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. 2023 Apr 19;15(1):2195517. doi: 10.1080/19420862.2023.2195517

Figure 5.

An image of a bispecific antibody with one chain as Fab and the other as an scFv or spFv. On SEC, the spFv bispecific antibody is mostly monomer, whereas the scFv bispecific contains mainly an oligomer species. Mass spectrometry data indicate that the stapling disulfide bonds are formed as designed.

Bispecifics with spFv show improved yields, product quality and expected disulfide formation in the stapled linker. (a) Schematic of BCMA (Fab) x CD3 (scFv/spFv) bispecific molecular architecture. HK in Fc regions indicate the knob-in-hole (K, knob; H, hole) mutations for Fc heterodimerization. RF (H435R and Y436F) mutations in the Fab containing heavy chain are introduced for purification to prevent binding to Protein A of RF containing chain monomers or homodimers. (b) SEC profiles post-CH1 of scFv/spFv Cris7b containing molecules with mAb1 indicate presence of oligomer species (labeled O) in scFv proteins that is absent in spFv proteins (monomer, M). (c) MS2-HCD spectrum of the peptide derived from non-reduced proalanase digestion representing the expected stapled disulfide linkage between Cys119-Cys237. The b- and – y type backbone fragments from each peptide half are annotated in the sequence map that is composed of Pep1 and Pep2 connected via the disulfide bridge.