Decreased pCO₂ accumulation by eliminating bicarbonate addition to high cell‐density cultures

Abstract

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 x 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.

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

“…A schematic which illustrates the various CO 2 sources in a perfusion bioreactor has been presented before. 31 Quantifying contributions from the medium and base on a mol/day basis is straightforward as their carbonate concentrations and flow rates are known. The flow rate of CO 2 gas into the reactor will help determine the amount of CO 2 gas added to the reactor (this is typically done only in the early stages of a perfusion culture to maintain bioreactor pCO 2 above a minimum threshold).…”

BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

“…Pluronic â F-68 is a polyol copolymer that acts as a surfactant, preventing cell damage by allowing cells to drain away from bubbles formed in bioreactors during stirring or sparging of cell cultures [8]. In addition, the BAY 81-8973 manufacturing process uses a 3-(N-morpholino)propanesulphonic acid (MOPS)-histidine buffer instead of sodium bicarbonate to reduce pCO 2 levels in the cell culture [10,11]. The final composition of the BAY 81-8973 protein-free cell culture medium was shown to enhance the specific rFVIII productivity of the baby hamster kidney (BHK) cells used to produce BAY 81-8973 [8].…”

Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

“…Moreover, the increased liquid volume per surface area involved in scale-up leads to accumulation of dissolved carbon dioxide (dCO 2 ) (Matsunaga et al 2009). This lowers the expression of MAbs in several cell types (deZengotita et al 1998;Goudar et al 2007;Zhu et al 2005) due to altered biochemical conditions within the cells, which in turn alters the deFuc%. We analyzed the relationship between the deFuc% and medium osmolality for four types of culture scales (1, 5, 30, and 400 L).…”