BHK-derived FVIII exhibits increased reactivity with αGal-KO serum and increased immunogenicity in αGal-KO mice. (A) Anti-αGal antibodies were evaluated by flow crossmatch analysis using αGal⁺ RBCs. Results are reported as mean fluorescence intensity (MFI). (B) Incubation of serum from αGal-KO mice with microarray containing BHK-derived FVIII (Helixate) or CHO-derived FVIII (Advate), followed by analysis of bound antibody. (C) Schematic diagram outlining the method used to absorb and elute αGal-specific antibodies from the serum of αGal-KO mice. (D) Evaluation of serum preabsorption, postabsorption on αGal RBCs, and antibody eluate obtained following elution of serum specifically absorbed onto αGal⁺ RBCs. (E) Evaluation of anti-FVIII antibody levels by ELISA following injection of BHK-derived FVIII into WT or αGal-KO recipients. (F) Evaluation of anti-FVIII antibody levels by ELISA following injection of CHO-derived FVIII into WT or αGal-KO recipients. ***P < .001, ****P < .0001, Student t test (A-B,E-F); ***P < .001, ****P < .0001, 1-way analysis of variance with Tukey’s post test (D). ns, not significant; RFU, relative fluorescence unit.