Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [≈2 biotins/ IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [¹²⁵I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200-300 kDa range. By contrast, direct mixing with [¹²⁵I]streptavidin rapidly gave larger oligomers of ≫669 and ≈440-669 kDa molecular mass, respectively. The biodistribution of the 200-300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440-669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.