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 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.     

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.     

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     

Magnetic seeding to aid recovery of biological precipitates

A new approach to solid/liquid separation for biological precipitates is presented. The precipitate is seeded with small dense and/or magnetic particles to increase the density difference between precipitate and liquid or to make the precipitate amenable to magnetic separation. Experiments on seeding of ammonium sulphate precipitates of casein and separation of the seeded precipitate by gravity settling and batch centrifugation have shown that the approach holds promise. Seeded precipitates exhibit hindered settling under gravity with typical settling velocities of 0.6 cm/min for iron oxide seed and 2.4 cm/min for nickel seed. Calculations indicate that seeding of these precipitates causes a 23 fold increase in density difference between precipitate particle and supernatant liquid. The design of a magnetic seeding step for use in an enzyme isolation process is discussed.     

Micro- and nanotechnology in cell separation

This review describes recent work in cell separation using micro- and nanoscale technologies. These devices offer several advantages over conventional, macroscale separation systems in terms of sample volumes, low cost, portability, and potential for integration with other analytical techniques. More importantly, and in the context of modern medicine, these technologies provide tools for point-of-care diagnostics, drug discovery, and chemical or biological agent detection. This review describes work in five broad categories of cell separation based on (1) size, (2) magnetic attraction, (3) fluorescence, (4) adhesion to surfaces, and (5) new emerging technologies. The examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field.     

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.     

One‐step magnetic modification of yeast cells by microwave‐synthesized iron oxide microparticles

Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non‐cross‐linked and glutaraldehyde cross‐linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole‐cell biocatalyst was stable; almost the same catalytic activity was observed after 1‐month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity.

Significance and Impact of the Study

Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave‐synthesized iron oxide microparticles is a low‐cost and easy‐to‐perform alternative to already existing magnetization techniques.     

在cDNA文库构建中探究癌症病人血浆外泌体miRNA的特异性扩增

血浆外泌体微小核糖核酸(microRNAs,miRNAs)与癌症的发生、诊断和治疗密切相关,但其分子机制尚不明晰。本研究探讨了癌症病人血浆外泌体miRNAs在cDNA文库构建中非特异性扩增的解决方案。在酶切法中,采用核酸外切酶T (exonuclease T, EXOT)和phi29 DNA聚合酶降解引物;在磁珠法中,利用DNA结合磁珠分离模板和引物。随后,采取琼脂糖凝胶电泳和变性聚丙烯酰胺凝胶电泳检测磁珠分离情况,运用RT-qPCR检测癌症病人血浆外泌体miRNA和不同连接物的含量变化。结果显示,非特异性扩增来源于miRNA的连接物USR5SR;核酸外切酶T (EXOT)和phi29 DNA聚合酶虽可降解USR5SR,但模板链也会发生降解;磁珠分离法中以9%PEG沉淀引物片段、15%PEG沉淀模板链效果最佳。综上所述,磁珠分离法能够高效解决cDNA文库构建中的非特异性扩增,从而实现293T细胞和癌症病人血浆外泌体miRNA cDNA文库的成功构建。     

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.     

Magnetic Precipitation: A New Platform for Protein Purification

One of the trends in downstream processing comprises the use of “anything‐but‐chromatography” methods to overcome the current downfalls of standard packed‐bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti‐TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.     

空间独立成分分析实现fMRI信号的盲分离

独立成分分析（ICA）在功能核磁共振成像（fMRI)技术中的应用是近年来人们关注的一个热点。简要介绍了空间独立成分分析（SICA）的模型和方法,将fMRI信号分析看作是一种盲源分离问题,用快速算法实现fMRI信号的盲源分离。对fMRI信号的研究大多是在假定已知事件相关时间过程曲线的情况下,利用相关性分析得到脑的激活区域。在不清楚有哪几种因素对fMRI信号有贡献、也不清楚其时间过程曲线的情况下,用SICA可以对fMRI信号进行盲源分离,提取不同独立成分得到任务相关成分、头动成分、瞬时任务相关成分、噪声干扰、以及其它产生fMRI信号的多种源信号。     

Quantification of non‐specific binding of magnetic micro‐ and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub‐optimal performance, one of the major challenges can be non‐specific binding of magnetic nano‐ or microparticles to non‐targeted cells. Depending on the type of separation, this non‐specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non‐specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we demonstrate that the number of MACS magnetic particles needed to impart a magnetization that is sufficient to cause non‐targeted cells to be retained in the column to be on the order of 500–1,000 nanoparticles. This number of non‐specifically bound particles was demonstrated experimentally with an instrument, cell tracking velocimeter, CTV, and it is demonstrated that the sensitivity of the CTV instrument for Fe atoms contained in magnetic nanoparticles on the order of 1 × 10^?15 g/mL of Fe. Biotechnol. Bioeng. 2010;105: 1078–1093. © 2009 Wiley Periodicals, Inc.     

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。     

Real-time PCR to study the sequence specific magnetic purification of DNA

The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations.     

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).     

Cell labelling and separation with polyglutaraldehyde microspheres

Non-magnetic and magnetic polyglutaraldehyde microspheres were used in the labelling and separation of mouse and human T and B lymphocytes. The enrichment of the separated T- and B-cell fractions of mouse and human cells was 30 and 40% respectively. After magnetic separation 79% of the mouse B-cell fraction and 23% of the T-cell fraction were surface Ig-positive. The corresponding figures for human T and B cells were 10 and 51–54%, respectively. The labelling of mouse spleen B cells with antibody-conjugated non-magnetic microspheres was between 27–53% depending on the labelling procedures. The best labelling was obtained when mouse spleen cells coated with rabbit anti-mouse Ig were incubated with protein A-coupled microspheres.     

Magnetic cell separation using nano-sized bacterial magnetic particles with reconstructed magnetosome membrane

Magnetic nanoparticles produced by magnetotactic bacterium, bacterial magnetic particles (BacMPs), covered with a lipid bilayer membrane (magnetosome membrane) can be used to separate specific target cells from heterogeneous mixtures because they are easily manipulated by magnets and it is easy to display functional proteins on their surface via genetic engineering. Despite possessing unique and valuable characteristics, the potential toxicity of BacMPs to the separated cells has not been characterized in detail. Here, a novel technique was developed for the reconstruction of magnetosome membrane of BacMPs expressing protein A (protein A-BacMPs) to reduce cytotoxicity and the newly developed nanomaterial was then used for magnetic cell separation. The development of the magnetosome membrane-reconstructed protein A-BacMP was based on the characteristics of the Mms13 anchor protein, which strongly binds to the magnetite surface of BacMPs. Treatment of protein A-BacMPs with detergents removed contaminating proteins but did not affect retention of Mms13-protein A fusion proteins. The particle surfaces were then reconstructed with phosphatidylcholine. The protein A-BacMPs containing reconstructed magnetosome membranes remained dispersible and retained the ability to immobilize antibody. In addition, they contained few membrane surface proteins and endotoxins, which were observed on non-treated protein A-BacMPs. Magnetic separation of monocytes and B-lymphocytes from the peripheral blood was achieved with high purity using magnetosome membrane-reconstructed protein A-BacMPs.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.