Large scale production and purification of human IL-2 from buffy coat lymphocytes stimulated with 12-O-tetradecanoylphorbol 13-acetate and calcium ionophore A23187.
Methods for the production of high titers of interleukin-2 (IL-2) from human buffy coat lymphocytes, and subsequent purification of the IL-2 are described. 50 buffy coats containing 1 X 10(11) leukocytes were first depleted of erythrocytes by batchwise leukapheresis using a Haemonetics model 15 blood wash centrifuge. Further lymphocyte enrichment was achieved using a one-step sedimentation in the presence of hydroxyethyl starch, which produced suspensions of more than 90% lymphocytes. This degree of lymphocyte purity was important since phagocytes were inhibitory to 12-O-tetradecanoylphorbol 13-acetate/calcium ionophore (TPA/A23187)-induced IL-2 production when their concentration exceeded 15% of the total cells. Cell culture was performed in stirred fermenters. Using TPA/A23187 induction, up to 500 micrograms of IL-2 per liter were produced. The IL-2 was purified by absorption from the supernatants onto controlled pore glass and elution with 50% ethylene glycol, followed by Fractogel chromatography, and then preparative high-performance liquid chromatography (HPLC) using an RP-6 column and elution with a gradient of n-propanol. A final HPLC rechromatography step using an analytical RP-6 column gave a homogeneous preparation with specific activity of 1.2 X 10(7) U/mg and a recovery from the starting supernatant of 22%.
D000001:Calcimycin / Q000494:pharmacology; D002469:Cell Separation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004357:Drug Synergism; D006801:Humans; D007376:Interleukin-2 / Q000302:isolation & purification*; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000032:analysis* / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007376:Interleukin-2; D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate