RESUMEN
This study introduced an automated long-term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed-batch fermentation, repeated-batch fermentation was implemented with the help of additional bioreactor internals ("sporulation supports"). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems (e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.
RESUMEN
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.
RESUMEN
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%.
