In vitro proliferation activity and EPOR binding activities of rHuEPO, darbepoetin alfa, AMG 114, and AMG 205 glycosylation analogs.
Notes: (A) H3-thymidine incorporation assays in UT-7/EPO cells demonstrates the inverse correlation between molecules with increasing numbers of N linked carbohydrate (rHuEPO = 3; darbepoetin alfa = 5; AMG 114 and AMG 205 = 7) and decreasing in vitro proliferation activity. Proliferation is measured as counts CPM of H3-thymidine incorporation into newly synthesized DNA and EC50 values of a representative experiment are shown. (B) Competitive binding assays were performed in UT-7/EPO cells whereby I125-rHuEPO was bound to cells and competed with increasing concentration of ESA for 2–3 hours. Cells were washed though phthalate oil, CMP was measured and IC50 calculated. A representative experiment is shown. Data kindly provided by Steve Elliott, Norma Rogers, and Tony Lorenzini, Amgen, Inc.
Abbreviations: CPM, counts per minute; EC50, 50% effective concentration; IC50, 50% inhibitory concentration; EPO, erythropoietin; EPOR, EPO receptor; rHuEPO, recombinant human EPO.