Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques
Introduction
Avidin is a glycosylated, homo-tetrameric protein. Each subunit can bind one molecule of the vitamin biotin, which features one of the strongest non-covalent binding known in nature (dissociation rate constant ka=10−15, Melamed and Green, 1963). The avidin–biotin interaction has been widely used in bioanalytical assays such as ELISAs, but also makes avidin an interesting molecule for the use as receptor specific targeting or receptor specific transport of biopharmaceuticals (Boado et al., 2008). Avidin is found in oviducts of birds, amphibians and reptiles and can be extracted from their egg whites. Besides of the extraction from egg white, the production of avidin using genetically modified organism, such as Escherichia coli (Hytönen et al., 2004), pichia pastoris (Schenk et al., 2008) or in transgenic plants (Hood et al., 1997) has been reported. Proteins constitute approximately 10% (w/w) of chicken egg white. In this protein fraction, the predominantly occurring proteins are ovalbumin (54%), ovotransferrin (12–13%), ovomucoid (11%) and lysozyme (3.5%). Only 0.5% of the total protein is avidin (Mine, 1995, Awade, 1996). Extensive studies on the purification of these proteins can be found in the literature as reviewed by Awade (1996) or reported in Guérin-Dubiard et al. (2005) and Omana et al. (2010). However, there is only a limited number of studies published addressing the purification of avidin.
For the purification of avidin, affinity chromatography e.g. using iminobiotin has been applied. This is, however, not favored regarding a large-scale production due to high costs and potentially short resin lifetimes. In most publications, an initial precipitation is followed by ion-exchange chromatography (Durance, 1994). The isoelectric point (pI) of avidin (10.0) and lysozyme (11.3) are similar and, therefore, an effective separation via ion exchange chromatography is challenging. Durance and Nakai (1988) and Li-Chan et al. (1986) reported of co-elution of avidin and lysozyme. However, besides precipitation, ion exchange chromatography and biotin-based affinity chromatography there have hardly been any work published in which separation techniques underlying different biochemical or biophysical mechanistics, e.g. hydrophobic interaction chromatography, were applied.
A separation using aqueous two-phase systems (ATPSs) which are composed of an aqueous polymer phase and an aqueous salt phase could pose an alternative to the chromatographic separation of avidin from egg white proteins. Protein separation in an ATPS is based on different affinity and hence distribution of proteins to either the salt-rich bottom phase or the more hydrophobic, polymer-rich top phase. ATPSs have been used effectively for the separation of a variety of proteins and are gaining increasing interest of industries for the purification of high valuable products as reviewed in the literature (Asenjo and Andrews, 2012, Rosa et al., 2011, Azevedo et al., 2009, Benavides and Rito-palomares, 2008). The technique provides several advantages such as good scalability, biocompatibility, tolerance of solid particles, solid particle removal, and selectivity. High selectivity, however, might only be identified by testing different ATPS compositions varying the polymer type and molecular weight, type of salt, tie-line length, phase ratio, concentration of neutral salts, pH or temperature. Furthermore, the protein concentration and solubility must be taken into account concerning high yields. Available models on phase formation and protein distribution do not comprehend all complexity inherent in the mechanistics of ATPS separations of proteins and therefore, the identification of suitable ATPSs is commonly still driven by heuristics. Even when applying Design of Experiments or optimization tools such as Simplex or Genetic Algorithm, ATPS process development still requires high experimental effort. The application of high-throughput experimentation (HTE) enables automated screenings for phase compositions and protein distribution with a reduced amount of sample material. If combined with fast analytics, HTE offers the researcher a fast identification of suitable systems, the possibility to test more ATPSs within the same timeline and thus, a rapid, initial determination of process robustness.
This study comprises three aims. The main purpose of this study was to present an evaluation of the distribution of the egg white protein avidin (AV) in polymer/phosphate ATPSs and focus on the differences in partitioning compared to the other egg white proteins ovomucoid (OM), lysozyme (LYS), ovotransferrin (OT) and ovalbumin (OV). While OM, LYS, OT and OV have been used as model proteins in aqueous two-phase partitioning experiments in several studies (de Oliveira et al., 2009, de Sousa et al., 2009, Su and Chiang, 2006, Asenjo et al., 1994, Andrews et al., 2005), AV partitioning in ATPSs is reported for the first time. The second aim was to demonstrate the beneficial application of a high-throughput approach in order to investigate the effects of changes in PEG molecular weight, sodium chloride concentration and pH on the distribution and recovery. A robotic screening method modified from Oelmeier et al. (2011) was therefore applied to perform the partitioning experiments on a liquid handling station. The third aim was to evaluate if extraction in ATPSs would pose a promising alternative to existing avidin purification process steps. Therefore, the screening was performed with a multi-component solution derived from partly purified egg white rather than using a model system of highly purified avidin. This required the application of an analytical technique different from photometric analysis, which otherwise would probably be the first choice for the analysis of the protein concentration. A tandem Reversed Phase (RP) HPLC method was developed for the fast quantitative analysis of AV and the impurities OM, LYS, OT and OV.
With using a real egg white solution as starting material we highlight the relevance of such an HTE approach as suitable for industrial early process development. The screening identified extraction being very promising for the separation of avidin from its impurities. An effective separation of the basic proteins avidin (pI=10.0) and lysozyme (pI=11.3) is presented, which is in particular beneficial compared to the challenging separation via ion exchange chromatography. This work therefore should also be considered as a basis for a further process development for the intermediate purification of AV via two-phase separation.
Section snippets
Material and methods
Unless stated differently percentage data given in the following correspond to weight per weight (w/w).
Results and discussion
In the following, the effects of the PEG molecular weight, the NaCl concentration and the pH-value on the distribution of avidin and its impurities are presented.
Conclusion and outlook
In this paper, the distribution of AV in ATPS was investigated for the first time. The study revealed large differences in partitioning of avidin and its impurities in egg white, ovomucoid, lysozyme, ovotransferrin, and ovalbumin in PEG/phosphate ATPSs. AV and OV strongly distributed into the salt-rich bottom phase, while OM, LYS, and OT could be distributed into the top phase when using low PEG MW. By the addition of 3% NaCl, a ten-fold increase in distribution values was obtained. Separation
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2017, Journal of Chromatography ACitation Excerpt :In addition, the majority of the phase-forming compounds are generally regarded as safe by regulatory agencies. However, there is still a significant knowledge gap in the multimodal physical and chemical effects influencing phase formation and partitioning, hindering the development of accurate theoretical models to predict the partitioning behavior of each individual biomolecule, thus making optimization procedures highly dependent on trial and error experiments [7–9]. Furthermore, even after extensive optimization, ATPE often suffers from a lack of selectivity, particularly when compared with established extraction procedures such as chromatography [10].