Ion-exchange purification of proteins using magnetic nanoclusters

Polymer-coated magnetic nanoclusters were used for recovery and purification of proteins from both model systems and cell-free Pichia pastoris fermentation broth. The nanoclusters exhibited extremely high capacity for proteins, up to 900 mg/mL adsorbent, and were recovered by high gradient magnetic separation (HGMS) at flow rates of up to 3,600 cm(3)/cm(2) h (flow rates up to 15,000 cm(3)/cm(2) h are possible). The nanoclusters were coated with a primary coating of poly(acrylic acid-co-styrenesulfonic acid-co-vinylsulfonic acid), which allowed both electrostatic and hydrophobic interactions with the protein to be used to enhance specificity for targeted products. With this dual mode separation, nearly pure protein could be recovered from complex mixtures, such as fermentation broth, in a few quick steps.     

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%.     

Ferritin as a label for high-gradient magnetic separation.

In three model systems, particles the size of cells or smaller have been surface labeled with ferritin to make them slightly paramagnetic, by virtue of the iron in the ferritin. In each case it was possible to show that labeled particles could be magnetically removed from a flowing suspension by the high-gradient magnetic separation (HGMS) technique. The first system of particles consisted of small (1 micron) carboxylate-modified latex spheres to which ferritin was covalently bound to create stable paramagnetic particles analogous to a ferritin-labeled subcellular membrane preparation. In the second system polyacrylamide beads that more closely approximated whole cells in size (5-50 microns) were labeled with immunoferritin. The third system was a biomembrane preparation: erythrocyte ghosts labeled with a ferritin-lectin conjugate. A field of 7 T (tesla) (70 kG) was used in each case, along with buffer flow rates through the HGMS column in the range 0.1-1.0 ml/min.     

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.     

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.     

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.     

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.     

Separation of yeast cells from aqueous solutions using magnetically stabilized fluidized beds

AIMS: To separate Saccharomyces cerevisiae cells from aqueous solutions using magnetically stabilized fluidized beds (MSFB) that utilize a horizontal magnetic field, and to study the effect of some parameters, such as bed porosity and height, liquid flow rate and inlet concentration on cell removal efficiency and breakthrough curves. METHODS AND RESULTS: The separation process was conducted in an MSFB under the effect of horizontal magnetic field. The magnetic particles used consist of a ferromagnetic core of magnetite (Fe3O4) covered by a stable layer of activated carbon to adsorb the yeast cells from the suspension. The yeast cell concentration in the effluent was determined periodically by measuring the absorbance at 610 nm. The effect of the magnetic field intensity on the bed porosity and consequently the exit-normalized cell concentration from the bed was studied. It was found that bed porosity increased by 75%, and the normalized cell concentration in the bed effluent decreased by 30%, when the magnetic field intensity was increased from 0 to 110 mT. In addition, increasing the magnetic field intensity and bed height delayed the breakthrough point, and allowed efficient cell removal. These results demonstrate an improved method to separate cells of low concentration from cell suspension. CONCLUSIONS: This study allows the continuous separation of yeast cells from aqueous solutions in an MSFB. The removal efficiency is affected by different parameters including the bed height, flow rate and initial concentration. The removal efficiency reaches 82%, and could be improved by varying the operational parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this investigation show that the MSFB using horizontal fields represents a potential tool for the continuous separation of cell suspension from aqueous solution. This study will contribute to a better understanding of the hydrodynamic parameters on the separation efficiencies of the cell.     

磁修饰酵母对直接大红染料的生物吸附

本研究采用水相合成的纳米级铁磁流体对灭活酿酒酵母细胞进行磁修饰,获得了具有良好磁响应的酵母。傅立叶变换红外光谱分析表明,该修饰酵母在Fe-O特征峰581cm1处的吸收明显加强。透射电镜观察表明,纳米磁性颗粒单个或成团聚集在酵母细胞表面。在本实验条件下,160μL磁修饰酵母对1mL浓度为0.4mg/mL直接大红染料吸附率达100%;8min达到吸附平衡;在70%乙醇中,染料脱吸附率为99.18%。由于磁修饰酵母吸附力强,吸附速度快,易于磁分离,是一种有前景的水溶性染料生物吸附剂。     

Improved 2D nano-LC/MS for proteomics applications: a comparative analysis using yeast proteome.

