A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc gamma4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method. Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3 g/L yeast extract, and 0.09% linoleic acid-BSA. Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove's modified Dulbecco's medium supplemented with these serum substitutes were comparable to those in the Ex-Cell 301 commercial serum-free medium. These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media. The low protein content of the developed medium facilitates downstream processing and product purification. (C) 2001 Elsevier Science Inc. All rights reserved.