Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent

The experimental conditions for the preparation of conjugates of ovalbumin (OA) and monomethoxypolyethylene glycol (mPEG) of a preselected average degree of conjugation, n, using cyanuric chloride as the coupling agent, have been investigated with emphasis on purification and characterization of the products. These conjugates served as prototypes of tolerogenic mPEG derivatives of antigenic proteins which were capable of suppressing in mammals the immunological response to the corresponding unmodified antigens. In other studies in this laboratory, the tolerogenicity of OA(mPEG)n conjugates was found to be a function of n. The reproducibility of the reaction leading to the production of OA(mPEG)n conjugates was shown to depend primarily on the reactivity of the mPEG-cyanuric chloride intermediate, which--for best results--had to be synthesized under completely anhydrous conditions. Isolation of the OA(mPEG)n conjugates was optimized by the use of ion-exchange chromatography whereby rapid removal of large amounts of uncoupled intermediate from the conjugate was achieved; the conditions of fractionation were affected by the degree of conjugation. This method of purification was superior to dialysis, ultrafiltration, and gel filtration. Furthermore, by the application of analytical hydrophobic interaction HPLC it was possible to differentiate among conjugates of different degrees of conjugation and to establish the absence of any detectable free OA in any of the preparations. The quantity of mPEG in the conjugates was determined directly by NMR.