Multistage aqueous two‐phase extraction of a monoclonal antibody from cell supernatant

This article presents results of continuous multistage aqueous two‐phase extraction of an immunoglobulin G1 from cell supernatant in a mixer‐settler unit. An aqueous two‐phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three‐stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:925–936, 2015     

Downstream antibody purification using aqueous two‐phase extraction

The extraction of antibodies using a polyethylene glycol (PEG)‐citrate aqueous two‐phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6‐fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product‐related impurities (aggregates and low molecular product fragments) from ～40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale‐up model for the use of counter‐current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1343–1353, 2013     

Effect of glycosylation on the partition behavior of a human antibody in aqueous two‐phase systems

Human proteins are expressed in some hosts wrongly glycosylated or nonglycosylated. Although it is accepted that glycosylation contributes to the stability of the protein in solution, the effect of glycosylation on the stability of human antibodies is not fully understood. In this work, we present solubility studies of two human antibodies that have the same primary structure but different glycosylation pattern. The studies were done by monitoring the partitioning behavior of both proteins in a series of aqueous two‐phase systems at and away the isoelectric point of the proteins and at different temperatures. Our studies show that in the absence of direct electrostatic forces, the partitioning behavior of the antibodies depends on the presence or absence of the polysaccharide chains. Overall, the nonglycosylated protein is less soluble than the glycosylated one. The potential of aqueous two‐phase systems for the separation of the glycosylated and nonglycosylated proteins was also explored. A simple series of extractions seems to be enough to separate the glycosylated variety from the nonglycosylated one at high purity but low yields. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:943–950, 2013     

Application of an aqueous two‐phase systems high‐throughput screening method to evaluate mAb HCP separation

Aqueous two‐phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud‐point method for binodal determination was developed. PEG4000–PO₄ binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000–PO₄ systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task. Biotechnol. Bioeng. 2011; 108:69–81. © 2010 Wiley Periodicals, Inc.     

Purification of human antibodies from transgenic corn using aqueous two‐phase systems

A recombinant human antibody expressed in corn was purified using aqueous two‐phase extraction. The antibody was an immunoglobulin G fully unglycosylated. Using systems of different compositions and/or pHs in each of one or two partitioning stages followed by one more stage in which the antibody was precipitated at the liquid/liquid interface facilitated the removal of different impurities in each stage. The best system yields a product 72% pure (22‐fold purification) with a yield of 49%. The optimum extraction was done in two partitioning stages followed by an interfacial precipitation stage using poly(ethylene)glycol/potassium phosphate systems. NaCl was added to the first stage to eliminate large molecular weight impurities. The pH in the first stage was kept at 6 but a pH of 8 was used in the second stage and in the precipitation stage. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Preparation and recycling of aqueous two‐phase systems with pH‐sensitive amphiphilic terpolymer PADB

In this study, a novel pH‐sensitive terpolymer PADB was synthesized by random terpolymerization of 2‐(dimethylamino) ethyl methacrylate, acrylic acid, and butyl methacrylate. The terpolymer PADB could form aqueous two‐phase systems (ATPS) with a light‐sensitive terpolymer PNBC, which was synthesized in our laboratory, using n‐isopropylacrylamide, n‐butyl acrylate, chlorophyllin sodium copper salt as monomers. More than 97% of the PADB terpolymer could be recovered by adjusting the pH to isoelectric point (PI) 4.1. The terpolymer PNBC could be recovered by using light radiation at 488 nm, with recovery ratio of 98%. BSA and lysozyme were partitioned in the PNBC–PADB ATPS to examine this new system. It was found that the partition coefficient of BSA and lysozyme could reach 4.46 and 0.49 in the systems, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014     

Concentration of mammalian genomic DNA using two‐phase aqueous micellar systems

The concentration of biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments in a two‐phase aqueous micellar system was investigated using both experiment and theory. The micellar system was generated using the nonionic surfactant Triton X‐114 and phosphate‐buffered saline (PBS). Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions. With this comparison, we demonstrated that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded‐volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle‐poor, DNA‐rich domains in the macroscopic micelle‐rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle‐poor phase divided by that of the bottom, micelle‐rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof‐of‐principle that the concentration of DNA fragments in the top phase could be increased two‐ to nine‐fold in a predictive manner. Biotechnol. Bioeng. 2009;102: 1613–1623. © 2008 Wiley Periodicals, Inc.     

