The aim of this paper was to characterize and to scale-up the CNBr chemical activation of Sepharose CL-4B in a stirred tank reactor to purify the recombinant Hepatitis B surface antigen for use in vaccines. The activation quality was assessed measuring the concentration of the cyanate esters and the percentage of nitrogen in the activated matrix. The Sepharose CL-4B incorporated less than 50% of the initial CNBr amount added to the matrix, but the monoclonal antibody CB.Hep-1, used as immunoligand to purify the rHBsAg, was efficiently immobilized in a weakly activated matrix (<8 ?mol/mL of matrix). The impellent type has a significant influence on activated matrix quality. The constant power per volume unit was a proper criterion to scale-up the Sepharose CL-4B March's modified chemical activation procedure with CNBr.