Phosphate feeding improves high-cell-concentration NSO myeloma culture performance for monoclonal antibody production

Phosphorus depletion was identified in high-cell-concentration fed-batch NSO myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10⁹ and 7 x 10⁹ cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10⁹ cells/L, which is much greater than ca. 7 x 10⁹ cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10⁹ cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10⁹ cells/L, as compared with 3 x 10⁹ cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NSO cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NSO cultures. (C) 2000 John Wiley and Sons, Inc.