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two‐phase polymer systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (K_C) was obtained in the presence of NaCl 0.1 M (K_C = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na₂SO₄ 0.5 M showed the highest K_C (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%η_TOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015     

Selection and optimization of an aqueous two‐phase system for the recovery of 1,3‐propandiol from fermentation broth

In this study a suitable alcohol/salt aqueous two‐phase (ATP) system was selected for the recovery of 1,3‐propandiol (1,3‐PD) from fermentation broth. From the different alcohol/salt systems studied the ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system the partition coefficient of 1,3‐PD in synthetic solutions was first optimized with the response surface methodology. The parameters studied were concentrations of ethanol (21.99–38.81% w/w), dipotassium hydrogen phosphate (14.99–31.81% w/w) and 1,3‐PD (6.36–73.64 g/L). The optimum conditions were found to be 35.39% w/w for ethanol, 28.40% w/w for dipotassium hydrogen phosphate and 73.6 g/L for 1,3‐PD. Under these conditions the maximum partition coefficient of 1,3‐PD and the extraction yield were determined as 23.14 and 97.82%, respectively. The optimum extraction conditions were then used to guide the recovery of 1,3‐PD from a real fermentation broth. The partition coefficient and extraction yield of 1,3‐PD reached 20.28–97.20% in this case, respectively. A favorable partition of the organic acids lactate, acetate and butyrate in the bottom phase was also achieved. We have also studied the removal of cells and macromolecules from the broth. Removal ratio of cells and proteins were 96.47 and 93.05%, respectively. Thus, the ethanol/dipotassium hydrogen phosphate ATP system appears to be an interesting alternative or can be used as one useful step in the downstream processing of 1,3‐PD from fermentation broth.     

Effects of chemical modifications in the partition behavior of proteins in aqueous two‐phase systems: A case study with RNase A

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two‐phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono‐PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)–potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer‐rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG‐rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 378–385, 2013     

Effect of aeration and agitation on extractive fermentation of clavulanic acid by using aqueous two‐phase system

In this work, the effects of agitation and aeration rates on aqueous two‐phase system (ATPS)‐based extractive fermentation of clavulanic acid (CA) by Streptomyces variabilis DAUFPE 3060 were investigated through a 2² full factorial design, where oxygen transfer rate (OTR) and oxygen uptake rate (OUR) were selected as the responses. Aeration rates significantly influenced cell growth, OUR, and CA yield, while OTR was practically the same in all the runs. Under the intermediate agitation (950 rpm) and aeration conditions (3.5 vvm) of the central point runs, it was achieved OTR of 1.617 ± 0.049 mmol L^?1 h^?1, OUR of 0.132 ± 0.030 mmol L^?1 h^?1, maximum CA production of 434 ± 4 mg L^?1, oxygen mass transfer coefficient of 33.40 ± 2.01 s^?1, partition coefficient of 66.5 ± 1.5, CA yield in the top and bottom phases of 75% ± 2% and 19% ± 1%, respectively, mass balance of 95% ± 4% and purification factor of 3.8 ± 0.1. These results not only confirmed the paramount role of O₂ supply, broth composition and operational conditions in CA ATPS‐extractive fermentation, but also demonstrated the possibility of effectively using this technology as a cheap tool to simultaneously produce and recover CA. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1444–1452, 2016     

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two‐phase extraction (with and without PEG recycle)

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two‐phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:126–133, 2016     

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