Albert D Donnenberg1, Tamir Kanias2, Darrell J Triulzi3, Catherine J Dennis4, Linda R Moore4, E Michael Meyer5, Derek Sinchar6, Joseph E Kiss3, Daniel P Normolle5, Mark T Gladwin6. 1. University of Pittsburgh School of Medicine, Department of Medicine, Pittsburgh, Pennsylvania, USA; University of Pittsburgh Medical Center Hillman Cancer Center, Pittsburgh, Pennsylvania, USA; McGowan Institute of Regenerative Medicine, Pittsburgh, Pennsylvania, USA. Electronic address: donnenbergad@upmc.edu. 2. Vitalant Research Institute, Denver, Colorado, USA. 3. University of Pittsburgh School of Medicine, Department of Medicine, Pittsburgh, Pennsylvania, USA; Vitalant, Pittsburgh, Pennsylvania, USA. 4. University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. 5. University of Pittsburgh Medical Center Hillman Cancer Center, Pittsburgh, Pennsylvania, USA. 6. University of Pittsburgh School of Medicine, Department of Medicine, Pittsburgh, Pennsylvania, USA.
Abstract
BACKGROUND: Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. METHODS: We have developed a closed-system, current good manufacturing practices (cGMP)-compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. RESULTS: The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin-labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. DISCUSSION: We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.
BACKGROUND: Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. METHODS: We have developed a closed-system, current good manufacturing practices (cGMP)-compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. RESULTS: The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin-labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. DISCUSSION: We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility.
Authors: Albert D Donnenberg; Tamir Kanias; Darrell J Triulzi; Catherine J Dennis; E Michael Meyer; Mark Gladwin Journal: Transfusion Date: 2019-06-06 Impact factor: 3.337
Authors: Cassandra D Josephson; Simone Glynn; Sunitha Mathew; Rebecca Birch; Sonia Bakkour; Lisa Baumann Kreuziger; Michael P Busch; Kathleen Chapman; Carla Dinardo; Jeanne Hendrickson; Eldad A Hod; Shannon Kelly; Naomi Luban; Alan Mast; Philip Norris; Brian Custer; Ester Sabino; Bruce Sachais; Bryan R Spencer; Mars Stone; Steve Kleinman Journal: Transfusion Date: 2022-04-19 Impact factor: 3.337