The most commonly used method for protein identification with two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS) involves the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration followed by reversed phase separation. However, in this approach ion exchange chromatography does not perform to its fullest extent, primarily because the injected volume of salt solution is not optimized to the SCX column. To improve the performance of strong cation exchange chromatography, we developed a new method for 2D online nano-LC/MS that replaces the injected salt step gradient with an optimized semicontinuous pumped salt gradient. The viability of this method is demonstrated in the results of a comparative analysis of a complex tryptic digest of the yeast proteome using the injected salt solution method and the semicontinuous pump salt method. The semicontinuous pump salt method compares favorably with the commonly used injection method and also with an offline 2D-LC method.     

Fractionation of a population ofSaccharomyces cerevisiae yeasts by centrifugation in a dextran gradient

A method of the fractionation of aSaccharomyces cerevisiae yeast population in dextran gradients is described. The elaboration of this method was based on the finding of a correlation between the size of individual cells and the number of bud scars on their surface and rapid indication of the scars by fluorescence microscopy. The basic conditions for fractionation (determined experimentally) were as follows: 2 ml. yeast suspension (100 mg. dry weight) was applied to the surface of a continuous dextran gradient of 9–16% concentration and was centrifuged at a relative centrifugal force of 200 G for 15 minutes. In fractionation of a whole population, the best fractionation was obtained in a linear gradient. Repeated separation of fractions obtained by centrifugation in a linear gradient in a concave gradient further separated cells without bud scars and accumulated cells with five scars and over. Three fractions were obtained by this technique. The first contained 90–98% cells without bud scars, the second 55–65% cells with 1–4 bud scars and the third 50% cells with five bud scars and over.     

Effect of static magnetic fields on the budding of yeast cells

The effect of static magnetic fields on the budding of single yeast cells was investigated using a magnetic circuit that was capable of generating a strong magnetic field (2.93 T) and gradient (6100 T² m^?1). Saccharomyces cerevisiae yeast cells were grown in an aqueous YPD agar in a silica capillary under either a homogeneous or inhomogeneous static magnetic field. Although the size of budding yeast cells was only slightly affected by the magnetic fields after 4 h, the budding angle was clearly affected by the direction of the homogeneous and inhomogeneous magnetic fields. In the homogeneous magnetic field, the budding direction of daughter yeast cells was mainly oriented in the direction of magnetic field B. However, when subjected to the inhomogeneous magnetic field, the daughter yeast cells tended to bud along the axis of capillary flow in regions where the magnetic gradient, estimated by B(dB/dx), were high. Based on the present experimental results, the possible mechanism for the magnetic effect on the budding direction of daughter yeast cells is theoretically discussed. Bioelectromagnetics 31:622–629, 2010. © 2010 Wiley‐Liss, Inc.     

The Influence of a gradient static magnetic field on an unstirred Belousov-Zhabotinsky reaction

It is believed that static magnetic fields (SMF) cannot affect the pattern formation of the Belousov-Zhabotinsky (BZ) reaction, which has been frequently studied as a simplified experimental model of a nonequilibrium open system, because SMF produces no induced current and the magnetic force of SMF far below 1 T is too low to expect the effects on electrons in the BZ reaction. In the present study, we examined whether the velocity of chemical waves in the unstirred BZ reaction can be affected by a moderate-intensity SMF exposure depending on the spatial magnetic gradient. The SMF was generated by a parallel pair of attracting rectangular NdFeB magnets positioned opposite each other. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)), and the magnetic force product of the magnetic flux density its gradient (a magnetic force parameter) were 206 mT, 37 mT/mm, and 3,000 mT(2)/mm. The ferroin-catalyzed BZ medium was exposed to the SMF for up to 16 min at 25 degrees C. The experiments demonstrated that the wave velocity was significantly accelerated primarily by the magnetic gradient. The propagation of the fastest wave front indicated a sigmoid increase along the peak magnetic gradient line, but not along the peak magnetic force product line. The underlying mechanisms of the SMF effects on the anomalous wave propagation could be attributed primarily to the increased concentration gradient of the paramagnetic iron ion complexes at the chemical wave fronts induced by the magnetic gradient.     

