Table 2.
Free cysteine analysis of EGFR-HER2 DuetMab
| Disulfide-bond linkages | Cysteines | % Free thiols |
|---|---|---|
| L-H (X) (WT) | LC Cys214 | ND |
| L-H (Y) (Mut) | LC Cys121 | 0.20% |
| HC Cys 126 | 0.35% | |
| H(X) - H(Y) Stabilizing disulfide-bond | HC Cys349 | 0.92% |
| HC Cys354 | 0.53% | |
| CH3 (X) hole | HC Cys367 | 0.84% |
| HC Cys425 | 0.69% | |
| HC Cys367 | 5.14% | |
| CH3 (Y) knob | HC Cys425 | 6.50% |
| Hinge (H-H) | HC Cys 226,229 | 0.49% |
X indicates cetuximab- and Y indicates transtuzumab-derived peptides. Only inter-chain and the engineered intra-chain disulfide bond-linked cysteines in the CH3 domain are shown. Free thiols are capped using N-ethyl-malimide (NEM) before Lys-C digestion. The percentage of free cysteines were calculated using selected ions of Cysteine containing peptides with NEM addition divided by the selected ions of reduced peptides. As low as 0.2% free thiol or disulfide-bond scrambling can be detected.