In-line microfiltration which is widely used for sterilization of therapeutic proteins prior to formulation is frequently operated at constant flux by pumping purified protein solution through a membrane. Increase in transmembrane pressure (TMP) due to membrane fouling is a major problem in such processes. This Study examines how resistance to filtration increases during in-fine microfiltration of concentrated protein solutions. The TMP was found to increase in three distinct phases, each characterized by its own rate and extent of reversibility. The major portion of the TNIP increase was found to be reversible upon front washing with buffer without any kind of physical or chemical cleaning. There was no evidence of concentration polarization in the processes Studied. Size exclusion chromatographic analysis of front wash samples suggested bovine serum albumin (BSA) monomer as the major foulant accountable for the reversible fouling. Aggregate free protein solutions prepared by pre-filtration showed negligible membrane fouling. We hypothesize that trace amounts of protein aggregates present in untreated feed Solutions were responsible for initiating membrane fouling. However, the actual fouling was caused by the protein monomer which was sequestered on and within the membrane by association with the retained aggregates. The location of foulant on and within the membrane was examined by confocal laser scanning microscopy. The deposition of the foulant was found to be restricted to a zone very close to the membrane surface. Intermittent protein filtration experiments were carried out in an effort to gain better insight on the membrane fouling process and to identify new strategies to maximize product throughput. (c) 2008 Elsevier B.V. All rights reserved.