Upscaling of Bio-Nano-Processes

Nanoparticles (NPs) are submicron moieties made of inorganic or organic materials, which have many novel properties compared with their bulk counterparts. The CO₂ laser pyrolysis of gas- and vapor-phase reactants offers an approach for the synthesis of uniform nanoparticles. The synthesis of iron oxide-based nanomaterials by laser pyrolysis has been achieved by a handling procedure, in which the oxidation process initiates and develops inside the laser-induced reaction zone. In a second step, a more complex experimental procedure is used, in which the iron precursor is allowed to dissociate alone in the flame with a surrounding oxidizing atmosphere. XRD and TEM analyses reveal a major content of maghemite/magnetite in samples SF. The power density and the nozzle diameter influence the particle size.

The building blocks for the fabrication of biocompatible magnetoresponsive carriers are the subdomain magnetite nanoparticles surface coated by biocompatible molecular layers which ensure their stabilization and dispersion in an appropriate carrier liquid, in order to obtain stable magnetic nanofluids that are the primary materials for the envisaged magnetic nanocomposites. The synthesis by chemical coprecipitation is the most simple and cost effective route to obtain hydrophobic and hydrophilic magnetite nanoparticles at industrial scale. Manifold physico-chemical characterization of the magnetic nanofluids is employed in order to certify their requested composition and structure.

We present an innovative processing route based on dynamic membrane pore extrusion and multiphase flow, which allows for the controlled production of particle and core-shell microstructures with tailored techno-functional properties. In the context of MagPro²Life, two scalable continuous processes based on this technology were specifically designed and implemented for the preparation of (i) surface functionalized magnetic composite particles and (ii) functional vesicles, using two respective devices: (i) a novel ROtating MEmbrane Reactor (ROMER) device and (ii) a NAno Membrane Pore EXtruder (NAMPEX) device. Extrusion and detachment of liquid drops and vesicles from pores in a surrounding immiscible continuous fluid phase under various flow conditions like co-flow, flow focusing and cross flow have been studied intensively at our Laboratory of Food process Engineering for a variety of different rheological and interfacial material characteristics within the past decade. Drop- and capsule deformation and breakup were studied in mechanistic detail utilizing in particular microfluidics technology. The coupling of drop/capsule formation with functionalization steps by either entrapment/encapsulation of components for controlled release or surface/interface modification for adjusted and more or less specific adsorption characteristics have been addressed with respect to the overarching goal of the project being the separation of specific protein fractions from soy whey streams.

The goal of Technische Universität Bergakademie Freiberg (TUBAF) in MagPro²life was to develop, based on Solution Process (SolPro), a low-cost scalable synthesis process as well as to optimize the efficiency of particle production regarding safety and ecological aspects. Depending on target protein charges in the extraction pH range, magnetic beads with cationic and anionic functionalities (CEX– and AEX–SolPro beads) were successfully synthesized and characterized. Both types of beads were equipped with the appropriate functionalities allowing their application in bioseparation (superparamagnetic, ion exchange properties). Characterization analyzes of the SolPro beads (CEX und AEX) showed the high potential of the developed processes. CEX–SolPro particles possess a lysozyme extraction capacity of approximately 280 mg/g, which is about three times higher than similar CEX-SolPro beads (same composition) produced by Hickstein and Peuker (2009) (77.6 mg/g). The newly designed and produced AEX–SolPro beads using poly(allyl)amine (PAAm) as anion exchanger exhibit extraction capacities of 85 mg/g for BSA as well as 145 mg/g for BBI. Several investigations proved that a quaternization of amino groups of poly(allyl)amine anchored on the beads surface increase their extraction capacity by a factor of 3. One kilogram of produced PAAm–SolPro was successfully tested by Solae Denmark in a pilot food line application.

Magnetite nanoparticles are produced in a continuous synthesis that allows the potentially unlimited production for large-scale applications. With the better control over the reaction parameters, the reproducibility is significantly improved. The particle size is tunable in a range of about 10–100 nm, and the distribution is significantly smaller than for those produced batch wise. The subsequent continuous surface modification of the pristine magnetite particles is difficult to date, especially if more than one chemical reaction is necessary. A batch preparation of kilogram of surface-modified ion-exchange magnetic particles for laboratory scale and pilot line purification experiments were realized. These ion-exchange particles could be recycled 50 times on an analytical scale without loss of efficiency. Several hundred grams of beads could be recovered 10 times with a stepwise efficiency of 99.9 %. A number of one-step protocols could be developed for the enrichment and purification of target proteins from different raw materials up to pilot-scale.

