Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

Purification of the total steroidal saponins from fenugreek seeds (Trigonella foenum-graecum L.) using aqueous two-phase system and determination of diosgenin content using micellar electrokinetic chromatography method

The total steroidal saponins, particularly its major steroidal sapogenin (diosgenin), are the main active principles of fenugreek seed extract. In this study, an ethanol-salt aqueous two-phase system (ATPS) was explored for the purification of the total steroidal saponins, and the process conditions were optimized by response surface methodology (RSM). Under the optimized conditions, the RSM predicted recovery of the total steroidal saponins in the top phase of ATPS was 97.9%, which agreed with the average experimental recovery (98.3 ± 4.2% (n = 6)). Moreover, a rapid micellar electrokinetic chromatography (MEKC) method was developed for the determination of diosgenin from extracts. The diosgenin content in the ATPS top phase extract was 3-fold higher than that in crude extract, suggesting this ATPS having a great potential for purification pharmacological active ingredients from fenugreek seeds.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

Partitioning of Porcine Pancreatic Lipase in a Two-Phase Systems of Polyethylene Glycol/Potassium Phosphate Aqueous

The hydrolysis of triglycerides at the oil–water interface, synthesis of esters and transesterification in microaqueous conditions are catalysed by lipase. For its application, a proper purification method was necessary. This study examined the application of an aqueous two-phase system to partition porcine pancreatic lipase. The influence of molecular weight and concentration of polyethylene glycol (PEG), tie line length (TLL), potassium phosphate concentration, sodium chloride (NaCl) addition and temperature in the partition was studied. The enzyme was more efficiently purified in PEG 8,000 at 14.5 °C (PF?=?3.89-fold), presenting more recoveries at the top phase with shorter TLL and lower concentrations of PEG and potassium phosphate. Moreover, the increase of these variables repressed the purification and the further addition of NaCl did not promote the purification of the enzyme. These results demonstrated the efficiency of the aqueous two-phase system on lipase purification.     

Aqueous two-phase partitioning of milk proteins

Milk of transgenic pigs secreting recombinant human Protein C (rHPC) was used as a model system to determine the utility of aqueous two-phase extraction systems (ATPS) for the initial step in the purification of proteins from milk. The major challenges in purification of recombinant proteins from milk are removal of casein micelles (that foul processing equipment) and elimination of the host milk proteins from the final product. When milk was partitioned in ATPS composed of polyethylene glycol (PEG) and ammonium sulfate (AS), the phases were clarified and most of the caseins precipitated at the interphase. The partition coefficients of the major milk proteins and rHPC were dependent upon the molecular weight of the PEG used in the ATPS. Higher-partition coefficients of the major whey proteins, Β-lactoglobulin, and α-lactalbumin were observed in ATPS made up of lower molecular-weight PEG (1000 or 1450) as compared to systems using higher molecular-weight PEG. Lowering the pH of the ATPS from 7.5 to 6.0 resulted in increased precipitation of the caseins and decreased their concentration in both phases. rHPC had a partition coefficient of 0.04 in a system composed of AS and PEG 1450. The rHPC in pig milk was shown to be highly heterogenous by two-dimensional gel electrophoresis. The heterogeneity was owing to inefficient proteolytic processing of the single chain to the heterodimeric form and differences in glycosylation and other post-translational processing. Differential partitioning of the multiple forms of purified rHPC in the ATPS was not observed. rHPC after processing in ATPS was recovered in a clear phase free of most major milk proteins. ATPS are useful as the initial processing step in the purification of recombinant proteins from milk because clarification and enrichment is combined in a single step.     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Partitioning of lactate dehydrogenase from bovine heart crude extract by polyethylene glycol-citrate aqueous two-phase systems

Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG)-citrate have been used for enzyme partitioning studies. The behavior of lactate dehydrogenase (LDH) from bovine heart crude extract was analyzed using a two-level factorial design in which the PEG molar mass and concentration, the citrate concentration were selected as independent variables, while the purification factor, the partition coefficient (K) and the activity yield were selected as responses. The statistical analysis revealed the effect of PEG molar mass on K. LDH exhibited a better partitioning toward PEG-rich phase and the highest K value (1079.81) was obtained with 42% (w/w) PEG 400 and 7.5% (w/w) citrate concentration. PEG molar mass also influenced the purification factor of the enzyme in the top phase. Possibly these ATPS remove inhibitors present in the extract affording higher enzyme yield.     

