Marco Rito-Palomares

Protein manipulation with insulator-based dielectrophoresis and direct current electric fields.

The present study demonstrates the manipulation of protein particles employing insulator-based dielectrophoresis (iDEP) and direct current (d.c.) electric fields. Fluorescently labeled bovine serum albumin (BSA) protein particles were concentrated inside a microchannel that contained an array of glass cylindrical insulating structures. d.c. electric fields were applied and the dielectrophoretic response of the particles was observed as a function of the suspending medium conductivity (25, 50 and 100 microS/cm) and pH (8 and 9). It was shown that the magnitude of the applied electric field (700-1600 V/cm) and suspending medium properties have a strong effect on the dielectrophoretic response of the protein particles. The results presented here are the first report on protein manipulation employing d.c.-iDEP.

Aqueous two-phase affinity partitioning systems: Current applications and trends ☆

Aqueous two-phase systems (ATPS) have been studied and used for product recovery and purification from diverse biological sources. ATPS are characterized by their versatility, easy scale up parameters, process integration capability and relative low cost. This technique is commonly regarded as a primary recovery stage mainly due to its low selectivity. However, the use of strategies involving the modification of ATPS with affinity ligands have resulted in significant increases in recovery yields and purification folds of biological products. The aim of this review is to highlight current applications, trends and challenges regarding affinity partitioning in aqueous two-phase systems for the fractionation, recovery and purification of biological products.

Process integration using aqueous two-phase partition for the recovery of intracellular proteins

This paper presents an integrated process for the primary recovery of intracellular glyceraldehyde 3-phosphate dehydrogenase (G3PDH) and other proteins from bakers’ yeast wherein cell disruption and aqueous two-phase systems (ATPS) were operated simultaneously. Polyethylene glycol and potassium phosphate were added to the cell suspension before disruption such that the bead mill was exploited as both cell disrupter and product extractor. The partition behaviour of both bulk protein and G3PDH for the integrated process was similar to that for a conventional process in which cell disruption and ATPS extraction were operated as discrete unit operations. The target products concentrated in the upper phase for all the systems evaluated. However, the cell debris generated during the integrated process also concentrated in the upper phase which limited the potential application of the system. Subsequent evaluation of the variation of system volume ratio (Vr) with the partition behaviour of cell debris recommended process conditions (i.e. PEG 12% (w/w), phosphate 28% (w/w), Vr=0.45, pH=7.0) suitable for the primary recovery of intracellular proteins from debris. The results are discussed in the context of the practical potential of the direct integration of ATPS with cell disruption processes.

Performance characterization of an insulator‐based dielectrophoretic microdevice

Dielectrophoresis (DEP), the motion of particles in nonuniform electric fields, is a nondestructive electrokinetic (EK) transport mechanism can be used to concentrate and separate bioparticles. Traditionally, DEP has been performed employing microelectrodes, an approach that is expensive due to the cost of microelectrode fabrication. An alternative is insulator‐based DEP (iDEP), an inexpensive method where nonuniform electric fields are created with arrays of insulating structures. This study presents the effects of operating conditions on the dielectrophoretic behavior of polystyrene microparticles under iDEP. Experiments were performed employing microchannels containing insulating structures that worked as insulators. The parameters varied were pH (8–9) and conductivity (25–100 μS/cm) of the bulk medium, and the magnitude of the applied field (200–850 V/cm). Optimal operating conditions in terms of pH and conductivity were obtained, and the microdevice performance was characterized in terms of concentration factor and minimum electric field required (minimum energy consumption). This is the first report on improving iDEP processes when EOF is present. DEP and EOF have been studied extensively, however, this study integrates the effect of suspending medium characteristics on both EK phenomena. These findings will allow improving the performance of iDEP microdevices achieving the highest concentration fold with the lowest energy consumption.

Influence of system and process parameters on partitioning of cheese whey proteins in aqueous two-phase systems

A practical study is described to characterise some problems encountered in the application of aqueous two-phase systems (ATPS) to protein recovery. These factors include practical design of extraction stages and the impact of ATPS compounding methods and biological suspension upon process performance. They were addressed using the recovery of whey proteins as a model. The known effects of system parameters (i.e. tie-line length, volume ratio and system pH) were exploited to define the specific operating conditions of a two-stage ATPS process for the recovery of whey proteins. The partition of whey proteins in ATPS assembled using different methods resulted in changes in the partition coefficient of the proteins. Such changes were associated with the initial location of the proteins in the polymer or salt-rich solutions of the ATPS. Cheese whey loaded into the ATPS caused the displacement of the binodal curve from the origin. Such behaviour was attributed to the residual fat present in the whey. These findings highlight those factors perceived as negative constraints on the wider adoption of ATPS processes for protein recovery from complex biological systems.

