Selective and predicable amine conjugation sites by kinetic characterization under excess reagents

The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]₀) based on the modified Michaelis–Menten equation, in which the initial rate constant per amine (k_NH2 = V_max/NH2/K_M) was determined by the maximum reaction rate (V_max/NH2) under saturated accessible sites and initial amine–linker affinity (1/K_M). Reductive amination (RA) displayed 3–4 times greater V_max/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]₀ or gradual addition to minimize the [L]₀/K_M ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/K_M, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.