Magnetically enhanced centrifugation is a new approach in high-gradient magnetic separation possible in continuous mode, which promises a large potential for industrial use. Magnetic particles are separated to a wire filter, from which they are cleaned by centrifugal forces to the chamber wall. The simulation of magnetic particles collected by a wire were possible by the discrete element method. The simulation shows the detachment of particles from the wires by the centrifugal force. Two prototype machines were set up to investigate the process. The discharge of particles from the wire filter depends on the magnetic field strength and the centrifugal velocity. It was possible to set up a machine reaching a separation efficiency of 99 % at a volume flow of 1 m³/h (excluding dead times) in a batch-wise mode. A second machine built as magnetically enhanced decanter separates particles and transports them out of the bowl by a screw conveyor in a completely continuous mode. Instead of the common electromagnets a permanent magnet assembly can be combined with a magnetically enhanced centrifuge to save costs.

The scalability of economic high-gradient magnetic separation (HGMS) technology is essential in order to demonstrate the feasibility of the concept. One of the means is the application of a permanent magnet with a hollow cylindrical volume made from identical magnetic blocks (e.g., Mandhala), another is the development of a High-Gradient Magnetic Filter (HGMF) with a new backwashing concept. The Mandhala (Magnetic Arrangement for Novel Discrete Halbach Layout) magnet produces a dipolar transversal magnetic field in the center of the bore and its usable volume is easily adaptable to the separation device’s extensions. The chapter presents the pilot scale design of the Mandhala magnet and the HGMF as well as experimental performance tests using a water—magnetic beads model system. Subsequently, experiments using soy–whey as a real feedstock demonstrate the purification of the protein Bowman-Birk inhibitor (BBI), an agent against cancer and multiples sclerosis.

Continuous Magnetic Extraction (CME) is a process for the separation and purification of proteins from crude feedstocks. Magnetic particles with functional surface groups, e.g., ion exchange ligands, serve as carriers for the target protein. Together with a mixture of thermoresponsive surfactants and binding buffer these carriers are added to the feed containing the target protein. After binding the target within minutes, the mixture is heated above the LCST of the surfactant and phase separation is induced. Through selective partitioning of the carrier particles to the top phase and partitioning of contaminants to the bottom phase of the system, purification is possible. The proof-of-concept of this bioseparation process has been recently released, and new devices for CME have been developed within the framework of the EU project MagPro²Life and the respective results are reported in this chapter. Several particle types and feed streams, representing a broad field of potential applications, were tested with regard to their suitability for the CME process.

This study describes the design and development of an in situ magnetic separation process (ISMS) based on the use of ion exchange functionalized magnetic particles. ISMS as a tool for in situ product removal (ISPR) has the potential to increase the performance of a biotechnological production process comprehensively by improving the bioprocess itself and the downstream processing simultaneously. The successful implementation of this concept requires the systematic examination and optimization of the different ISMS subsystem. Hence, this report presents detailed characterization data for the magnetic particle system, the magnetic separator, and the bioprocess. For each system, specific requirements were defined and subsequently applied to identify suitable process components. In this context always economic considerations were also accounted to enable the application of this new process as cost-efficiently as possible. Especially the selection and modification of a magnetic particle system and the development of a low-cost magnetic separator were subjected to these basic criteria. For the final verification of the ISMS effects, all components were brought together in a pilot scale system and used to perform cultivation with integrated ISPR. The results of this cultivation were compared to reference cultivations without ISMS to quantify the effects on the upstream processing. In addition, a detailed analysis of the efficiency of purification was performed to evaluate the consequences on the downstream processing.

High-gradient magnetic filtration bases on the separation of target media by synthetic particles with magnetic core and an adsorbent immobilized on the surface. The process was used in a pilot line at liter-scale for the separation of a protein Bowman-Birk protease Inhibitor (BBI) out of an industrial coproduct stream in the soy industry. The target protein has potential applications as pharmaceutical or food additive. The starting medium is challenging as the product cost is low, its concentration is low and its purity needs to be high. The main method tested was magnetically enhanced centrifugation in a batch-wise mode with anion exchange ligands. The separation of the target protein at large scale was successful and economical. The efficiency was low due to the low ligand selectivity and the batch-wise processing. For commercialization, high ligand selectivity and continuous processing would make this new magnetic separation process very attractive. An economic study was performed to determine the influence of different parameters on prices. Competing technologies were evaluated. A risk analysis of nanoparticles used in the pilot line was performed.

The separation of proteins by High Gradient Magnetic Filtration is a promising process for the purification of media. Its success depends on four different elements: the synthesis of coated particles with magnetic core, a selective functionalization which needs to be adapted to adsorb the target matter and exclude contamination, the separation devices, which need to fit for the process in scale, budget and separation characteristics, and the process design. All of these elements need to fit together and to be available at a reasonable price.