1Research & Development, TRC Biotech, Huntingdon Valley, PA, USA; ²University of Miami School of Medicine, Miami, FL, USA; ³Research & Development, Immunicon Corporation, Huntingdon Valley, PA, USA

A new magnetic particle is described that is superior to current magnetic separation technologies based on superparamagnetism for certain applications in the field of cell separation. The particles are particularly suited to studies in diluted and undiluted blood and bone marrow especially for purging unwanted cells. The particles are ferromagnetic and composed of nickel. Remarkably, ferromagnetic particles (FMP) (0.2 -1.4 micron) can be dispersed following exposure to a magnetic field (1.4 micron particles aggregate to 5 micron particles following exposure to a magnetic field but simple vortexing returns the particles to 1.4 micron). This property was formerly attributed only to superparamagnetic particles. As a result key features of FMP include (1) extremely rapid magnetic separation times on the order of seconds, (2) rapid cell capture times on the order of seconds to minutes and (3) because the particles are metallic, almost complete lack of non-specific binding/trapping of non-targeted cells which leads to very high recovery of desired cells, a particularly important attribute when recovery of rare cells, i.e. stem cells is the desired goal.

Magnetic collection time was investigated using a spectrophotometer with a modified cuvette that had magnets attached to the two sides of the cuvette not in the light path. Magnetic collection time was analyzed using light scatter as a function of time. FMP separated in 3-5 seconds. Large superparamagnetic particles (4.5 micron) collected in 20 seconds while nanometer size particles required >250 seconds. Binding kinetics is also extremely rapid with FMP. Using CD15-FMP as a model system in undiluted whole blood, mixing times as rapid as three seconds was sufficient to capture CD15 positive granulocytes. Analysis of the depleted sample revealed > 99% depletion of granulocytes (23,230 prior to depletion vs 206 post depletion). Total mixing plus magnetic separation time: <10 seconds. Most importantly, the metallic nature of FMP results in the elimination of nonspecific binding/trapping of non-targeted cells often seen with separation technologies based on superparamagnetic particles. In the above example on removal of CD15 positive cells no lymphocytes were depleted (12,694 pre-depletion vs. 12,815 post depletion). Using a rare event model system, 40 PC3 cells (prostate cancer cell line) were spiked into 7 ml whole blood. Following depletion of leukocytes using CD45-FMP 35 PC3 cells were recovered (PC3 cells were counted using Immunicons rare event cell assay). In conclusion, a new magnetic particle has been discovered that is uniquely suited to purging unwanted cells while yielding high recovery of desired, non-targeted cells. The procedure is extremely user friendly and suited to both research studies and large scale separations such as those used for stem cell transplantation. Finally, because of the extremely rapid reaction kinetics and magnetic separation times, the particles permit immediate isolation of cells for down stream processing. These rapid isolation times are not available with current magnetic separation technologies.

Quick-Sep^TM Ferromagnetic Cell Selection Technology is based on particles made of NICKEL. The technology is superior to current technologies because of three key properties of NICKEL:

Density Nickel has a density of 8.9 g/cm³ which is ~8 times the density of cells and ~6 times the density of current magnetic particles. This difference in density results in very high mixing efficiency which in turn yields very rapid reaction kinetics often on the order of seconds.

Magnetic Properties Because Quick-Sep^TM particles are composed of solid Nickel, the particles are ferromagnetic. This results in magnetic separation times up to ten times faster than current technologies that are based on superparamagnetic particles.

Particle Surface Non-targeted cells do not stick to Quick-Sep^TM particles because of the characteristics of the particle surface. As a result only targeted cells are captured.

Magnetic separation technology evolved around superparamagnetic particles since ferromagnetic particles remain magnetized following placement in a magnetic field. The key to Quick-Sep^TM ferromagnetic particle technology is that, remarkably, particles less than 1.8 micron in diameter can be easily dispersed by simple vortexing after the particles have been magnetized.

Since Quick-Sep^TM particles are ferromagnetic, they are more magnetic than superparamagnetic particles. As a result of this difference, Quick-Sep^TM particles demonstrate faster magnetic collection times.

A desirable attribute of Quick-Sep^TM FMP is the lack of nonspecific binding of non-targeted cells. FMP coated with CD15-antibody were used to deplete >99% of the granulocytes and a subset of monocytes known to be CD15 positive from whole blood with quantitative recovery of the non-targeted lymphocytes (top figure next page). A separate experiment demonstrated that FMP coated with CD15, CD45, and CD4 depleted greater than 99.5% of the leukocyte population (top figure next page).