Diafiltrations by altered operational modes were applied to improve protease purity during ultrafiltration of pre-treated tuna spleen extract. The performance of mode 1 (pre-diafiltration followed by post-concentration) and mode 2 (pre-concentration followed by post-diafiltration) was compared. A critical flux operation was involved in mode 1. Severe fouling was avoided in mode 1 while 12-fold purification of protease was achieved after an initial diafiltration volume of 3 and a final volume concentration factor of 2 were obtained with a total operational time of about 4 h. The conventional operation, that is mode 2, provided 2-fold purification of protease after having obtained an initial volume concentration factor of 2 and a final diafiltration volume of 3. Fouling was much more severe in mode 2 than in mode 1. Consequently, low flux caused by fouling led to a long operational time of about 9 h in mode 2. The internal membrane fouling was due to the adsorption of molecules onto the membrane pore wall and pore blocking by protein or other solutes in both mode I and mode 2. The difference in resistance between mode I and mode 2 was mainly due to concentration polarization and external membrane fouling. In this investigation it was established that operation of diafiltration with critical flux concept could provide a more efficient purifying process than the conventional diafiltration operation. The reduction of fouling not only improved process efficiency and protease purity, it also possibly further reduced the cost of membrane cleaning. (C) 2009 Elsevier B.V. All rights reserved.