Direct purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel using a PEG/salt-based Aqueous Two Phase System

An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.     

Separation and purification of aloe polysaccharides by a combination of membrane ultrafiltration and aqueous two-phase extraction

A two-step process was developed for the purification of polysaccharides from the pulp of Aloe varavia using aqueous two-phase system (ATPS) extraction and a novel copolymer ultrafiltration membrane. The first step was ATPS under optimal separations conditions using a total composition of 18% PEG2000, 25% ammonium sulfate, pH 3.0, and 0.3 M NaCl. To form the copolymer membrane, poly(acrylonitrile-acrylamide-styrene) was prepared by solution polycondensation using azoisobutyronitrile as initiator. Then, membranes were formed from the dissolved copolymer by the phase inversion method. Copolymer structure was investigated by infrared spectrum and thermogravimetric analysis (TGA). The copolymer membrane surface and cross section were observed by scanning electron microscopy. The water flux of this membrane was 26.33 mL/(cm² h), and retention was 96% for bovine serum albumin and 34% for dextran T40000. The separation and purification of aloe polysaccharide were carried using this copolymer membrane following ATPS. The TGA of aloe polysaccharide demonstrated a high purity of the polysaccharide. By gas chromatographic analysis, it was shown that mannose is the main monosaccharide in the aloe polysaccharide, and only a few glucose residues are present.     

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.     

Solvent systems for countercurrent chromatography: an aqueous two phase liquid system based on a room temperature ionic liquid

A new aqueous two phase liquid system (ATPS) based on the ionic liquid 1-butyl-3-methyl imidazolium chloride (BMIM Cl), potassium dibasic phosphate (K(2)HPO(4)) and water was recently proposed in the literature. The full phase diagram of this ATPS was prepared and some tie lines were fully determined. It was compared to classical ATPSs based on polyethylene glycol with an average molecular mass of 1000 (PEG 1000) and 10,000 (PEG 10000) and K(2)HPO(4). Two countercurrent chromatography (CCC) columns, a hydrostatic Sanki and a J type hydrodynamic CCC columns were used to test the liquid phase retention of these ATPSs in all possible configurations. It was found that the BMIM Cl ATPS liquid phases were much easier to retain in the two CCC columns than the PEG 1000 ATPS phases. Using protein and alcohol solutes, it was established that the BMIM Cl ATPS has a polarity completely different from that of the PEG 1000 ATPS. For example, ovalbumin partitions equally between the two phases of the PEG 1000 ATPS (K(D)=1.4) when it is completely located in the BMIM Cl upper phase of the ionic liquid ATPS (K(D)=180). The discrimination factor of the ionic liquid system and its intrinsic hydrophobicity were respectively found three times higher and ten times lower than the respective values of the PEG 1000 ATPS.     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

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.     

Liquid–Liquid Equilibrium and Thermodynamic Modeling of Aqueous Two-Phase System Containing Polypropylene Glycol and NaClO<Subscript>4</Subscript> at <Emphasis Type="Italic">T</Emphasis> = (288.15 and 298.15) K

