Conjugatability and Physicochemical Stability of an Infliximab–vcMMAE Antibody–Drug Conjugate

Transmembrane tumor necrosis factor (tmTNF) can support internalization of anti-TNF antibodies, providing a mechanistic rationale for exploring anti-TNF antibody-drug conjugate formats. We evaluated infliximab as a scaffold for conventional interchain-cysteine conjugation using an MC-valine-citrulline-PABC-MMAE linker-payload (vcMMAE). Increasing tris(2-carboxyethyl)phosphine (TCEP) from 2.2 to 2.6 equivalents increased the average drug-to-antibody ratio (DAR) from 3.12 to 3.65 by reduced RP-HPLC and from 3.5 to 4.1 by hydrophobic interaction chromatography (HIC). SEC-detectable high-molecular-weight (HMW) species remained approximately 1% across the conjugates, comparable to unconjugated infliximab (1.1%). A selected preparation with an average DAR of approximately 4 showed no substantial change in its SEC profile after five freeze-thaw cycles or incubation at 37°C for 2 weeks. These results demonstrate that infliximab is amenable to conventional interchain-cysteine conjugation to approximately DAR = 4 without an evident increase in SEC-detectable HMW species and provide an initial chromatographic assessment of its post-conjugation physicochemical stability.