Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.     

L-asparaginase release from Escherichia coli cells with aqueous two-phase micellar systems

A method was proposed to release and separate L-asparaginase (EC 3.5.1.1) from Escherichia coli ATCC 11303 cells with aqueous two-phase micellar systems. The systems were composed of K2HPO4 and Triton X-100. The method combines enzyme release with enzyme purification. The influence of Triton X-100 concentration, K2HPO4 concentration, and pH on the release and partition of L-asparaginase was investigated. Experimental results showed that E. coli cells treated with 9.4% (w/v) K2HPO4 and 15% (w/v) Triton X-100 at 25 degrees C for 15-20 h released nearly 80% of the enzyme. Most of the released enzyme was partitioned to the bottom phase (phosphate-rich phase). The effects of Triton X-100 concentration, K2HPO4 concentration, and pH on cloud point were also studied. Electron micrography indicated that the chemical treatment altered the inner structure of E. coli cells significantly.     

Cutinase-peptide fusions in thermoseparating aqueous two-phase systems. Prediction of partitioning and enhanced tag efficiency by detergent addition

It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide. Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K= 1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data.     

Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers

In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50 degrees C, HM-EOPO, 14 degrees C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance.     

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.     

In situ extraction/preconcentration of PCBs and PAHs from aqueous samples using polytetrafluoroethylene tubing

In situ extraction/preconcentration of organics from water samples was accomplished using polytetrafluoroethylene (PTFE) tubing. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were concentrated inside the tubing by flowing aqueous samples through it. The adsorbed PCBs and PAHs were then recovered by solvent desorption. The eluent was subsequently analyzed using gas chromatography with electron capture detection (GC-ECD) or gas chromatography with flame ionization detection (GC-FID). Multiple samples were simultaneously processed to concentrate organics onto several PTFE tubings. Analyses of seawater and surface microlayers using this technique demonstrated that organics in the surface microlayers were elevated with respect to those in the water column, consistent with previous findings.     

Enantioseparation of mandelic acid enantiomers in ionic liquid aqueous two-phase extraction systems

In the past decade, ionic liquids have received great attention owing to their potential as green solvent alternatives to conventional organic solvents. In this work, hydrophobic achiral ionic liquids (1-butyl-3-methylimidazolium-hexafluorophosphate([bmim][PF₆]), 1-octyl-3-methylimidazolium tetrafluoroborate([omim][BF₄])) were used as solvents in chiral liquid-liquid extraction separation of mandelic acid (MA) enantiomers with β-cyclodextrin (β-CD) derivatives as hydrophilic chiral selectors preferentially forming complexes with (R)-enantiomers. Factors affecting the separation efficiency were optimised, namely the type of the extraction solvents and β-CD derivatives, concentrations of the β-CD derivatives and MA enantiomers, pH, and temperature. Excellent enantioseparation of MA enantiomers was achieved in the ionic liquid aqueous two-phase extraction systems under the optimal conditions of pH 2.5 and temperature of 5°C with the maximum enantioselectivity (α) of 1.74. The experimental results demonstrated that the ionic liquid aqueous two-phase extraction systems with a β-CD derivative as the chiral selector have a strong chiral recognition ability, which might extend the application of ionic liquids in chiral separation.     

Microfluidics with aqueous two-phase systems

An overview is given about research activities in which aqueous two phase systems (ATPSs) are utilized in microfluidic setups. ATPSs consist of two immiscible aqueous phases and have traditionally been used for the separation and purification of biological material such as proteins or cells. Microfluidic implementations of such schemes are usually based on a number of co-flowing streams of immiscible phases in a microchannel, thereby replacing the standard batch by flow-through processes. Some aspects of the stability of such flow patterns and the recovery of the phases at the channel exit are reviewed. Furthermore, the diffusive mass transfer and sample partitioning between the phases are discussed, and corresponding applications are highlighted. When diffusion is superposed by an applied electric field normal to the liquid/liquid interface, the transport processes are accelerated, and under specific conditions the interface acts as a size-selective filter for molecules. Finally, the activities involving droplet microflows of ATPSs are reviewed. By either forming ATPS droplets in an organic phase or a droplet of one aqueous phase inside the other, a range of applications has been demonstrated, extending from separation/purification schemes to the patterning of surfaces covered with cells.     

Spiral coils for counter-current chromatography using aqueous polymer two-phase systems

Retention properties of polyethylene glycol-phosphate aqueous two-phase systems in a spiral coil (5 mm I.D.) on Type-J synchronous counter-current chromatographic devices have been compared for the elution mode where the lower phase is the mobile phase and flows from the inside head terminal. This was achieved with the aid of digital imaging under stroboscopic illumination, an image analysis and measurement of the displaced volume of the stationary phase. For the spiral coil, high and stable stationary phase retention at mobile phase flow rates up to 64 ml/min has been obtained. Wave-like disturbance of the interface near the proximal point was observed and analyses have been made for possible use in protein separation.     

Tobacco protein separation by aqueous two-phase extraction

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.     

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.     

Drop formation in aqueous two-phase systems

Extraction using aqueous two-phase systems (ATPSs) is a versatile technique for the downstream processing of various proteins/enzymes. The study of drop formation deals with the fundamental understanding of the behavior of liquid drops under the influence of various external body as well as surface forces. These studies provide a basis for designing of the extractions in column contactors in which liquid drops play a major role. Most of the drop formation studies reported so far is restricted to aqueous-organic systems. ATPSs, differ from aqueous-organic systems in their physical properties. In view of this, an attempt was made to develop a model for drop formation in ATPSs adopting the information available on aqueous-organic systems. In order to validate the model, experiments were performed by using polyethylene glycol (PEG)/salt systems of different phase compositions at various flow rates. At low flow rates the single stage model and at high flow rates the two stage model are able to predict the drop volume during its formation from tip of capillary. The experimental results were found to agree reasonably well with those predicted by the model.     

Extraction of the antimicrobial peptide cerein 8A by aqueous two-phase systems and aqueous two-phase micellar systems

Cerein 8A is an antimicrobial peptide with potential application against food spoilage and pathogenic bacteria. The partitioning of cerein 8A was investigated in two liquid-liquid extraction systems that are considered promising for bioseparation and purification purposes. Aqueous two-phase systems (ATPSs) were prepared with polyethylene glycol (PEG) and inorganic salts, and the addition of NaCl was investigated in this system. The best results concerning partition coefficients (K (b)) were obtained with PEG?+?ammonium sulphate, and K (b) value significantly increases when NaCl was added. Cerein 8A was effectively extracted into the micelle-rich phase in a 4% Triton X-114 medium. Recovery yield was higher for ATPS compared to micellar systems. Cerein 8A can be isolated from a crude suspension containing the bioactive molecule by ATPSs. Successful implementation of peptide partitioning represents an important step towards developing a low-cost effective separation method for cerein 8A.     

聚乙二醇-硫酸铵双水相萃取次甲基蓝的研究

利用分光光度法研究了次甲基蓝在聚乙二醇-硫酸铵双水相体系的萃取行为,探索了质量配比(mPEc/m(NH4)2SO4)、温度和次甲基蓝的浓度对双水相及次甲基蓝萃取率(Y)的影响.实验表明:(1)在一定的温度和浓度下,随着mPEC/m(NH4)2SO4的减小,分配系数K( cup/clow)增加,萃取率Y略有减小;(2)在一定的质量配比和浓度下,分配系数K(cup/clow)随温度的升高而增大,但萃取率Y却基本保持不变;(3)在一定的质量配比和温度下,随着次甲基蓝浓度的增加,分配系数K(cup/clow)增大;萃取率Y略有增加.在mPEG/m( NH4) 2SO4为1.50∶1,c次甲基蓝=8.915×10-5mol/L、t=30℃的实验条件下,K(cup/clow)=22.14,萃取率(y)可达95.43％.     

A new extraction method for the preparation of zinc oxide nanoparticles in aqueous two-phase systems

An extraction method was suggested for the preparation of differently shaped nano- and micrometer- sized zinc oxide particles at the interface boundary of aqueous two-phase systems based on polyethylene glycol (polyethylene oxide) and sodium sulfate using NaOH and aqueous ammonia as precipitating agents.     

Xylanase recovery effect of extraction conditions on the aqueous two-phase system using experimental design

The partitioning of xylanase produced byPenicillium janthinellum in aqueous two-phase systems (ATPS) using poly(ethylene glycol) (PEG) and phosphate (K₂HPO₄/KH₂PO₄) was studied employing a statistical experimental design. The aim was to identify the key factors governing xylanase partitioning. The interactions of five factors (PEG concentration molecular weight, concentration of buffer K₂HPO₄/KH₂PO₄, pH, and NaCl concentration) and their main effects on the partition coefficient (K) were evaluated by means of a 2⁵ full-factorial experimental design with four center points. The %PEG, %NaCl, and pH were the most important factors affecting the response variable (K). Response surface methodology (RSM) was adopted and an empirical second-order polynomial model was constructed on the basis of the results. The optimum partition conditions were pH 7.0, PEG = 8.83% and NaCl = 6.02%. Adequacy of the model for predicting optimum response value was tested under these conditions. The experimental xylanase partition coefficient (K) was 2.21, whereas its value predicted by the model was 2.33. These results indicate that the predicted model was adequate for the process. PEG molecular weight and phosphate concentration did not affect the xylanase partition coefficient.     

Acoustic field assisted demixing of aqueous two-phase systems

Acoustic field assisted demixing was employed to decrease the demixing time in aqueous two-phase systems (polyethylene glycol-maltodextrin and polyethylene glycol-potassium phosphate). Application of acoustic field has decreased the demixing time in polyethylene glycol-maltodextrin by around twofold and up to about 3.2-fold in polyethylene glycol-potassium phosphate systems. Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time. In the polyethylene glycol-maltodextrin system, phase demixing was found to depend greatly on which of the phases iscontinuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence controlling the phase demixing rate. In case of the polyethylene glycol-potassium phosphate system, droplet coalescence was found to play a critical role in phase demixing. Addition of NaCl increased the demixing time and presence of Escherichia coli cells did not seem to have any influence on phase demixing.     

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.     

Separation of endo-polygalacturonase using aqueous two-phase partitioning

The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)-polyvinyl alcohol (PVA10000) and PEG-hydroxypropyl starch (Reppal PES100) aqueous two-phase systems was studied, and revealed the possibility of using aqueous two-phase extraction to purify and concentrate endo-PG from its clarified fermentation broth. For the PEG8000-PVA10000 system, endo-PG presented in the fermentation broth (at concentration that is more than 40% of total protein) mainly dominates in the top phase with a partitioning coefficient of 6, while total protein concentrates in the bottom phase. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: a first extraction in polyethylene glycol (PEG8000)-polyvinyl alcohol system, followed by a second extraction in PEG8000-(NH4)2SO4 system. This allowed the separation of endo-PG from polymer and the recycling of PEG polymer, since endo-PG was very strongly partitioned into the bottom phase of the PEG8000-(NH4)2SO4 system. Laboratory-scale experiments were performed to test the efficiency of this scheme. It was found that enzyme recovery was up to 91% with a total purification factor of about 1.9 and a concentration factor of more than 5. About 90% of the total PEG added into the systems can be recovered, and no reduction was obtained in the purification factor using recycled PEG.