Large-scale separation of magnetic bioaffinity adsorbents

Flat magnetic separator was used to separate magnetic bioaffinity adsorbents from litre volumes of suspensions. Both magnetic cross-linked erythrocytes and magnetic chitosan were efficiently separated; at least 95% adsorbent recovery was achieved at maximum flow rate (1680 ml min^–1). Using this system low amounts of trypsin were concentrated from large sample volumes using magnetic erythrocytes as affinity adsorbent.     

磁分离仪分离磁性细菌的新方法

磁性细菌胞内可以产生磁性颗粒,因此具有趋磁性,基于这种特性,利用磁分离的原理,本研究开发了一种磁性细菌分离仪,提供了一种分离磁性细菌的新方法。以氧化亚铁硫杆菌为例,使用磁性细菌分离仪进行分离,可以得到强磁菌和弱磁菌。利用透射电镜观察,强磁菌胞内磁性颗粒明显多于弱磁菌;半固体平板磁泳实验也表明强磁菌趋磁性明显强于弱磁菌。各项实验结果表明磁性细菌分离仪可以有效地分离磁性细菌,这是一种分离磁性细菌的新方法,将促进磁性细菌分离培养的研究。     

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.     

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.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

磁纳米识别探针的构造及其在真菌毒素检测中的应用进展

真菌毒素是一种由真菌产生的具有毒性的次级代谢产物,易引发严重的食品安全问题,不断探索更为高效准确的新型检测方法具有重要意义。磁纳米识别探针具有高效易分离、结合容量大、识别效果好、功能性强等优势,为复杂基质中痕量真菌毒素检测研究带来新方向。本文对磁纳米识别探针构造,由内向外对构成探针的磁纳米核心颗粒及其表面修饰物的种类及特点进行总结分析,在此基础上进而从探针的选择与功能、检测条件、检测灵敏度及特异性等方面,对近年来磁纳米识别探针在食品体系真菌毒素检测中的应用研究进行概述归纳,并对其未来的应用前景与发展方向进行展望。     

High gradient magnetic separation of rosette-forming cells

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (<2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20–30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by, the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20–50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90–100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80–90%.     

Fluorescent detection of cyanobacterial DNA using bacterial magnetic particles on a MAG-microarray

Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-microm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated.     

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.     

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     

Characterization of Heavy Metals in Soil by High Gradient Magnetic Separation

In recent years forecasting soil cleaning efficiencies of polluted soil, especially those contaminated with heavy metals, has become an important issue. Failure of the methods to predict the right efficiency has resulted in financial losses and penalties. This article describes an addition to the traditional characterization methods for soils contaminated by heavy metals, improving the quality of the basic decisions to be made. The method is based on magnetic separation using a Frantz Isodynamic Separator (FIS) for this study. The FIS isolates soil particles containing heavy metals so that these particles, which are relevant for soil cleaning, can be studied in more detail. Four contaminated soils were tested, for example, soils that were a problem for the soil-cleaning industry. The experiments indicate that each soil has its own magnetic properties that should be regarded as a fingerprint. Density measurements of two soils show that densities approach the quartz density separated at moderate and high magnetic fields suggesting that some of the heavy metals cannot be removed by density separation techniques. A pilot plant scale test supports this conclusion. It also shows that a part of the heavy metals are found in particles lighter than quartz. Based on the results, a qualitative model is proposed to account for the presence of the heavy metals in soil.     

Direct magnetic separation of immune cells from whole blood using bacterial magnetic particles displaying protein G

Direct separation of target cells from mixed population, such as peripheral blood, umbilical cord blood, and bone marrow, is an essential technique for various therapeutic or diagnosis applications. In this study, novel particles were fabricated, and direct magnetic separation of immune cells from whole blood using such particles was performed. The magnetotactic bacterium Magnetospirillum magneticum AMB‐1 synthesizes intracellular bacterial magnetic particles (BacMPs), and protein G was expressed on the surface of the BacMPs by gene fusion techniques with anchor proteins isolated from BacMP membrane. The BacMPs displaying protein G (protein G‐BacMPs) had high binding capabilities to a wide range of antibody types, and various versions of protein G‐BacMPs binding with different anti‐CD monoclonal antibodies were constructed. Consequently, direct magnetic separation of immune cells from whole blood using protein G‐BacMPs binding with anti‐CD monoclonal antibodies was demonstrated. B lymphocytes (CD19⁺ cells) or T lymphocytes (CD3⁺ cells), which represent less than 0.05% in whole blood cells, were successfully separated at a purity level of more than 96%. This level was superior to that from previous reports using other magnetic separation approaches. The results of this study demonstrate the utility of protein G‐BacMP and this particle may become a powerful tool for various therapeutic or diagnosis applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Geometrical optimisation of a biochip microchannel fluidic separator

