AlloStim® and CRCL are manufactured in clean rooms under Good Manufacturing Practices. The process for making AlloStim® consists of four steps:
1. PURIFICATION OF CD4 HELPER CELLS FROM NORMAL BLOOD
Blood is collected from a normal, healthy blood donor. The donor is screened to be free from infection or viruses that can be transmitted by blood, including HIV, hepatitis and syphilis. The blood contains many types of cells. In order to make AlloStim®, it is necessary to purify the immune cells called “CD4” cells from the blood sample. In order to purify CD4 cells from the blood, monoclonal antibodies that are designed to attach only to CD4 cells are used. The antibody is then attached to a small magnetic bead. These small magnetic beads are mixed with the blood cells, the beads will attach only to CD4 cells and no other cells.
The mixture of cells and beads is then placed into a column, a magnet is then placed on the column. All the cells with magnetic beads attached will be attracted to the magnet. When the bottom of the column is released the cells without magnetic beads attached will flow out of the column. However, all CD4 cells attached to the beads will be held in the column by the magnet. These purified CD4 cells are the source cells used to make AlloStim®
2. CELL CULTURE FOR 9 DAYS
The CD4 T-cells purified from blood of normal donors are the source cells for producing AlloStim®. The purified CD4 cells are mixed with another type of special microbead. These beads are coated with two different monoclonal antibodies. These antibodies attach to the CD4 cells and provide growth and differentiation signals to these cells. The cells mixed with these special beads are inoculated into cell culture bioreactors. The mixture of cells and beads are incubated for 9-days in the bioreactor. Over the 9-days the cells expand and differentiate into T-Stim® intermediary cells.
- The cells mixed with antibody-coated microbeads are incubated for 9 days. After 9 days in culture, the source CD4 cells expand and change into an intermediate cell type called T-StimTM
- The T-StimTM cells are harvested from the bioreactor using automated cell processing equipment
- Since the antibody-coated microbeads are paramagnetic, they will be attacted to a magnetic field. Thus by passing the bead/cell mixture over a powerful magnet, the T-StimTM cells can be separated from the microbeads.
- The T-StimTM cells are then aliquoted into single dose cryovials and stored frozen in liquid nitrogen in the laboratory. The intermediate T-StimTM cells can be stored in liquid nitrogen for several years.
In the fourth step of the AlloStimTM production process, the intermediate T-StimTM cells must be formulated and transformed into the final AlloStim® product prior to administration to a patient.
- Frozen T-StimTM dose vials are removed from frozen storage and thawed. T-StimTM cells are placed in an incubator located in a clean room
- Monoclonal antibody-coated microbeads are added to the cells (2 beads:cell)
- T-StimTM cells are incubated with the microbeads for 4h in the clean room. After 4h, the cells activate and change into AlloStim®
- The AlloStim® cells, with the beads still attached, are washed and placed in formulation media. These cells with beads are then aliquoted into syringes of 1ml, 3ml or 5ml. These syringes are placed in validated, temperature-controlled packages designed to maintain 40C.
The refrigerated syringes can be transported by overnight courier to clinic destinations anywhere in the world. The AlloStim® cells remain viable and functional for at least 72h after formulation.