Final Report Summary - MAGPRO²LIFE (Advanced Magnetic nanoparticles deliver smart Processes and Products for Life)
Executive Summary:
The project was very challenging and ambitious in depth and breath. A new technology platform was established through the execution of the several work packages in a coordinated effort. With no doubts, the new developments are cutting edge and the research team established world leadership in the area with this project. It was a complex challenge with an interdisciplinary technology solution. The project demonstrates a successful combination of bio and nanotechnology.
The project was technically successfully and fulfilled all the necessary requirements for safety and health. Numerous tests and LCA show that the developed magnetic particle system is close for commercial use. In addition to the technical success, the project has shown that this new technology platform is also economical successfully compared to other technologies. Consequent process scale-up with cost evaluations led to a new process with lower cost footprint.
The technology in its different layers (particles, particle production processes, process technology and equipment) was demonstrated in scale. The project had included research work but most of the time was spent on commercialization and scale-up of the technology. New equipment was developed, like the magnetic centrifuge, which are in a pilot scale today. Discovered knowledge in this project will bring this magnetic centrifuge concept to a commercialization. Different methods to produce magnetic particles were discovered. Particles are smartly functionalized. Models were developed and built. The results were established and demonstrated not with academic material but with real product streams from the industry.
Challenges of the project were the work on the details and the connection of the different layers of technology so that a whole new technology platform could be established. The researcher and the industrial partners believe the technology is ready for the market.
The technology is economically viable for Food, Bio and Pharma applications. The research found it is very challenging to be economically successful for Feed applications. Many aspects were researched in depth and solutions were found. Analytic methods were successfully developed and are ready for future commercialization.
The new technology platform could have impact on the next generation of healthy food, biotechnology and pharma applications and very interesting future options arrived out of the project, like enzyme use for industrial bio-processes.
Project Context and Objectives:
Publishable summary MagPro²Life
« Advanced Magnetic nano-particles deliver smart Processes and Products for Life «
Researchers from Denmark, Germany, Ireland, Romania, Spain, Switzerland and UK kicked off the EU MagPro²Life project on July 1, 2009. The consortium brings together universities, research institutes, SME and enterprises and is coordinated by Solae. The primary objective of this project is to develop pilot lines to introduce nanotechnology based processes into the value chain of existing industries.
Implementation of functional magnetic particles as adsorbents in the bio-processing industry requires the synergistic interplay of a host of components. The two major challenges for implementation of magnetic (nano) particles and composites in industry are the cost-effective large-scale manufacturing of appropriately functionalized super-paramagnetic particles, and the lack of large-scale process technology to separate these particles from the production streams. For this technology to be accepted in industry, this technology must be sustainable, safe, capable of scaling to high production rates, reliable, robust, and economically competitive to current existing downstream processing techniques. The goal of the MagPro²Life project is to address these challenges and to demonstrate the use of functional magnetic (nano)particle separation at pilot-scale for selected feed, food, and pharmaceutical products. The MagPro²Life project carefully builds on knowledge gained in the NanoBioMag project, funded by the EU under the FP6 program, through a series of focused objectives: (i) development of scalable magnetic particles production technologies; (ii) development of scalable surface functionalizing technologies; (iii) development of large-scale processing technologies to be used with the adsorbents produced by objectives (i) and (ii); and (iv) integration of the previous three objectives to demonstrate that this technology can be implemented for safe, industrial scale bio-separation.
Biotechnologically derived substances for large scale feed, food and pharma applications represent one of the most important sources of new products due to their precisely controlled structural and functional properties, potential for economic and responsible production and overall broad benefits to society through bio-compatibility and sustainability. The costs of producing bio-materials are in many cases dominated by separation processes, which can constitute up to 80% of the total cost of production. Using smart magnetic adsorbent particles to selectively separate the target product out of a complex product mixture like the fermentation broth or bio-feed stock can drastically reduce costs. By using magnetic separation and extraction technologies to separate the magnetic carrier particles, novel processing ways emerge.
While the developments of modern gene technology promise to make an increasing number of sophisticated pharmaceuticals available, their enormous production prices will inevitably place strong pressures upon health care systems, even those in wealthier countries. Higher value proteins derived from natural and recombinant sources represent main target bio-products within this project. The range of prices of various industrial protein products vary astronomically up to several million € per kg. High-value pharmaceutical proteins are only affordable for highly developed countries in the western hemisphere, but even in these privileged countries the high cost of these drugs exerts a strongly negative impact on their social health care system. By reducing the production costs of new pharmaceuticals the deliverables of this project will contribute to solving this dilemma.
The project will likely deliver exploitable findings directly applicable to individual products. Practical exploitation will be undertaken by the industrial partners as the first end users. The knowledge database produced during MagPro²Life shall guarantee fast market readiness and fast and easy transfer to products other than those employed in the pilot line demonstrations.
The knowledge gained in the areas of process and equipment development and applications will form a basis for creating new process equipment and new high-value products. Equipment when commercialized will be available to industrial sectors such as pharmaceutical, food, chemical and technical industries. It is planned to sell licences for the patented equipment to biotech manufactures.
In the first 18 months of the project, several distinct manufacturing routes were developed to produce assemblies based on inexpensive super paramagnetic particles combined with bio-materials, functional polymers or self-adapting polymers. Recognition systems are constructed to make the adsorbents selective and/or endow them with multifunctional properties. Bio-materials, representing each of the 3 industries for pilot scale demonstration, have been selected and characterized. Focus in the next period will be the comparative characterization of the particle systems and subsequent scale-up of the most promising ones. To successfully compete with classical production processes it is crucial to provide high efficient process equipment especially regarding the recovery of the magnetic adsorbent particles. In the following are the technologies, the consortium is focusing on: Continuous Magnetic Extraction, Magnetic Field Enhanced Centrifugation, and Magnetic Classification. Lab equipment has been used to model the separation systems and preliminary screening of the particle systems has been performed. Focus has been on the pilot scale design of the separation equipment, while efforts for the next period will be the manufacture and optimization of this equipment.
In the second 18 project months, the bio-systems for production and capturing the target molecules have been studied and optimized in all three areas of interest. The separation equipment was built, optimized and bench-marked. The pilot lines where set-up and are ready to run and generate data for the risk assessment, the economic studies, Life Cycle Analysis and technology bench-marking. Pro-actively, particular focus was set to Anion Exchanger and Cation Exchanger functionalization of the magnetic beads. Sufficient quantities of particle systems were made available for all three pilot plants to trial and run. Additional bead systems of interest are planned in smaller quantities and will be tested on lab scale. They will be scaled up using SuperPro Designer® software in order to offer the possibility to compare these data with the data from the pilot lines.
In the third and last reporting period, the pilot lines have run and demonstrated the MagPro²Life technology. Full data analysis and modeling made an assessment possible on all the aspects of potential successful exploitation. Scale, particle cost and binding capacity have the most significant impact on production cost. We showed that although at current state of the art, still optimization is needed, it is possible to commercially exploit the technology at competitive product prices, with distinct advantages against immediate competing technologies as packed bed and expanded bed technologies. A full comprehensive confidential report has been issued on the technology as explored in this project. A book on the subject matter will be published (Springer) in second quarter of 2014.
Project Results:
Final Report 4.1 (SESAM) – Results of S&T foregrounds
SOL
Solae was the overall coordinator of the MagPro2Life project with 15 industrial and university partners from Denmark, Germany, Ireland, Romania, Spain, Switzerland and the U.K.
Solae was work package leader for WP0 (Organization and Coordination), WP1 (Specifications and Conceptual Design), WP7 (Food Pilot Line, RTD and Demonstration), WP8 (Economic, Market, Safety, Health and Life Cycle Analysis), and WP9 (Dissemination and Training).
The main objective for the MagPro2Life project was to use advanced magnetic (nano) particles to extract valuable components during food, pharma and feed processing.
Solae and Merck successfully developed a process that captures proteins in soy whey streams with silica.
Solae, Merck and DTU successfully developed processes that capture protein in soy whey streams using the magnetic fishing technology. This was done with and without silica pretreatment of the soy whey. A group of proteins were separated basis ion exchange functionalized magnetic (nano) particles, resulting in a protein concentrate containing a broad band of proteins, and enriched in the protein if interest. A single protein was separated basis antibody affinity ligand magnetic (nano) particles, resulting in a very pure high value protein product. This enabled Solae to evaluate the feasibility of protein extraction and production by magnetic fishing.
Solae successfully scaled the magnetic fishing process capturing proteins in a soy whey stream. The scale-up, full process set-up and protein production was done in a food pilot line magnetic fishing process setup at Solae in Aarhus, Denmark. The food pilot plant included scale-up of a pilot scale magnetic centrifugation process using MEC200 produced by AKMPT.
Solae and KIT-MVM executed a full performance testing of the magnetic centrifuge (MEC200). The tests were done in the food pilot line at Solae in Aarhus, Denmark.
Solae developed process models (SuperPro) for the protein production by magnetic fishing to understand major cost drivers and key points of process improvement (yield, magnetic particles, equipment).
The process models enabled Solae to do an economic analysis of the magnetic fishing technology at production scale in one food application.
The process models also enabled Solae to benchmark the magnetic fishing technology against competing technologies like packed bed chromatography (PB), expanded bed chromatography (EBA) and ultrafiltration (UF). DTU provided the model input for the competing technologies basis protein production done at DTU in Lyngby, Denmark.
Solae collaborated with DuPont to establish and compare a Life Cycle Analysis (LCA) on the magnetic fishing technology against expanded bed chromatography (EBA) as the most relevant competing technology.
A full risk assessment was conducted for the magnetic fishing process basis the DuPont NanoRisk Framework program. The potential risks (safety, health and environmental) associated with the use of magnetic nano particles in the food pilot line at Solae in Aarhus, Denmark was evaluated. The risk assessment included some precautionary actions in the work environment, some toxicological studies of the magnetic nano particles used and an evaluation of the environmental fate of the magnetic nano particles when used in the food pilot line.
DTU
The main focus of the project for DTU was to benchmark the performance of magnetic adsorbents, for BBI capture from Soy whey, against the closest competing technologies, i.e. expanded bed adsorption (EBA), packed bed chromatography (PBC), and ultra filtration (U.F). The data from this was used in the lifecycle analysis of full scale BBI production performed by Solae Denmark. Part of the project was also to take part in identifying relevant targets for demonstration of the magnetic adsorbent based technology. One of these was lunasin from soy whey. Furthermore as a spin off from evaluation of purification targets, a potential new application not foreseen in the original application has been identified, i.e. for recyclable immobilized enzymes.
Experiments in the laboratory with each of the competing technologies, i.e. packed bed chromatography, expanded bed adsorption and ultrafiltration, plus two magnetic adsorbent (Merck TMAP, and Orica) based systems, were used to provide data for the modelling in SuperPro Designer. Each technology approach was optimised for BBI purification from soy whey obtained from the Solae factory in Iper Belgium and both crude and pre-treated soy whey were used. This is the first time that such an extensive evaluation of the performance of technologies competing with magnetic separations has been undertaken. The data has been made available to the consortium and selected data has been included in a chapter of the book being written by the consortium which will be made available to the public. A spinoff from these studies has been a much better knowledge of the behavior of magnetic adsorbents and their interactions with feedstock’s. With respect to concrete results and foregrounds from this work, the following have been developed: SOPs for bench-marking performance of magnetic adsorbents in soy feedstock, BBI assay in micro titre plates, lab data on performance of TMAP and Orica magnetic adsorbents in model and real (soy whey) feedstock’s for use in economic and LCA modelling, lab data on performance of EBA, packed bed chromatography and UF for BBI purification from soy whey for use in economic and LCA modelling.
Experiments to develop an assay for quantification of lunasin were conducted, as a prerequisite for developing a purification procedure for the target from soy whey. A preliminary lunasin assay (HPLC based) was developed. Further work was down prioritised in favour of BBI purification.
Studies of an alternative target for demonstration purification, beta-glucosidase (from Novozymes) on the Orica and TMAP adsorbents showed that it was extremely difficult to elute it. Rather than dismiss this system, its potential as a magnetic immobilized enzyme for bio-fuels and bio refineries was examined in the lab and subsequently in a demonstration with the MEC in collaboration with Solae Denmark. This involved immobilisation of the enzyme by binding to 400 g Orica particles, followed by 4 cycles of use in ca. 20 L of cellulose containing feedstock, each time for 20 h at 50°C. This unforeseen spin out opens up a vast new range of applications for the MEC and magnetic adsorbents used and developed in the consortium. Two journal articles are in preparation with document the lab and demonstration studies.
KIT-IFG
Within the MagPro²Life the partner KIT-IFG followed two main tasks. First, the further development of a capture step isolating proteins from crude feedstocks by means of a combination of Aqueous Micellar Two-Phase Systems (AMTPS) and magnetic micro- or nanoparticles, called Continuous Magnetic Extraction (CME). The basic idea for this process was developed in a preceding STREP project called NanoBioMag within FP6; however, it was shown within MagPro²Life that the process can be run continuously using nontoxic, cheap surfactants. The second task was dealing with a new isocratic chromatography process based on thermoresponsive media and a newly designed column featuring a traveling temperature zone (Traveling Cooling Zones Reactor, TCZR). The thermoresponsive media were delivered by the partner UBI, who started the development of magnetic and non-magnetic beads with thermoresponsive coating within the FP6 project NanoBioMag.
In the course of the work on Continuous Magnetic Extraction the following main milestones and deliverables were achieved: i) characterization of suitable combinations of phase forming surfactant and magnetic sorbents; ii) investigation of the physico-chemical background of the partitioning behavior of the particles in the AMPTS, iii) setup and characterization of the CME process; iv) application of CME to purify proteins from various feed streams; v) removal of the remaining surfactant from the product stream.
Prototypes of the magnetic extraction set-up allowed the continuous separation of magnetic particles delivered by the partners Merck and NIIMT of sizes from 2 µm down to 25 nm with separation efficiencies > 99 % at flow rates up to 9 liters per hour. In close collaboration with the partners responsible for the pharma and food pilot lines the CME process was tested for continuous separation of an antibody fragment in 15 liter scale. The process resulted in a total yield of 67 % of the target protein and a purification factor of 6.3. The continuous purification of the Bowman-Birk protease inhibitor (BBI) from soy whey feed streams was carried out with different anion exchange particles. The trials returned around 50 % of the total BBI at a purity of up to 99 %. For dissemination the results of the CME work were published in five high ranking peer reviewed journal articles (e.g. J Chrom. A, Langmuir, Sep. Sc. Tech.) and presented at several national and international conferences. For exploitation KIT has a granted patent on the CME process and is looking for industrial partners further developing the CME applications in frame of a license agreement.
In case of the TCZR the following milestones and deliverables were achieved in order to develop the system towards a truly continuous chromatographic process: (i) characterization of adsorption properties of thermo-responsive sorbents delivered by the partner UBI; (ii) construction and hydrodynamic testing of the Traveling Cooling Zone Reactor (TCZR); (iii) cyclic experiments within the TCZR; (iv) proof-of-concept of continuous TCZR operation; (v) investigation of properties and the parameter space of the TCZR by means of a simulation program. The core of the TCZR is a linear movable temperature zone, surrounding a fixed-bed column filled with thermo-responsive sorbents. At a velocity of 0.1 mm s-1 the temperature zone is able to generate a temperature drop of more than 20 K within a narrow 2 cm section. Moving the zone along the column up to the end, sharp protein peaks can be eluted while the feed enters the column continuously. In close collaboration with the partner UBI, thermo-responsive cation and anion exchange sorbents were tested within the TCZR. For this, a PhD student and a second TCZR setup were transferred from KIT to UBI. Like in the case of the CME system dissemination of the TCZR work was done through several peer reviewed journal publications and international presentations. Exploitation is based on a filed PCT patent and it is planned to test the TCZR principle for specific applications together with two large European pharma companies.
In total the resources being provided within the project MagPro²Life were used for development of two radically new bio-separation processes, both being able to continuously purify and concentrate target proteins form different feed streams. For each of the processes two fully operable set-ups were realized and tested for different applications in close cooperation with the project partners. Finally, the work included the training of three Ph.D. and numerous master and bachelor students.
KIT-MVM
Currently HGMS devices are either based on electromagnets and are hence large and expensive, or do not provide the possibility of washing and elution steps in the cell. A Halbach permanent magnet arrangement was set up at KIT-MVM in combination with a HGMS filter cell. This allows reduction of investment and operating costs in comparison with common HGMS devices based on electromagnets. Common permanent magnets with pole yokes have a rectangular geometry and therefore cannot be stirred and hence used for washing and elution steps. An important advantage of the Halbach-HGMS is the round geometry of the magnetic field, allowing the implementation of a stirrer and the use of the device for washing and elution steps.
Magnetically Enhanced Centrifugation (MEC) was set up in three different devices. Additionally a lab machine set up in the previous project Nanobiomag was initially used for investigation at KIT-MVM. The design of magnetic wires was optimized on this machine. A first important result consists in the optimization of magnetic wires for MEC. A design comprising laser-cut wires of rectangular, longish shape arranged in flow direction was worked out to optimize separation. Wire distances were optimized to avoid bridge building of particles, which is in a centrifuge a function of rotational velocity. The design was implemented in all three pilot machines.
Additionally a model was derived predicting separation efficiency of magnetically enhanced centrifugation. The model is based on particle properties, i.e. size distribution, density and magnetization, as well as on device and process parameters such as volume flow, magnetic field, magnetic wire data etc. The model was developed supported by simulation of the flow by computational fluid dynamics, of the magnetic field by Finite Element Modeling, and of the magnetic particles by Discrete Element Modeling. It was validated by comparison with experiments and serves for the layout of processes and for the optimization of magnetic filters.
A permanent magnet design was developed creating a magnetic field in axial direction. The device was tested with MEC. Separation is similar compared to an electromagnet used alternatively on a MEC. This strongly reduces investment and operating costs of MEC, as electromagnets consume tremendous amounts of energy. In particular in combination with a continuous machine design, e.g. the magnetic decanter, the option of a permanent magnet is interesting.
An automatically discharging machine concept was targeted for use in pilot lines. A study on a variety of discharge principles was done in cooperation of KIT-MVM and Andritz KMPT, with machine concepts designed in cooperation. First a batch-wise academic peeler-centrifuge was set up at KIT-MVM and used as workhorse for academic investigation. It is based on a structured wire matrix produced by laser-cutting out of ferromagnetic stainless steel. It is used in a batch-wise manner, can be first filled batch-wise at up to 200 g of magnetic particles. The machine can be drained, with magnetic particles being kept in the device as it is filled from top and discharged at the bottom. Particles are detached from the wall and dispersed by stirring the peeler knives in a new liquid. Separation, washing and elution steps were performed subsequently in the machine. The machine was used in the pharma line for protein separation, showing the possibility of selective separation and high yield.
A continuous machine was developed and set up at KIT-MVM. It is based on a decanter principle, but changed in design to be sealed. Its main purpose is demonstration of a continuous design to be set up of completely continuous production lines. The machine was tested separating particles in a cycle for several hours at high concentration of 20 g/l and volume flow of 50 up to 120 l/h. This is to our knowledge the first full-continuous device in HGMS and potentially the most interesting device for HGMS in pilot lines at large scale.
A batch-wise large-scale industry centrifuge was set up by Andritz KMPT. It is based on a similar principle as the academic batch-wise MEC, yet in this case the wire matrix serves as stirring device. The machine was optimized for high volume flow of up to 1 m³/h at a 0.4 T magnetic flux density. All process steps, i.e. separation, washing and elution, can be performed in this machine. It was set up, tested and used successfully in the food line for protein separation. The wire matrix was developed in a 3D design to achieve constant distance between magnetic wires. Additionally a matrix featuring bi-metal design was developed by Andritz KMPT to avoid particle agglomeration in a specific area. In this design two different steal kinds are welded together in a plate, a magnetic and non-magnetic kind. The wire matrix is laser-cut out of these joint plates.
UBI
The bio-separation technology used in industry today is based on principles first discovered around 70 years ago and improvements are needed at all stages of processing, i.e. from the pre-treatment of raw materials prior to fermentation, the fermentative product itself, and during subsequent purification and modification to yield the final product. Functional magnetic adsorbents have the potential to enhance the physical and chemical properties of bio-separation process, i.e. (nano) particles have extremely high surface areas, rapid binding kinetics, and unique physical and chemical properties. One of the goals within the MagPro²Life project was to demonstrate a process for the economic manufacture of a relevant biopharmaceutical recombinant antibody fragment from E. coli and to entrench a new purification process based on magnetic adsorbents to purify the antibody fragment from the fermentation broth. Antibodies are widely used in the treatment of many cancers by specifically binding to target the tumor cell, thus making them more visible and susceptible to the immune system. E. coli is a well know host and is commonly used for the production of recombinant proteins. For the creation of a suitable feedstock for downstream processing (DSP), the fermentation and release conditions were optimized in different terms. After reaching an adequate titre of the recombinant antibody fragment, the first step in developing a new process in bio-separation technology was the successful demonstration of a magnetic benchmarking process before start working in a pilot plant using magnetic separation technology. The different magnetic separation devices used for the realisation of the project were specially designed for protein purification using magnetic adsorbents. Capturing of the recombinant target antibody fragment was realised by magnetic IEX (ion exchange) adsorbents, produced by different partners of the MagPro²Life project.
As a reference process to the magnetic purification process of recombinant antibody fragment, the purification using normal commercial IEX and affinity packed bed columns was chosen. Chromatography on packed bed is still the most common method for protein purification. An advantage for the usage of affinity adsorbents is the saving of process steps, like adjusting the ionic strengths of the feedstock, as crude fermentation broths have a high ionic strength. A disadvantage of affinity adsorbents are there comparatively high purchase costs.
Among the realisation of the experiments calculations regarding the economic efficiency of the magnetic process using appropriate software (SuperPro Designer®, Intelligent, Inc., USA) were executed, to proof the contestability of the magnetic process.
KIT-IBLT
Focus of the work of KIT IBLT within the MagPro2Life consortium was to setup a pilot scale cultivation process for phytase production with integrated in-situ magnetic separation (ISMS). Based on this process the potential beneficial application should be demonstrated. In the course of this development several exploitable results and foreground were produced which should be explained in the following.
Main exploitable result of the work of KIT IBLT is a set of design specifications for the industrial application of magnetic separation for in-situ separation of extra-cellular products like for example functional proteins. Basis for this design specification is the experimental representation and detailed evaluation of the ISMS concept based on the developed phytase example processes. In this context ISMS cultivations in different scales up to 100 L total fermenter volume were conducted to establish an economic balancing model. Furthermore a comprehensive catalog of requirements was specified for the ISMS subsystems magnetic particle system, magnetic separator and bioprocess. Especially for metal-chelate and ion-exchange functionalized magnetic particles, extensive characterization data in form of adsorption isotherms and kinetics as function of different media properties was generated. In doing so focus was put on the special requirements of direct broth extraction of functional proteins without negatively affecting the bio-process. As a consequence of these characterization experiments a particle coating procedure was developed for ion-exchange functionalized magnetic particles to avoid unwanted biomass adsorption. In the course of this development process also diverse characterization results for agarose, agar-agar and alginate coated magnetic particles were obtained. With respect to the magnetic separation device design specifications for a pilot scale high gradient magnetic filter (HGMF) device were obtained by testing of different small scale HGMF devices regarding particle capacity and particle separation. Based on this data for the pilot scale HGMF in addition also a modified particle loading strategy was developed and successfully tested enabling a more selective solid-solid-liquid separation. For the implementation of this separator a sterilizable separation circuit was developed connecting the bio-reactor, the magnetic separator and the required feed vessel. Regarding the bio-process, layout data for the design of a reproducible process control strategy was developed and applied on the extra-cellular phytase production of E.coli and B.amyloliquefaciens strains. Using the hereby produced phytase species and those from other (fungal) origins characterization data was generated with respect to enzymatic properties like stability, substrate specificity and pH / temperature dependency of the catalytic turn-over.
MEK
Core of the project was the production of kg-quantities of surface-modified magnetic particles to purify selected target proteins in combination with an efficient magnetic separator.
The continuous synthesis of magnetite with the potential for unlimited production on a large scale was the basis for all MagPrep particle species. The principle of this method has already been developed/patented before the start of MagPro2Life and was refined/up-scaled within the project to kg-quantities per day. In addition, the tightly controlled reaction conditions allow the production of narrow-sized particles within the range of about 10–100 nm. Depending on the intended application smaller beads with a high surface area/binding capacity or larger resins with a better magnetization can be selected. The number size distribution of this material can be determined by automated image analysis of electron micrographs of specially prepared particle mono-layers (patent filed).
The surface modification included a standard coating with silica as a basis for the subsequent ATRP (Atom Transfer Radical Polymerization) modification. This technology allows to fine-tune the density and length of surface-initiated polymer tentacles with almost any kind of functionalities to influence the binding capacity and magnetization of the resulting particles. The batch production of kg-quantities of anion and cation exchange resins (MagPrep TMAP and MagPrep SO3) was established and the particles used for the capture/purification of target proteins on an analytical and pilot scale. In addition carboxylated MagPrep particles have been coated with anti-BBI peptides and a monoclonal anti-BBI antibody for the affinity capture of BBI directly from untreated raw material.
MagPrep TMAP was used for the purification of BBI (Bowman-Birk Inhibitor) from soy whey in combination with conventional heat- and silica-treatment as well as the enrichment of antibodies from cell culture supernatant and human plasma by removing unwanted protein components. MagPrep SO3 could be applied for the specific capture/elution of recombinant Fabs from bacterial cell lysate and antibodies from cell culture supernatant. However, the ultimate purification of BBI could be achieved with MagPrep anti-BBI based on a covalently immobilized monoclonal BBI-specific antibody, allowing a one-step protocol for the isolation of the target protein from untreated soy whey. Moreover, the specific activity was about twice as high as with the three-step purification or commercially available samples.
From the economic point of view the particle costs, binding capacity and recyclability (including chemical and mechanical stability) is of decisive importance. Analytically the beads have been reused up to 50 times without loss of performance, on a pilot scale a 10 times recycling with 99,9% step-wise recovery could be demonstrated. Due to the current multi-step surface modification the production costs need to be reduced further to be competitive to conventional chromatography.
Based on a toxicity study the strong anion exchange resin MagPrep TMAP turned out to be harmless with respect to skin and eye irritation as well as acute oral toxicity.
FZMB
The used magnetic reader system, developed by FZMB works on basis of susceptometry and allows off-line measurements in cylindrical cuvettes (100 µl sample volume). In order to realize a flow through mode and to enhance the measurement signals the geometry and placement of the coils were changed and the complete sample measurement head redesigned. The magnetic reader system output was measured using serially diluted suspensions of different particles. A comparison of the detection limits for the 100 µl and 2 ml measurement head illustrates that the increase of the sample volume did not effect a signal enhancement. Furthermore the Merck particles MagPrep Silica have very high detection limits and so they are not applicable for the used susceptometric system. The reason for that is probably their remanence (residual magnetism). Because signal enhancement was not successful in the 2 ml head, the cuvette system of the small measurement head has been rebuilt and flow through mode has been realized. For the measured particles (TUBAF, 7,5 % magnetite) a detection limit of 181.1 µg iron per liter (18 ng iron absolute) could be reached.
Within the work for antibody generation (WP 2) an antibody was produced, which is specifically directed against human Fab from UBI. Because of the absence of a second antibody it was attempted to find a commercial antibody to build up a sandwich assay. Therefore several anti-Fab antibodies have been tested and an appropriate detection antibody was found (Bethyl). Based on these antibodies an ELISA could be established (working range: 5 – 1000 ng/ml). The functionality of this ELISA for the Fab' a33 was shown by the work at UBI with real Fab samples.
Sandwich-ELISA for Lunasin was generated using the polyclonal antibody as capture antibody and the biotinylated form as detector. Standard peptide was the synthetic full-length Lunasin. The working range of the ELISA is 0,1 to 20 ng/ml. Within WP 2 Lunasin antibodies were generated. Several combinations were studied using the polyclonal antibody as capture antibody and monoclonal antibodies as detector antibodies. Only one combination showed consistent values and a low zero value. After optimization a ELISA based on this combination was established, which works well with standard peptide and purified lunasin. The working range of the ELISA is 5 to 500 ng/ml.
Based on the successful production of monoclonal antibodies the development of a sandwich ELISA for BBI was possible. In the characterization one antibody has been shown a particularly good binding: mab F2. Therefore, this antibody has been selected as capture and the other clones were used as detection antibodies. Two combinations were selected and tested in sandwich ELISA. The best combination (F2/F3) was further investigated to optimize the concentration of the reagents. BBI from Sigma was used as standard material. The ELISA has a detection range of 2,5 to 100 ng/ml. Validation was done successfully. The limit of detection is 3,4 ng/ml and the limit of quantification is 5,8 ng/ml. Additional the robustness of the ELISA was examined by variation of pH and temperature. Tests with soy samples were successful and showed a good correlation to the enzymatic chymotrypsin assay. In opposite to the enzymatic assay the ELISA is able to measure much more samples within 5-6 hours. For the development of a rapid BBI assay the principle of the Sandwich-ELISA was transferred to the ABICAP system. Different detection systems were tested. Best results showed the set up with biotinylated detection antibody in interaction with streptavidin-poly-HRP as enzymatic detection system. Calibration was done with BBI standard from Sigma. Tests with soy samples (eluates from magnetic separation) showed a good correlation to ELISA values.
UCD
Deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles” and deliverable 47, originally titled “Produce phage that bind to Bowman-Birk inhibitor and Lunasin proteins” but later changed to “Produce lab scale quantities of peptide-immobilized magnetic beads with affinity for BBI” represent the work undertaken by UCD.
Towards the originally titled “deliverable 47”, UCD successfully identified phage-based, antibody-based and peptide-based affinity ligands for BBI purification applications in the consortium. Synthetic peptide-based probes targeting the Bowman-Birk inhibitor (BBI) have been recognized by screening a bacteriophage dodecapeptide library against the purified antigen (Bowman-Birk inhibitor). Monovalent peptides were synthesized as branched, multimeric peptides. These peptides - peptide 1 (GAMHLPWHMGTL)4K2KGSGCG) display avidity effects as a result of their tetrameric nature, and therefore improve binding characteristics. Synthesis of peptide was performed by solid phase peptide synthesis at greater than 95% purity. Subsequent characterization of the peptides and grafting onto magnetic beads, using heterobifunctional SMCC cross linkers, allowed the one-step purification of BBI from crude soy whey mixtures. The nature of the eluted protein was confirmed to be BBI by SDS-PAGE gel electrophoresis, mass spectrometry, and ability to inhibit chymotrypsin and trypsin. Using 10mg of functionalized magnetic particles, we estimated a purification of ~100 μg Bowman Birk inhibitor from 50mL of crude soy whey extracts [1]. Additionally, antibody engineering of a pre-existing monoclonal antibody (mAb238, Brandon et al 1989) has been performed. This has resulted in the production of antibody fragments (Scfv and diabody), in bacteria, with the capacity to recognize and bind to the Bowman-Birk inhibitor. These BBI probes have been subsequently used in magnetic-based assays via grafting through direct amine coupling protocols, ultimately allowing the rapid purification of BBI from crude soy whey mixtures [2].Efforts towards the newly appointed deliverable “Produce lab scale quantities of peptide immobilized magnetic beads with affinity for BBI” were unsuccessful as a result of peptide degradation over the time course between the two deliverables. Peptide degradation was confirmed by ELISA and surface plasmon resonance (Biacore) measurements.
Towards deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles”, the pIII phage coat proteins were genetically engineered to modify the N-terminus with either a –CHKKPSKSC (M13Si) or –HHHHHH (M13His) peptide sequence and phage were successfully assembled on SPMs coated with either silica or nitrilotriacetic acid, respectively. This assembly permitted end-on attachment of the phage to magnetic particles. In an alternative side-on orientation, the pVIII phage proteins were modified NHS chemistry to generate M13 biotin phage. These phage were then successfully coupled to streptavidin-coated magnetic particles. In this case, the whole length of the phage is immobilized onto the bead in a side-on orientation [3]
[1] Fields, Conor, Paul Mallee, Julien Muzard, and Gil U. Lee. "Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation." Food chemistry 134, no. 4 (2012): 1831-1838.
[2] Muzard, Julien, Conor Fields, James John O’Mahony, and Gil U. Lee. "Probing the soybean Bowman–Birk inhibitor using recombinant antibody fragments."Journal of agricultural and food chemistry 60, no. 24 (2012): 6164-6172.
[3] Muzard, Julien, Mark Platt, and Gil U. Lee. "M13 Bacteriophage‐Activated Superparamagnetic Beads for Affinity Separation." Small 8, no. 15 (2012): 2403-2411.
ETH
The S&T results achieved by ETH Zürich, in collaboration with the MP2L project partners are divided into two main sections (A) New Magnetic Containers/Carriers and (B) a new processing technology for producing the same.
In (A) so-called MANACOs (Magnetic Nano Containers) were designed and further developed. MANACOs represent magneto responsive carriers for adsorbed or encapsulated functional components. For the latter preference was given to hydrophobic proteins to be harvested from soy whey waste streams (e.g Bowman-Birk Inhibitor, BBI and Lunasin). The backbone structure of the MANACOs consists of lipids, preferably (i) phospholipids for nano-/meso-scale containers/carriers forming uni- or multi-lamellar vesicles or bicelles and (ii) triglycerides or waxes for macroscopic ones, forming semi-solid or solid particles. The magneto-responsiveness of these containers/carriers has been successfully applied by doting with paramagnetic atoms (lanthanides: thullium Tu or Dysprosium Dy) in case of the nano-/meso-scale MANACOS and with super paramagnetic fatty acid coated magnetite nanoparticles for the macroscopic MANACOs.
For the separation processing within the MP2L project further preference was given to these macroscopic MANACOs in the diameter range of 1-50 microns. Related surface modifications of the triglyceride based macroscopic MANACOs with polymers were adapted to address anionic or cationic exchange (AEX, CEX). Both were produced following the same two-step approach based on a new rotating membrane process (ROME) developed for the production of “naked” MANACOs (see B). Briefly, a functional polymeric material is pre-synthesized (1) and subsequently used as a surface-active ingredient for the preparation of coated MANACO particles via a mechanically gentle emulsification in the ROME process (2). Functional block copolymers were specifically designed with a molecular structure such that they coat the wax droplets during emulsification and form, after drop solidification, an external functional layer, which is subsequently chemically bound onto the matrix of the resulting MANACOs via in-situ radical polymerization. A scalable procedure allowing for the chemical synthesis of both cationic and anionic block copolymers with varying molecular composition and structure was developed, based on Atom Transfer Radical Polymerization (ATRP).
The newly developed processing technology (B) for the generation of different types of MANACOs is based on a dynamic membrane emulsification principle, coupled with a surface modification-/functionalization-step. The device in which this coupled processes are run is denoted as ROtating MEmbrane Reactor (ROMER). A triglyceride or wax melt with suspended paramagnetic nanoparticles was pressed through the rotating membrane forming drops being detached from the membrane surface by the acting wall shear stress generated by the continuous watery emulsion fluid phase flow across the membrane. The continuous fluid phase contained the functional block copolymers attaching to the drop surface. The wall shear stress controls the size of the MANACOs being adjusted by the rotational membrane velocity and the shear gap width between membrane and housing wall and is practically decoupled from the total throughput rate of the continuous process. Material characteristics of influence to the MANACO are coupled with the acting wall shear stress in the critical dimensionless Capillary Number Ca*, which was used for process scaling.
An up-scaled ROMER II emulsification chamber was constructed using a cone-plate geometry (cone: rotor, plate: static membrane) in order to allow for the utilization of flat micro engineered membranes. In a first stage, the particle production process was developed using silicon-based membranes with a pore size of 10 µm, which were routinely produced in-house, in ETH clean room facilities. In parallel, we developed an alternative process for the fabrication of silicon nitride-based membranes with a pore size in the range from 0.4 to 5µm, and suitable for utilization in the ROME II device.
MANACOs were successfully coated with a CEX functional layer utilizing the same production process than for the AEX functionalized MANACOs. The anchoring of the CEX functional layer as well as the achieved ligand binding selectivity and binding strength have to be further improved.
BUH
The Romer I Device was constructed, manufactured and implemented for research at the ETH producing Manacos .
The new concept, design and construction of the pilot Romer II device were fulfilled till the end of 2010. The pilot plant was manufactured and put into operation till June 2011. Afterwards it was transferred to the ETH for the production of Manacos.
Based on the experiences made with the Romer I apparatus a new concept of the process room was elaborated in order to produce Manacos.
Essentially the process room is flushed by a flow against the centrifugal forces in order to guarantee the build-up of a shear flow. The process room consists of two discs, the one rotating with a rotation speed which allows to build up a shear rate of 106 s-1 . In order to avoid any instabilities a second process room consisting o a Couette flow between two cones was constructed.
The device is instrumented with the necessary sensors (pressure, throughput, temperature, gap-measurement) and can be operated hy pressure feedback control or through put control.The operation panel is separated from the control panel for a better handling.
The mechanical simulation of the process for the generation of vesicles by pressing a membrane of amphiphilic molecules through a hole was realized with FEM. In order to simulate the mechanical properties of the membrane the constitutive equation of Skalak has to be implemented into the FEM program (LS-Dyna). We are now able to simulate the process of the bud building with different amphiphilic molecules.
Additionally to the tasks mentioned it was decided in the consortium meeting in Zürich at January 2010 to try to produce nano scaled magnetite particles by a top-down strategy, i.e. by comminution of magnetite pigment (BASF) in a ball mill with addition of an adequate surfactant. After having comminuted the pigment introducing 104 kWh/to the mean particle size was 1.4 microns. Further trials were not carried out due to the high energy input needed.
TUBAF
The goal of TUBAF in MagPro2life was to develop, based on SolPro, a low-cost scale up able synthesis process as well as to optimize the efficiency of particles production having regard to safety and ecological aspects. Depending on target protein charges in the extraction pH range, CEX and AEX SolPro beads were successfully synthesized and characterized. Both types of beads were equipped with the appropriate functionalities allowing their application in the bio separation (super paramagnetic, ion exchange properties). To ensure the industrial application of SolPro, it was important by designing the product to choose or to develop synthesis processes getting scale up potential. Therefore the SolPro beads were synthesized using spray drying process, which is a well-established method to particle design in many technological fields like pharmaceutical, cosmetic, food or chemical industry.
The SolPro beads were fitted with super paramagnetic behavior by using nano-sized iron oxides material called magnetite. The nanoparticles were produced via co precipitation of iron salts. During the first project period, a continuously operating process was being successfully developed to produce water-based magnetic fluid made of dispersed iron oxides nanoparticles using sono-precipitation. By doing that, two types of reactors were used: a conical and a cavitation reactor. In each type of reactors, 120 g of super paramagnetic iron oxides nanoparticles were produced continuously as water dispersion with high flow rates up to 6000 ml/h. It was found that, the energy input by ultrasound had no significant positive influence on the nanoparticles quality (size, magnetization). Therefore, the use of a T-mixer like in the batch process, which is more competitive and easier to scale up than ultrasonication, would be sufficient to achieve this.
Another component making SolPro particles suitable for the bio separation is the ion exchange resin. For positively charged target protein in extraction milieu, sulfonated copolymer polystyrene divinylbenzene was used. To be better integrated in the beads and thereby ensure their mechanical stability, cation exchangers should be nano- or submicrosized. Mini-emulsion polymerization was found to be adequate to produce these. Their synthesis in a pilot scale implied the use of chemical sophisticated equipment. To overcome this limitation, a new production process for cation exchanger following the same specifications formulated above was being evolved. Sulfonated copolymers polystyrene divinylbenzene in micron size (x50,3≈. 650 μm) are available on the market in large scale as low-cost material. Established milling technologies were exploited to mill these low cost cation exchangers down to sub micron sized. Thereby, their adsorption capacity stays quite unchanged. The technological innovation that should worth pointing out here is the use of milling technologies for the production of low cost submicron sized cation exchange resins. To extract negatively charged protein, weak ployelectrolytes was chosen as anion exchangers. A basic technology for their incorporation in SolPro beads was being developed during the project too. The technique used to synthesize AEX SolPro was combined functional groups activation, particles synthesis followed by surface modification. All these process steps can be operated in the large scale.
Characterization analyses of the SolPro beads (CEX und AEX) showed the high potential of the developed processes. CEX SolPro particles possess a lysozyme extraction capacity of approx. 280 mg/g, which is about a factor three higher than similar CEX-SolPro beads (same composition) produced by Hickstein (77.6 mg/g). The novel designed and produced AEX SolPro beads using poly(allyl)amine (PAAm) as anion exchanger exhibit extraction capacities of 85 mg/g as well as 145 mg/g for BSA and BBI respectively. Several instigations proved that a quaternization of amino groups of poly(allyl)amine anchored on the beads surface increase their extraction capacity by a factor of three. One kilogram of produced PAAm Solpro was being successfully tested by Solae Denmark in food pilot line. application of PAAm Solpro.
In future works, the basic chemistry used for the synthesis and modification especially the quaternization of PAAm-SolPro beads should be improved. This would further increase their BBI extraction capacity in a real extraction milieu (non-treated or pre-treated soy whey) considerably. The Application of such adsorbents to water conditioning and rare earth elements processing can also be investigated.
RATB
A chemical co-precipitation method was applied for the preparation of primary magnetite nano-particles suitable for magnetic fluid and magnetic nano-composite preparation. The relative strengths and ranges of various interaction potentials were controlled by the diameter of magnetite cores and the thickness of the stabilizing layer in order to ensure the dimensionless magnetic interaction energy to be below 1 and the mean particle size less than 10 nm. Consequently, the resulting magnetite nanoparticle systems, such as nanofluids, microgels and nanocontainers will show superparamagnetic behaviour.
Depending on the clustering and functionalizing procedures applied by partners, magnetite nano-particles both with hydrophobic and hydrophilic coatings were synthesized. The surfacted magnetite nano-particles dispersed in light hydrocarbon or water, depending on the nature of the coating, serve as primary materials for the preparation of various functionalized magnetic nano-composites-magnetic beads- for magnetic separation purposes.The main requirements refering to biocompatibility, as well as efficient coating and functionalizing capacity were ensured by vegetable origin fatty acids used. The strictly applied synthesis conditions, such as the precipitation temperature, pH value and the excess amount of NH4OH ensure the formation of Fe3O4 over Fe2O3, as well as the very good reproducibility of the procedures on lab-scale.
TEM, VSM, magnetogranulometry, rheometry and dynamic light scattering investigations offered data on the magnetic response, as well as on the magnetic, solid and hydrodynamic size distributions in case of hydrophobic and hydrophilic nanoparticle systems up to close packing and evidence the superparamagnetic behavior, the efficiency of surface coating of magnetic nanoparticles and the degree of clustering. The evaluation of the mean thickness of fatty acid coating and effective and mean ellipticities of particles conducted to the mean cluster size which is rather small and practically does not affect the very good dispersability of hydrophobic and hydrophilic magnetite nanoparticles in light organic non-polar solvents and water, respectively. These primary materials were used by partners to prepare magnetic nanocomposite particles for magnetic separation purposes which usually involve HGMF. Among the required properties are the high magnetic moment density (several tens of emu/g) and the superparamagnetic behavior (no remanence). The adequate size range and efficient surface coating of primary magnetite NPs are essential in order to ensure the required magnetic response of closely packed magnetite NPs in nano-composites, such as MANACO’s and magnetic micro-gels designed for magnetic separation. The investigations concerning the character of magnetic response at close packing (at minimum distance between surface coated magnetic nano-particles) were extended to polymer coated magnetite NP clusters obtained by NIIMT applying a controlled clusterization procedure. The static magnetization data analysis using the Ivanov & Kuznetsova magnetization model for close packed MNPs evidence the efficient screening of magnetic dipole-dipole interactions. The full magnetization curve does not show any hysteresis as the magnetite nano-particle clusters encapsulated into polymer shell have no magnetic moment in the absence of an external magnetic field. The superparamagnetic behavior of surfacted magnetite nano-particle clusters, relevant for their use in magnetic separation, is demonstrated by the above analysis.
Optimized procedures were developed and experimented for kg-scale synthesis of hydrophobic and hydrophilic magnetite nano-particles.
NIIMT
The research activities of NIIMT focused mainly on the development and optimization of the synthesis methods of hybrid magnetic nano-structures with controlled morphology, such as core-shell particles or micro-gels with the required properties for magnetic separation: superparamagnetic behavior, high magnetization, chemical and mechanical stability, high concentration of surface functional groups, good re-dispersability.
Synthesis procedures have been developed for polycaprolactone or polylactic acid coating of surface modified magnetite nanoparticles using either classical polymerization reaction or microwave irradiation. Magnetite nanoparticles prepared using a common co-precipitation process, were surface-functionalized in situ by the addition of glycolic acid or acrylic acid. For the attachment of the polymer chain on the preformed functionalized magnetite the “grafting-from” strategy was applied, where the polymerization is initiated directly from the particles surface. HRTEM evidences the formation of core-shell nano-structures with the magnetite core coated with a thin shell of either polymer or co-polymer. The polymer/co-polymer coated magnetite nano-particles show superparamagnetic behavior of magnetization at room temperature and high saturation magnetization values.
An important part of NIIMT research activity was devoted to the synthesis and characterization of magnetic microgels with controlled size, anion exchange and cation exchange properties, chemical and mechanical stability, and good re-dispersability. Lab-scale preparation methods for magnetic microgels have been developed using magnetic nanofluids containing surfacted magnetite nanoparticles dispersed either in water or in light hydrocarbon provided by the partner Romanian Academy-Timisoara Branch (RATB). Physical properties of magnetic microgels have been investigated by TEM, HRTEM, DLS, Zeta potential, FTIR, XPS and magnetic measurements, to determine de relevant synthesis parameters for adjusting the particles size and dispersion properties, for the increase of magnetization and concentration of functional groups (carboxyl or amino) on the surface.
Clusters of magnetite nanoparticles with hydrophilic coating from water based magnetic nano-fluid are embedded into polymers such as poly(N-isopropylacrylamide), polyacrylic acid, poly(3-acryloamido-propyl trimethylammonium chloride) applying free radical polymerization method. TEM and DLS investigations show that the morphology and size distribution of magnetic microgels depends strongly on the polymerization conditions and physico-chemical properties of the water based magnetic nanofluid.
The preparation of magnetic micro-gels based on magnetite nano-particles with hydrophobic coating from toluene based magnetic nanofluid (provided by RATB) was performed using mini-emulsion method. This method allows the reproducible preparation of size controlled magnetic nano-particles clusters which later on are encapsulated into polymers.
The magnetic microgels prepared using either water or organic carrier magnetic nanofluids show superparamagnetic behaviour at room temperature and high magnetization values, proving that the developed synthesis procedures favors close packing of magnetite nanoparticles and allows the encapsulation of a relatively high fraction of magnetite nano-particles into the polymers. These magnetic beads have potential applications in biotechnology and nanomedicine.
NILPRP
Laboratory and semi-pilot scale continuous synthesis procedures of either maghemite-based nanoparticles or metallic iron - iron carbide core / carbon shell nanoparticles with high saturation magnetization and near superparamagnetic behaviour were developed using optimized laser pyrolysis technique. The iron-oxide based nano-particles were dispersed in aqueous media at near neutral pH by functionalization with L-DOPA bio-molecule and can have potential application in drug delivery or MRI contrast agents.
USAL
The main scientific and technological advance was the proof that magnetic classification works in wet-mode and gives the desired and expected class differences. In fact, the results obtained show that a practical experimental difference of magnitudes as low as 1.3 - 2.0 between classes is achieved, which represents a major step in the magnetic processing area. This indicates that future lab and pilot-scale versions of the wet-mode MAGCLA will be able to classify different particles present in an initial mixture according to their magnetic susceptibility values, with as close as 2 in order of magnitude.
The proof was first made through an indirect experimental proof (using particles with similar characteristics but under different magnetic fields influence), then with a comparison of the trajectories obtained in contrast with the ones predicted by theory (numerical simulations were obtained that showed the working principles) and finally with a direct proof by using particles with different magnetic susceptibilities (the difference in their magnetic susceptibilities is much lower than 50 in magnitude) and achieving their classification (division of the feed into three outcomes with a difference in their magnetic susceptibilities magnitudes much lower than 50). The proofs were made with and without the presence of a classifying exit.
The development of the theoretical background and the simulations of the process were also a main achievement, as it allows to optimize the process. These preliminary theoretical results were validated against experimental results. The first preliminary version of this theory was also needed to make possible the full design of the device, which was only viable after dimensions were known.
A full microfluidics image view system was built to capture and analyze the trajectories followed by the particles on a small part of the new device (proof of concept).
The small-scale proof-of-concept system was tested for two different kinds of particles (well-shaped and defined Cospheric and bad-defined and bad-behaved Orica particles) and the results have shown magnetic classification in wet-mode to be possible even in harsh conditions.
We also obtained results concerning the influence of velocity and distance between the magnet array and the surface of the pipe, in what concerns the separating point; and also determined the existing differences in the separating magnetic susceptibilities ratio between the top and bottom positions in the inner wall of the pipe for the same radial position.
A full version of the large-scale wet-mode prototype was built, assembled and tested, except for the superconducting magnetic generator. The preliminary tests allowed us to conclude about size dependence, flow characteristics, etc. They also show that secondary flow was not that important. Very preliminary trials of the large lab-scale device by using two permanent magnet arrays in some well-chosen position, and using feeds of (magnetic) magnetite, hematite, wolframite and Cospheric particles were done, and a classification in between 60-80 % was achieved.
In addition USAL analyzed several magnetic separating devices that, using several cycles, could reach magnetic classification, in order to compare costs and efficiency. The idea was to determine which comparing devices may be built and applied (HGMS, HGMF, MSFB's, etc.) in order to validate the future results obtained by the MAGCLA and determine the real improvement made by the new device.
Potential Impact:
Final Report 4.1 (SESAM) – Dissemination and Potential Impact
SOL
The MagPro²Life Project successfully demonstrated the magnetic fishing technology to be sustainable, safe, and capable of scaling to high production rates, reliable, robust and economically competitive to current existing downstream processing techniques. As DuPont acquired Solae in full on May 1, 2012, and became part of the Nutrition and Health Business Unit, a wide range of opportunities becomes available in terms of applications for this technology. Areas are as wide as digestive, immune, bone, heart, oral and weight management products, taste, and texture and appearance components. Areas are as wide as digestive, immune, bone, heart, oral and weight management products as well as taste, texture and appearance components, protective ingredients, and reduced food waste.
The outcome of the 4-years study on the Magnetic Fishing will be presented on the F2F DuPont T&I Leadership meeting in Europe, November 2013. In the mean time we will approach the scientific community through targeted groups within DuPont by the means of F2F or webinar meetings. DuPont has the worldwide patent on the Magnetic separation technology.
DTU
The data, procedures and models used for comparing magnetic adsorbent based purification of BBI with competing technologies and for scale up production are an extremely important part of marketing the technologies developed in the consortium. It is expected to stimulate widespread commercial interest in the biotech and food industry on the potential of the technology and is expected to spark demand for the particles and equipment developed in the consortium.
The bio-based economy with bio-refineries and bio-fuels at the forefront are the hottest topics in society today. These technologies rely on enzymatic hydrolysis of the renewable resources used for substrates. Free enzymes are used in these processes which are lost during processing and contribute to making the technologies expensive or with extremely marginal profitability in the case of bio-fuels. Magnetic recyclable enzymes can be expected to revolutionize the production processes, and the MagPro2Life consortium partners involved in particle manufacture and separator design have a marked competitive advantage that they can exploit.
Exploitation of the results from DTU in the public arena has been/is in the process of being done in the following ways:
• Alftren, J., Søndergaard, W., Hobley, T.J. (2013) Immobilization of beta-glucosidase by adsorption on anion exchange magnetic particles enables enzyme re-use during hydrolysis of pretreated wheat straw. Manuscript submitted to J. Biotechol., 17th July 2013.
• Søndergaard, W., Alftren, J., Hobley, T.J. (2013) Pilot scale demonstration of magnetic immobilised beta-glucosidase for repeated lignocellulosic hydrolysis. Manuscript in preparation for submission August 2013.
• Søndergaard, W., Ottow, K and Hobley, T.J. (2013) Benchmarking magnetic separation against competing technologies. Manuscript in preparation for the book: Advancing magnetic separations for food, feed and farma, Edited by Nirschl, H.
• Cerff, M., Scholz, A., Käppler, T., Ottow, K.E. Hobley, T.J. Posten, C. (2013) Semi-continuous in situ magnetic separation for enhanced extracellular protease production – modeling and experimental validation. Biotechnol. Bioeng, 110: 2161-2172
• Maury, T.L.M Ottow, K.E. Brask, J., Villadsen, J., Hobley, T. (2012) Use of High-Gradient Magnetic Fishing for Reducing Proteolysis During Fermentation, Biotechnology Journal, 7: 909-918
• Ottow, K.E. Lund-Olesen, T., Maury, T.L. Hansen, M.F. Hobley, T.J. (2011) A magnetic adsorbent based process for semi-continuous PEGylation of proteins. Biotechnol. Journal. 6:396-409.
• Peuker, U., Thomas, O., Hobley, T., Franzreb, M., Berensmeier, S., Schäfer, M., Hickstein, B. (2010) Bioseparation, Magnetic Particle Adsorbents. In Wiley Encyclopedia of Industrial Biotechnology, Flickinger, Michael C (Ed), ISBN 978-0471799306.
