Protein Attachment Mechanism for Improved Functionalization of Affinity Monolith Chromatography (AMC).

This work aims at understanding the attachment mechanisms and stability of proteins on a chromatography medium to develop more efficient functionalization methodologies, which can be exploited in affinity chromatography. In particular, the study was focused on the understanding of the attachment mechanisms of bovine serum albumin (BSA), used as a ligand model, and protein G on novel amine-modified alumina monoliths as a stationary phase. Protein G was used to develop a column for antibody purification. The results showed that, at lower protein concentrations (i.e., 0.5 to 1.0 mg·mL-1), protein attachment follows a 1st-order kinetics compatible with the presence of covalent binding between the monolith and the protein. At higher protein concentrations (i.e., up to 10 mg·mL-1), the data preferably fit a 2nd-order kinetics. Such a change reflects a different mechanism in the protein attachment which, at higher concentrations, seems to be governed by physical adsorption resulting in a multilayered protein formation, due to the presence of ligand aggregates. The threshold condition for the prevalence of physical adsorption of BSA was found at a concentration higher than 1.0 mg·mL-1. Based on this result, protein concentrations of 0.7 and 1.0 mg·mL-1 were used for the functionalization of monoliths with protein G, allowing a maximum attachment of 1.43 mg of protein G/g of monolith. This column was then used for IgG binding-elution experiments, which resulted in an antibody attachment of 73.5% and, subsequently, elution of 86%, in acidic conditions. This proved the potential of the amine-functionalized monoliths for application in affinity chromatography.

Molecular insights on NaCl crystal formation approaching PVDF membranes functionalized with graphene.

Membrane-assisted crystallization is an emerging technology where microporous hydrophobic membranes are used not as selective barriers but to promote the water vapor transfer between phases inducing supersaturation in solution. This has been successfully tested in the crystallization of ionic salts, low molecular weight organic acids and proteins. In this work, molecular dynamics simulations were used to study the crystal nucleation and growth of sodium chloride in contact with hydrophobic polymer surfaces at a supersaturated concentration of salt. A pristine polyvinylidene fluoride (PVDF) surface and PVDF containing different concentrations of graphene platelets were studied. Membrane crystallization tests were performed in parallel, in order to compare the experimental results with the computational ones. Here, with an integrated experimental-computational approach, we demonstrate that graphene-containing membranes assisted the crystal growth of NaCl, speeding up crystal nucleation in comparison with the pristine PVDF membranes. The computational results agreed with the experimental data, allowing the possibility of exploring the behavior of nanomaterials in membrane processes at a microscopic level.

Influence of Lipase Immobilization Mode on Ethyl Acetate Hydrolysis in a Continuous Solid-Gas Biocatalytic Membrane Reactor.

Solid-gas biocatalysis was performed in a specially designed continuous biocatalytic membrane reactor (BMR). In this work, lipase from Candida rugosa (LCR) and ethyl acetate in vapor phase were selected as model enzyme and substrate, respectively, to produce acetic acid and ethanol. LCR was immobilized on functionalized PVDF membranes by using two different kinds of chemical bond: electrostatic and covalent. Electrostatic immobilization of LCR was carried out using a membrane functionalized with amino groups, while covalent immobilization was carried out using membrane, with or without surface-immobilized polyacrylamide (PAAm) microgels, functionalized with aldehyde groups. These biocatalytic membranes were tested in a solid-gas BMR and compared in terms of enzyme specific activity, catalytic activity, and volumetric reaction rate. Results indicated that lipase covalently immobilized is more effective only when the immobilization is mediated by microgels, showing catalytic activity doubled with respect to the other system with covalently bound enzyme (4.4 vs 2.2 µmol h-1). Enzyme immobilized by ionic bond, despite a lower catalytic activity (3.5 vs 4.4 µmol h-1), showed the same specific activity (1.5 mmol·h-1·g-1ENZ) of the system using microgels, due to a higher enzyme degree of freedom coupled with an analogously improved enzyme hydration. Using the optimized operating conditions regarding immobilized enzyme amount, ethyl acetate, and molar water flow rate, all three BMRs showed continuous catalytic activity for about 5 months. On the contrary, the free enzyme (in water/ethyl acetate emulsion) at 50 °C was completely inactive and at 30 °C (temperature optimum) has a specific activity 2 orders of magnitude lower (8.4 × 10-2 mmol h-1 g-1) than the solid-gas biocatalytic membrane reactor. To the best of our knowledge, this is the first example of solid-gas biocatalysis, working in the gaseous phase in which a biocatalytic membrane reactor, with the enzyme/substrate system lipase/ethyl acetate, was used.

Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with Improved Enzyme Activity in a Biocatalytic Membrane Reactor.

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.

Preparation of Drug-Loaded PLGA-PEG Nanoparticles by Membrane-Assisted Nanoprecipitation.

PURPOSE: The aim of this work is to develop a scalable continuous system suitable for the formulation of polymeric nanoparticles using membrane-assisted nanoprecipitation. One of the hurdles to overcome in the use of nanostructured materials as drug delivery vectors is their availability at industrial scale. Innovation in process technology is required to translate laboratory production into mass production while preserving their desired nanoscale characteristics. METHODS: Membrane-assisted nanoprecipitation has been used for the production of Poly[(D,L lactide-co-glycolide)-co-poly ethylene glycol] diblock) (PLGA-PEG) nanoparticles using a pulsed back-and-forward flow arrangement. Tubular Shirasu porous glass membranes (SPG) with pore diameters of 1 and 0.2 µm were used to control the mixing process during the nanoprecipitation reaction. RESULTS: The size of the resulting PLGA-PEG nanoparticles could be readily tuned in the range from 250 to 400 nm with high homogeneity (PDI lower than 0.2) by controlling the dispersed phase volume/continuous phase volume ratio. Dexamethasone was successfully encapsulated in a continuous process, achieving an encapsulation efficiency and drug loading efficiency of 50% and 5%, respectively. The dexamethasone was released from the nanoparticles following Fickian kinetics. CONCLUSIONS: The method allowed to produce polymeric nanoparticles for drug delivery with a high productivity, reproducibility and easy scalability.

Tyrosinase immobilized on a hydrophobic membrane.

Polyvinylidene fluoride (PVDF) membrane surfaces were ad hoc functionalized chemically to make them suitable for enzymatic immobilization. The process was performed by grafting the membrane surface with 1,4-diaminobutane and subsequently by activating it with glutarhaldehyde. The chemico-physical properties of the original PVDF membrane and of the modified membranes were studied by infrared spectroscopy, scanning electron microscopy, and static contact angle measurements. The activated membranes were used as a support for covalent immobilization of tyrosinase. The activity of free and immobilized enzyme was studied and compared. The experimental data showing the specific activity of the immobilized enzyme are similar to the value obtained with the free one. This means that the immobilization procedure did not alter the catalytic properties of the tyrosinase. In addition, the surface modification of the PVDF made it a promising material to use in enzyme or biomolecule immobilization processes.

Use of a Ceramic Membrane to Improve the Performance of Two-Separate-Phase Biocatalytic Membrane Reactor.

Biocatalytic membrane reactors (BMR) combining reaction and separation within the same unit have many advantages over conventional reactor designs. Ceramic membranes are an attractive alternative to polymeric membranes in membrane biotechnology due to their high chemical, thermal and mechanical resistance. Another important use is their potential application in a biphasic membrane system, where support solvent resistance is highly needed. In this work, the preparation of asymmetric ceramic hollow fibre membranes and their use in a two-separate-phase biocatalytic membrane reactor will be described. The asymmetric ceramic hollow fibre membranes were prepared using a combined phase inversion and sintering technique. The prepared fibres were then used as support for lipase covalent immobilization in order to develop a two-separate-phase biocatalytic membrane reactor. A functionalization method was proposed in order to increase the density of the reactive hydroxyl groups on the surface of ceramic membranes, which were then amino-activated and treated with a crosslinker. The performance and the stability of the immobilized lipase were investigated as a function of the amount of the immobilized biocatalytst. Results showed that it is possible to immobilize lipase on a ceramic membrane without altering its catalytic performance (initial residual specific activity 93%), which remains constant after 6 reaction cycles.

Characterization of olive mill wastewater fractions treatment by integrated membrane process.

