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

ABSTRACT

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.