Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems

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Abstract

The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid–liquid extraction with poly(ethylene glycol) (PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG 1000, 2000, 4000 and 6000), PEG and potassium citrate concentration, BSA concentration (CBSA) and pH on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG 1000. The yield of the BSA, 99%, was obtained in the top phase under the following conditions: 19% (w/w) PEG 1000, 20% (w/w) potassium citrate and 0.75 mg/g CBSA at pH 7.0 and 30 °C. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a 92% yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Ye + Ybe) is up to 91%.

Introduction

Aqueous two-phase systems (ATPSs) are formed when two polymers or a polymer and a salt are solubilised in water above certain concentrations. Although the application of ATPS at process scale has been hampered by the complexity of the system combined with the fact that partition mechanisms are poorly understood and method development is fairly empirical (Huddleston et al., 1991a, Huddleston et al., 1991b), aqueous two-phase extraction (ATPE) is a versatile method suitable for separating biological particles and macromolecules. Compared with other commonly used separation and purification techniques, this technique has a number of advantages, such as scaling up feasibility, ease of continuous process, lower interfacial tension, and biocompatibility (Li et al., 2002, Przybycien et al., 2004, Thommes and Etzel, 2007, Azevedo et al., 2009a, Azevedo et al., 2009b). Some successful applications of aqueous two-phase extraction on large/industrial scales have been demonstrated (Kula et al., 1984, Aguilar et al., 2006). Several reports on the application of ATPE for the separation and purification of biological active substances are also available (Silva and Franco, 2000, Albertsson, 1986, Walter and Johansson, 1986, Srinivas et al., 2002). Polymer/phosphate salts and polymer/sulfate salt systems have been most commonly used. Those salts, however, led to a strong negative impact on the environment. As the citrate anion is both biodegradable and nontoxic (Vernau and Kula, 1990), we employed potassium citrate as a substitute for phosphate and sulfate salts. Similar to a few recent ones, the separation and purification of penicillin acylase (Marcos et al., 1999), human antibodies (Azevedo et al., 2009a, Azevedo et al., 2009b), hexokinase (Oliveira et al., 2003), insulin (Alves et al., 2000), and plasmid DNA (Rahimpour et al., 2006, Gabriela et al., 2009) were successful using in the PEG/citrate systems.

Nowadays, bovine serum albumin (BSA) has aroused the interest of researchers greatly due to its great abundance in blood plasma proteins, as well as its wide applications in many fields. Extraction of BSA has been studied by employing the reversed micelle method (Zhang et al., 2002, Zhang et al., 1999, Umesh Hebbar and Aaghavarao, 2007). It is well known that the backward transfer of protein from reverse micelles to the aqueous solution is relatively slow due to high interfacial resistance to the mass transfer (Umesh Hebbar and Aaghavarao, 2007, Dekker et al., 1989, Duungan et al., 1991). Zhang et al. (1999) reported that the recovery of the BSA was higher than 90% by adding 2-propanol to the aqueous phase in the back-extraction step, however, the back-extraction of protein was more difficult to be accomplished. The behaviors of BSA partitioning were also studied in PEG/dextran aqueous two-phase system (ATPS) (Gunduz and Korkmaz, 2000). In their work, they used BSA as a model protein to examine the effects of pH and concentration of NaCl salt on partitioning in aqueous PEG–dextran two-phase systems. However, up till now, there is no research on the separation of BSA using PEG/potassium citrate system.

In the present work, we described the partitioning features of BSA in PEG/potassium citrate ATPS. The aim of this work was to study the effect of these factors on partitioning of the BSA and to separate the BSA from PEG. In order to enhance the yield of the protein in the top phase, the influences of process parameters like molecular weight of PEG, concentration of PEG and potassium citrate, CBSA and pH on BSA partitioning were studied. In the back-extraction step, the BSA could be easily removed from the polymer-rich phase and the PEG could be recycled.

Section snippets

Materials

Bovine serum albumin and Coomassie Blue G250 (made by Sinopharm Chemical Reagent Co., Ltd.); PEG (made by Shanghai Lingfeng); potassium citrate and citric acid (made by Tianjin Guangcheng Chemical Company). All other reagents were of analytical grade. Double distilled and deionized water were used throughout.

Aqueous two-phase diagrams

Binodal curves were determined by the turbidimetric titration method as the one used previously (Zafarani-Moattar and Sadeghi, 2001, Zafarani-Moattar et al., 1995). The temperature was

Effect of PEG molecular weight on the protein partitioning

The systems of different PEG (1000, 2000, 4000 and 6000)/20% (w/w) potassium citrate concentrations were used in the experiment. As shown in Fig. 2, the increase of the PEG molecular weight results in less available space for BSA in the upper phase, which leads to the decrease of partition coefficient (K). This behavior is in agreement with an exclusion effect owing to the diminution of the free volume available in the top phase. Exclusion volume effect (Almedia et al., 1998, Rabelo et al., 2004

Conclusions

Aqueous two-phase extraction was successfully used in the partitioning of BSA in extraction and back-extraction step. Various factors were investigated in order to find the suitable conditions for the BSA partitioning. Statistically valid models were obtained using SAS software for both partition coefficient and recovery of the BSA. The optimal system consisted of 19% (w/w) PEG 1000 and 21% (w/w) potassium citrate at pH 7.0 and 30 °C. On these conditions, a high recovery (99%) was obtained in

Acknowledgment

The authors acknowledge the financial support for this work from The National Natural Science Foundation of China (No. 20876089).

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