Decreased pCO 2 accumulation by eliminating bicarbonate addition to high cell‐density cultures

High‐density perfusion cultivation of mammalian cells can result in elevated bioreactor CO 2 partial pressure (pCO 2 ), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 × 10 6 cells/mL, the bioreactor pCO 2 exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO 3 , and 40% from NaHCO 3 added for pH control. Recognizing the limitations to the practicality of gas sparging for CO 2 removal in perfusion systems, a strategy based on CO 2 reduction at the source was investigated. The NaHCO 3 in the medium was replaced with a MOPS–Histidine buffer, while Na 2 CO 3 replaced NaHCO 3 for pH control. These changes resulted in 63–70% pCO 2 reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing‐scale. Bioreactor pCO 2 values after these modifications were in the 68–85 mm Hg range, pCO 2 reductions consistent with those theoretically expected. Low bioreactor pCO 2 was accompanied by both 68–123% increased growth rates and 58–92% increased specific productivity. Bioreactor pCO 2 reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO 2 reduction at the source through medium and base modification should be readily applicable to large‐scale fed‐batch cultivation of mammalian cells. Biotechnol. Bioeng. 2007;96:1107–1117. © 2006 Wiley Periodicals, Inc.