In this work the phase equilibrium of an aqueous two phase system (ATPS) containing polypropylene glycol (PPG, molecular weight = 425 kg·mol^?1) and NaClO₄ was investigated at atmospheric pressure and at 288.15 and 298.15 K. Two phase regions and composition of phases were determined. Our results show that as the temperature increases, the two-phase region expands. Also, the extended UNIQUAC (E-UNIQUAC) equation was used to correlate the equilibrium data. To reduce the number of adjustable parameters, ATPSs composed of PEG and PPG were collected from the literature and simultaneously correlated using the E-UNIQUAC model. Also, the effect of temperature on the liquid–liquid equilibrium (LLE) was considered by using temperature-dependent parameters. In the modeling, two different scenarios were supposed. In the first, polymer and salt were treated as solutes (Case A), while in the second, the pseudo-solvent approach was considered (Case B). The results showed good agreement with experimental data in both cases. The average absolute deviation of the model using Case B was about 0.2% and that for Case A was about 2% in the ATPS composed of PEG. Meanwhile, the reported errors in the ATPS containing PPG for Case A and Case B were almost equal.     

Synthesis of pentablock and multibranched copolymers bearing poly(ethylene glycol), hyperbranched polyglycidol,and poly(L‐lactide) with biocompatibility for controlled drug release

A series of multibranched pentablock copolymer (mBr5BlC), poly(L ‐lactide)‐b‐HBP‐b‐poly(ethylene glycol)‐b‐HBP‐b‐poly(L ‐lactide) (HBP = hyperbranched polyglycidol), has been synthesized by ring‐opening multibranching polymerization of glycidol using bifunctional poly(ethylene glycol) [PEG; molecular weight (MW) = 1000] as an initiator, followed by ring‐opening polymerization (ROP) of L ‐lactide in the presence of stannous octoate. The ROP of different amounts of L ‐lactide on HBP‐b‐PEG‐b‐HBP [MW = 2550; polydispersity index (PDI) = 1.08] yielded a series of the targeted mBr5BlCs of the MW range of 4360–15,300 with narrow PDI. All the mBr5BlCs have been well demonstrated to be in possession of good biocompatibility as biomaterials for various applications in biological medicine areas. The mBr5BlCs with tunable MW exhibited promising controllability in self‐assembly into spherical micellar structures with an average diameter range of 59–140 nm, which have no acute and intrinsic cytotoxicity against normal cells and provide a convenient strategy for drug loading. The anticancer drug doxorubicin was demonstrated to have a good affinity with the copolymer system, and its controlled release was performed in various pHs. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Interfacial tension in aqueous biopolymer-surfactant mixtures

The current study offers a first insight into the interfacial properties of pullulan-sodium dodecyl sulphate (SDS) aqueous two-phase systems (ATPS) in the presence of sodium chloride (NaCl). The effect of composition on the interfacial tension (sigma) in these ATPS was investigated over a wide range of pullulan, SDS and NaCl concentrations. An increase in the interfacial tension was observed with increasing pullulan and SDS concentrations and a small increase was also observed as the NaCl concentration was increased. In both cases the interfacial tension increases were closely related to the phase behaviour of these systems; as a consequence of increasing the pullulan, SDS and/or NaCl concentrations, the system moves further away from the critical point. In all systems interfacial tensions (of the order of muN/m) were comparable with those reported for polymer-polymer ATPS. Interfacial tensions sigma can be well correlated with the difference in pullulan and SDS concentrations between the phases (DeltaC pul and DeltaC SDS) and also the tie-line length (TLL); all yield straight lines on a log-log scale.     

Downstream processing of antibodies: Single-stage versus multi-stage aqueous two-phase extraction

