Agglomeration behaviour of magnetic microparticles during separation and recycling processes in mRNA purification

Abstract

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.

Development of a Single‐Use Device and an Associated Optical Measurement Method for Automated Magnetic Bioseparation

“…Due to the relatively open structure of the separator matrix for allowing optical measurements, magnetic induced agglomeration within the separation chamber is beneficial. As described in literature [11,31], magnetic beads attract each other when a magnetic field is applied. This agglomeration behavior is amplified with higher concentrations due to higher contact possibilities of single particles.…”

“…For performing separation experiments, a special batch of M-PVA-C22 magnetic beads (PerkinElmer chemagen Technologie GmbH, Germany) in deionized water was chosen. With a carboxylic group the magnetic beads can be functionalized with enzymes [2,14] or oligonucleotides, making them suitable for mRNA purification [11,28]. Magnetic bead concentrations of c B,0 = 0.2-4 g L -1 were used for performing HGMS.…”

“…High-gradient magnetic separation (HGMS) is a separation process in which magnetic particles can be selectively separated from a multi-component suspension in a flowthrough process using a magnetizable matrix (inside an HGMS separation chamber) and an external magnetic field. Appropriately suited magnetic particles are used for immobilization of enzymes in biocatalysis [1][2][3][4][5] or for separation of biomolecules [6][7][8][9], such as antibodies [10] and mRNA from cell lysates, respectively [11,12].…”

Numerical simulation of contact and separation of magnetic particles under uniform magnetic field

“…Due to the high surface-to-volume ratio and the control of surface properties, magnetic particles are very suitable for cell capture [5]. In addition, the dynamic behavior of magnetic particles can be precisely controlled using an external magnetic field, such as performing buffer exchange washing step [6], rapid immunoassay [7,8], or achieving effective fluid mixing [9][10][11][12]. With the increasing demand for the design of artificial external control structures, the precise control of magnetic particles under the magnetic field has become very important.…”