Hemolysis lysis assays for cynomolgus monkey or C5hu/hu mice PK/PD studies

For hemolysis assays, sheep and rabbit red blood cells were obtained from Colorado serum (cat#31112 and 31081, respectively). The hemolytic activity in serum samples from C5^hu/hu mice or cynomolgus monkey following SC injection of REGN3918, in-house eculizumab or IgG4^P control antibody was evaluated ex vivo using a hemolysis assay. Hemolysin-sensitized sheep red blood cells (SRBCs) were used for the classical complement pathway initiated hemolysis assay. Briefly, SRBCs were washed and resuspended at 1x10⁹ cells/mL in gelatin veronal buffer with CaCl₂ and MgCl₂ (GVB⁺⁺). To sensitize the SRBCs, cells were incubated with 0.75 mg/mL hemolysin at 37°C for 20 minutes. Sensitized SRBCs were further diluted in GVB⁺⁺ to 2x10⁸ cells/mL. Sera from C5^hu/hu mice were used at a 1:10 final dilution and were ex vivo supplemented with hC3 at final concentrations of 90 μg/mL. Monkey sera were diluted at 1:20 final dilution. A total of 1x10⁸ sensitized SRBCs in a volume of 100 μL were plated into round bottom 96-well plates followed by addition of 100 μL of diluted test serum. Sensitized SRBCs without addition of test serum (GVB⁺⁺ only) were included to determine background hemolysis, or were lysed by addition of distilled water to determine maximal hemolysis signal. Cells were gently mixed and incubated at 37°C for 10 or 60 minutes. Cells were pelleted by centrifugation at 1,250xg at 4°C for 7 minutes. A total of 100 μL of the supernatant was transferred to a fresh 96-well flat bottom plate and the OD412 was read on a Spectramax microplate reader. The hemolytic activity was calculated as follows:

Rabbit RBCs (RbRBC) were used for the alternative pathway initiated hemolysis assay. In brief, RbRBCs were washed and resuspended at 2x10⁸ cells/mL in gelatin veronal buffer with 40mM MgCl₂ and 40mM EGTA (GVB-Mg²⁺/EGTA). A total of 2x10⁷ RbRBCs in a volume of 100 μL were plated into each well of 96 well round bottom plates followed by addition of 100 μL of test serum (final 10%). RbRBCs without addition of test serum (GVB Mg^2+/EGTA only) were included to determine background hemolysis. The maximal hemolysis signal was determined by addition of distilled water to lyse the cells. Cells were gently mixed and incubated at 37°C for 60 minutes. Subsequently, hemolysis was quantified as described above.