Rapid and efficient selection of yeast displaying a target protein using thermo-responsive magnetic nanoparticles

Magnetic separation provides a relatively quick and easy-to-use method for cell isolation and protein purification. We have developed a rapid and efficient procedure to isolate yeast cells displaying a target polypeptide, namely, the Staphylococcus aureus ZZ domain, which serves as s model for protein interactions and can bind immunoglobulin G (IgG). We optimized selection of ZZ-displaying yeast cells using thermoresponsive magnetic nanoparticles. A model library was prepared by mixing various proportions of target yeast displaying the ZZ domain with control cells. Target cells in the model library that bound to the ZZ-specific binding partner, biotinylated IgG, were selected with biotinylated thermoresponsive magnetic nanoparticles using the biotin-avidin sandwich system. We determined ZZ expression levels and optimized the concentrations of both magnetic nanoparticles and avidin for efficient selection of target cells. After optimization, we successfully enriched the target cell population 4700-fold in a single round of selection. Moreover, only two rounds of selection were required to enrich the target cell population from 0.001% to nearly 100%. Our results suggest that magnetic separation will be useful for efficient exploration of novel protein-protein interactions and rapid isolation of biomolecules with novel functions.     

Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies

Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes. Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques. To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae. The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation. Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF. By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing. This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption.     

New concepts for rapid yeast settling. I. Flocculation with an inert powder

A novel technique for settling microorganisms has been described. The technique involves adding a dense, inert powder to a suspension of microorganisms under conditions where flocculation of the microorganism with the inert poweder occurs. The flocs formed are small and relatively dense and settle rapidly. Suspensions of Saccharomyces cerevisiae yeast have been flocculated with several different inert seed materials achieving rapid settling and separations of up to 99.9%. Nickel powder was used as a seed material for most experiments described here, and iron sand showed promise as a cheaper seed for large-scale use. The degree of flocculation and cell separation obtained depended largely on the seed concentration and the components in solution. Temperature and pH had little effect. When the method was initially applied to a practical fermentation, flocculation was poor because of inhibiting compounds in the fermentation medium, but modification of the technique produced good flocculation in the medium.     

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%.     

出芽短梗霉膨大细胞分选及产糖分析

本研究运用Percoll密度梯度离心的方法对出芽短梗霉Aureobasidium pullulans的两种细胞形态进行分选,并对两种形态的细胞进行多糖产量的分析。通过对转速、分选时间、Percoll分离液浓度的优化,确定了两种细胞形态分选效果最佳的条件是Percoll分离液浓度为60%、转速为5 000r/min、离心时间为30min。经过光学显微镜和透射电子显微镜观察发现上层为酵母状细胞（YL）、下层为膨大细胞（SC）,并发现膨大细胞外有明显的薄膜包被,且产大量多糖。也为今后在相应状态下研究出芽短梗霉膨大细胞的其他代谢机理提供了可行的方法,满足后续研究的需要。     

Porcine blood cell separation by porous cellulose acetate membranes

Leukocytes were separated from whole porcine blood using laboratory prepared polymeric asymmetric porous membranes from cellulose acetate (CA) and by applying standard blood cell separation methods: centrifugation in a Ficoll solution gradient and in sucrose solution concentration gradient. Leukocytes, obtained by different separation methods were characterised by their quantity, type, viability and growth ability. Membranes prepared by a wet phase inversion process from different cellulose acetate/acetone/water and magnesium chlorate VII systems, were characterised according to: permeability to deionised water, surface morphology and by the determination of the flux of the permeate during the whole porcine blood separation. Cellulose acetate membranes prepared from 300 μm thick cast solution (14.8 wt% of cellulose acetate, 19.9 wt% of water, 2.3 wt% of Magnesium perchlorate, and 63.0 wt% of acetone), have separation characteristics comparable with the standard separation methods; in the dead-end mode filtration, 21.3% of leukocytes from porcine whole blood are separated. The leukocyte number in peripheral blood before separation was 450,000 ml^-1; the number passed through after was 95,000±6620. The main interest of the study was to introduce the CA membrane filters for the continus technological separation of the leukocyte/lymphocytes from animal (= porcine, bovine, horse..) blood. This revised version was published online in June 2006 with corrections to the Cover Date.