High-gradient magnetic affinity separation of trypsin from porcine pancreatin

We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined submicron-sized magnetic supports are presented. These support materials exhibit unique features, which facilitate their large-scale processing using high magnetic field gradients, namely sufficiently high magnetization, a relatively narrow particle size distribution and ideal superparamagnetism. Following systematic optimization with respect to activation chemistry, spacer length and ligand density, conditions for preparation of effective high capacity (Q(max) = 120 mg g(-1)) strongly interacting (Kd < 0.3 microm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies approximately 95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximately 4.1 at the expense of only a 4% loss in alpha-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly increased scale using a high-gradient magnetic separation system to capture loaded benzamidine-linked adsorbents following batch adsorption. With the aid of a simple recycle loop over 80% of the initially adsorbed trypsin was recovered in-line with an overall purification factor of approximately 3.5.     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Carbon nanotube clusters as universal bacterial adsorbents and magnetic separation agents

The magnetic susceptibility and high bacterial affinity of carbon nanotube (CNT) clusters highlight their great potential as a magnetic bio‐separation agent. This article reports the CNT clusters' capability as “universal” bacterial adsorbents and magnetic separation agents by designing and testing a multiwalled carbon nanotube (MWNT) cluster‐based process for bacterial capturing and separation. The reaction system consisted of large clusters of MWNTs for bacterial capture and an external magnet for bio‐separation. The designed system was tested and optimized using Escherichia coli as a model bacterium, and further generalized by testing the process with other representative strains of both gram‐positive and gram‐negative bacteria. For all strains tested, bacterial adsorption to MWNT clusters occurred spontaneously, and the estimated MWNT clusters' adsorption capacities were nearly the same regardless of the types of strains. The bacteria‐bound MWNT clusters also responded almost instantaneously to the magnetic field by a rare‐earth magnet (0.68 Tesla), and completely separated from the bulk aqueous phase and retained in the system. The results clearly demonstrate their excellent potential as highly effective “universal” bacterial adsorbents for the spontaneous adsorption of any types of bacteria to the clusters and as paramagnetic complexes for the rapid and highly effective magnetic separations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Optimizing a rotor‐stator filter matrix for high‐gradient magnetic separation of functionalized magnetic particles

The processing of wines with enzymes is a process chain in which losses of biocatalyst are unavoidable. A promising technique for the minimization of these losses and for the reduction of processing time is the high‐gradient magnetic separation in combination with enzymes, which are immobilized onto functionalized magnetic particles. When magnetizable particles are used and magnetic separation is applied to separate these particles from nonmagnetizable particles and solutes, the enzymes can be recycled and used for several production batches. The magnetic filter used in this study had a filter matrix with concentrically stacked circular rotor and stator plates which are arranged in an alternating order. Different geometries of the filter plate notches were examined to optimize the reproducibility of particle retention. In computational fluid dynamic studies, the influence of the notch geometries on the shear rate generation was analyzed for the rinsing procedure. Separation experiments with an optimized geometry of the filter plates were carried out in water and white wine suspensions.     

In situ magnetic separation for extracellular protein production

A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production.     

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high‐gradient magnetic separation

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed‐bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed ‘rotor‐stator’ type High Gradient Magnetic Fishing (HGMF) system for large‐scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single‐cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:78–89, 2015     

Magnetic chitosan composite particles: Evaluation of thorium and uranyl ion adsorption from aqueous solutions

Magnetic chitosan composite particles with 40 μm average size and 24 emu/g saturation magnetization obtained by an in situ procedure were evaluated as a new low-cost adsorbent for radioactive wastewater decontamination. Sorbent characterization by SEM, EDX, FTIR and magnetization measurements proved that the target ions were bound and their surface distribution was uniform. The 18 emu/g magnetization of the metal loaded particles was high enough to ensure their easy magnetic field separation and recovery. The parameters influencing the sorption process were optimized with respect to sorbent mass, target ion concentration and contact time. The material under study had superior adsorption capacity both for uranyl (666.67 mg/g) and thorium (312.50 mg/g) ions when compared to other low-cost adsorbents reported in literature. The adsorption process is spontaneous and endothermic. The material may be regenerated and re-used.     

Recovery of lysozyme from hen egg white by selective magnetic cake filtration

A new concept for the improvement of the downstream processing and purification is the so‐called magnetic separation. By using surface functionalized magnetic substrate particles, which selectively adsorb the target product, it can be directly separated out of a crude bioprocess stream. These methods are already used for analytical purposes, where only small amounts of functionalized particles are necessary. To apply the same concept at a larger scale, effective and economical procedures have to be provided. First, suitable process equipment has to be developed. Second, the magnetic particles have to be manufactured with a stable surface functionalization and long‐term stability for their reuse. Up to now mainly high‐gradient magnetic separation filter devices are applied for selective magnetic separation. They consist of a magnetic matrix in which the magnetic particles are trapped. In this work, a new magnetic filter is introduced that overcomes the capacity limitations of the current high‐gradient magnetic separation technology. The principle is demonstrated by selective recovery of lysozyme from hen egg white. Prior to the separation experiments magnetic beads with a strong acid cation‐exchange surface functionalization are synthesized. The separation procedure is implemented in only one unit operation. With the implementation of the displacement elution sequence lysozyme can be separated out of a hen egg white solution with a purification factor of PF=36 and a purity P=0.83.     