Single-stage and multi-stage strategies have been evaluated and compared for the purification of human antibodies using liquid–liquid extraction in aqueous two-phase systems (ATPSs) composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH). The performance of single-stage extraction systems was firstly investigated by studying the effect of pH, TEG-COOH concentration and volume ratio on the partitioning of the different components of a Chinese hamster ovary (CHO) cells supernatant. It was observed that lower pH values and high TEG-COOH concentrations favoured the selective extraction of human immunoglobulin G (IgG) to the PEG-rich phase. Higher recovery yields, purities and percentage of contaminants removal were always achieved in the presence of the ligand, TEG-COOH. The extraction of IgG could be enhanced using higher volume ratios, however with a significant decrease in both purity and percentage of contaminants removal. The best single-stage extraction conditions were achieved for an ATPS containing 1.3% (w/w) TEG-COOH with a volume ratio of 2.2, which allowed the recovery of 96% of IgG in the PEG-rich phase with a final IgG concentration of 0.21 mg/mL, a protein purity of 87% and a total purity of 43%. In order to enhance simultaneously both recovery yield and purity, a four stage cross-current operation was simulated and the corresponding liquid–liquid equilibrium (LLE) data determined. A predicted optimised scheme of a counter-current multi-stage aqueous two-phase extraction was hence described. IgG can be purified in the PEG-rich top phase with a final recovery yield of 95%, a final concentration of 1.04 mg/mL and a protein purity of 93%, if a PEG/dextran ATPS containing 1.3% (w/w) TEG-COOH, 5 stages and volume ratio of 0.4 are used. Moreover, according to the LLE data of all CHO cells supernatant components, it was possible to observe that most of the cells supernatant contaminants can be removed during this extraction step leading to a final total purity of about 85%.     

Aqueous two-phase systems for protein separation: a perspective

Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the properties of the two phase system. The mechanism of partitioning is complex and not very easy to predict but, as this review paper shows, some very clear trends can be established. Hydrophobicity is the main determinant in the partitioning of proteins and can be measured in many different ways. The two methods that are more attractive, depending on the ATPS used (PEG/salt, PEG/polymer), are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one based on precipitation with ammonium sulphate (parameter 1/m*). The effect of charge has a relatively small effect on the partitioning of proteins in PEG/salt systems but is more important in PEG/dextran systems. Protein concentration has an important effect on the partitioning of proteins in ATPS. This depends on the higher levels of solubility of the protein in each of the phases and hence the partitioning observed at low protein concentrations can be very different to that observed at high concentrations. In virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. Furthermore, true partitioning behavior, which is independent of the protein concentration, only occurs at relatively low protein concentration. As the concentration of a protein exceeds relatively low values, precipitation at the interface and in suspension can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. Regarding the effect of protein molecular weight, no clear trend of the effect on partitioning has been found, apart from PEG/dextran systems where proteins with higher molecular weights partitioned more readily to the bottom phase. Bioaffinity has been shown in many cases to have an important effect on the partitioning of proteins. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This separation and purification has also been successfully used for the separation of virus and virus-like particles.     

Integrated Downstream Processing of Lactoperoxidase from Milk Whey Involving Aqueous Two-Phase Extraction and Ultrasound-Assisted Ultrafiltration

The present work involves the adoption of an integrated approach for the purification of lactoperoxidase from milk whey by coupling aqueous two-phase extraction (ATPE) with ultrasound-assisted ultrafiltration. The effect of system parameters of ATPE such as type of phase system, polyethylene glycol (PEG) molecular mass, system pH, tie line length and phase volume ratio was evaluated so as to obtain differential partitioning of contaminant proteins and lactoperoxidase in top and bottom phases, respectively. PEG 6000-potassium phosphate system was found to be suitable for the maximum activity recovery of lactoperoxidase 150.70% leading to 2.31-fold purity. Further, concentration and purification of enzyme was attempted using ultrafiltration. The activity recovery and purification factor achieved after ultrafiltration were 149.85% and 3.53-fold, respectively. To optimise productivity and cost-effectiveness of integrated process, influence of ultrasound for the enhancement of permeate flux during ultrafiltration was also investigated. Intermittent use of ultrasound along with stirring (2 min acoustic and 2 min stirring) resulted in increased permeate flux from 0.94 to 2.18 l/m² h in comparison to the ultrafiltration without ultrasound. The use of ultrasound during ultrafiltration resulted in increase in flux, but there was no significant change in activity recovery and purification factor. The integrated approach involving ATPE and ultrafiltration may prove to be a feasible method for the downstream processing of lactoperoxidase from milk whey.