Generic application of an aqueous two-phase process for protein recovery from animal blood

The recovery of proteins from animal blood using an established two-stage extraction process was selected as a practical model system to study the generic application of polyethylene glycol (PEG)-phosphate aqueous two-phase systems (ATPS). Processing of whole bovine blood in the ATPS two-stage process resulted in the partition of soluble protein (e.g. bovine serum albumin (BSA), haemoglobin, IgG; partition coefficient K=55) into a PEG-rich top phase and cell debris into a phosphate-rich bottom phase. Subsequent back extraction of soluble protein into a second phosphate-rich bottom phase resulted in a maximum overall protein recovery of 62%. The increased protein concentration within the ATPS (from 1.2 to 7.0 mg/g) caused a decreased in the recovery to 44%. Recycling of PEG into the initial extraction stage did not significantly influence the partition behaviour of protein over the equivalent of 20 operational cycles, but protein recovery decreased from 44 to 37%. The extreme conditions (waste material characterised by the presence of solids and impurities) in which the implementation of this ATPS process was tested, confirms the potential for the generic application of ATPS for processing complex biological suspensions to achieve a simple primary recovery and partial purification of target protein solutes.

Recovery of crocins from saffron stigmas (Crocus sativus) in aqueous two-phase systems

Crocins are carotenoid derivates that have recently attracted the interest of the scientific community due to their nutraceutical properties. Saffron (dry Crocus sativus stigmas) is one of the main known sources of crocins. In this study the potential use of aqueous two-phase system (ATPS) for the extraction of crocins from C. sativus stigmas was evaluated. The partitioning behavior of crocins in different types of ATPS (polymer-polymer, polymer-salt, alcohol-salt and ionic liquid-salt) was evaluated. Ethanol-potassium phosphate ATPS were selected based on their high top phase recovery yield and low cost of system constituents. The evaluation and optimization of system parameters rendered conditions (V(R)=3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl and 2% (w/w) of sample load) under which more than 75% of total crocins were recovered in the top (ethanol rich) phase, whereas the wasted stigmas accumulated in the bottom phase. Lastly, a comparison between an optimized solid-liquid extraction using ethanol:water as solvent and ATPS was conducted demonstrating that similar yields are achieved with both strategies (76.89 ± 18% and 79.27 ± 1.6%, respectively). However, ATPS rendered a higher extraction selectivity of 1.3 ± 0.04 mg of crocins for each mg of phenolic compound, whereas ethanolic extraction showed a selectivity of 0.87 ± 0.01. The results reported herein demonstrate the potential application of ATPS, particularly ethanol-potassium phosphate systems, for the recovery of crocins from C. sativus stigmas.

Aqueous two‐phase systems strategies for the recovery and characterization of biological products from plants

The increasing interest of the biopharmaceutical industry to exploit plants as economically viable production systems is demanding the development of new downstream strategies to maximize product recovery. Aqueous two-phase systems (ATPSs) are a primary recovery technique that has shown great potential for the efficient extraction and purification of biological compounds. The present paper gives an overview of the efficient use of ATPS-based strategies for the isolation and partial purification of bioparticles from plant origin. Selected examples highlight the main advantages of this technique, i.e. scaling-up feasibility, process integration capability and biocompatibility. An overview of the recent approach of coupling ATPSs with traditional techniques to increase bioseparation process performance is discussed. A novel approach to characterization protein from plants combining ATPSs and two-dimensional electrophoresis (2-DE) is introduced as a tool for process development. In the particular case of products from plant origin, early success has demonstrated the potential application of ATPS-based strategies to address the major disadvantages of the traditional recovery and purification techniques. This literature review discloses the relevant contribution of ATPSs to facilitate the establishment of bioprocesses in the growing field of high-value products from plants.

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl--pyrone produced by Trichoderma harzianum

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.

Hydrophobic interaction chromatography for purification of monoPEGylated RNase A

The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ~85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.