Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

Aqueous two-phase fractionation of biological suspensions for protein recovery from bovine blood

The practical application of a two-stage aqueous two-phase systems (ATPS) for the fractionation and recovery of proteins from biological suspensions is described. A model process for the recovery of proteins from whole bovine blood was selected to study the implementation of an ATPS process. Recycling of used PEG into the initial extraction stage did not significantly influence the partition behaviour of serum albumin in subsequent cycles. © Rapid Science Ltd. 1998     

Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems

The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and system pH was carried out to determine under which conditions the laccase concentrates predominantly to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w), phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 95%. The results reported here demonstrated the potential application of ATPS for the valorisation of residual material and the potential establishment of a downstream process to obtain value added products with commercial application.     

Novel approach towards recovery of glycosaminoglycans from tannery wastewater

Poly ethylene glycol (PEG)-poly acrylic acid (PAA) based aqueous two-phase system (ATPS) was selected as a practical model to recover glycosaminoglycans (GAGs) from tannery wastewater. The influence of PEG molecular weight, tie line length (TLL), pH, temperature and NaCl concentration on the partition coefficient of glycosaminoglycans from tannery wastewater was studied. Partition coefficient of glycosaminoglycan decreases on increase of PEG molecular weight, NaCl concentration and temperature, whereas it increases with increase of pH. In the PEG-rich phase, increased partitioning of GAGs was observed with increase in TLL. The partitioning of GAGs was better in PEG 4000 at pH 8.0, 20 °C with a yield of 91.50%. This study demonstrates the potential application of ATPS processes for the recovery of GAGs from complex biological suspensions.     

An integrated practical implementation of continuous aqueous two‐phase systems for the recovery of human IgG: From the microdevice to a multistage bench‐scale mixer‐settler device

Aqueous two‐phase systems (ATPS) are a liquid‐liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer‐settler device in one stage, multistage and multistage with recirculation configuration. Single‐stage pure IgG extraction with a polyethylene glycol (PEG) 3350‐phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems

Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 microg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.     

High‐throughput downstream process development for cell‐based products using aqueous two‐phase systems (ATPS) – A case study

The availability of preparative‐scale downstream processing strategies for cell‐based products presents a critical juncture between fundamental research and clinical development. Aqueous two‐phase systems (ATPS) present a gentle, scalable, label‐free, and cost‐effective method for cell purification, and are thus a promising tool for downstream processing of cell‐based therapeutics. Here, the application of a previously developed robotic screening platform that enables high‐throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high‐throughput screening (HTS)‐data and countercurrent distribution (CCD)‐modeling, and validated the CCD‐model experimentally is designed. The obtained data are shown an excellent congruence between CCD‐model and experimental data, indicating that CCD‐models in combination with HTS‐data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge‐dependent separation of HL‐60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.     

Economic simulation of batch and continuous aqueous two-phase purification for viral products

Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid–liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry.     

Partition behavior of CD133+ stem cells from human umbilical cord blood in aqueous two‐phase systems: In route to establish novel stem cell primary recovery strategies

Aqueous two‐phase systems (ATPS) represent a promising strategy for the recovery of CD133⁺ stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer–polymer ATPS were exploited to study the partitioning behavior of CD133⁺ stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133⁺ sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133⁺ stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133⁺ in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133⁺ stem cells and contaminants from complex samples was as follows: 59% of CD133⁺ stem cells fractionated to the top phase when employing ficoll 400,000–DEX 70,000 or 100% to the bottom phase with Ucon‐DEX 75,000 and PEG 8,000‐DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000‐DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon‐DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000‐DEX 500,000. Cell viability was at least 98% after ATPS recovery. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:700–707, 2014     

Extractive disruption process integration using ultrasonication and an aqueous two‐phase system for protein recovery from Chlorella sorokiniana

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time‐consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100‐fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.     

Characterization of aqueous two-phase partition systems by distribution analysis of radiolabeled analytes (DARA): application to process definition and control in biorecovery.

In this article, we describe a characterization method applicable to aqueous two-phase systems (ATPS) heavily loaded with complex biological feed-stocks. We also studied the partition behavior of mixtures of traceable and quantifiable radiolabeled amino acids, selected on the basis of their relative hydrophobicity A unique linear relation was established between the tie-line length (TLL: commonly determined by graphical methods) and the hydrophobic factor (HF) for ATPS comprising potassium phosphate and PEG alone, and validated for polymer molecular weights from 300 to 8000 Da in systems operated at an apparent pH value of 7.5. Radiolabeled amino acids were subsequently applied to the characterization of ATPS loaded with whole bovine blood by the determination of effective tie-line lengths (TLL(e)). The addition of biomass to ATPS increased TLL(e) relative to that of blank ATPS of equivalent original composition of PEG and phosphate. In addition, an increase of biomass loading (variously sourced from blood, yeast, and E. coli) contributed to phase formation and stabilization of loaded ATPS in respect of system sensitivity toward operational conditions. The controlled application of sensitive ATPS (adjacent to the binodal curve) could thus be reconsidered for further application of aqueous two-phase partitioning as a primary purification process. The application of effective tie-line determinations by distribution analysis of radiolabeled analytes (DARA) as a process-aid in the design and operation of ATPS in biorecovery is discussed.     

Impact of cell disruption and polymer recycling upon aqueous two-phase processes for protein recovery

A practical study is presented of the influence of cell debris and polymer recycling upon the operation of two-stage acqueous two-phase systems (ATPS) for the recovery of yeast bulk protein, pyruvate kinase and fumarase. Brewers' yeast was disrupted using one of two types of high-pressure homogenisers or a bead mill. The different cell debris suspensions were partitioned in a single PEG-phosphate ATPS extraction and the efficiency of solid-liquid separation was examined. A continuously operated two-stage ATPS process, using spray columns, is presented and practical problems of polymer recycling are discussed. Conclusions are drawn concerning the generic implementation and operational stability of ATPS in practical protein recoveries.     

A novel and environment-friendly bioprocess of 1,3-propanediol fermentation integrated with aqueous two-phase extraction by ethanol/sodium carbonate system

An integrated fermentation–separation process for the production of 1,3-propanediol (1,3-PD) was investigated. Aqueous two-phase system (ATPS) not only recovered 97.9% of 1,3-PD, but simultaneously also removed 99.1% cells, 81.9% proteins, 75.5% organic acids, and 78.7% water. Furthermore, after extraction the bottom phase of ATPS was used to adjust the pH of the culture during fermentation, leading to 16% and 126% increases in the concentrations of 1,3-PD and lactic acid, and dramatic decreases in the concentration of acetic acid and formic acid. The total mass conversion yield of three main products (1,3-PD, 2,3-butanediol, and lactic acid) from glycerol reached 81.6%. The salt-enriched phase could also be used to absorb carbon dioxide (CO₂), resulting in 94% recovery for carbonate. Finally, process simulation using the program PRO/II showed the use of ATPS reduced 75.1% of the energy expenditure and 89.0% of CO₂ emissions.