Oral conference presentations:
• Using magnetic adsorbents for protein modifications. 8th European Symposium on Biochemical Engineering Science (ESBES), September 5th – 8th, 2010. Oral presentation.
KIT-IFG-MVM-IBLT
Biotechnological production processes compared to chemical ones have certain benefits like reduced energy consumption and increased selectivity. However due to the usually diluted product streams downstream processing is often related to multi step processes with huge buffer consumption and accordingly a high number of waste streams. The introduction of new integrated processes based on magnetic separation is helping to increase the sustainability significantly. Through partial substitution of multiple process steps (product capture, concentration and purification) by one magnetic separation step, resource consumption and waste generation are reduced. Furthermore also the reduction of product losses during the downstream procedure helps to increase the yield of a given process, thus further improving the economic viability and sustainability. The magnetic separators and processes developed by KIT IFG, MVM and IBLT proved to be efficient in this context. Through the setup and operation of the feed pilot line as well as through the integration of the different magnetic separation devices into other pilot lines, it could be demonstrated that specific procedural advantages are achievable by the respective magnetic separation processes. In detail increased productivity of the bio-process was achieved in the feed pilot line process. For the pharma pilot line efficient product purification was attained using ion-exchange functionalized magnetic particles in combination with the magnetic enhanced centrifuge (MEC) and the aqueous two phase separator. Furthermore also the implementation of the MEC into the food pilot line process revealed potential economic benefits.
The knowledge generated by all three KIT sub partners during the project was presented in several university lectures and student courses. In addition research results were disseminated on 11 national and 13 international meetings and conferences in terms of oral and poster presentations. Overall 17 scientific publications and conference proceedings are representing the basis for the continued dissemination of the gained results. This should enable the fast and easy transfer of the knowledge generated by KIT onto other processes hereby contributing to the common welfare of society.
UBI
Conventional bioprocess manufacture shows heavy reliance on adsorption-desorption processes many if not most of which are chromatographic. Despite the key manufacturing roles ‘bind and elute’ chromatography processes play their future sustainability is cause for concern. They are: (i) costly; (ii) suffer from low productivity; (iii) employ high volumes of buffer in turn generating (iv) excessive amounts of waste. To address these issues UoB, in close collaboration with MagPro2Life partners (esp. KIT-IFG, KIT-MVM, MEK) advanced four integrated processes promising leaner, greener, and more sustainable manufacturing of valuable bio-commodities – three of them magnetically driven and the fourth chromatography based.
The main focus of MagPro2Life was the development and demonstration of magnet technology based processes for protein purification for 3 different product pilot-lines, i.e.: animal feed, food and biopharmaceuticals. All of UoB’s activities addressed MagPro2Life’s biopharma product pilot-line, and its primary roles were to: (i) set up and manage the complete (USP and DSP) biopharma pilot lines for demonstration of continuous magnetic extraction (CME), magnetically enhanced centrifugation (MEC) and high-gradient magnetic fishing (HGMF); (ii) design, manufacture and characterize (physically, chemically, functionally) novel and/or improved adsorbent materials specifically tailored for use in new engineering equipment, and scaling up their manufacture if required; (iii) integrate use of the new materials in the engineering equipment invented and furnished by KIT; and finally (iv) benchmark the new processes against conventional packed bed chromatographic purification routes.
Platform technologies for making a great many different polymer brush modified magnetic and chromatographic adsorbents were developed at UoB, and some of the high capacity and/or intelligent adsorbents made were tested in KIT-IFG’s engineering equipment. The concepts of all three magnet based approaches (CME, MEC, HGMF) were rigorously tested in UoB’s Biopharma pilot-line and benchmarked for the recovery of an antibody fragment product from crude periplasmic extracts of E. coli using magnetic cation exchange particles. All three MagPro2Life pilot-lines were subjected to economic feasibility studies, and the most favorable results were obtained for the bio-pharma line. Moreover, efficient and economic product purification was attained for all three magnetic particle based separation approaches employed for the bio-pharma product.
UoB has published 3 original full research papers (with KIT-IFG) in top journals thus far, and a book chapter on magnetic particle based separations (with TUBAF, KIT-IFG and DTU). A further 6 papers will be submitted in due course along with 2 PhD theses later this year. Moreover, UoB shall seek to gain IP related to its portfolio of adsorbent manufacturing methodologies, and especially some of the newer smart polymer brush chemistry it has invented recently. Additional UoB outputs include: (i) a total of 11 presentations at prestigious international conferences in Europe and the US; and (ii) the direct use of some UoB’s knowledge and experience gained during the running of MagPro2Life in its teaching of the MSc Biochemical Engineering Course (i.e. in its lectures, tutorials and even exam questions). No active efforts to enhance engagement with the wider general public were instituted by UoB during the course of this grant. We are nevertheless confident that the new adsorbent manufacturing methods, the materials themselves, and the new processes employing them, represent important scientific and technological advances in their own right, and further that their collective impact on the future well-being of the greater society will be positive, significant and lasting. The first example of this is likely to come from HGMF. Indeed, one of the most important of MagPro2Life’s outputs is the maturation of this technique to the point where it is now poised on the brink of its first true industrial application, and a bright future ahead.
MEK
Magnetic particles can preferably be used in an early stage of downstream processing with only little or partially cleared raw materials that cannot be directly loaded on conventional chromatography columns. Ideally one such capture/elution step might replace several traditional centrifugation/filtration/dialysis procedures.
The continuous synthesis of the basic magnetite particles (patented by Merck in 2008) will be transferred to our regular production unit to replace the current less reliable batch procedure. This will reduce the off-specification numbers and thus increase the overall economy and ecology of the process.
The ion exchange MagPrep TMAP and MagPrep SO3 particles are planned to be commercialized for the manual and/or automated capture/purification of biomolecules. The marketing is supported by the project applications/publications and strengthens the overall magnetic bead business of Merck.
Due to the increasing public awareness of the potential health hazards of nanoparticles, many commercial products (e.g. cosmetics) already need to be labeled accordingly. A new method to prepare and automatically scan/determine the number size distribution of (magnetic) beads from electron micrographs has been filed for patenting and might replace current less reliable procedures. The MagPrep TMAP toxicity study indicating the non-toxic nature of the particles further contributes to the acceptance of the material in commercial industrial processes.
FZMB
Within the project a Kappa light chain (kappa-IgLC) ELISA Kit (reactivity: human) was developed and is ready for commercialization. This ELISA is very interesting for the pharmaceutical industry (in-process-control) and R&D laboratories. The market for Fab development is still growing. For this reason the market potential of the ELISA is high. There are different competitors. The market price for an ELISA (96 well) is 600-800 €. Time to market is 12-14 months (stability charges). To improve exploitability the generation of a complementary “Lambda light chain ELISA Kit” is planned.
The Lunasin ELISA has a low marketing potential at the moment in a niche market for soy industry and R&D laboratories. More interesting is the production and commercialization of the native (purified) Lunasin as standard material. Purification was demonstrated by using a procedure consisting of anion exchange, reduction, ultrafiltration and reversed phase chromatography.
The developed Bowman Birk Inhibitor ELISA and also the mouse anti-BBI antibody are ready for commercialization. BBI is more common than Lunasin, but it is still a niche market (soy industry, R&D). A commercially available BBI ELISA was not found in the market (only patents and publications). There is one competitor for the BBI antibody (price: 375 €/mg). Time to market is 9-10 months for the BBI ELISA and 3-5 months for anti-BBI antibody.
Fzmb is owner of the cell lines for the capture antibody for the Kappa light chain ELISA Kit and for both antibodies of the BBI ELISA.
UCD
UCD has three publications coming out of the magpro2life project. Two of the papers relate to D47 where antibody-derived and peptide-based probes were identified.:
[1] Fields, Conor, Paul Mallee, Julien Muzard, and Gil U. Lee. "Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation." Food chemistry 134, no. 4 (2012): 1831-1838.
[2] Muzard, Julien, Conor Fields, James John O’Mahony, and Gil U. Lee. "Probing the soybean Bowman–Birk inhibitor using recombinant antibody fragments."Journal of agricultural and food chemistry 60, no. 24 (2012): 6164-6172.
The other publication is a protocol paper involving the techniques, genetic engineering and steps required to generate recombinant antibody fragments starting from hybridoma cells:
[3] Fields, Conor, David O'Connell, Sujing Xiao, Gil U. Lee, Philippe Billiald, and Julien Muzard. "Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies." Nature protocols 8, no. 6 (2013): 1125-1148.
Material for deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles” was presented at the ACS national meeting in San Diego on the 27th March 2012. Material surrounding the first publication [1] was presented at Trinity College Dublin on the 31st May 2012
Exploitable foreground in the form of a patent entitled “Bowman-Birk protease inhibitor (BBI) binding peptides, and uses thereof in the separation, detection or purification of BBI” has been submitted. The described patent may be of interest to the feed and food industry.
ETH
The developed and up-scaled ROMER processing technology in particular if micro-engineered membranes with tailored pore and surface characteristics are applied is expected to have large potential in being a key process component for future industrial continuous and controlled productions of complex multiphase composite particle structures and their functionalization by layer adsorption and/or encapsulation. The processing principle was approved to work equally well for nano-/meso-scale entities like vesicles or bicelles as well as on macro-scale for solid triglyceride or wax particles. Economic potential for applications in food, pharma and cosmetics systems has been discussed with industrial partners and ROMER device commercialization is on the way /1,2/.
MANACO systems with magneto-responsive properties have been successfully developed on different length scales. Vesicles and bicelles doted with lanthanide atoms were shown to react in magnetic fields concerning permeability or orientation. Magneto-responsive bicelles were embedded in thermo-reversible gel structures from which temperature-time sensors or -integrators could be developed for which simple birefringence measurements provide clear information on the degree of sustained anisotropic oriented structure. The application of such elements is under approval for applications in intelligent packaging solutions. A patent has been filed and publications concerning the fundamental results were published /3,4/.
For the macroscopic composite particle based MANACOs tuned for separation processing of specific proteins from waste streams successful steps in surface coating and ligand attachment have been done, however binding selectivity and binding strength have to be further improved. Related further research work is planned, particularly for the area of nutrient and drug delivery systems with tailored gastro-intestinal release characteristics.
/1/ S. Holzapfel, E. Rondeau, P. Mühlich and E. J. Windhab (2013); Drop Detachment from a Micro-Engineered Membrane Surface in a Dynamic Membrane Emulsification; Process Chem. Eng. Technol. 2013, 36, No. 00, 1–11; DOI: 10.1002/ceat.201300256
/2/ Kaspar, P., Holzapfel, S., Windhab, E. W. and Jäckel, H. (2011). "Self-aligned mask renewal for anisotropically etched circular micro- and nanostructures." Journal of Micromechanics and Microengineering, 21, 115003.
/3/ Liebi, M., van Rhee, P.G. Christianen, P.C.M. Kohlbrecher, J., Fischer, P., Walde, P. and Windhab, E.J. (2013) "Alignment of Bicelles Studied with High-Field Magnetic Birefringence and Small-Angle Neutron Scattering Measurements" Langmuir, Vol. 29, 3467-3473.
/4/ Liebi, M., Kohlbrecher, J., Ishikawa, T., Fischer, P., Walde, P. and Windhab, E. J. (2012) "Cholesterol Increases the Magnetic Aligning of Bicellar Disks from an Aqueous Mixture of DMPC and DMPE–DTPA with Complexed Thulium Ions" Langmuir, Vol. 28, 10905-10915.
BUH
The aim of the ROMER II device was the production of Manacos (Magnetic Nano Containers in the form of beads or vesicles) in the µm range with a narrow particle size distribution. For this purpose the fluid which is transformed into beads is pressed through a fine membrane with fine pores into the shear field of a second fluid on the other side of the membrane. In doing so the buds which are build up on the membrane are broken away and the beads are formed.
This technology could be used commercially for the production of emulsions. There are different technologies used nowadays depending on the application.
The state of the art for the production of emulsions is the high pressure homogenization. This technology is based on the dispersion of a pre-emulsion under high pressure (>1000 bar). The produced emulsions have a broad particle size distribution.
There are other technologies like ultrasonic homogenizer or high shear dispersers which however need higher energy input or are not able to break down the droplets to the range under 1 µm .
TUBAF
During the whole project period, TUBAF has held several educational courses, seminars and oral presentations with international audience about technological implementation and application of the nanotechnology in the bio separation. The safety and ecological of improved SolPro (Solution Process) was pointed out to improve the acceptance of safe manipulation of nanotechnology and also to pave the way for industrial applications. The main process steps of SolPro to produce magnetic beads and their scale up potential for industrial application were presented too. It was demonstrated how the precipitation of magnetite, the phase transfer, the min-emulsion polymerization, the milling of macro-porous cation exchanger down to sub-micron scale and spray drying works on the lab scale. Technological innovation like the use of milling technologies for the production of low cost submicron sized cation exchange resins was particularly highlighted.
Posters and oral presentations have been given at different national (Fachausschuss Grenzflächenbestimmter Systeme und Prozesse, 2010 and 2012) and international (World Congress of Particle Technology 6, 2010 as well as European Congress of Chemical Engineering, 2012) conferences. In addition to publications in national and international journals (Chemie Ingenieur Technik, Encyclopedia of Industrial Biotechnology), TUBAF has filed a patent on the production of sub micron macro-porous ion exchange resins using mechanical milling technologies (Verfahren zur Herstellung von submikronen makroporösen Ionenaustauschermaterialien durch mechanische Zerkleinerung, 2012).
In MagPro²life, TUBAF has improved “Solution Process” for the bio separation. Due to the incorporation of several new synthesis techniques getting scale up potential, CEX and AEX magnetic beads were successfully synthesized. Beads characterization has proved, that both types were equipped with the appropriate functionalities allowing their application in the bio separation (super paramagnetic, ion exchange properties). Additional to this, the synthesis techniques and materials used are low cost. Therefore, the novel magnetic beads production technologies offer wide range of applications. The low cost adsorbents show for example a high potential for water conditioning as well as rare earth elements processing.
RATB
Highly reproducible procedures were developed for the kg-scale synthesis of magnetite nanoparticles with hydrophobic and hydrophilic surface coating by applying the method of chemical co-precipitation and using vegetable origin oleic acid surfactant (Merck product). The low mean size (below 10 nm) and surfactant coating ensure superparamagnetic behavior up to close packing of nano-particles which are essential features for their use as primary particles in manufacturing functionalized magnetoresponsive nano-composites for magnetic separation of bio-materials, magnetic drug targeting and MRI & MPI contrast agents. The procedures developed refer also to large scale synthesis of magnetic nano-fluids with water and light organic carrier liquids with potential impact on magnetic fluid based technologies.
There were 4 papers (2 Coll&Interface Sci 2012, Soft Matter 2013; 2 AIP Proceedings ), and 1 book chapter (Karger Publ. Co.,Basel), 19 presentations at conferences, symposiums and workshops, among them 4 invited talks.
It is worth to evidence the participation at several outstanding European scientific events such as Colloids and Materials 2011 Amsterdam, Colloids and Nanomedicine 2012 Amsterdam, IMAGINENANO 2013 Bilbao.
NIIMT
NIIMT obtained different hybrid magnetic nanostructures with controlled morphology, superparamagnetic behaviour, high magnetization, chemical and mechanical stability: polylactones coated magnetite nanoparticles, surfacted cluster of magnetite nanoparticles, magnetic microgel with anion and cation exchange properties.
Depending on the functionality and size these bio-compatible magneto-responsive nano-materials developed in the project have several applications with potential economic impact: new technologies of magnetic separation of bio-materials that can be taken by the pharmaceutical industry; nano-medicine - contrast agents in high resolution MRI imaging and magnetic carriers in drug transportation and release to the target.
The dissemination of the results have been done by: Publications - 2 papers (1 published in ISI journal Soft Matter and 1 published in American Institute of Physics Conference Proceedings); 1 book chapter Ed. Karger Publ Co. and participations at international conferences and workshops: 12. Among the conferences it is worth to mention our participation to large European events in nanoscience like Colloids and Materials 2011 Amsterdam, Colloids and Nanomedicine 2012 Amsterdam, IMAGINENANO 2013 Bilbao, EuroNanoForum 2013 Dublin.
NILPRP
The iron-oxide based nanoparticles were dispersed in aqueous media at near neutral pH by functionalization with L-DOPA biomolecule and can have potential application in drug delivery or MRI contrast agents.
The Fe-FexC@C nanocomposites have the potential to by introduced in PNIPAA-PAA microgels as magneto-responsive composites for magnetic separation of targeted high-value proteins from complex mixtures. Also, these Fe-C nano-composites were functionalized with CMCNa poly-electrolyte as stable aqueous dispersions and could be used in hyperthermia anticancer therapy. The maghemite L-DOPA coated nano-particles based aqueous suspensions (20 g/l) have potential for advanced thermal transfer nanofluids due to their enhanced thermal conductivity and low viscosity. Two ISI-quoted papers in Applied Surface Science resulted from this research along with 11 presentations and posters at conferences, symposia and workshops, such as EMRS European Materials Research Society Spring or Fall Meetings (Strasbourg or Warsaw) and PARTEC - International Congress on Particle Technology (Nurnberg).
USAL
From the scientific point-of-view the main potential impact was to provide a proof on the MAGCLA concept. MAGCLA may impact in many fields. In fact, with magnetic classification we are potentially capable of selectively separating substances from each other using only one single step and one machine. The Proof-of-concept given shows this potential to be real. Applications are very broad, ranging from medical analysis until the treatment of diseases, from protein recovery to cell separation, from water decontamination to catalysis improvement, or from mining industries to recycling industries and reaction engineering.
The main dissemination activities were: organization of seminars, presentations in conferences, seminars, media briefings, press conferences, media divulgation, etc., presentations for students, scientific community and general public, and divulgation of investigation in nanotechnologies, magnetic separation and of the generated knowledge.