One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles

Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by nonspecific binding of the particles as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly.     

Protein purification using magnetic adsorbent particles

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.     

Magnetic quantum dots in biotechnology – synthesis and applications

Quantum dots (QDs) have great promise in biological imaging, and as this promise is realized, there has been increasing interest in combining the benefits of QDs with those of other materials to yield composites with multifunctional properties. One of the most common materials combined with QDs is magnetic materials, either as ions (e.g. gadolinium) or as nanoparticles (e.g. superparamagnetic iron oxide nanoparticles, SPIONs). The fluorescent property of the QDs permits visualization, whereas the magnetic property of the composite enables imaging, magnetic separation, and may even have therapeutic benefit. In this review, the synthesis of fluorescent–magnetic nanoparticles, including magnetic QDs is explored; and the applications of these materials in imaging, separations, and theranostics are discussed. As the properties of these materials continue to improve, QDs have the potential to greatly impact biological imaging, diagnostics, and treatment.     

Magnetic supports for immobilized enzymes and bioaffinity adsorbents

Magnetic separation of immobilized enzymes or bioaffinity adsorbents allows their selective recovery from liquors containing other suspended solids, and gives easier handling of large numbers of samples in analysis. Non-porous magnetic supports seem to be more resistant to fouling, diffusional limitation and attrition than conventional porous supports. A variety of magnetic powders and linkage methods have been used in the preparation of supports, though it is not clear how many of these have the required properties for potential applications (including linkage stability, particle size and magnetic properties). Non-porous magnetic supports are attractive for use in liquors containing fouling materials or suspended solids, either for bioaffinity adsorbents or for immobilized enzymes acting on small molecular substrates. Magnetic supports also offer considerable advantages in analysis based on bioaffinity interactions, such as immunoassay.     

Separation of magnetic affinity biopolymer adsorbents in a Davis tube magnetic separator

A Davis tube (a matrix-free, flow-through magnetic separator used mainly in mineral processing) has been tested for separation of magnetic affinity biopolymer adsorbents from larger volumes of suspensions. Both magnetic chitosan and magnetic cross-linked erythrocytes could be efficiently separated from litre volumes of suspensions. Up to 90% adsorbent recovery was achieved under optimised separation conditions.     

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the loaded adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h^–1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h^–1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h^–1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.     

Concanavalin A‐immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D ‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP‐ready magnetic separator

Magnetic separation processes are known as integrated bioanalytical protein purification method since decades and are well described. However, use of magnetic separation processes in a regulated industrial production environment has been prevented by the lack of suitable process equipment and prejudice against the productivity of the process and its qualification for cleaning‐in‐place operation. With the aim of overcoming this prejudice, a comprehensive process development approach is presented, based on a GMP‐compliant magnetic separator, including an optimization of the batch adsorption process, implementation into a technical‐scale, and the development and validation of cleaning routines for the device. By the implementation of a two‐step counter‐current binding process, it was possible to raise the yields of the magnetic separation process even for very low concentrated targets in a vast surplus of competing proteins, like the hormone equine chorionic gonadotropin in serum, from 74% to over 95%. For the validation of the cleaning process, a direct surface swabbing method combined with a total organic carbon analysis was established for the determination of two model contaminants. The cleanability of the process equipment was proven for both model contaminants by reliably meeting the 10 ppm criteria.     

Protein separations using colloidal magnetic nanoparticles

Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 8 nm are shown to be effective ion exchange media for the recovery and separation of proteins from protein mixtures. These particles have high adsorptive capacities (up to 1200 mg protein/mL adsorbent, an order of magnitude larger than the best commercially available adsorbents) and exhibit none of the diffusional resistances offered by conventional porous ion exchange media. Protein-laden particles are readily recovered from the feed solution using high-gradient magnetic filtration.     

Preparative separation of nebivolol isomers by improved throughput reverse phase tandem two column chromatography

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)⁻¹ and productivity of 20 g L⁻¹ day⁻¹.     

The enzymatic synthesis of glucosylmyristate as a reaction model for general considerations on 'sugar esters' production

'Sugar esters' are non-ionic biodegradable surfactant which are potentially attractive for the cosmetic and food market. Unfortunately, the formation of by-products by chemical synthesis affects the quality of the surfactant. Enzymatic synthesis is a promising and environmentally friendly approach preventing this problem but requiring a proper setting up of the reaction system (membrane reactor, azeotropic mixture, pervaporation system, etc.) for the control of the water activity so that the enzyme efficiency is often negatively affected. In this paper, the synthesis of glucosylmyristate by Novozym 435 is taken as a model to illustrate the general features of the 'sugar esters' synthesis, with particularly regard to the influence on the synthesis of the pre-reaction treatment of the enzyme and reaction mixture, water adsorbents, reaction solvents, products and reagents. The aim is reached by placing the water adsorbents in contact with the enzyme preparation, so that high dehydration efficiency is achieved and the other factors affecting the kinetic of the synthesis are better highlighted.