High-gradient magnetic separation for technical scale protein recovery using low cost magnetic nanoparticles
Graphical abstract
Introduction
Nowadays, as in many other industrial sectors, biotechnological production volumes are continuously increasing while product prices are falling. Therefore, large-scale sustainable bioprocessing requires maximizing efficiency together with reducing energy consumption and waste [1]. The lack of revolutionary advances in downstream processing is slowing biotechnological progress, preventing adequate responses to the increasing demand for biotechnological products. This situation has long been recognized [2], but the processing chain following fermentation remains a time and cost intensive sequence with many steps. The concentration of biomolecules and the removal of impurities are critical, decisively influencing the efficiency of the entire processing chain [3]. Highly-integrated separation processes and innovative concepts are necessary to provide a strong impetus to this industrial sector, making it more advanced and competitive [4]. New alternatives are needed at all stages and have to compete economically with the unit operations currently applied, e.g. solid/liquid separation and subsequent chromatographic purification. In this context, high-gradient magnetic separation (HGMS) is a quite promising fast and comparably simple technology, which enables the direct isolation of target biomaterial from raw biological suspensions by means of functionalized magnetic carriers [5]. This technology, also called high-gradient magnetic fishing [6], allows a significant reduction in the number of processing steps while increasing total product yield. Even though the advantages of magnetic separation for bioprocessing applications are obvious [7], nowadays it is primarily employed only at a laboratory scale, mainly due to the lack of adequate liter-scale separators, the high cost of the industrial systems and the complex design [8].
The working principle of HGMS is the application of large static magnetic fields (approx. 1 T) to columns containing ferrous matrices. Magnetic gradients as high as 104 T/m create forcefields capable of capturing even weakly magnetic particles in a flow stream [9]. These processes have been applied in some industrial sectors, such as wastewater treatment [10], food processing or steel production for more than 50 years [11]. Nowadays, other uses like removal of contaminants from mineral oils are also being explored [12]. In most cases, the magnetic separators are primarily used in continuous flow processes. Their use for biotechnological purposes began in the early 1970s [13] and is still common today though generally in the form of batch separators [14], [15], although in a recently published work Lindner and Nirschl report on the combination of HGMS and centrifugation to generate a continuous process [16]. The number of published biotechnological large-scale HGMS applications is still low [17], [18], [19], [20], [21]. Specifically Franzreb and different collaborators have been working intensively in the field, reporting on HGMS methodology and parameters [6] as well as concrete application examples with micron beads as carriers e.g. for lactoferrin capture [22], superoxide dismutase recovery [23] as well as the integrated multiple use of immobilized lipase [24]. More recently reports on other areas, such as algae separation, have been the aim of studies [25].
Within the broad pool of magnetic beads available for biomolecular capture by magnetic forces, smaller iron oxide magnetic nanoparticles (MNPs) are a special group which is being intensively studied nowadays. They are highly interesting due to their large surface-to-volume ratio and their biocompatibility. However, there are some difficulties with their use in HGMS: (1) large magnetic field gradients are needed to manipulate the small particles; (2) flow conditions have to be optimized to retain all magnetic material; (3) the applied MNPs for the separation process must first be functionalized with the appropriate chemistries, a task that usually requires intensive developing processes and high costs. One classical method for bioseparation, and more specifically for histidine (His)-tagged proteins, is the use of chelate ligands decorated with immobilized metal ions [26]. This technique, called immobilized metal ion affinity (IMA), employs typically tri- or tetradentated ligands. In this paper we present a separation method based on MNPs functionalized with a pentadentate chelate ligand (ethylene diamine tetraacetic acid, EDTA) later loaded with copper ions. The characterization of the applied Cu-EDTA-MNP system as well as the separation results on a laboratory scale were recently published by our group [27]. While IMA techniques are established for the purification of proteins, the application on an industrial scale is quite new because of worries regarding the effects of the His-tag as well as metal ion leaching [28]. In our previous paper, we reported on the lack of metal ion leaching with EDTA as a chelate ligand. This system yields promising results with very good efficiency also at a pilot-plant scale, as we show with the present report on the isolation of His-tagged green fluorescent protein (His-GFP) as a model biomolecule by means of high-gradient magnetic separation from an E. coli cell lysate.
The work presented here was carried out on a HGMS rotor stator prototype [13]. First results from a pilot plant similar to ours were published very recently and dealt with the multicycle purification of equine chorionic Gonadotropin (eCG) using microparticles [29]. Previous publications had already reported on studies carried out with a smaller rotor stator system: Brown and collaborators showed the suitability of HGMS for a technical application [30], whereas Müller and co-authors isolated eCG and compared the HGMS process with the technically established fixed-bed chromatography [31]. Our focus lies on the application of nanoparticles (instead of micro-) and also on the optimization of relevant processing parameters to improve final purification and concentration factors. Although Moeser and collaborators [32] demonstrated that MNPs coated with a bifunctional polymer or a phospholipid layer were retained by the magnetic field despite their small size, the HGMS experiments in their work were run at a mL scale. To the best of our knowledge, the only previous report on the suitability of large-scale HGMS for nanoparticulate systems in bioseparation processes is the one by Holschuh and Schwämmle [33]. They applied Protein A coated beads of about 100 nm in diameter to purify antibodies and showed the high potential of the method for the bioprocessing of up to 100 L. Their device included four Neodyn® magnetic rods which were manually driven. In our work, we have used even smaller particles than Holschuh and Schwämmle (about 30 nm in diameter). The fact that HGMS with small nanoparticles has now been successfully applied at an industrial level is of great importance because the downstream industry can then benefit directly from the current enormous developments in nanoresearch. Moreover, the applied nanoparticles are low-cost carriers thanks to the easier and shorter synthesis in comparison to common polymer microparticles; another advantage is their better long term stability because no degradation takes place.
Our example demonstrates that HGMS offers a real, promising alternative for the recovery field in an industrial environment with the additional advantages of being a fast, robust and comparably simple processing technique.
Section snippets
Materials
The plasmid vector pET-28a (Novagen, Madison, USA) was used for the cloning and overexpression of His-GFP (model protein) from an E. coli BL21 (DE3) strain. After fermentation, the cell suspension was concentrated in a disk separator (GEA Westfalia Separator Group, Germany), then disrupted by high-pressure homogenization in two passages at 900 bar (GEA Niro Soave, Italy) and finally stored at −20 °C. The dry biomass content was between 0.8% and 1.5%. The target protein concentration in the lysate
Preliminary studies
Prior to any separation processing, the applied HGMS prototype had been characterized with respect to maximal filter capacity, pump capacity, separation efficiency and residence time behavior. According to the manufacturer, maximum capacity was approximately 100 g magnetic beads. In fact, this value depends strongly on the type of particles applied. We achieved a capacity of more than 200 g with our MNPs (Tris buffer, pH 8.5) without causing a breakthrough of the filter for particle suspensions
Conclusions and outlook
Enhanced downstream processing is of high relevance in current biotechnological research. High capacity and selectivity together with easy and quick processing steps are the preferred properties for efficient separation processes. Bioseparation using magnetic nanoparticles is of special importance for protein purification compared to more common processes such as affinity chromatography, in particular due to its simplicity, speed and low cost [34]. We report here on an improved HGMS process for
Acknowledgements
We would like to thank Dr. J. Bauer from the Merck KGaA for providing the EDTA-MNPs. M. Wolf and S. Reinlein conducted part of the research for their master thesis.
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