BACKGROUND: Up to now, the management of olive mill wastewaters, a three-phase mill by-product, remains an unsolved problem, in particular for those regions where huge quantities of vegetable water are produced. Olive mill wastewaters were therefore treated to evaluate the characteristics of permeate and retentate fractions produced by an integrated membrane system working at two different volume concentration factors. RESULTS: The effect of two membrane-based filtration steps (microfiltration and nanofiltration) on the content of chemical oxygen demand, dry matter, sensory quality, phenolic compounds and antioxidant activity of permeate and retentate samples was evaluated. Furthermore, the effect of two different volume concentration factors (VCF), in the nanofiltration step, were investigated. At high VCF values, the total phenolic content in the retentate fraction was found to be 3.7-fold higher than the starting one, while the reduction of chemical oxygen demand in the permeate fraction was greater than 97% also at lower VCF values. CONCLUSION: Each filtration step has provided useful information concerning the utility and appropriateness of the processes chosen, suggesting a sustainable hypothesis of 'normal industrial practice' that can be included in current processes of oil extraction, in order to purify water and recover phenolic compounds with high added value.

Scalable production of chitosan sub-micron particles by membrane ionotropic gelation process.

Ionotropic gelation (IG) is a highly attractive method for the synthesis of natural water-soluble polymeric nanoparticles (NPs) and sub-micron particles (sMP) due to its relatively simple procedure and the absence of organic solvents. The method involves the electrostatic interaction between two ionic species of opposite charge. Although it is well studied at the laboratory scale, the difficulty to achieve size control in conventional bench-top process is actually a critical aspect of the technology. The aim of this work is to study the membrane dispersion technology in combination with IG as a suitable scalable method for the production of chitosan sub-micron particles (CS-sMPs). The two phases, one containing chitosan (CS) and the other containing sodium tripolyphosphate (TPP), were put in contact using a tubular hydrophobic glass membrane with a pore diameter of 1 µm. TPP (dispersed phase) was permeated through the membrane pores into the lumen side along which the CS solution (the continuous phase) flowed in batch recirculation or continuous single-pass operation mode. The influence of chemical variables (i.e. pH, concentration and mass ratio of polyelectrolyte species, emulsifier) and fluid-dynamic parameters (i.e. polyelectrolyte solution flow rate and their relative mass ratio) was studied to precisely tune the size of CS-Ps.

Membrane Cascade Fractionation of Tomato Leaf Extracts-Towards Bio-Based Crop Protection.

Promising initial results from the use of membrane-fractionated extracts of tomato leaf as crop protection agents have recently been reported. This paper provides additional evidence from larger scale experiments that identify an efficient pipeline for the separation of tomato leaf extracts to generate a fraction with significant defence elicitor activity. A UF tubular membrane 150 kDa, with an internal diameter of 5 mm, proved appropriate for initial extract clarification, whereas afterwards a UF 10 kDa and three NF membranes (200-800 Da) in sequence were evaluated for the subsequent fractionation of this tomato extract. The compositions of sugars, proteins and total biophenols were changed in these fractions with respect to the initial extract. The initial extract ratio of sugars: proteins: biophenols was 1:0.047:0.052, whereas for the retentate of the 800 Da NF membrane, which has the higher crop protection activity, this ratio was 1:0.06:0.1. In this regard, it appears that the main crop protection effect in this fraction was due to the sugars isolated. It was found that with the appropriate membrane cascade selection (UF 150 kDa, UF 10 kDa and NF 800 Da) it was possible to produce (easily and without the need of additional chemicals) a fraction that has significant activity as an elicitor of disease resistance in tomato, whereas the remaining fractions could be used for other purposes in a biorefinery. This is very promising for the wider application of the proposed approach for the relatively easy formulation of bio-based aqueous streams with bio-pesticide activities.

Biorefinery of Tomato Leaves by Integrated Extraction and Membrane Processes to Obtain Fractions That Enhance Induced Resistance against Pseudomonas syringae Infection.

