A Modified Process for Preparation of Pathogen Inactivated RBC Using S-303 in Multiple Additive Solutions Maintains RBC Function and Reduces Immunologic Activity.
Authors: Erickson A.C. , Lee V.A. , Schott M.A. , Castro G.M. , Peterson T. , North A. 
Published: October 2019

Abstract
Background: Treatment of RBC with S-303 (acridine derivative) inactivates pathogens and leukocytes. The original process for S-303 treatment of RBC (oSRBC) for pathogen inactivation (PI) used 0.2mM S-303 and 2mM glutathione (GSH). Two randomized, controlled Phase III trials were voluntarily discontinued when antibodies, specific to residual acridine on oSRBC, were detected in 2 patients. Following this observation, a modified PI process using 0.2mM S-303 and 20mM GSH (mSRBC) was developed to reduce S-303 binding to the RBC surface which inactivates a broad spectrum of pathogens and maintains good RBC function.
Aims: This study was designed to demonstrate the reduction in binding of S-303 to the RBC surface and preservation of in vitro RBC function with MSRBC in preparation for Phase I clinical trials.
Methods: Leukoreduced RBC were prepared in Erythrosol, AS-3, AS-5, SAG-M or PAGGS-M additive solution. mSRBC were treated with 20mM GSH, pH 9.0, plus 0.2mM S-303 and incubated for 20 hours at RT. Post-treatment binding of S-303 to the RBC surface was assessed by gel agglutination test using a mouse monoclonal antibody specific to acridine. In vitro function was assayed pre- and 20 hours post-treatment, and weekly for 35 days (d) of storage. Parameters assessed for in vitro RBC function include intracellular ATP, 2,3-DPG, hemolysis, and extracellular potassium, glucose and lactate.
Results: No agglutination was detected by gel column for mSRBC compared to 4+ readings for oSRBC indicating a reduction in S-303 bound to the RBC surface. While S-303 treatment results in higher initial ATP levels compared to pre-treatment, the mSRBC ATP level is maintained throughout 35d of storage. At 35d, oSRBC displayed glucose and lactate concentrations comparable to control RBC. In contrast, mSRBC had lower glucose and higher lactate concentrations than control RBC, consistent with observed higher ATP levels from increased glucose metabolism. After 35d of storage, hemolysis for oSRBC, Erythrosol mSRBC, AS-5 mSRBC, SAG-M mSRBC, and PAGGS-M mSRBC was comparable to controls whereas AS-3 mSRBC hemolysis was similar with the exception of 1 donor in AS-3 that showed a higher hemolysis level. Extracellular potassium levels were unaffected by oSRBC and mSRBC treatment.
Conclusion: A modified PI process has been developed for RBC that significantly reduces S-303 bound to the RBC surface compared to the original process while retaining the critical RBC functions. The reduction in bound S-303 offers the potential for PI treatment for RBC without immunologic reactivity.
ATP (μmol/g Hb) % Hemolysis GAT Mean (SD) Mean (SD) oSRBC t=0 3.9 (0.2) 0.1 (0.0) t=20h 4.8 (0.3) 0.2 (0.0) 4+ 35 day 2.6 (0.4) 0.4 (0.0) mSRBC Erythrosol t=0 3.4 (0.2) 0.1 (0.0) t=20h 5.7 (0.1) 0.1 (0.0) 0 35 day 3.8 (0.2) 0.1 (0.0) mSRBC AS-3 t=0 3.4 (0.5) 0.2 (0.1) t=20h 6.2 (0.4) 0.3 (0.1) 0 35 day 3.7 (0.4) 0.7 (0.8) mSRBC AS-5 t=0 3.3 (0.2) 0.1 (0.1) t=20h 5.4 (0.7) 0.3 (0.4) 0 35 day 3.3 (0.4) 0.5 (0.3) mSRBC SAG-M t=0 3.1 0.0 t=20h 5.0 0.0 0 35 day in progress in progress mSRBC PAGGS-M t=0 3.1 0.0 t=20h 5.1 0.0 0 35 day in progress in progress