List of Websites:
www.magpro2life.eu
The project was very challenging and ambitious in depth and breath. A new technology platform was established through the execution of the several work packages in a coordinated effort. With no doubts, the new developments are cutting edge and the research team established world leadership in the area with this project. It was a complex challenge with an interdisciplinary technology solution. The project demonstrates a successful combination of bio and nanotechnology.
The project was technically successfully and fulfilled all the necessary requirements for safety and health. Numerous tests and LCA show that the developed magnetic particle system is close for commercial use. In addition to the technical success, the project has shown that this new technology platform is also economical successfully compared to other technologies. Consequent process scale-up with cost evaluations led to a new process with lower cost footprint.
The technology in its different layers (particles, particle production processes, process technology and equipment) was demonstrated in scale. The project had included research work but most of the time was spent on commercialization and scale-up of the technology. New equipment was developed, like the magnetic centrifuge, which are in a pilot scale today. Discovered knowledge in this project will bring this magnetic centrifuge concept to a commercialization. Different methods to produce magnetic particles were discovered. Particles are smartly functionalized. Models were developed and built. The results were established and demonstrated not with academic material but with real product streams from the industry.
Challenges of the project were the work on the details and the connection of the different layers of technology so that a whole new technology platform could be established. The researcher and the industrial partners believe the technology is ready for the market.
The technology is economically viable for Food, Bio and Pharma applications. The research found it is very challenging to be economically successful for Feed applications. Many aspects were researched in depth and solutions were found. Analytic methods were successfully developed and are ready for future commercialization.
The new technology platform could have impact on the next generation of healthy food, biotechnology and pharma applications and very interesting future options arrived out of the project, like enzyme use for industrial bio-processes.
Project Context and Objectives:
Publishable summary MagPro²Life
« Advanced Magnetic nano-particles deliver smart Processes and Products for Life «
Researchers from Denmark, Germany, Ireland, Romania, Spain, Switzerland and UK kicked off the EU MagPro²Life project on July 1, 2009. The consortium brings together universities, research institutes, SME and enterprises and is coordinated by Solae. The primary objective of this project is to develop pilot lines to introduce nanotechnology based processes into the value chain of existing industries.
Implementation of functional magnetic particles as adsorbents in the bio-processing industry requires the synergistic interplay of a host of components. The two major challenges for implementation of magnetic (nano) particles and composites in industry are the cost-effective large-scale manufacturing of appropriately functionalized super-paramagnetic particles, and the lack of large-scale process technology to separate these particles from the production streams. For this technology to be accepted in industry, this technology must be sustainable, safe, capable of scaling to high production rates, reliable, robust, and economically competitive to current existing downstream processing techniques. The goal of the MagPro²Life project is to address these challenges and to demonstrate the use of functional magnetic (nano)particle separation at pilot-scale for selected feed, food, and pharmaceutical products. The MagPro²Life project carefully builds on knowledge gained in the NanoBioMag project, funded by the EU under the FP6 program, through a series of focused objectives: (i) development of scalable magnetic particles production technologies; (ii) development of scalable surface functionalizing technologies; (iii) development of large-scale processing technologies to be used with the adsorbents produced by objectives (i) and (ii); and (iv) integration of the previous three objectives to demonstrate that this technology can be implemented for safe, industrial scale bio-separation.
Biotechnologically derived substances for large scale feed, food and pharma applications represent one of the most important sources of new products due to their precisely controlled structural and functional properties, potential for economic and responsible production and overall broad benefits to society through bio-compatibility and sustainability. The costs of producing bio-materials are in many cases dominated by separation processes, which can constitute up to 80% of the total cost of production. Using smart magnetic adsorbent particles to selectively separate the target product out of a complex product mixture like the fermentation broth or bio-feed stock can drastically reduce costs. By using magnetic separation and extraction technologies to separate the magnetic carrier particles, novel processing ways emerge.
While the developments of modern gene technology promise to make an increasing number of sophisticated pharmaceuticals available, their enormous production prices will inevitably place strong pressures upon health care systems, even those in wealthier countries. Higher value proteins derived from natural and recombinant sources represent main target bio-products within this project. The range of prices of various industrial protein products vary astronomically up to several million € per kg. High-value pharmaceutical proteins are only affordable for highly developed countries in the western hemisphere, but even in these privileged countries the high cost of these drugs exerts a strongly negative impact on their social health care system. By reducing the production costs of new pharmaceuticals the deliverables of this project will contribute to solving this dilemma.
The project will likely deliver exploitable findings directly applicable to individual products. Practical exploitation will be undertaken by the industrial partners as the first end users. The knowledge database produced during MagPro²Life shall guarantee fast market readiness and fast and easy transfer to products other than those employed in the pilot line demonstrations.
The knowledge gained in the areas of process and equipment development and applications will form a basis for creating new process equipment and new high-value products. Equipment when commercialized will be available to industrial sectors such as pharmaceutical, food, chemical and technical industries. It is planned to sell licences for the patented equipment to biotech manufactures.
In the first 18 months of the project, several distinct manufacturing routes were developed to produce assemblies based on inexpensive super paramagnetic particles combined with bio-materials, functional polymers or self-adapting polymers. Recognition systems are constructed to make the adsorbents selective and/or endow them with multifunctional properties. Bio-materials, representing each of the 3 industries for pilot scale demonstration, have been selected and characterized. Focus in the next period will be the comparative characterization of the particle systems and subsequent scale-up of the most promising ones. To successfully compete with classical production processes it is crucial to provide high efficient process equipment especially regarding the recovery of the magnetic adsorbent particles. In the following are the technologies, the consortium is focusing on: Continuous Magnetic Extraction, Magnetic Field Enhanced Centrifugation, and Magnetic Classification. Lab equipment has been used to model the separation systems and preliminary screening of the particle systems has been performed. Focus has been on the pilot scale design of the separation equipment, while efforts for the next period will be the manufacture and optimization of this equipment.
In the second 18 project months, the bio-systems for production and capturing the target molecules have been studied and optimized in all three areas of interest. The separation equipment was built, optimized and bench-marked. The pilot lines where set-up and are ready to run and generate data for the risk assessment, the economic studies, Life Cycle Analysis and technology bench-marking. Pro-actively, particular focus was set to Anion Exchanger and Cation Exchanger functionalization of the magnetic beads. Sufficient quantities of particle systems were made available for all three pilot plants to trial and run. Additional bead systems of interest are planned in smaller quantities and will be tested on lab scale. They will be scaled up using SuperPro Designer® software in order to offer the possibility to compare these data with the data from the pilot lines.
In the third and last reporting period, the pilot lines have run and demonstrated the MagPro²Life technology. Full data analysis and modeling made an assessment possible on all the aspects of potential successful exploitation. Scale, particle cost and binding capacity have the most significant impact on production cost. We showed that although at current state of the art, still optimization is needed, it is possible to commercially exploit the technology at competitive product prices, with distinct advantages against immediate competing technologies as packed bed and expanded bed technologies. A full comprehensive confidential report has been issued on the technology as explored in this project. A book on the subject matter will be published (Springer) in second quarter of 2014.
Project Results:
Final Report 4.1 (SESAM) – Results of S&T foregrounds
SOL
Solae was the overall coordinator of the MagPro2Life project with 15 industrial and university partners from Denmark, Germany, Ireland, Romania, Spain, Switzerland and the U.K.
Solae was work package leader for WP0 (Organization and Coordination), WP1 (Specifications and Conceptual Design), WP7 (Food Pilot Line, RTD and Demonstration), WP8 (Economic, Market, Safety, Health and Life Cycle Analysis), and WP9 (Dissemination and Training).
The main objective for the MagPro2Life project was to use advanced magnetic (nano) particles to extract valuable components during food, pharma and feed processing.
Solae and Merck successfully developed a process that captures proteins in soy whey streams with silica.
Solae, Merck and DTU successfully developed processes that capture protein in soy whey streams using the magnetic fishing technology. This was done with and without silica pretreatment of the soy whey. A group of proteins were separated basis ion exchange functionalized magnetic (nano) particles, resulting in a protein concentrate containing a broad band of proteins, and enriched in the protein if interest. A single protein was separated basis antibody affinity ligand magnetic (nano) particles, resulting in a very pure high value protein product. This enabled Solae to evaluate the feasibility of protein extraction and production by magnetic fishing.
Solae successfully scaled the magnetic fishing process capturing proteins in a soy whey stream. The scale-up, full process set-up and protein production was done in a food pilot line magnetic fishing process setup at Solae in Aarhus, Denmark. The food pilot plant included scale-up of a pilot scale magnetic centrifugation process using MEC200 produced by AKMPT.
Solae and KIT-MVM executed a full performance testing of the magnetic centrifuge (MEC200). The tests were done in the food pilot line at Solae in Aarhus, Denmark.
Solae developed process models (SuperPro) for the protein production by magnetic fishing to understand major cost drivers and key points of process improvement (yield, magnetic particles, equipment).
The process models enabled Solae to do an economic analysis of the magnetic fishing technology at production scale in one food application.
The process models also enabled Solae to benchmark the magnetic fishing technology against competing technologies like packed bed chromatography (PB), expanded bed chromatography (EBA) and ultrafiltration (UF). DTU provided the model input for the competing technologies basis protein production done at DTU in Lyngby, Denmark.
Solae collaborated with DuPont to establish and compare a Life Cycle Analysis (LCA) on the magnetic fishing technology against expanded bed chromatography (EBA) as the most relevant competing technology.
A full risk assessment was conducted for the magnetic fishing process basis the DuPont NanoRisk Framework program. The potential risks (safety, health and environmental) associated with the use of magnetic nano particles in the food pilot line at Solae in Aarhus, Denmark was evaluated. The risk assessment included some precautionary actions in the work environment, some toxicological studies of the magnetic nano particles used and an evaluation of the environmental fate of the magnetic nano particles when used in the food pilot line.
DTU
The main focus of the project for DTU was to benchmark the performance of magnetic adsorbents, for BBI capture from Soy whey, against the closest competing technologies, i.e. expanded bed adsorption (EBA), packed bed chromatography (PBC), and ultra filtration (U.F). The data from this was used in the lifecycle analysis of full scale BBI production performed by Solae Denmark. Part of the project was also to take part in identifying relevant targets for demonstration of the magnetic adsorbent based technology. One of these was lunasin from soy whey. Furthermore as a spin off from evaluation of purification targets, a potential new application not foreseen in the original application has been identified, i.e. for recyclable immobilized enzymes.
Experiments in the laboratory with each of the competing technologies, i.e. packed bed chromatography, expanded bed adsorption and ultrafiltration, plus two magnetic adsorbent (Merck TMAP, and Orica) based systems, were used to provide data for the modelling in SuperPro Designer. Each technology approach was optimised for BBI purification from soy whey obtained from the Solae factory in Iper Belgium and both crude and pre-treated soy whey were used. This is the first time that such an extensive evaluation of the performance of technologies competing with magnetic separations has been undertaken. The data has been made available to the consortium and selected data has been included in a chapter of the book being written by the consortium which will be made available to the public. A spinoff from these studies has been a much better knowledge of the behavior of magnetic adsorbents and their interactions with feedstock’s. With respect to concrete results and foregrounds from this work, the following have been developed: SOPs for bench-marking performance of magnetic adsorbents in soy feedstock, BBI assay in micro titre plates, lab data on performance of TMAP and Orica magnetic adsorbents in model and real (soy whey) feedstock’s for use in economic and LCA modelling, lab data on performance of EBA, packed bed chromatography and UF for BBI purification from soy whey for use in economic and LCA modelling.
Experiments to develop an assay for quantification of lunasin were conducted, as a prerequisite for developing a purification procedure for the target from soy whey. A preliminary lunasin assay (HPLC based) was developed. Further work was down prioritised in favour of BBI purification.
Studies of an alternative target for demonstration purification, beta-glucosidase (from Novozymes) on the Orica and TMAP adsorbents showed that it was extremely difficult to elute it. Rather than dismiss this system, its potential as a magnetic immobilized enzyme for bio-fuels and bio refineries was examined in the lab and subsequently in a demonstration with the MEC in collaboration with Solae Denmark. This involved immobilisation of the enzyme by binding to 400 g Orica particles, followed by 4 cycles of use in ca. 20 L of cellulose containing feedstock, each time for 20 h at 50°C. This unforeseen spin out opens up a vast new range of applications for the MEC and magnetic adsorbents used and developed in the consortium. Two journal articles are in preparation with document the lab and demonstration studies.
KIT-IFG
Within the MagPro²Life the partner KIT-IFG followed two main tasks. First, the further development of a capture step isolating proteins from crude feedstocks by means of a combination of Aqueous Micellar Two-Phase Systems (AMTPS) and magnetic micro- or nanoparticles, called Continuous Magnetic Extraction (CME). The basic idea for this process was developed in a preceding STREP project called NanoBioMag within FP6; however, it was shown within MagPro²Life that the process can be run continuously using nontoxic, cheap surfactants. The second task was dealing with a new isocratic chromatography process based on thermoresponsive media and a newly designed column featuring a traveling temperature zone (Traveling Cooling Zones Reactor, TCZR). The thermoresponsive media were delivered by the partner UBI, who started the development of magnetic and non-magnetic beads with thermoresponsive coating within the FP6 project NanoBioMag.
In the course of the work on Continuous Magnetic Extraction the following main milestones and deliverables were achieved: i) characterization of suitable combinations of phase forming surfactant and magnetic sorbents; ii) investigation of the physico-chemical background of the partitioning behavior of the particles in the AMPTS, iii) setup and characterization of the CME process; iv) application of CME to purify proteins from various feed streams; v) removal of the remaining surfactant from the product stream.
Prototypes of the magnetic extraction set-up allowed the continuous separation of magnetic particles delivered by the partners Merck and NIIMT of sizes from 2 µm down to 25 nm with separation efficiencies > 99 % at flow rates up to 9 liters per hour. In close collaboration with the partners responsible for the pharma and food pilot lines the CME process was tested for continuous separation of an antibody fragment in 15 liter scale. The process resulted in a total yield of 67 % of the target protein and a purification factor of 6.3. The continuous purification of the Bowman-Birk protease inhibitor (BBI) from soy whey feed streams was carried out with different anion exchange particles. The trials returned around 50 % of the total BBI at a purity of up to 99 %. For dissemination the results of the CME work were published in five high ranking peer reviewed journal articles (e.g. J Chrom. A, Langmuir, Sep. Sc. Tech.) and presented at several national and international conferences. For exploitation KIT has a granted patent on the CME process and is looking for industrial partners further developing the CME applications in frame of a license agreement.
In case of the TCZR the following milestones and deliverables were achieved in order to develop the system towards a truly continuous chromatographic process: (i) characterization of adsorption properties of thermo-responsive sorbents delivered by the partner UBI; (ii) construction and hydrodynamic testing of the Traveling Cooling Zone Reactor (TCZR); (iii) cyclic experiments within the TCZR; (iv) proof-of-concept of continuous TCZR operation; (v) investigation of properties and the parameter space of the TCZR by means of a simulation program. The core of the TCZR is a linear movable temperature zone, surrounding a fixed-bed column filled with thermo-responsive sorbents. At a velocity of 0.1 mm s-1 the temperature zone is able to generate a temperature drop of more than 20 K within a narrow 2 cm section. Moving the zone along the column up to the end, sharp protein peaks can be eluted while the feed enters the column continuously. In close collaboration with the partner UBI, thermo-responsive cation and anion exchange sorbents were tested within the TCZR. For this, a PhD student and a second TCZR setup were transferred from KIT to UBI. Like in the case of the CME system dissemination of the TCZR work was done through several peer reviewed journal publications and international presentations. Exploitation is based on a filed PCT patent and it is planned to test the TCZR principle for specific applications together with two large European pharma companies.
In total the resources being provided within the project MagPro²Life were used for development of two radically new bio-separation processes, both being able to continuously purify and concentrate target proteins form different feed streams. For each of the processes two fully operable set-ups were realized and tested for different applications in close cooperation with the project partners. Finally, the work included the training of three Ph.D. and numerous master and bachelor students.
KIT-MVM
Currently HGMS devices are either based on electromagnets and are hence large and expensive, or do not provide the possibility of washing and elution steps in the cell. A Halbach permanent magnet arrangement was set up at KIT-MVM in combination with a HGMS filter cell. This allows reduction of investment and operating costs in comparison with common HGMS devices based on electromagnets. Common permanent magnets with pole yokes have a rectangular geometry and therefore cannot be stirred and hence used for washing and elution steps. An important advantage of the Halbach-HGMS is the round geometry of the magnetic field, allowing the implementation of a stirrer and the use of the device for washing and elution steps.
Magnetically Enhanced Centrifugation (MEC) was set up in three different devices. Additionally a lab machine set up in the previous project Nanobiomag was initially used for investigation at KIT-MVM. The design of magnetic wires was optimized on this machine. A first important result consists in the optimization of magnetic wires for MEC. A design comprising laser-cut wires of rectangular, longish shape arranged in flow direction was worked out to optimize separation. Wire distances were optimized to avoid bridge building of particles, which is in a centrifuge a function of rotational velocity. The design was implemented in all three pilot machines.
Additionally a model was derived predicting separation efficiency of magnetically enhanced centrifugation. The model is based on particle properties, i.e. size distribution, density and magnetization, as well as on device and process parameters such as volume flow, magnetic field, magnetic wire data etc. The model was developed supported by simulation of the flow by computational fluid dynamics, of the magnetic field by Finite Element Modeling, and of the magnetic particles by Discrete Element Modeling. It was validated by comparison with experiments and serves for the layout of processes and for the optimization of magnetic filters.
A permanent magnet design was developed creating a magnetic field in axial direction. The device was tested with MEC. Separation is similar compared to an electromagnet used alternatively on a MEC. This strongly reduces investment and operating costs of MEC, as electromagnets consume tremendous amounts of energy. In particular in combination with a continuous machine design, e.g. the magnetic decanter, the option of a permanent magnet is interesting.
An automatically discharging machine concept was targeted for use in pilot lines. A study on a variety of discharge principles was done in cooperation of KIT-MVM and Andritz KMPT, with machine concepts designed in cooperation. First a batch-wise academic peeler-centrifuge was set up at KIT-MVM and used as workhorse for academic investigation. It is based on a structured wire matrix produced by laser-cutting out of ferromagnetic stainless steel. It is used in a batch-wise manner, can be first filled batch-wise at up to 200 g of magnetic particles. The machine can be drained, with magnetic particles being kept in the device as it is filled from top and discharged at the bottom. Particles are detached from the wall and dispersed by stirring the peeler knives in a new liquid. Separation, washing and elution steps were performed subsequently in the machine. The machine was used in the pharma line for protein separation, showing the possibility of selective separation and high yield.
A continuous machine was developed and set up at KIT-MVM. It is based on a decanter principle, but changed in design to be sealed. Its main purpose is demonstration of a continuous design to be set up of completely continuous production lines. The machine was tested separating particles in a cycle for several hours at high concentration of 20 g/l and volume flow of 50 up to 120 l/h. This is to our knowledge the first full-continuous device in HGMS and potentially the most interesting device for HGMS in pilot lines at large scale.
A batch-wise large-scale industry centrifuge was set up by Andritz KMPT. It is based on a similar principle as the academic batch-wise MEC, yet in this case the wire matrix serves as stirring device. The machine was optimized for high volume flow of up to 1 m³/h at a 0.4 T magnetic flux density. All process steps, i.e. separation, washing and elution, can be performed in this machine. It was set up, tested and used successfully in the food line for protein separation. The wire matrix was developed in a 3D design to achieve constant distance between magnetic wires. Additionally a matrix featuring bi-metal design was developed by Andritz KMPT to avoid particle agglomeration in a specific area. In this design two different steal kinds are welded together in a plate, a magnetic and non-magnetic kind. The wire matrix is laser-cut out of these joint plates.
UBI
The bio-separation technology used in industry today is based on principles first discovered around 70 years ago and improvements are needed at all stages of processing, i.e. from the pre-treatment of raw materials prior to fermentation, the fermentative product itself, and during subsequent purification and modification to yield the final product. Functional magnetic adsorbents have the potential to enhance the physical and chemical properties of bio-separation process, i.e. (nano) particles have extremely high surface areas, rapid binding kinetics, and unique physical and chemical properties. One of the goals within the MagPro²Life project was to demonstrate a process for the economic manufacture of a relevant biopharmaceutical recombinant antibody fragment from E. coli and to entrench a new purification process based on magnetic adsorbents to purify the antibody fragment from the fermentation broth. Antibodies are widely used in the treatment of many cancers by specifically binding to target the tumor cell, thus making them more visible and susceptible to the immune system. E. coli is a well know host and is commonly used for the production of recombinant proteins. For the creation of a suitable feedstock for downstream processing (DSP), the fermentation and release conditions were optimized in different terms. After reaching an adequate titre of the recombinant antibody fragment, the first step in developing a new process in bio-separation technology was the successful demonstration of a magnetic benchmarking process before start working in a pilot plant using magnetic separation technology. The different magnetic separation devices used for the realisation of the project were specially designed for protein purification using magnetic adsorbents. Capturing of the recombinant target antibody fragment was realised by magnetic IEX (ion exchange) adsorbents, produced by different partners of the MagPro²Life project.
As a reference process to the magnetic purification process of recombinant antibody fragment, the purification using normal commercial IEX and affinity packed bed columns was chosen. Chromatography on packed bed is still the most common method for protein purification. An advantage for the usage of affinity adsorbents is the saving of process steps, like adjusting the ionic strengths of the feedstock, as crude fermentation broths have a high ionic strength. A disadvantage of affinity adsorbents are there comparatively high purchase costs.
Among the realisation of the experiments calculations regarding the economic efficiency of the magnetic process using appropriate software (SuperPro Designer®, Intelligent, Inc., USA) were executed, to proof the contestability of the magnetic process.
KIT-IBLT
Focus of the work of KIT IBLT within the MagPro2Life consortium was to setup a pilot scale cultivation process for phytase production with integrated in-situ magnetic separation (ISMS). Based on this process the potential beneficial application should be demonstrated. In the course of this development several exploitable results and foreground were produced which should be explained in the following.
Main exploitable result of the work of KIT IBLT is a set of design specifications for the industrial application of magnetic separation for in-situ separation of extra-cellular products like for example functional proteins. Basis for this design specification is the experimental representation and detailed evaluation of the ISMS concept based on the developed phytase example processes. In this context ISMS cultivations in different scales up to 100 L total fermenter volume were conducted to establish an economic balancing model. Furthermore a comprehensive catalog of requirements was specified for the ISMS subsystems magnetic particle system, magnetic separator and bioprocess. Especially for metal-chelate and ion-exchange functionalized magnetic particles, extensive characterization data in form of adsorption isotherms and kinetics as function of different media properties was generated. In doing so focus was put on the special requirements of direct broth extraction of functional proteins without negatively affecting the bio-process. As a consequence of these characterization experiments a particle coating procedure was developed for ion-exchange functionalized magnetic particles to avoid unwanted biomass adsorption. In the course of this development process also diverse characterization results for agarose, agar-agar and alginate coated magnetic particles were obtained. With respect to the magnetic separation device design specifications for a pilot scale high gradient magnetic filter (HGMF) device were obtained by testing of different small scale HGMF devices regarding particle capacity and particle separation. Based on this data for the pilot scale HGMF in addition also a modified particle loading strategy was developed and successfully tested enabling a more selective solid-solid-liquid separation. For the implementation of this separator a sterilizable separation circuit was developed connecting the bio-reactor, the magnetic separator and the required feed vessel. Regarding the bio-process, layout data for the design of a reproducible process control strategy was developed and applied on the extra-cellular phytase production of E.coli and B.amyloliquefaciens strains. Using the hereby produced phytase species and those from other (fungal) origins characterization data was generated with respect to enzymatic properties like stability, substrate specificity and pH / temperature dependency of the catalytic turn-over.
MEK
Core of the project was the production of kg-quantities of surface-modified magnetic particles to purify selected target proteins in combination with an efficient magnetic separator.
The continuous synthesis of magnetite with the potential for unlimited production on a large scale was the basis for all MagPrep particle species. The principle of this method has already been developed/patented before the start of MagPro2Life and was refined/up-scaled within the project to kg-quantities per day. In addition, the tightly controlled reaction conditions allow the production of narrow-sized particles within the range of about 10–100 nm. Depending on the intended application smaller beads with a high surface area/binding capacity or larger resins with a better magnetization can be selected. The number size distribution of this material can be determined by automated image analysis of electron micrographs of specially prepared particle mono-layers (patent filed).
The surface modification included a standard coating with silica as a basis for the subsequent ATRP (Atom Transfer Radical Polymerization) modification. This technology allows to fine-tune the density and length of surface-initiated polymer tentacles with almost any kind of functionalities to influence the binding capacity and magnetization of the resulting particles. The batch production of kg-quantities of anion and cation exchange resins (MagPrep TMAP and MagPrep SO3) was established and the particles used for the capture/purification of target proteins on an analytical and pilot scale. In addition carboxylated MagPrep particles have been coated with anti-BBI peptides and a monoclonal anti-BBI antibody for the affinity capture of BBI directly from untreated raw material.
MagPrep TMAP was used for the purification of BBI (Bowman-Birk Inhibitor) from soy whey in combination with conventional heat- and silica-treatment as well as the enrichment of antibodies from cell culture supernatant and human plasma by removing unwanted protein components. MagPrep SO3 could be applied for the specific capture/elution of recombinant Fabs from bacterial cell lysate and antibodies from cell culture supernatant. However, the ultimate purification of BBI could be achieved with MagPrep anti-BBI based on a covalently immobilized monoclonal BBI-specific antibody, allowing a one-step protocol for the isolation of the target protein from untreated soy whey. Moreover, the specific activity was about twice as high as with the three-step purification or commercially available samples.
From the economic point of view the particle costs, binding capacity and recyclability (including chemical and mechanical stability) is of decisive importance. Analytically the beads have been reused up to 50 times without loss of performance, on a pilot scale a 10 times recycling with 99,9% step-wise recovery could be demonstrated. Due to the current multi-step surface modification the production costs need to be reduced further to be competitive to conventional chromatography.
Based on a toxicity study the strong anion exchange resin MagPrep TMAP turned out to be harmless with respect to skin and eye irritation as well as acute oral toxicity.
FZMB
The used magnetic reader system, developed by FZMB works on basis of susceptometry and allows off-line measurements in cylindrical cuvettes (100 µl sample volume). In order to realize a flow through mode and to enhance the measurement signals the geometry and placement of the coils were changed and the complete sample measurement head redesigned. The magnetic reader system output was measured using serially diluted suspensions of different particles. A comparison of the detection limits for the 100 µl and 2 ml measurement head illustrates that the increase of the sample volume did not effect a signal enhancement. Furthermore the Merck particles MagPrep Silica have very high detection limits and so they are not applicable for the used susceptometric system. The reason for that is probably their remanence (residual magnetism). Because signal enhancement was not successful in the 2 ml head, the cuvette system of the small measurement head has been rebuilt and flow through mode has been realized. For the measured particles (TUBAF, 7,5 % magnetite) a detection limit of 181.1 µg iron per liter (18 ng iron absolute) could be reached.
Within the work for antibody generation (WP 2) an antibody was produced, which is specifically directed against human Fab from UBI. Because of the absence of a second antibody it was attempted to find a commercial antibody to build up a sandwich assay. Therefore several anti-Fab antibodies have been tested and an appropriate detection antibody was found (Bethyl). Based on these antibodies an ELISA could be established (working range: 5 – 1000 ng/ml). The functionality of this ELISA for the Fab' a33 was shown by the work at UBI with real Fab samples.
Sandwich-ELISA for Lunasin was generated using the polyclonal antibody as capture antibody and the biotinylated form as detector. Standard peptide was the synthetic full-length Lunasin. The working range of the ELISA is 0,1 to 20 ng/ml. Within WP 2 Lunasin antibodies were generated. Several combinations were studied using the polyclonal antibody as capture antibody and monoclonal antibodies as detector antibodies. Only one combination showed consistent values and a low zero value. After optimization a ELISA based on this combination was established, which works well with standard peptide and purified lunasin. The working range of the ELISA is 5 to 500 ng/ml.
Based on the successful production of monoclonal antibodies the development of a sandwich ELISA for BBI was possible. In the characterization one antibody has been shown a particularly good binding: mab F2. Therefore, this antibody has been selected as capture and the other clones were used as detection antibodies. Two combinations were selected and tested in sandwich ELISA. The best combination (F2/F3) was further investigated to optimize the concentration of the reagents. BBI from Sigma was used as standard material. The ELISA has a detection range of 2,5 to 100 ng/ml. Validation was done successfully. The limit of detection is 3,4 ng/ml and the limit of quantification is 5,8 ng/ml. Additional the robustness of the ELISA was examined by variation of pH and temperature. Tests with soy samples were successful and showed a good correlation to the enzymatic chymotrypsin assay. In opposite to the enzymatic assay the ELISA is able to measure much more samples within 5-6 hours. For the development of a rapid BBI assay the principle of the Sandwich-ELISA was transferred to the ABICAP system. Different detection systems were tested. Best results showed the set up with biotinylated detection antibody in interaction with streptavidin-poly-HRP as enzymatic detection system. Calibration was done with BBI standard from Sigma. Tests with soy samples (eluates from magnetic separation) showed a good correlation to ELISA values.
UCD
Deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles” and deliverable 47, originally titled “Produce phage that bind to Bowman-Birk inhibitor and Lunasin proteins” but later changed to “Produce lab scale quantities of peptide-immobilized magnetic beads with affinity for BBI” represent the work undertaken by UCD.
Towards the originally titled “deliverable 47”, UCD successfully identified phage-based, antibody-based and peptide-based affinity ligands for BBI purification applications in the consortium. Synthetic peptide-based probes targeting the Bowman-Birk inhibitor (BBI) have been recognized by screening a bacteriophage dodecapeptide library against the purified antigen (Bowman-Birk inhibitor). Monovalent peptides were synthesized as branched, multimeric peptides. These peptides - peptide 1 (GAMHLPWHMGTL)4K2KGSGCG) display avidity effects as a result of their tetrameric nature, and therefore improve binding characteristics. Synthesis of peptide was performed by solid phase peptide synthesis at greater than 95% purity. Subsequent characterization of the peptides and grafting onto magnetic beads, using heterobifunctional SMCC cross linkers, allowed the one-step purification of BBI from crude soy whey mixtures. The nature of the eluted protein was confirmed to be BBI by SDS-PAGE gel electrophoresis, mass spectrometry, and ability to inhibit chymotrypsin and trypsin. Using 10mg of functionalized magnetic particles, we estimated a purification of ~100 μg Bowman Birk inhibitor from 50mL of crude soy whey extracts [1]. Additionally, antibody engineering of a pre-existing monoclonal antibody (mAb238, Brandon et al 1989) has been performed. This has resulted in the production of antibody fragments (Scfv and diabody), in bacteria, with the capacity to recognize and bind to the Bowman-Birk inhibitor. These BBI probes have been subsequently used in magnetic-based assays via grafting through direct amine coupling protocols, ultimately allowing the rapid purification of BBI from crude soy whey mixtures [2].Efforts towards the newly appointed deliverable “Produce lab scale quantities of peptide immobilized magnetic beads with affinity for BBI” were unsuccessful as a result of peptide degradation over the time course between the two deliverables. Peptide degradation was confirmed by ELISA and surface plasmon resonance (Biacore) measurements.
Towards deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles”, the pIII phage coat proteins were genetically engineered to modify the N-terminus with either a –CHKKPSKSC (M13Si) or –HHHHHH (M13His) peptide sequence and phage were successfully assembled on SPMs coated with either silica or nitrilotriacetic acid, respectively. This assembly permitted end-on attachment of the phage to magnetic particles. In an alternative side-on orientation, the pVIII phage proteins were modified NHS chemistry to generate M13 biotin phage. These phage were then successfully coupled to streptavidin-coated magnetic particles. In this case, the whole length of the phage is immobilized onto the bead in a side-on orientation [3]
[1] Fields, Conor, Paul Mallee, Julien Muzard, and Gil U. Lee. "Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation." Food chemistry 134, no. 4 (2012): 1831-1838.
[2] Muzard, Julien, Conor Fields, James John O’Mahony, and Gil U. Lee. "Probing the soybean Bowman–Birk inhibitor using recombinant antibody fragments."Journal of agricultural and food chemistry 60, no. 24 (2012): 6164-6172.
[3] Muzard, Julien, Mark Platt, and Gil U. Lee. "M13 Bacteriophage‐Activated Superparamagnetic Beads for Affinity Separation." Small 8, no. 15 (2012): 2403-2411.
ETH
The S&T results achieved by ETH Zürich, in collaboration with the MP2L project partners are divided into two main sections (A) New Magnetic Containers/Carriers and (B) a new processing technology for producing the same.
In (A) so-called MANACOs (Magnetic Nano Containers) were designed and further developed. MANACOs represent magneto responsive carriers for adsorbed or encapsulated functional components. For the latter preference was given to hydrophobic proteins to be harvested from soy whey waste streams (e.g Bowman-Birk Inhibitor, BBI and Lunasin). The backbone structure of the MANACOs consists of lipids, preferably (i) phospholipids for nano-/meso-scale containers/carriers forming uni- or multi-lamellar vesicles or bicelles and (ii) triglycerides or waxes for macroscopic ones, forming semi-solid or solid particles. The magneto-responsiveness of these containers/carriers has been successfully applied by doting with paramagnetic atoms (lanthanides: thullium Tu or Dysprosium Dy) in case of the nano-/meso-scale MANACOS and with super paramagnetic fatty acid coated magnetite nanoparticles for the macroscopic MANACOs.
For the separation processing within the MP2L project further preference was given to these macroscopic MANACOs in the diameter range of 1-50 microns. Related surface modifications of the triglyceride based macroscopic MANACOs with polymers were adapted to address anionic or cationic exchange (AEX, CEX). Both were produced following the same two-step approach based on a new rotating membrane process (ROME) developed for the production of “naked” MANACOs (see B). Briefly, a functional polymeric material is pre-synthesized (1) and subsequently used as a surface-active ingredient for the preparation of coated MANACO particles via a mechanically gentle emulsification in the ROME process (2). Functional block copolymers were specifically designed with a molecular structure such that they coat the wax droplets during emulsification and form, after drop solidification, an external functional layer, which is subsequently chemically bound onto the matrix of the resulting MANACOs via in-situ radical polymerization. A scalable procedure allowing for the chemical synthesis of both cationic and anionic block copolymers with varying molecular composition and structure was developed, based on Atom Transfer Radical Polymerization (ATRP).
The newly developed processing technology (B) for the generation of different types of MANACOs is based on a dynamic membrane emulsification principle, coupled with a surface modification-/functionalization-step. The device in which this coupled processes are run is denoted as ROtating MEmbrane Reactor (ROMER). A triglyceride or wax melt with suspended paramagnetic nanoparticles was pressed through the rotating membrane forming drops being detached from the membrane surface by the acting wall shear stress generated by the continuous watery emulsion fluid phase flow across the membrane. The continuous fluid phase contained the functional block copolymers attaching to the drop surface. The wall shear stress controls the size of the MANACOs being adjusted by the rotational membrane velocity and the shear gap width between membrane and housing wall and is practically decoupled from the total throughput rate of the continuous process. Material characteristics of influence to the MANACO are coupled with the acting wall shear stress in the critical dimensionless Capillary Number Ca*, which was used for process scaling.
An up-scaled ROMER II emulsification chamber was constructed using a cone-plate geometry (cone: rotor, plate: static membrane) in order to allow for the utilization of flat micro engineered membranes. In a first stage, the particle production process was developed using silicon-based membranes with a pore size of 10 µm, which were routinely produced in-house, in ETH clean room facilities. In parallel, we developed an alternative process for the fabrication of silicon nitride-based membranes with a pore size in the range from 0.4 to 5µm, and suitable for utilization in the ROME II device.
