovered in the flow through while 66-71% of impurities remained bound (∼2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm3) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm3 of alkaline lysate per hour per dm3 of PB- CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration. [ABSTRACT FROM AUTHOR]

The structural stability of human IgG was studied throughout the extraction process with thermo-responsive UCON/dextran aqueous two-phase systems. Systems composed by 8% UCON and 5% dextran were assessed in both acetate pH 5 and phosphate pH 7. The extraction of IgG to the top phase was found to be favored at pH 5 reaching 85% yield with UCON 50HB-3520. During the extraction process conducted at 25°C no alteration in the secondary structure, evaluated by circular dichroism, was observed. The back-extraction step was induced by the thermo-precipitation of the UCON polymers at 59°C. At both pH values, mild changes in the secondary structure of IgG were observed for the 50HB-3520 and 50HB-5100 polymers while 50HB-2000 showed an increase in β-sheet content. The overall yield of the process was as high as 82% determined for the UCON 50HB-3520 system at pH 5. [ABSTRACT FROM AUTHOR]

Background: Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo. Results: In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium. Conclusion: The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications. [ABSTRACT FROM AUTHOR]

Aqueous two-phase extraction (ATPE) has been used since the 1960s, but even having advantages as high recovery yields and selectivity and relatively low cost, it has not been extensively adopted by the biotechnological industry. Recently, the production of bioproducts is entering a new era, where the continuous mode processes are spreading, and the high degree of integration of the ATPE makes them suitable for this kind of processes, however, few studies were done. The following review presents the brief history and the key challenges of continuous ATPE until nowadays and the main devices used, discussing the importance of the microfluidics in the study and development of continuous ATPE, and it analyses the future challenges. In the end, it can be concluded that continuous ATPE has not overcome some major challenges, but it will be an important tool in the downstream processing of biomolecules. [ABSTRACT FROM AUTHOR]

The purification of monoclonal antibodies anti-CD34 produced in hybridoma cells was accomplished by aqueous two phase extraction, using an integrated process that allowed to clarify and partially purify the produced mAb in just one step. The feasibility of using polyethylene-glycol (PEG)/dextran systems was studied at different ionic strengths (0-300 mM NaCl) and at different pH values (pH 3, 4 and 7). The effect of molecular weight (MW) of PEG (3350 and 6000 Da) was also evaluated. For all the conditions studied, it was observed that antibodies partition preferentially to the PEG-rich phase, cells to the interface and soluble proteins to the bottom dextran-rich phase. The best recovery yield was obtain with an ATPS composed by 7% PEG 6000 Da, 5% dextran 500,000 Da, 150 mM NaCl at pH 3. In this system, it was possible to recover 84 ± 6.5% IgG with 0.1 ± 0.2 % of cells in the top phase. [ABSTRACT FROM AUTHOR]