High-gradient Magnetic Fishing Research Articles

Magnetic One-Step Purification of His-Tagged Protein by Bare Iron Oxide Nanoparticles.

Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His6-tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.

Magnetic hydrophobic-charge induction adsorbents for the recovery of immunoglobulins from antiserum feedstocks by high-gradient magnetic fishing.

BACKGROUNDThe extraction of biopharmaceuticals from plasma and serum often employs overly complicated antiquated procedures that can inflict serious damage on especially prone protein targets and which afford low purification power and overall yields. This paper describes systematic development of a high‐gradient magnetic fishing process for recovery of immunoglobulins from unclarified antiserum.RESULTSNon‐porous superparamagnetic particles were transformed into hydrophobic‐charge induction adsorbents and then used to recover immunoglobulins from rabbit antiserum feedstocks. Comprehensive characterisation tests conducted with variously diluted clarified antiserum on a magnetic rack revealed that immunoglobulin binding was rapid (equilibrium reached in <45 s), strong (Kd < 0.1 mg mL‐1), of high capacity (Qmax = 214 mg g‐1), and pH and ionic strength dependent. In a high‐gradient magnetic fishing process conducted with the same adsorbent, and a conventional ‘magnetic filter + recycle loop’ arrangement, >72% of the immunoglobulin present in an unclarified antiserum feed was recovered in 0.5 h in >3‐fold purified form.CONCLUSIONSFast magnetic particle based capture of antibodies from an unclarified high‐titre feed has been demonstrated. Efficient product recovery from ultra‐high titre bioprocess liquors by high‐gradient magnetic fishing requires that improved magnetic adsorbents displaying high selectivity, ultra‐high capacity and operational robustness are used with ‘state‐of‐the‐art’ rotor–stator magnetic separators. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

M13 Bacteriophage‐Activated Superparamagnetic Beads for Affinity Separation

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage.

Use of high‐gradient magnetic fishing for reducing proteolysis during fermentation

Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation. Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K(d) = 1.94 micromolar; Q(max) = 222.8 mg/g), which obeyed the Langmuir isotherm and had rapid binding kinetics (equilibrium in <300 s). When applied directly in shake-flask cultures or in a 1-L fermenter and then removed by HGMF, the degradation of the model protein bovine serum albumin was stopped. The adsorbents could be recycled and reused during the same fermentation to remove freshly produced proteases, extending the life of the model protein in the fermenter. HGMF may provide an efficient method of stabilizing heterologous proteins produced in cultivation processes.

Demonstration of a Strategy for Product Purification by High-Gradient Magnetic Fishing: Recovery of Superoxide Dismutase from Unconditioned Whey

A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high-gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.15-0.6 mg L(-1)) in whey. The first part of the process design consisted of ligand screening in which metal chelate supports charged with copper(II) ions were found to be the most suitable. The second stage involved systematic and sequential optimization of conditions for the following steps: product adsorption, support washing, and product elution. Next, the capacity of a novel high-gradient magnetic separator (designed for biotechnological applications) for trapping and holding magnetic supports was determined. Finally, all of the above elements were assembled to deliver a HGMF process for the isolation of SOD from crude sweet whey, which consisted of (i) binding SOD using Cu2+ -charged magnetic metal chelator particles in a batch reactor with whey; (ii) recovery of the "SOD-loaded" supports by high-gradient magnetic separation (HGMS); (iii) washing out loosely bound and entrained proteins and solids; (iv) elution of the target protein; and (v) recovery of the eluted supports from the HGMF rig. Efficient recovery of SOD was demonstrated at approximately 50-fold increased scale (cf magnetic rack studies) in three separate HGMF experiments, and in the best of these (run 3) an SOD yield of >85% and purification factor of approximately 21 were obtained.

Characterization of magnetic ion-exchange composites for protein separation from biosuspensions

Downstream processing is a major issue in biotechnological production. A multitude of unit operations with nonsatisfying yield are often used to reach the desired product purity. Direct recovery technologies such as high-gradient magnetic fishing (HGMF) are advantageous because of their ability to separate the desired product in early stages from crude cultivation broths. However, the use of magnetic particles to capture valuable biotechnological products is often linked to the drawback that support particles are expensive and not available in greater quantities. This current work presents new composite magnetic particles that can be used in biotechnology. They are manufactured by a spray drying process. During this process, the nanosized magnetite particles as well as functional ion-exchange nanoparticles are integrated into one particle in which they are linked by a matrix polymer. The production procedure is flexible, scalable, and therefore economical. These particles have good adsorption capacities of up to 85 mg/g adsorbed protein and good binding kinetics. They are resistant to harsh conditions such as short ultrasonic treatment or extreme pHs. In order to test their usefulness in biosuspensions, model proteins were separated using these particles. The anion and cation exchanger particles separated lysozyme (LZ) or BSA from cultivation suspensions. The selectivity of recovery was dependent on other proteins present as is usual for ion-exchange binding mechanisms.

Avoiding proteolysis during fermentation by using high gradient magnetic fishing

Avoiding proteolysis during fermentation by using high gradient magnetic fishing

Superparamagnetic Cation–Exchange Adsorbents for Bioproduct Recovery from Crude Process Liquors by High-Gradient Magnetic Fishing

ABSTRACTDifferent routes were screened for the preparation of superparamagnetic cation–exchange adsorbents for the capture of proteins using high-gradient magnetic fishing. Starting from a polyglutaraldehyde-coated base particle, the most successful of these involved attachment of sulphite to oligomers of epichlorohydrin formed on the particle surface. The resultant cation-exchanger had a maximum lysozyme binding capacity of 272 mg g–1 and a dissociation constant of 0.73 µM. Using lysozyme as a model protein in small-scale studies, appropriate conditions were then selected for the capture of lactoperoxidase from sweet bovine whey. Subsequently, a high-gradient magnetic fishing process was constructed for the fractionation of whey, in which lactoperoxidase was purified 36-fold and concentrated 4.7-fold.

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g −1 combined with a dissociation constant of 0.60 μM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g −1 with a dissociation constant of 0.042 μM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (≤0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4–1 M. The anion-exchanger adsorbed β-lactoglobulin (β-LG) selectively allowing separation from the remaining protein.