MANACOs were successfully coated with a CEX functional layer utilizing the same production process than for the AEX functionalized MANACOs. The anchoring of the CEX functional layer as well as the achieved ligand binding selectivity and binding strength have to be further improved.
BUH
The Romer I Device was constructed, manufactured and implemented for research at the ETH producing Manacos .
The new concept, design and construction of the pilot Romer II device were fulfilled till the end of 2010. The pilot plant was manufactured and put into operation till June 2011. Afterwards it was transferred to the ETH for the production of Manacos.
Based on the experiences made with the Romer I apparatus a new concept of the process room was elaborated in order to produce Manacos.
Essentially the process room is flushed by a flow against the centrifugal forces in order to guarantee the build-up of a shear flow. The process room consists of two discs, the one rotating with a rotation speed which allows to build up a shear rate of 106 s-1 . In order to avoid any instabilities a second process room consisting o a Couette flow between two cones was constructed.
The device is instrumented with the necessary sensors (pressure, throughput, temperature, gap-measurement) and can be operated hy pressure feedback control or through put control.The operation panel is separated from the control panel for a better handling.
The mechanical simulation of the process for the generation of vesicles by pressing a membrane of amphiphilic molecules through a hole was realized with FEM. In order to simulate the mechanical properties of the membrane the constitutive equation of Skalak has to be implemented into the FEM program (LS-Dyna). We are now able to simulate the process of the bud building with different amphiphilic molecules.
Additionally to the tasks mentioned it was decided in the consortium meeting in Zürich at January 2010 to try to produce nano scaled magnetite particles by a top-down strategy, i.e. by comminution of magnetite pigment (BASF) in a ball mill with addition of an adequate surfactant. After having comminuted the pigment introducing 104 kWh/to the mean particle size was 1.4 microns. Further trials were not carried out due to the high energy input needed.
TUBAF
The goal of TUBAF in MagPro2life was to develop, based on SolPro, a low-cost scale up able synthesis process as well as to optimize the efficiency of particles production having regard to safety and ecological aspects. Depending on target protein charges in the extraction pH range, CEX and AEX SolPro beads were successfully synthesized and characterized. Both types of beads were equipped with the appropriate functionalities allowing their application in the bio separation (super paramagnetic, ion exchange properties). To ensure the industrial application of SolPro, it was important by designing the product to choose or to develop synthesis processes getting scale up potential. Therefore the SolPro beads were synthesized using spray drying process, which is a well-established method to particle design in many technological fields like pharmaceutical, cosmetic, food or chemical industry.
The SolPro beads were fitted with super paramagnetic behavior by using nano-sized iron oxides material called magnetite. The nanoparticles were produced via co precipitation of iron salts. During the first project period, a continuously operating process was being successfully developed to produce water-based magnetic fluid made of dispersed iron oxides nanoparticles using sono-precipitation. By doing that, two types of reactors were used: a conical and a cavitation reactor. In each type of reactors, 120 g of super paramagnetic iron oxides nanoparticles were produced continuously as water dispersion with high flow rates up to 6000 ml/h. It was found that, the energy input by ultrasound had no significant positive influence on the nanoparticles quality (size, magnetization). Therefore, the use of a T-mixer like in the batch process, which is more competitive and easier to scale up than ultrasonication, would be sufficient to achieve this.
Another component making SolPro particles suitable for the bio separation is the ion exchange resin. For positively charged target protein in extraction milieu, sulfonated copolymer polystyrene divinylbenzene was used. To be better integrated in the beads and thereby ensure their mechanical stability, cation exchangers should be nano- or submicrosized. Mini-emulsion polymerization was found to be adequate to produce these. Their synthesis in a pilot scale implied the use of chemical sophisticated equipment. To overcome this limitation, a new production process for cation exchanger following the same specifications formulated above was being evolved. Sulfonated copolymers polystyrene divinylbenzene in micron size (x50,3≈. 650 μm) are available on the market in large scale as low-cost material. Established milling technologies were exploited to mill these low cost cation exchangers down to sub micron sized. Thereby, their adsorption capacity stays quite unchanged. The technological innovation that should worth pointing out here is the use of milling technologies for the production of low cost submicron sized cation exchange resins. To extract negatively charged protein, weak ployelectrolytes was chosen as anion exchangers. A basic technology for their incorporation in SolPro beads was being developed during the project too. The technique used to synthesize AEX SolPro was combined functional groups activation, particles synthesis followed by surface modification. All these process steps can be operated in the large scale.
Characterization analyses of the SolPro beads (CEX und AEX) showed the high potential of the developed processes. CEX SolPro particles possess a lysozyme extraction capacity of approx. 280 mg/g, which is about a factor three higher than similar CEX-SolPro beads (same composition) produced by Hickstein (77.6 mg/g). The novel designed and produced AEX SolPro beads using poly(allyl)amine (PAAm) as anion exchanger exhibit extraction capacities of 85 mg/g as well as 145 mg/g for BSA and BBI respectively. Several instigations proved that a quaternization of amino groups of poly(allyl)amine anchored on the beads surface increase their extraction capacity by a factor of three. One kilogram of produced PAAm Solpro was being successfully tested by Solae Denmark in food pilot line. application of PAAm Solpro.
In future works, the basic chemistry used for the synthesis and modification especially the quaternization of PAAm-SolPro beads should be improved. This would further increase their BBI extraction capacity in a real extraction milieu (non-treated or pre-treated soy whey) considerably. The Application of such adsorbents to water conditioning and rare earth elements processing can also be investigated.
RATB
A chemical co-precipitation method was applied for the preparation of primary magnetite nano-particles suitable for magnetic fluid and magnetic nano-composite preparation. The relative strengths and ranges of various interaction potentials were controlled by the diameter of magnetite cores and the thickness of the stabilizing layer in order to ensure the dimensionless magnetic interaction energy to be below 1 and the mean particle size less than 10 nm. Consequently, the resulting magnetite nanoparticle systems, such as nanofluids, microgels and nanocontainers will show superparamagnetic behaviour.
Depending on the clustering and functionalizing procedures applied by partners, magnetite nano-particles both with hydrophobic and hydrophilic coatings were synthesized. The surfacted magnetite nano-particles dispersed in light hydrocarbon or water, depending on the nature of the coating, serve as primary materials for the preparation of various functionalized magnetic nano-composites-magnetic beads- for magnetic separation purposes.The main requirements refering to biocompatibility, as well as efficient coating and functionalizing capacity were ensured by vegetable origin fatty acids used. The strictly applied synthesis conditions, such as the precipitation temperature, pH value and the excess amount of NH4OH ensure the formation of Fe3O4 over Fe2O3, as well as the very good reproducibility of the procedures on lab-scale.
TEM, VSM, magnetogranulometry, rheometry and dynamic light scattering investigations offered data on the magnetic response, as well as on the magnetic, solid and hydrodynamic size distributions in case of hydrophobic and hydrophilic nanoparticle systems up to close packing and evidence the superparamagnetic behavior, the efficiency of surface coating of magnetic nanoparticles and the degree of clustering. The evaluation of the mean thickness of fatty acid coating and effective and mean ellipticities of particles conducted to the mean cluster size which is rather small and practically does not affect the very good dispersability of hydrophobic and hydrophilic magnetite nanoparticles in light organic non-polar solvents and water, respectively. These primary materials were used by partners to prepare magnetic nanocomposite particles for magnetic separation purposes which usually involve HGMF. Among the required properties are the high magnetic moment density (several tens of emu/g) and the superparamagnetic behavior (no remanence). The adequate size range and efficient surface coating of primary magnetite NPs are essential in order to ensure the required magnetic response of closely packed magnetite NPs in nano-composites, such as MANACO’s and magnetic micro-gels designed for magnetic separation. The investigations concerning the character of magnetic response at close packing (at minimum distance between surface coated magnetic nano-particles) were extended to polymer coated magnetite NP clusters obtained by NIIMT applying a controlled clusterization procedure. The static magnetization data analysis using the Ivanov & Kuznetsova magnetization model for close packed MNPs evidence the efficient screening of magnetic dipole-dipole interactions. The full magnetization curve does not show any hysteresis as the magnetite nano-particle clusters encapsulated into polymer shell have no magnetic moment in the absence of an external magnetic field. The superparamagnetic behavior of surfacted magnetite nano-particle clusters, relevant for their use in magnetic separation, is demonstrated by the above analysis.
Optimized procedures were developed and experimented for kg-scale synthesis of hydrophobic and hydrophilic magnetite nano-particles.
NIIMT
The research activities of NIIMT focused mainly on the development and optimization of the synthesis methods of hybrid magnetic nano-structures with controlled morphology, such as core-shell particles or micro-gels with the required properties for magnetic separation: superparamagnetic behavior, high magnetization, chemical and mechanical stability, high concentration of surface functional groups, good re-dispersability.
Synthesis procedures have been developed for polycaprolactone or polylactic acid coating of surface modified magnetite nanoparticles using either classical polymerization reaction or microwave irradiation. Magnetite nanoparticles prepared using a common co-precipitation process, were surface-functionalized in situ by the addition of glycolic acid or acrylic acid. For the attachment of the polymer chain on the preformed functionalized magnetite the “grafting-from” strategy was applied, where the polymerization is initiated directly from the particles surface. HRTEM evidences the formation of core-shell nano-structures with the magnetite core coated with a thin shell of either polymer or co-polymer. The polymer/co-polymer coated magnetite nano-particles show superparamagnetic behavior of magnetization at room temperature and high saturation magnetization values.
An important part of NIIMT research activity was devoted to the synthesis and characterization of magnetic microgels with controlled size, anion exchange and cation exchange properties, chemical and mechanical stability, and good re-dispersability. Lab-scale preparation methods for magnetic microgels have been developed using magnetic nanofluids containing surfacted magnetite nanoparticles dispersed either in water or in light hydrocarbon provided by the partner Romanian Academy-Timisoara Branch (RATB). Physical properties of magnetic microgels have been investigated by TEM, HRTEM, DLS, Zeta potential, FTIR, XPS and magnetic measurements, to determine de relevant synthesis parameters for adjusting the particles size and dispersion properties, for the increase of magnetization and concentration of functional groups (carboxyl or amino) on the surface.
Clusters of magnetite nanoparticles with hydrophilic coating from water based magnetic nano-fluid are embedded into polymers such as poly(N-isopropylacrylamide), polyacrylic acid, poly(3-acryloamido-propyl trimethylammonium chloride) applying free radical polymerization method. TEM and DLS investigations show that the morphology and size distribution of magnetic microgels depends strongly on the polymerization conditions and physico-chemical properties of the water based magnetic nanofluid.
The preparation of magnetic micro-gels based on magnetite nano-particles with hydrophobic coating from toluene based magnetic nanofluid (provided by RATB) was performed using mini-emulsion method. This method allows the reproducible preparation of size controlled magnetic nano-particles clusters which later on are encapsulated into polymers.
The magnetic microgels prepared using either water or organic carrier magnetic nanofluids show superparamagnetic behaviour at room temperature and high magnetization values, proving that the developed synthesis procedures favors close packing of magnetite nanoparticles and allows the encapsulation of a relatively high fraction of magnetite nano-particles into the polymers. These magnetic beads have potential applications in biotechnology and nanomedicine.
NILPRP
Laboratory and semi-pilot scale continuous synthesis procedures of either maghemite-based nanoparticles or metallic iron - iron carbide core / carbon shell nanoparticles with high saturation magnetization and near superparamagnetic behaviour were developed using optimized laser pyrolysis technique. The iron-oxide based nano-particles were dispersed in aqueous media at near neutral pH by functionalization with L-DOPA bio-molecule and can have potential application in drug delivery or MRI contrast agents.
USAL
The main scientific and technological advance was the proof that magnetic classification works in wet-mode and gives the desired and expected class differences. In fact, the results obtained show that a practical experimental difference of magnitudes as low as 1.3 - 2.0 between classes is achieved, which represents a major step in the magnetic processing area. This indicates that future lab and pilot-scale versions of the wet-mode MAGCLA will be able to classify different particles present in an initial mixture according to their magnetic susceptibility values, with as close as 2 in order of magnitude.
The proof was first made through an indirect experimental proof (using particles with similar characteristics but under different magnetic fields influence), then with a comparison of the trajectories obtained in contrast with the ones predicted by theory (numerical simulations were obtained that showed the working principles) and finally with a direct proof by using particles with different magnetic susceptibilities (the difference in their magnetic susceptibilities is much lower than 50 in magnitude) and achieving their classification (division of the feed into three outcomes with a difference in their magnetic susceptibilities magnitudes much lower than 50). The proofs were made with and without the presence of a classifying exit.
The development of the theoretical background and the simulations of the process were also a main achievement, as it allows to optimize the process. These preliminary theoretical results were validated against experimental results. The first preliminary version of this theory was also needed to make possible the full design of the device, which was only viable after dimensions were known.
A full microfluidics image view system was built to capture and analyze the trajectories followed by the particles on a small part of the new device (proof of concept).
The small-scale proof-of-concept system was tested for two different kinds of particles (well-shaped and defined Cospheric and bad-defined and bad-behaved Orica particles) and the results have shown magnetic classification in wet-mode to be possible even in harsh conditions.
We also obtained results concerning the influence of velocity and distance between the magnet array and the surface of the pipe, in what concerns the separating point; and also determined the existing differences in the separating magnetic susceptibilities ratio between the top and bottom positions in the inner wall of the pipe for the same radial position.
A full version of the large-scale wet-mode prototype was built, assembled and tested, except for the superconducting magnetic generator. The preliminary tests allowed us to conclude about size dependence, flow characteristics, etc. They also show that secondary flow was not that important. Very preliminary trials of the large lab-scale device by using two permanent magnet arrays in some well-chosen position, and using feeds of (magnetic) magnetite, hematite, wolframite and Cospheric particles were done, and a classification in between 60-80 % was achieved.
In addition USAL analyzed several magnetic separating devices that, using several cycles, could reach magnetic classification, in order to compare costs and efficiency. The idea was to determine which comparing devices may be built and applied (HGMS, HGMF, MSFB's, etc.) in order to validate the future results obtained by the MAGCLA and determine the real improvement made by the new device.
Potential Impact:
Final Report 4.1 (SESAM) – Dissemination and Potential Impact
SOL
The MagPro²Life Project successfully demonstrated the magnetic fishing technology to be sustainable, safe, and capable of scaling to high production rates, reliable, robust and economically competitive to current existing downstream processing techniques. As DuPont acquired Solae in full on May 1, 2012, and became part of the Nutrition and Health Business Unit, a wide range of opportunities becomes available in terms of applications for this technology. Areas are as wide as digestive, immune, bone, heart, oral and weight management products, taste, and texture and appearance components. Areas are as wide as digestive, immune, bone, heart, oral and weight management products as well as taste, texture and appearance components, protective ingredients, and reduced food waste.
The outcome of the 4-years study on the Magnetic Fishing will be presented on the F2F DuPont T&I Leadership meeting in Europe, November 2013. In the mean time we will approach the scientific community through targeted groups within DuPont by the means of F2F or webinar meetings. DuPont has the worldwide patent on the Magnetic separation technology.
DTU
The data, procedures and models used for comparing magnetic adsorbent based purification of BBI with competing technologies and for scale up production are an extremely important part of marketing the technologies developed in the consortium. It is expected to stimulate widespread commercial interest in the biotech and food industry on the potential of the technology and is expected to spark demand for the particles and equipment developed in the consortium.
The bio-based economy with bio-refineries and bio-fuels at the forefront are the hottest topics in society today. These technologies rely on enzymatic hydrolysis of the renewable resources used for substrates. Free enzymes are used in these processes which are lost during processing and contribute to making the technologies expensive or with extremely marginal profitability in the case of bio-fuels. Magnetic recyclable enzymes can be expected to revolutionize the production processes, and the MagPro2Life consortium partners involved in particle manufacture and separator design have a marked competitive advantage that they can exploit.
Exploitation of the results from DTU in the public arena has been/is in the process of being done in the following ways:
• Alftren, J., Søndergaard, W., Hobley, T.J. (2013) Immobilization of beta-glucosidase by adsorption on anion exchange magnetic particles enables enzyme re-use during hydrolysis of pretreated wheat straw. Manuscript submitted to J. Biotechol., 17th July 2013.
• Søndergaard, W., Alftren, J., Hobley, T.J. (2013) Pilot scale demonstration of magnetic immobilised beta-glucosidase for repeated lignocellulosic hydrolysis. Manuscript in preparation for submission August 2013.
• Søndergaard, W., Ottow, K and Hobley, T.J. (2013) Benchmarking magnetic separation against competing technologies. Manuscript in preparation for the book: Advancing magnetic separations for food, feed and farma, Edited by Nirschl, H.
• Cerff, M., Scholz, A., Käppler, T., Ottow, K.E. Hobley, T.J. Posten, C. (2013) Semi-continuous in situ magnetic separation for enhanced extracellular protease production – modeling and experimental validation. Biotechnol. Bioeng, 110: 2161-2172
• Maury, T.L.M Ottow, K.E. Brask, J., Villadsen, J., Hobley, T. (2012) Use of High-Gradient Magnetic Fishing for Reducing Proteolysis During Fermentation, Biotechnology Journal, 7: 909-918
• Ottow, K.E. Lund-Olesen, T., Maury, T.L. Hansen, M.F. Hobley, T.J. (2011) A magnetic adsorbent based process for semi-continuous PEGylation of proteins. Biotechnol. Journal. 6:396-409.
• Peuker, U., Thomas, O., Hobley, T., Franzreb, M., Berensmeier, S., Schäfer, M., Hickstein, B. (2010) Bioseparation, Magnetic Particle Adsorbents. In Wiley Encyclopedia of Industrial Biotechnology, Flickinger, Michael C (Ed), ISBN 978-0471799306.