Tomato leaves have been shown to contain significant amounts of important metabolites involved in protection against abiotic and biotic stress and/or possessing important therapeutic properties. In this work, a systematic study was carried out to evaluate the potential of a sustainable process for the fractionation of major biomolecules from tomato leaves, by combining aqueous extraction and membrane processes. The extraction parameters (temperature, pH, and liquid/solid ratio (L/S)) were optimized to obtain high amounts of biomolecules (proteins, carbohydrates, biophenols). Subsequently, the aqueous extract was processed by membrane processes, using 30-50 kDa and 1-5 kDa membranes for the first and second stage, respectively. The permeate from the first stage, which was used to remove proteins from the aqueous extract, was further fractionated in the second stage, where the appropriate membrane material was also selected. Of all the membranes tested in the first stage, regenerated cellulose membranes (RC) showed the best performance in terms of higher rejection of proteins (85%) and lower fouling index (less than 15% compared to 80% of the other membranes tested), indicating that they are suitable for fractionation of proteins from biophenols and carbohydrates. In the second stage, the best results were obtained by using polyethersulfone (PES) membranes with an NMWCO of 5 kDa, since the greatest difference between the rejection coefficients of carbohydrates and phenolic compounds was obtained. In vivo bioactivity tests confirmed that fractions obtained with PES 5 kDa membranes were able to induce plant defense against P. syringae.

Preparation of stimulus responsive multiple emulsions by membrane emulsification using con a as biochemical sensor.

In this work, a novel strategy for the controlled fabrication of biomolecular stimulus responsive water-in-oil-in-water (W/O/W) multiple emulsion using the membrane emulsification process was investigated. The emulsions interface was functionalized with a biomolecule able to function as a receptor for a target compound. The interaction between the biomolecular receptor and target stimulus activated the release of bioactive molecules contained within the structured emulsion. A glucose sensitive emulsion was investigated as a model study case. Concanavalin A (Con A) was used as the biomolecular glucose sensor. Various physicochemical strategies for stimulus responsive materials formulation are available in literature, but the preparation of biomolecule-responsive emulsions has been explored for the first time in this paper. The development of novel drug delivery systems requires advanced and highly precise techniques to obtain their particular properties and targeting requirements. The present study has proven the flexibility and suitability of membrane emulsification for the preparation of stable and functional multiple emulsions containing Con A as interfacial biomolecular receptor able to activate the release of a bioactive molecule as a consequence of interaction with the glucose target molecule. The influence of emulsion interfacial composition and membrane emulsification operating conditions on droplets stability and functional properties have been investigated. The release of the bioactive molecule as a function of glucose stimulus and its concentration has been demonstrated.

Comparison between Lipase Performance Distributed at the O/W Interface by Membrane Emulsification and by Mechanical Stirring.

Multiphase bioreactors using interfacial biocatalysts are unique tools in life sciences such as pharmaceutical and biotechnology. In such systems, the formation of microdroplets promotes the mass transfer of reagents between two different phases, and the reaction occurs at the liquid-liquid interface. Membrane emulsification is a technique with unique properties in terms of precise manufacturing of emulsion droplets in mild operative conditions suitable to preserve the stability of bioactive labile components. In the present work, membrane emulsification technology was used for the production of a microstructured emulsion bioreactor using lipase as a catalyst and as a surfactant at the same time. An emulsion bioreaction system was also prepared by the stirring method. The kinetic resolution of (S,R)-naproxen methyl ester catalyzed by the lipase from Candida rugosa to obtain (S)-naproxen acid was used as a model reaction. The catalytic performance of the enzyme in the emulsion systems formulated with the two methods was evaluated in a stirred tank reactor and compared. Lipase showed maximum enantioselectivity (100%) and conversion in the hydrolysis of (S)-naproxen methyl ester when the membrane emulsification technique was used for biocatalytic microdroplets production. Moreover, the controlled formulation of uniform and stable droplets permitted the evaluation of lipase amount distributed at the interface and therefore the evaluation of enzyme specific activity as well as the estimation of the hydrodynamic radius of the enzyme at the oil/water (o/w) interface in its maximum enantioselectivity.

Enzyme catalysis coupled with artificial membranes towards process intensification in biorefinery- a review.

In this review, for the first time, the conjugation of the major types of enzymes used in biorefineries and the membrane processes to develop different configurations of MBRs, was analyzedfor the production of biofuels, phytotherapics and food ingredients. In particular, the aim is to critically review all the works related to the application of MBR in biorefinery, highlighting the advantages and the main drawbacks which can interfere with the development of this system at industrial scale. Alternatives strategies to overcome main limits will be also described in the different application fields, such as the use of biofunctionalized magnetic nanoparticles associated with membrane processes for enzyme re-use and membrane cleaning or the membrane fouling control by the use of integrated membrane process associated with MBR.

Production of Plant-Derived Oleuropein Aglycone by a Combined Membrane Process and Evaluation of Its Breast Anticancer Properties.

