Biacore 8k

Surface plasmon resonance (SPR) assay was performed on a Bio-Rad ProteOn XPR36 (Bio-Rad, Hercules, CA, USA) or a Biacore 8K (formerly GE Healthcare, now part of Cytiva, Marlborough, MA, USA). Human complement C5 proteins (wt or R885 variants) were immobilized on a Bio-Rad GLH sensor chip docked in ProteOn or on a CM5 sensor chip docked in Biacore 8K, followed by flowing of zilucoplan or eculizumab biosimilar at varying concentrations in 1× HEPES buffer (pH 7.4, 150 mM of NaCl, 1 mM of MgCl₂, 0.005% surfactant P-20, and 1% dimethyl sulfoxide (DMSO) or 1× phosphate-buffered saline (PBS) buffer (pH 7.4, 0.005% P-20, and 1% DMSO). The resulting SPR sensorgrams were recorded and analyzed using the software provided by the vendors to extract the association and dissociation rate constants (k_a and k_d) and the binding affinity (KD).
For the binding of C5 and C3b, human C3b (Complement Technology) was site-specifically biotinylated via the thioester using Thermo Fisher EZ-link™ maleimide-PEG2-biotin. Biotinylated C3b was immobilized on a Bio-Rad neutravidin sensor chip. C5 in the absence and presence of zilucoplan in 1×HEPES buffer pH 7.4 was flowed over the immobilized C3b, and the resulting SPR signals were recorded using a Bio-Rad ProteOn XPR36.

Surface plasmon resonance (SPR) assay was performed on a Bio-Rad ProteOn XPR36 (Bio-Rad, Hercules, CA, USA) or a Biacore 8K (formerly GE Healthcare, now part of Cytiva, Marlborough, MA, USA). Human complement C5 proteins (wt or R885 variants) were immobilized on a Bio-Rad GLH sensor chip docked in ProteOn or on a CM5 sensor chip docked in Biacore 8K, followed by flowing of zilucoplan or eculizumab biosimilar at varying concentrations in 1× HEPES buffer (pH 7.4, 150 mM of NaCl, 1 mM of MgCl₂, 0.005% surfactant P-20, and 1% dimethyl sulfoxide (DMSO) or 1× phosphate-buffered saline (PBS) buffer (pH 7.4, 0.005% P-20, and 1% DMSO). The resulting SPR sensorgrams were recorded and analyzed using the software provided by the vendors to extract the association and dissociation rate constants (k_a and k_d) and the binding affinity (KD).
For the binding of C5 and C3b, human C3b (Complement Technology) was site-specifically biotinylated via the thioester using Thermo Fisher EZ-link™ maleimide-PEG2-biotin. Biotinylated C3b was immobilized on a Bio-Rad neutravidin sensor chip. C5 in the absence and presence of zilucoplan in 1×HEPES buffer pH 7.4 was flowed over the immobilized C3b, and the resulting SPR signals were recorded using a Bio-Rad ProteOn XPR36.