Oral conference presentations:
• Using magnetic adsorbents for protein modifications. 8th European Symposium on Biochemical Engineering Science (ESBES), September 5th – 8th, 2010. Oral presentation.
KIT-IFG-MVM-IBLT
Biotechnological production processes compared to chemical ones have certain benefits like reduced energy consumption and increased selectivity. However due to the usually diluted product streams downstream processing is often related to multi step processes with huge buffer consumption and accordingly a high number of waste streams. The introduction of new integrated processes based on magnetic separation is helping to increase the sustainability significantly. Through partial substitution of multiple process steps (product capture, concentration and purification) by one magnetic separation step, resource consumption and waste generation are reduced. Furthermore also the reduction of product losses during the downstream procedure helps to increase the yield of a given process, thus further improving the economic viability and sustainability. The magnetic separators and processes developed by KIT IFG, MVM and IBLT proved to be efficient in this context. Through the setup and operation of the feed pilot line as well as through the integration of the different magnetic separation devices into other pilot lines, it could be demonstrated that specific procedural advantages are achievable by the respective magnetic separation processes. In detail increased productivity of the bio-process was achieved in the feed pilot line process. For the pharma pilot line efficient product purification was attained using ion-exchange functionalized magnetic particles in combination with the magnetic enhanced centrifuge (MEC) and the aqueous two phase separator. Furthermore also the implementation of the MEC into the food pilot line process revealed potential economic benefits.
The knowledge generated by all three KIT sub partners during the project was presented in several university lectures and student courses. In addition research results were disseminated on 11 national and 13 international meetings and conferences in terms of oral and poster presentations. Overall 17 scientific publications and conference proceedings are representing the basis for the continued dissemination of the gained results. This should enable the fast and easy transfer of the knowledge generated by KIT onto other processes hereby contributing to the common welfare of society.
UBI
Conventional bioprocess manufacture shows heavy reliance on adsorption-desorption processes many if not most of which are chromatographic. Despite the key manufacturing roles ‘bind and elute’ chromatography processes play their future sustainability is cause for concern. They are: (i) costly; (ii) suffer from low productivity; (iii) employ high volumes of buffer in turn generating (iv) excessive amounts of waste. To address these issues UoB, in close collaboration with MagPro2Life partners (esp. KIT-IFG, KIT-MVM, MEK) advanced four integrated processes promising leaner, greener, and more sustainable manufacturing of valuable bio-commodities – three of them magnetically driven and the fourth chromatography based.
The main focus of MagPro2Life was the development and demonstration of magnet technology based processes for protein purification for 3 different product pilot-lines, i.e.: animal feed, food and biopharmaceuticals. All of UoB’s activities addressed MagPro2Life’s biopharma product pilot-line, and its primary roles were to: (i) set up and manage the complete (USP and DSP) biopharma pilot lines for demonstration of continuous magnetic extraction (CME), magnetically enhanced centrifugation (MEC) and high-gradient magnetic fishing (HGMF); (ii) design, manufacture and characterize (physically, chemically, functionally) novel and/or improved adsorbent materials specifically tailored for use in new engineering equipment, and scaling up their manufacture if required; (iii) integrate use of the new materials in the engineering equipment invented and furnished by KIT; and finally (iv) benchmark the new processes against conventional packed bed chromatographic purification routes.
Platform technologies for making a great many different polymer brush modified magnetic and chromatographic adsorbents were developed at UoB, and some of the high capacity and/or intelligent adsorbents made were tested in KIT-IFG’s engineering equipment. The concepts of all three magnet based approaches (CME, MEC, HGMF) were rigorously tested in UoB’s Biopharma pilot-line and benchmarked for the recovery of an antibody fragment product from crude periplasmic extracts of E. coli using magnetic cation exchange particles. All three MagPro2Life pilot-lines were subjected to economic feasibility studies, and the most favorable results were obtained for the bio-pharma line. Moreover, efficient and economic product purification was attained for all three magnetic particle based separation approaches employed for the bio-pharma product.
UoB has published 3 original full research papers (with KIT-IFG) in top journals thus far, and a book chapter on magnetic particle based separations (with TUBAF, KIT-IFG and DTU). A further 6 papers will be submitted in due course along with 2 PhD theses later this year. Moreover, UoB shall seek to gain IP related to its portfolio of adsorbent manufacturing methodologies, and especially some of the newer smart polymer brush chemistry it has invented recently. Additional UoB outputs include: (i) a total of 11 presentations at prestigious international conferences in Europe and the US; and (ii) the direct use of some UoB’s knowledge and experience gained during the running of MagPro2Life in its teaching of the MSc Biochemical Engineering Course (i.e. in its lectures, tutorials and even exam questions). No active efforts to enhance engagement with the wider general public were instituted by UoB during the course of this grant. We are nevertheless confident that the new adsorbent manufacturing methods, the materials themselves, and the new processes employing them, represent important scientific and technological advances in their own right, and further that their collective impact on the future well-being of the greater society will be positive, significant and lasting. The first example of this is likely to come from HGMF. Indeed, one of the most important of MagPro2Life’s outputs is the maturation of this technique to the point where it is now poised on the brink of its first true industrial application, and a bright future ahead.
MEK
Magnetic particles can preferably be used in an early stage of downstream processing with only little or partially cleared raw materials that cannot be directly loaded on conventional chromatography columns. Ideally one such capture/elution step might replace several traditional centrifugation/filtration/dialysis procedures.
The continuous synthesis of the basic magnetite particles (patented by Merck in 2008) will be transferred to our regular production unit to replace the current less reliable batch procedure. This will reduce the off-specification numbers and thus increase the overall economy and ecology of the process.
The ion exchange MagPrep TMAP and MagPrep SO3 particles are planned to be commercialized for the manual and/or automated capture/purification of biomolecules. The marketing is supported by the project applications/publications and strengthens the overall magnetic bead business of Merck.
Due to the increasing public awareness of the potential health hazards of nanoparticles, many commercial products (e.g. cosmetics) already need to be labeled accordingly. A new method to prepare and automatically scan/determine the number size distribution of (magnetic) beads from electron micrographs has been filed for patenting and might replace current less reliable procedures. The MagPrep TMAP toxicity study indicating the non-toxic nature of the particles further contributes to the acceptance of the material in commercial industrial processes.
FZMB
Within the project a Kappa light chain (kappa-IgLC) ELISA Kit (reactivity: human) was developed and is ready for commercialization. This ELISA is very interesting for the pharmaceutical industry (in-process-control) and R&D laboratories. The market for Fab development is still growing. For this reason the market potential of the ELISA is high. There are different competitors. The market price for an ELISA (96 well) is 600-800 €. Time to market is 12-14 months (stability charges). To improve exploitability the generation of a complementary “Lambda light chain ELISA Kit” is planned.
The Lunasin ELISA has a low marketing potential at the moment in a niche market for soy industry and R&D laboratories. More interesting is the production and commercialization of the native (purified) Lunasin as standard material. Purification was demonstrated by using a procedure consisting of anion exchange, reduction, ultrafiltration and reversed phase chromatography.
The developed Bowman Birk Inhibitor ELISA and also the mouse anti-BBI antibody are ready for commercialization. BBI is more common than Lunasin, but it is still a niche market (soy industry, R&D). A commercially available BBI ELISA was not found in the market (only patents and publications). There is one competitor for the BBI antibody (price: 375 €/mg). Time to market is 9-10 months for the BBI ELISA and 3-5 months for anti-BBI antibody.
Fzmb is owner of the cell lines for the capture antibody for the Kappa light chain ELISA Kit and for both antibodies of the BBI ELISA.
UCD
UCD has three publications coming out of the magpro2life project. Two of the papers relate to D47 where antibody-derived and peptide-based probes were identified.:
[1] Fields, Conor, Paul Mallee, Julien Muzard, and Gil U. Lee. "Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation." Food chemistry 134, no. 4 (2012): 1831-1838.
[2] Muzard, Julien, Conor Fields, James John O’Mahony, and Gil U. Lee. "Probing the soybean Bowman–Birk inhibitor using recombinant antibody fragments."Journal of agricultural and food chemistry 60, no. 24 (2012): 6164-6172.
The other publication is a protocol paper involving the techniques, genetic engineering and steps required to generate recombinant antibody fragments starting from hybridoma cells:
[3] Fields, Conor, David O'Connell, Sujing Xiao, Gil U. Lee, Philippe Billiald, and Julien Muzard. "Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies." Nature protocols 8, no. 6 (2013): 1125-1148.
Material for deliverable 30 “Apply pIII phage display to select phage that bind to the magnetic particles” was presented at the ACS national meeting in San Diego on the 27th March 2012. Material surrounding the first publication [1] was presented at Trinity College Dublin on the 31st May 2012
Exploitable foreground in the form of a patent entitled “Bowman-Birk protease inhibitor (BBI) binding peptides, and uses thereof in the separation, detection or purification of BBI” has been submitted. The described patent may be of interest to the feed and food industry.
ETH
The developed and up-scaled ROMER processing technology in particular if micro-engineered membranes with tailored pore and surface characteristics are applied is expected to have large potential in being a key process component for future industrial continuous and controlled productions of complex multiphase composite particle structures and their functionalization by layer adsorption and/or encapsulation. The processing principle was approved to work equally well for nano-/meso-scale entities like vesicles or bicelles as well as on macro-scale for solid triglyceride or wax particles. Economic potential for applications in food, pharma and cosmetics systems has been discussed with industrial partners and ROMER device commercialization is on the way /1,2/.
MANACO systems with magneto-responsive properties have been successfully developed on different length scales. Vesicles and bicelles doted with lanthanide atoms were shown to react in magnetic fields concerning permeability or orientation. Magneto-responsive bicelles were embedded in thermo-reversible gel structures from which temperature-time sensors or -integrators could be developed for which simple birefringence measurements provide clear information on the degree of sustained anisotropic oriented structure. The application of such elements is under approval for applications in intelligent packaging solutions. A patent has been filed and publications concerning the fundamental results were published /3,4/.
For the macroscopic composite particle based MANACOs tuned for separation processing of specific proteins from waste streams successful steps in surface coating and ligand attachment have been done, however binding selectivity and binding strength have to be further improved. Related further research work is planned, particularly for the area of nutrient and drug delivery systems with tailored gastro-intestinal release characteristics.
/1/ S. Holzapfel, E. Rondeau, P. Mühlich and E. J. Windhab (2013); Drop Detachment from a Micro-Engineered Membrane Surface in a Dynamic Membrane Emulsification; Process Chem. Eng. Technol. 2013, 36, No. 00, 1–11; DOI: 10.1002/ceat.201300256
/2/ Kaspar, P., Holzapfel, S., Windhab, E. W. and Jäckel, H. (2011). "Self-aligned mask renewal for anisotropically etched circular micro- and nanostructures." Journal of Micromechanics and Microengineering, 21, 115003.
/3/ Liebi, M., van Rhee, P.G. Christianen, P.C.M. Kohlbrecher, J., Fischer, P., Walde, P. and Windhab, E.J. (2013) "Alignment of Bicelles Studied with High-Field Magnetic Birefringence and Small-Angle Neutron Scattering Measurements" Langmuir, Vol. 29, 3467-3473.
/4/ Liebi, M., Kohlbrecher, J., Ishikawa, T., Fischer, P., Walde, P. and Windhab, E. J. (2012) "Cholesterol Increases the Magnetic Aligning of Bicellar Disks from an Aqueous Mixture of DMPC and DMPE–DTPA with Complexed Thulium Ions" Langmuir, Vol. 28, 10905-10915.
BUH
The aim of the ROMER II device was the production of Manacos (Magnetic Nano Containers in the form of beads or vesicles) in the µm range with a narrow particle size distribution. For this purpose the fluid which is transformed into beads is pressed through a fine membrane with fine pores into the shear field of a second fluid on the other side of the membrane. In doing so the buds which are build up on the membrane are broken away and the beads are formed.
This technology could be used commercially for the production of emulsions. There are different technologies used nowadays depending on the application.
The state of the art for the production of emulsions is the high pressure homogenization. This technology is based on the dispersion of a pre-emulsion under high pressure (>1000 bar). The produced emulsions have a broad particle size distribution.
There are other technologies like ultrasonic homogenizer or high shear dispersers which however need higher energy input or are not able to break down the droplets to the range under 1 µm .
TUBAF
During the whole project period, TUBAF has held several educational courses, seminars and oral presentations with international audience about technological implementation and application of the nanotechnology in the bio separation. The safety and ecological of improved SolPro (Solution Process) was pointed out to improve the acceptance of safe manipulation of nanotechnology and also to pave the way for industrial applications. The main process steps of SolPro to produce magnetic beads and their scale up potential for industrial application were presented too. It was demonstrated how the precipitation of magnetite, the phase transfer, the min-emulsion polymerization, the milling of macro-porous cation exchanger down to sub-micron scale and spray drying works on the lab scale. Technological innovation like the use of milling technologies for the production of low cost submicron sized cation exchange resins was particularly highlighted.
Posters and oral presentations have been given at different national (Fachausschuss Grenzflächenbestimmter Systeme und Prozesse, 2010 and 2012) and international (World Congress of Particle Technology 6, 2010 as well as European Congress of Chemical Engineering, 2012) conferences. In addition to publications in national and international journals (Chemie Ingenieur Technik, Encyclopedia of Industrial Biotechnology), TUBAF has filed a patent on the production of sub micron macro-porous ion exchange resins using mechanical milling technologies (Verfahren zur Herstellung von submikronen makroporösen Ionenaustauschermaterialien durch mechanische Zerkleinerung, 2012).
In MagPro²life, TUBAF has improved “Solution Process” for the bio separation. Due to the incorporation of several new synthesis techniques getting scale up potential, CEX and AEX magnetic beads were successfully synthesized. Beads characterization has proved, that both types were equipped with the appropriate functionalities allowing their application in the bio separation (super paramagnetic, ion exchange properties). Additional to this, the synthesis techniques and materials used are low cost. Therefore, the novel magnetic beads production technologies offer wide range of applications. The low cost adsorbents show for example a high potential for water conditioning as well as rare earth elements processing.
RATB
Highly reproducible procedures were developed for the kg-scale synthesis of magnetite nanoparticles with hydrophobic and hydrophilic surface coating by applying the method of chemical co-precipitation and using vegetable origin oleic acid surfactant (Merck product). The low mean size (below 10 nm) and surfactant coating ensure superparamagnetic behavior up to close packing of nano-particles which are essential features for their use as primary particles in manufacturing functionalized magnetoresponsive nano-composites for magnetic separation of bio-materials, magnetic drug targeting and MRI & MPI contrast agents. The procedures developed refer also to large scale synthesis of magnetic nano-fluids with water and light organic carrier liquids with potential impact on magnetic fluid based technologies.
There were 4 papers (2 Coll&Interface Sci 2012, Soft Matter 2013; 2 AIP Proceedings ), and 1 book chapter (Karger Publ. Co.,Basel), 19 presentations at conferences, symposiums and workshops, among them 4 invited talks.
It is worth to evidence the participation at several outstanding European scientific events such as Colloids and Materials 2011 Amsterdam, Colloids and Nanomedicine 2012 Amsterdam, IMAGINENANO 2013 Bilbao.
NIIMT
NIIMT obtained different hybrid magnetic nanostructures with controlled morphology, superparamagnetic behaviour, high magnetization, chemical and mechanical stability: polylactones coated magnetite nanoparticles, surfacted cluster of magnetite nanoparticles, magnetic microgel with anion and cation exchange properties.
Depending on the functionality and size these bio-compatible magneto-responsive nano-materials developed in the project have several applications with potential economic impact: new technologies of magnetic separation of bio-materials that can be taken by the pharmaceutical industry; nano-medicine - contrast agents in high resolution MRI imaging and magnetic carriers in drug transportation and release to the target.
The dissemination of the results have been done by: Publications - 2 papers (1 published in ISI journal Soft Matter and 1 published in American Institute of Physics Conference Proceedings); 1 book chapter Ed. Karger Publ Co. and participations at international conferences and workshops: 12. Among the conferences it is worth to mention our participation to large European events in nanoscience like Colloids and Materials 2011 Amsterdam, Colloids and Nanomedicine 2012 Amsterdam, IMAGINENANO 2013 Bilbao, EuroNanoForum 2013 Dublin.
NILPRP
The iron-oxide based nanoparticles were dispersed in aqueous media at near neutral pH by functionalization with L-DOPA biomolecule and can have potential application in drug delivery or MRI contrast agents.
The Fe-FexC@C nanocomposites have the potential to by introduced in PNIPAA-PAA microgels as magneto-responsive composites for magnetic separation of targeted high-value proteins from complex mixtures. Also, these Fe-C nano-composites were functionalized with CMCNa poly-electrolyte as stable aqueous dispersions and could be used in hyperthermia anticancer therapy. The maghemite L-DOPA coated nano-particles based aqueous suspensions (20 g/l) have potential for advanced thermal transfer nanofluids due to their enhanced thermal conductivity and low viscosity. Two ISI-quoted papers in Applied Surface Science resulted from this research along with 11 presentations and posters at conferences, symposia and workshops, such as EMRS European Materials Research Society Spring or Fall Meetings (Strasbourg or Warsaw) and PARTEC - International Congress on Particle Technology (Nurnberg).
USAL
From the scientific point-of-view the main potential impact was to provide a proof on the MAGCLA concept. MAGCLA may impact in many fields. In fact, with magnetic classification we are potentially capable of selectively separating substances from each other using only one single step and one machine. The Proof-of-concept given shows this potential to be real. Applications are very broad, ranging from medical analysis until the treatment of diseases, from protein recovery to cell separation, from water decontamination to catalysis improvement, or from mining industries to recycling industries and reaction engineering.
The main dissemination activities were: organization of seminars, presentations in conferences, seminars, media briefings, press conferences, media divulgation, etc., presentations for students, scientific community and general public, and divulgation of investigation in nanotechnologies, magnetic separation and of the generated knowledge.
List of Websites:
www.magpro2life.eu