Natural products and herbal therapies represent a thriving field of research, but methods for the production of plant-derived compounds with a significative biological activity by synthetic methods are required. Conventional commercial production by chemical synthesis or solvent extraction is not yet sustainable and economical because toxic solvents are used, the process involves many steps, and there is generally a low amount of the product produced, which is often mixed with other or similar by-products. For this reason, alternative, sustainable, greener, and more efficient processes are required. Membrane processes are recognized worldwide as green technologies since they promote waste minimization, material diversity, efficient separation, energy saving, process intensification, and integration. This article describes the production, characterization, and utilization of bioactive compounds derived from renewable waste material (olive leaves) as drug candidates in breast cancer (BC) treatment. In particular, an integrated membrane process [composed by a membrane bioreactor (MBR) and a membrane emulsification (ME) system] was developed to produce a purified non-commercially available phytotherapic compound: the oleuropein aglycone (OLA). This method achieves a 93% conversion of the substrate (oleuropein) and enables the extraction of the compound of interest with 90% efficiency in sustainable conditions. The bioderived compound exercised pro-apoptotic and antiproliferative activities against MDA-MB-231 and Tamoxifen-resistant MCF-7 (MCF-7/TR) cells, suggesting it as a potential agent for the treatment of breast cancer including hormonal resistance therapies.

Integration of organic electrochemical transistors and immuno-affinity membranes for label-free detection of interleukin-6 in the physiological concentration range through antibody-antigen recognition.

We demonstrate the label-free and selective detection of interleukin-6 (IL-6), a key cell-signaling molecule in biology and medicine, by integrating an OECT with an immuno-affinity regenerated cellulose membrane. The objective of the membrane is to increase the local concentration of IL-6 at the sensing electrode and, thereby, enhance the device response for concentrations falling within the physiological concentration range of cytokines. The OECT gate electrode is functionalized with an oligo(ethylene glycol)-terminated self-assembled alkanethiolate monolayer (SAM) for both the immobilization of anti IL-6 antibodies and the inhibition of non-specific biomolecule binding. The OECT gate/electrolyte interface is exploited for the selective detection of IL-6 through the monitoring of antigen-antibody binding events occurring at the gate electrode.

Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.

Pharmaceutical Particles Design by Membrane Emulsification: Preparation Methods and Applications in Drug Delivery.

Nowadays, the rational design of particles is an important issue in the development of pharmaceutical medicaments. Advances in manufacturing methods are required to design new pharmaceutical particles with target properties in terms of particle size, particle size distribution, structure and functional activity. Membrane emulsification is emerging as a promising tool for the production of emulsions and solidified particles with tailored properties in many fields. In this review, the current use of membrane emulsification in the production of pharmaceutical particles is highlighted. Membrane emulsification devices designed for small-scale testing as well as membrane-based methods suitable for large-scale production are discussed. A special emphasis is put on the important factors that contribute to the encapsulation efficiency and drug loading. The most recent studies about the utilization of the membrane emulsification for preparing particles as drug delivery systems for anticancer, proteins/peptide, lipophilic and hydrophilic bioactive drugs are reviewed.

Membrane Bioreactors for Pharmaceutical Applications: Optically Pure Enantiomers Production.

In biological systems, recognition at molecular level is governed by chiral interactions. Therefore, optical isomers have very different effect in natural systems. For example, one can have beneficial effect while the other can be very harmful. For these reasons, chiral drugs nowadays are mainly admitted in the optically pure form. Given these requirements, it is clear why demand for chiral drugs has grown dramatically and the singleenantiomer drug segment has become an important part of the overall pharmaceutical market. As a consequence, the development of new chiral separation techniques is a very hot topic in both academic research and industrial innovation. Membrane bioreactors have proven their feasibility in the production of optically pure enantiomers by combining enantiospecific biochemical reactions with mass transport through membranes. The principles and the applications of enantioselective membrane bioreactors in kinetic resolution for pharmaceutical applications will be discussed. Various membrane bioreactors configurations and operation mode will be illustrated. The type of enzymes utilized to produce chiral drugs or their intermediates will be also reported. Multistep syntheses, conducted in sequential reactions catalysed by spatially aligned biocatalysts, as promising technology for the synthesis of fine chemicals will be highlighted.