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.     

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%.     

Magnetic separation in biotechnology

New developments in magnetic labelling techniques for cells and microspheres have extended the useful range of magnetic separation, particularly high gradient magnetic separation, into biotechnical areas. The basic magnetic principles involved are reviewed and representative samples of labelling techniques and results drawn from the past three years are presented. Illustrative examples of large scale operation in other industries are also presented, demonstrating the potential of the biotechnological applications.     

Semi‐continuous in situ magnetic separation for enhanced extracellular protease production—modeling and experimental validation

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Enhancing ligand–protein binding in affinity thermoprecipitation: Elucidation of spacer effects

Copolymers of N‐isopropylacrylamide and N‐acryloyl amino acid spacers of varying chain length were synthesized. p‐Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (K_i) of these polymers for trypsin decreased, i.e., the efficiency of PABA–trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower K_i than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N‐acyl‐p‐aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 418–425, 1999.     

Effects of strong static magnetic fields used in magnetic resonance imaging on insulin-secreting cells

The magnetic flux density of MRI for clinical diagnosis has been steadily increasing. However, there remains very little biological data regarding the effect of strong static magnetic fields (SMFs) on human health. To evaluate the effects of strong SMFs on biological systems, we cultured insulin-secreting cells under exposure to sham and SMF conditions (3-10 T of magnetic flux density, and 0-41.7 T/m of magnetic field gradient) for 0.5 or 1 h, and analyzed insulin secretion, mRNA expression, glucose-stimulated insulin secretion, insulin content, cell proliferation and cell number. Exposure to SMF with a high magnetic field gradient for 1 h significantly increased insulin secretion and insulin 1 mRNA expression. Exposure to SMF with a high magnetic flux density for 0.5 h significantly enhanced responsiveness to glucose stimulation. Exposure to SMF did not affect the insulin content, cell proliferation or cell number. Our results suggested that MRI systems with a higher magnetic flux density might not cause cell proliferative or functional damages on insulin-secreting cells, and that SMF with a high magnetic field gradient might be used clinically after thorough in vivo investigations are conducted.     

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     

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     

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.     

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     

Agglomeration behaviour of magnetic microparticles during separation and recycling processes in mRNA purification

Purification of mRNA with oligo(dT)‐functionalized magnetic particles involves a series of magnetic separations for buffer exchange and washing. Magnetic particles interact and agglomerate with each other when a magnetic field is applied, which can result in a decreased total surface area and thus a decreased yield of mRNA. In addition, agglomeration may also be caused by mRNA loading on the magnetic particles. Therefore, it is of interest how the individual steps of magnetic separation and subsequent redispersion in the buffers used affect the particle size distribution. The lysis/binding buffer is the most important buffer for the separation of mRNA from the multicomponent suspension of cell lysate. Therefore, monodisperse magnetic particles loaded with mRNA were dispersed in the lysis/binding buffer and in the reference system deionized water, and the particle size distributions were measured. A concentration‐dependent agglomeration tendency was observed in deionized water. In contrast, no significant agglomeration was detected in the lysis/binding buffer. With regard to magnetic particle recycling, the influence of different storage and drying processes on particle size distribution was investigated. Agglomeration occurred in all process alternatives. For de‐agglomeration, ultrasonic treatment was examined. It represents a suitable method for reproducible restoration of the original particle size distribution.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

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.     

Chromatographic separation of maltopentaose from maltooligosaccharides

An experimental study on the chromatographic separation of maltopentaose from a mixture, including glucose, maltose, maltotriose, and maltopentaose, was carried out in a nonionic polymeric sorbent column while varying the operating conditions, such as the solution pH, buffer contents, and isopropyl alcohol (IPA) concentration. Unlike the pH and buffer contents, the IPA concentration had a significant impact on the single component chromatograms for maltopentaose. The retention times of the maltooligosaccharides with the nonionic polymeric sorbent SP207 were in the following order: glucose<maltose<maltotriose<maltopentaose. From the experimental binary, ternary, and quaternary chromatograms, gradient chromatographic separation with a changing IPA concentration as a function of time was required to obtain high-purity maltopentaose and reduce the elution time.     

Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides.

Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.     

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.