IRX-2 modified manufacturing process
A highly efficient method of making a primary cell derived biologic by purifying mononuclear cells (MNCs) in a automated cell processor to remove contaminating cells by loading leukocytes onto lymphocyte separation medium (LSM) and centrifuging the medium to obtain purified MNCs, storing the MNCs overnight in a closed sterile bag system, stimulating an induction mixture of the MNCs with phytohemagglutinin (PHA) or other mitogen and ciprofloxacin in a scalable cell culture device and producing a primary cell derived biologic from the MNCs, removing the mitogen from the induction mixture by filtering, incubating the induction mixture, clarifying the induction mixture by filtering to obtain a primary cell derived biologic supernatant, and clearing the primary cell derived biologic supernatant from adventitious agents by anion exchange chromatography, filtration. A closed system prevents contamination of the resulting primary cell derived biologic. An automated method of purifying cells. A method of scalably inducing cells.
1. A method of making a primary cell derived biologic, including the steps of:
(a) removing contaminating cells from mononuclear cells (MNCs) by loading leukocytes onto lymphocyte separation medium (LSM), and washing and centrifuging the medium with an automated cell processing and washing system;
(b) storing the MNCs overnight in a closed sterile bag system;
(c) stimulating the MNCs with a mitogen and ciprofloxacin in a disposable cell culture system to produce cytokines;
(d) removing the mitogen from the mononuclear cells by filtering;
(e) incubating the filtered MNCs in a culture medium;
(f) producing a clarified supernatant by filtering the MNCs from the culture medium;
(g) producing a chromatographed supernatant by removing DNA from the clarified supernatant by anion exchange chromatography; and
(h) removing viruses from the chromatographed supernatant by filtering with dual 15 nanometer filters in series,
thereby producing a primary cell derived biologic, wherein the primary cell derived biologic comprises IL-1β, IL-2, and IFN-γ.
2. The method of claim 1 , wherein step (a) further comprises removing contaminating cells from MNCs from multiple donors simultaneously.
3. The method of claim 1 , wherein step (a) further comprises centrifuging at 1500 to 3000 rpm for 20 minutes to optimize removal of granulocytes and red blood cells.
4. The method of claim 1 , wherein the mitogen is phytohaemagglutinin (PHA).
5. The method of claim 4 , wherein step (d) comprises lowering the level of PHA to less than 150 ng/mL.
6. The method of claim 5 , wherein step (d) comprises filtering in tangential flow mode.
7. The method of claim 1 , wherein step (d) comprises filtering in tangential flow mode.
8. The method of claim 1 , wherein the incubation in a culture medium is for 24 hours.
9. The method of claim 1 , wherein the filter of step (f) is a 0.45 micrometer filter.
10. The method of claim 1 , wherein step (h) further comprises applying ultraviolet-C (UVC) light to the chromatographed supernatant to clear the chromatographed supernatant of adventitious agents.
11. The method of claim 10 , wherein the UVC is applied uniformly by spirally flowing the chromatographed supernatant along an UVC irradiation source.
12. The method of claim 1 , wherein the ciprofloxacin is present in an amount of 80 micrograms/mL in step (c).
13. The method of claim 1 , wherein the culture medium of step (e) includes 80 micrograms/mL of ciprofloxacin.
14. The method of claim 1 , wherein the purified mononuclear cells (MNCs) in step (a) have a granulocyte content below 5%.
15. The method of claim 1 , wherein step (a) further comprises removing platelets to a level of below 1.2×10 10 cells.
16. The method of claim 1 , wherein the primary cell derived biologic comprises the cytokines IL-1β, IL-2, IL-6, IL-8, TNF-αand IFN-γ.
17. The method of claim 1 , wherein at least 4 L of the primary cell derived biologic is produced.