Assuring viral safety of bioreactors in upstream bioprocessing is an important issue in manufacturing biologicals. Nanofiltration of cell culture media components is a robust yet expensive method of assuring biosafety. The present article investigates the performance of mAb-producing Chinese hamster ovary (CHO) cells using 20 nm-filtered chemically defined medium in WAVE Cellbag™ bioreactor in a 10L pilot scale. For 20-nm virus retentive filtration nanocellulose-based non-woven filter paper developed at Uppsala University was used. The cell performance in WAVE Cellbag™ bioreactor was quantified with respect to cell viability, metabolic activity, and protein yield and compared to internal reference data from stirred tank-type bioreactor. The results revealed that 20-nm filtered supplemented chemically-defined media supported the growth of mAb-producing CHO cells, with desirable cell metabolic activity and mAb yield. The observed differences between the reference data from unfiltered media in the stirred tank bioreactor and nanofiltered media in WAVE Cellbag™ bioreactor were mainly due to the configuration of the equipment rather than due to nanofiltraion. The results represent an important step forward for scaling up bioprocesses using cost-efficient nanocellulose-based nanofilters in upstream bioprocessing.