66 Chemische Verfahrenstechnik
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- Magnetisches Trennverfahren (2)
- high-gradient magnetic separation (2)
- mRNA-Impfstoff (2)
- magnetic beads (2)
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Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)-functionalized magnetic beads after cell lysis. The mRNA-loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high-gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow-through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)-functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of cB = 1.6–2.1 g·L–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min–1 with the developed SU-HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min–1, the retained particle mass is even more than 99.99%.
Purification of mRNA with oligo(dT)-functionalized magnetic particles involves a series of magnetic separations for buffer exchange and washing. Magnetic particles interact and agglomerate with each other when a magnetic field is applied, which can result in a decreased total surface area and thus a decreased yield of mRNA. In addition, agglomeration may also be caused by mRNA loading on the magnetic particles. Therefore, it is of interest how the individual steps of magnetic separation and subsequent redispersion in the buffers used affect the particle size distribution. The lysis/binding buffer is the most important buffer for the separation of mRNA from the multicomponent suspension of cell lysate. Therefore, monodisperse magnetic particles loaded with mRNA were dispersed in the lysis/binding buffer and in the reference system deionized water, and the particle size distributions were measured. A concentration-dependent agglomeration tendency was observed in deionized water. In contrast, no significant agglomeration was detected in the lysis/binding buffer. With regard to magnetic particle recycling, the influence of different storage and drying processes on particle size distribution was investigated. Agglomeration occurred in all process alternatives. For de-agglomeration, ultrasonic treatment was examined. It represents a suitable method for reproducible restoration of the original particle size distribution.
The implementation of single-use technologies offers several major advantages, e.g. prevention of cross-contamination, especially when spore-forming microorganisms are present. This study investigated the application of a single-use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040-09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease-inhibiting substance in the single-use bioreactor were similar to those in the glass bioreactor.
The present work aimed at investigating an extraction protocol based on consecutive steps of isoelectric point (pH ~ 4.25) mediated gum swelling and deproteinisation as an alternative method to produce flaxseed gum extracts of enhanced techno-functional characteristics. The osidic and proximate composition, structure conformation, flow behaviour, dynamic rheological and thermal properties of gums isolated from brown and golden flaxseeds were assessed. Gum extraction under near-to-isoelectric point conditions did not impair the extraction yield, residual protein and ash content, whilst it resulted in minor changes in the sugar composition of the flaxseed gum extracts. The deconvolution of the GPC/SEC chromatographs revealed the presence of four major polysaccharidic populations corresponding to arabinoxylans, rhamnogalacturonan–I and two AX-RG-I composite fractions. The latter appeared to minimise the intra- and interchain polymer non-covalent interactions (hydrogen bonding) leading to a better solvation affinity in water and lyotropic solvents. Golden flaxseed gums exerted higher molecular weight (Mw = 1.34–1.15 × 106 Da) and intrinsic viscosities (6.63–5.13 dL g−1) as well as better thickening and viscoelastic performance than the brown flaxseed gum exemplars. Golden flaxseed gums exhibited a better thermal stability compared to the brown flaxseed counterparts and therefore, they are suitable for product applications involving severe heat treatments.
Cryotropic gelation is one of the most common approaches to design novel hydrogels with multifaceted technological and biological functionalities. In the present paper, we studied the ability of highly galactosyl-substituted galactomannans, i.e. fenugreek and alfalfa gum, to form physically crosslinked hydrogels via cryogenic processing. Cycling of the galactomannan solutions (0.25 to 4% wt) from 25 to −20 to 25 °C induced the physical crosslinking of the galactomannan chains leading to the formation of different cryogel structures, i.e. filamentous aggregates (c* < c < 1%), cellular-like gel networks (1 ≤ c < 4%) or a homogeneously swollen gel (c ≥ 4%), depending on the total biopolymer content. Alfalfa gum-based cryogels exhibited higher elasticity and stiffness, better uniformity of the structure and a lower macropore size than their fenugreek counterparts. The physical blending of alfalfa or fenugreek gum with locust bean gum (2% total biopolymer) led to the reinforcement of the mechanical properties of the cryogels without significantly altering their microstructural aspects.