Filtern
Dokumenttyp
Sprache
- Englisch (4)
Volltext vorhanden
- ja (4)
Gehört zur Bibliographie
- nein (4) (entfernen)
Schlagworte
- Magnetisches Trennverfahren (4) (entfernen)
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.
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%.
The introduction of functionalized magnetizable particles for the purification of enzymes or for the multi-use of pre-immobilized biocatalysts offers a great potential for time and cost savings in biotechnological process design. The selective separation of the magnetizable particles is performed for example by a high-gradient magnetic separator. In this study FEM and CFD simulations of the magnetic field and the fluid flow field within a filter chamber of a magnetic separator were carried out, to find an optimal separator design. The motion of virtual magnetizable particles was calculated with a one-way coupled Lagrangian approach in order to test many geometric and parametric variations in reduced time. It was found that a flow homogenisator smoothed the fluid flow, so that the linear velocity became nearly equal over the cross section in the direction of flow. Furthermore the retention of magnetizable particles increases with a high total edge length within the filter matrix.
The introduction of functionalized magnetizable particles and high-gradient magnetic separation represents a time and money saving alternative to conventional purification and separation unit operations in the biotechnical sector. This technique has some advantages especially for the recycling of immobilized enzymes. A new magnetic filter with sight glasses was constructed and produced to study the performance of high-gradient magnetic separation at varied parameters. By optical analysis the buildup of a clogging was identified as the major parameter which affected the separation performance. For the cleaning procedure, a two-phase flow of water with highly dispersed air bubbles was tested which led to a nearly complete cleaning of the filter chamber.