High‐Affinity Rabbit Antibodies Directed Against Methotrexate: Production, Purification, Characterization, and Pharmacokinetics in the Rat

Abstract

We describe the production, purification, characterization, and disposition of rabbit polyclonal methotrexate antibodies. These antibodies are prepared for subsequent testing of a drug delivery approach to reduce systemic toxicity upon regional administration of methotrexate. The polyclonal antibodies were raised in New Zealand white female rabbits immunized with a methotrexate–bovine serum albumin conjugate. The anti–methotrexate antibodies were sequestered from rabbit serum through the use of a protein‐G affinity column which allowed for purification of up to 100mg of rabbit IgG in 30–40min. The extent of purification was demonstrated through SDS–PAGE and calculation of specific binding activity relative to total protein concentration. The purified antibodies have been shown to have high affinity (Keqrange: 1.8 × 108to 8.75 × 109M−1) and high selectivity for methotrexate. Preliminary pharmaco‐kinetic studies of the purified antibodies in the rat following a 6 mg/kg intravenous infusion (n= 4) indicate a steady state volume of distribution of 38.0 ±11.2mLkg−1, a systemic clearance of 0.92 ±0.67mLkg−1h−1and an elimination half life of 28.9 ± 7.9h (mean ± SD).