Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; Mr=5000), benzotriazole carbonate methoxyPEG (BTCmPEG; Mr=5000 or 20,000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; Mr=2000, 5000, or 20,000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; Mr=20,000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (≤2 mmol/L). The effective immunocamouflaging of RBC antigens coupled with efficient purification of the immunocamouflaged population provides encouragement for the further development of the stealth erythrocyte.