Continuous aqueous two-phase extraction of human antibodies using a packed column

The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350 Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson-Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.     

Partition coefficient of bovine serum albumin in two-phase aqueous systems

Summary We measured partition coefficients of bovine serum albumin in the following two-phase aqueous systems: polyethylene glycol-dextran and polyethylene glycol-potassium phosphate. We report the effects on partition coefficients of variables such as relative molecular masses of polyethylene glycol and dextran, phase composition and temperature.Contribution of National Bureau of Standards, not subject to copyright.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment.     

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350 g h⁻¹. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.     

Temperature dependence of the partition coefficient of proteins in aqueous two-phase systems.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.     

Hydrodynamics and mass transfer in two-phase aqueous extraction using spray columns

Spray columns can be used to isolate and purify proteins using the two-phase aqueous extraction technique based on polyethylene glycol (PEG) and dextran. The fractional dispersed phase (PEG) holdup and overall mass transfer coefficients were measured in a 9.7 mm i.d. spray column. We found that the dispersed phase holdup increased with increasing PEG phase velocity. The overall mass transfer coefficients for bovine serum albumin, normalized for the PEG holdup, were found to be independent of the PEG phase velocity. This result was expected, since true mass transfer coefficients do not vary with phase velocity.     

Enzyme release from heat-stressed cell membranes as a function of hydrophobicity evaluated by using aqueous two-phase systems

The release behavior of a periplasmic enzyme, acid phosphatase, from heat-stressed Escherichia coli cells was characterized by using kinetic analyses when the cells were treated by Triton X-100–EDTA. The hydrophobicity of the cell surface and the release-rate of the enzyme were not influenced by heat treatment at temperatures between 30 and 50°C. However, these values varied above 55°C. The release-rate constants were found to correspond to the net and local hydrophobicity of the outer membrane surface, evaluated by aqueous two-phase partitioning.     

Drowning-out crystallisation of sodium sulphate using aqueous two-phase systems

A novel method to obtain crystals of pure, anhydrous salt, using aqueous two-phase systems was studied. A concentrated salt solution is mixed with polyethylene glycol (PEG), upon which three phases are formed: salt crystals, a PEG-rich liquid and a salt-rich liquid. After removal of the solid salt, a two-phase system is obtained. Both liquid phases are recycled, allowing the design of a continuous process, which could be exploited industrially. The phase diagram of the system water–Na₂SO₄–PEG 3350 at 28°C was used. Several process alternatives are proposed and their economic potential is discussed. The process steps needed to produce sodium sulphate crystals include mixing, crystallisation, settling and, optionally, evaporation of water. The yield of sodium sulphate increases dramatically if an evaporation step is used.     

Technological aspects of extractive fermentation using aqueous two-phase systems

World Journal of Microbiology and Biotechnology -     

Optimization of conditions for bacteriocin extraction in PEG/salt aqueous two-phase systems using statistical experimental designs

The bacteriocin nisin was extracted in PEG/salt aqueous two-phase systems (ATPS) using the property that the systems can extract hydrophobic proteins. The concentrations of the phase-forming components, PEG 4000 and Na(2)SO(4), were optimized for nisin recovery by means of statistical experimental designs, and it was found that they strongly influenced nisin recovery. The optimal composition of ATPS was found to be 15.99% (w/w) PEG 4000 and 15.85% (w/w) Na(2)SO(4) (pH 2), and the optimal ATPS allowed an 11.60% increase of nisin recovery compared to the standard method of nisin assay.     

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.     

Continuous counter-current two-phase aqueous extraction.

Continuous counter-current column operation provides operating convenience for contacting two-phase aqueous partitioning systems for protein extraction. We discuss in detail the important parameters for designing spray, packed, plate and York-Scheibel columns for protein recovery using both polymer-polymer and polymer-salt two-phase aqueous systems. We compare the various contractors for their operating and extraction efficiency. The work also provides a step-by-step design procedure and specific recommendations for future data needs.     

Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.     

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest.     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.