This article reports on the geometric optimisation of a T-shaped biochip microchannel fluidic separator aiming to maximise the separation efficiency of plasma from blood through the improvement of the unbalanced separation performance among different channel bifurcations. For this purpose, an algebraic analysis is firstly implemented to identify the key parameters affecting fluid separation. A numerical optimisation is then carried out to search the key parameters for improved separation performance of the biochip. Three parameters, the interval length between bifurcations, the main channel length from the outlet to the bifurcation region and the side channel geometry, are identified as the key characteristic sizes and defined as optimisation variables. A balanced flow rate ratio between the main and side channels, which is an indication of separation effectiveness, is defined as the objective. It is found that the degradation of the separation performance is caused by the unbalanced channel resistance ratio between the main and side channel routes from bifurcations to outlets. The effects of the three key parameters can be summarised as follows: (a) shortening the interval length between bifurcations moderately reduces the differences in the flow rate ratios; (b) extending the length of the main channel from the main outlet is effective for achieving a uniformity of flow rate ratio but ineffective in changing the velocity difference of the side channels and (c) decreasing the lengths of side channels from upstream to downstream is effective for both obtaining a uniform flow rate ratio and reducing the differences in the flow velocities between the side branch channels. An optimisation process combining the three parameters is suggested as this integration approach leads to fast convergent process and also offers flexible design options for satisfying different requirements.     

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.     

Determination of proteolytic activity with magnetic dye-stained gelatine

A procedure for the determination of proteolytic activity with dyed magnetic gelatine as an insoluble chromolytic substrate is described. The magnetic nature of the substrate enables magnetic separation of unhydrolysed substrate from the hydrolysed dyed peptide fragments. Such type of substrates could enable the development of new automated protease assays based on the principle of Flow Injection Analysis (FIA).     

Modelling and simulation of the behaviour of a biofluid in a microchannel biochip separator

This paper reports an investigation into the flow behaviour of a biofluid in a microchannel systems through conceptual analysis and modelling. The application is the design of a microfluidic chip developed for the separation of plasma from blood. The effect of key design features of the microchannels on the flow behaviour of the biofluid is explored. These include geometric features such as the constriction, bending channel, bifurcation and the channel length ratio between the main and side channels. The performance of each design is discussed in terms of separation efficiency of the red blood cells with respect to the rest of the medium. Particular phenomena such as the Fahraeus and Fahraeus–Lindqvist effects, the Zweifach–Fung bifurcation law and the cell-free layer are discussed. In this paper, the fluid is modelled as a single-phase flow assuming either Newtonian or Non-Newtonian behaviour to investigate the effect of the fluid viscosity on both flow and separation efficiency. For a flow rate-controlled Newtonian flow system, it is found that viscosity and outlet pressure have little effect on the velocity distribution through each of the microchannels. For a diluted fluid where the flow in the whole channel system is modelled with a uniform viscosity, less plasma is separated from blood than observed in the non-Newtonian case. This results in an increase in the flow rate ratio between the main and side channels. A comparison of Newtonian and non-Newtonian flows shows that both flows tend to behave identically with an increase in the shear strain rate.     

High-efficiency bioaffinity separation of cells and proteins using novel thermoresponsive biotinylated magnetic nanoparticles

Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max, avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4 antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max with a UCST in bioaffinity separation of cells and biomolecules.     

Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube

Metallothionein (MT) isoforms from various liver tissues were separated with capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH. The electrophoresis was performed on MT-1 and MT-2 purified from mouse, rat, rabbit and human livers. The retention times of mouse and rat MT-1 coincided, while the retention times of rabbit and human MT-1 were longer. The retention times of MT-2 purified from the four sources were the same. MT-1 and MT-2 separated more definitely with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-Tris buffer (25 mM, pH 7.4) than with N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS)-Tris buffer (25 mM, pH 7.7) or with N-(2-acetamido)iminodiacetic acid (ADA)-Tris buffer (25 mM, pH 7.4). In addition, liver MT isoforms prepared from Zn- or Cd-administered mice could be separated.