Size-based analysis of virus removal filter fouling using fractionated protein aggregates

Fouling by protein aggregates reduces virus removal filter performance. In the present study, we investigated the effects of different-sized protein aggregates on fouling and aggregate retention in order to better understand the fouling mechanisms. Human immunoglobulin G was denatured by heating to produce aggregates of various sizes and then fractionated by size exclusion chromatography into different-sized aggregates with a narrow size distribution. The fractionated aggregates were filtered on Planova 20N, a virus removal filter known for its stable filtration capability. Analysis of flux behavior demonstrated different flux decrease patterns for different-sized aggregates. Observation of aggregate retention by staining revealed that larger aggregates were captured closer to the inner surface of the membrane while smaller aggregates penetrated farther into the membrane. These findings demonstrate that Planova 20N has a gradient structure with decreasing pore size from the inner to the outer surface of the membrane. This structure minimizes fouling and enables stable filtration by protecting the smaller pores located closer to the outer surface from clogging by large aggregates. Applying the predominant clogging models to the present filtrations revealed that clogging behavior transitioned from complete blocking to cake filtration as filtration progressed. In this combination model, after a certain number of pores are blocked by complete blocking, newly arrived aggregates begin to accumulate on previously captured aggregates, generating cake between capture layers within the membrane. Application of the approaches described here will facilitate elucidation of membrane fouling and virus removal mechanisms.