Manipulation of the sodium‐potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures

Abstract

Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed.

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

“…Suppression of cell growth at high viabilities and productivities is especially important when bleed rates in steady‐state perfusion need to be minimized in order to prevent product loss. Such attempts to decouple productivity from growth were already proven by media additives such as small molecule cell growth inhibitors, or altered sodium–potassium levels to arrest cells in the G0/G1 phase . Alternatively, process control strategies were used to keep cells in a physiological state using lactate consumption and the resulting rise in culture pH to regulate perfusion media addition or minimizing growth and maximize productivity by reduced culture temperatures or limiting CSPR .…”

“…Such attempts to decouple productivity from growth were already proven by media additives such as small molecule cell growth inhibitors, or altered sodium-potassium levels to arrest cells in the G0/G1 phase. 17 Alternatively, process control strategies were used to keep cells in a physiological state using lactate consumption and the resulting rise in culture pH to regulate perfusion media addition 18 or minimizing growth and maximize productivity by reduced culture temperatures 19,20 or limiting CSPR. 21 Culture pH, pCO 2 levels, and osmolality were also shown to profoundly impact cell growth and productivity 22,23 and may be used for future perfusion control strategies.…”

Improving the viability of tissue‐resident stem cells using an organ‐preservation solution

“…There exist more than 100 types of preservation solutions in the field of organ transplantation , and we included only Euro‐Collins solution in this study and did not compare other widely used preservation solutions such as University of Wisconsin, histidine‐tryptophan‐ketoglutarate, Celsior or Perfadex . These preservation solutions, which have demonstrated the ability to facilitate the organ viability and function during refrigerated storage, also can be beneficial during tissue transport for the isolation and culture of adult cells; however, considering the potential impairment of cell proliferation in high extracellular potassium concentration conditions substantiated by our results and previous investigations , care should be taken when using ICF‐type solution for tissue preservation. Another limitation is that the tissue stem cell function after preservation was examined only by comparing the colony formation ability of lung mesenchymal stem cells whose origin and function are still controversial .…”

Case study: The characterization and implementation of dielectric spectroscopy (biocapacitance) for process control in a commercial GMP CHO manufacturing process

“…The time‐course potassium concentration was also observed to be lower than historical process experience and further review of the formulation changes showed an adjustment in the potassium phosphate concentration as well. It was hypothesized that the change in the ratio of sodium and potassium may have resulted in a change in the function of the Na + ‐K + ‐ATPase, which modulates a variety of cellular functions from growth to nutrient uptake such as glutamate and aspartate . As a confirmation, potassium phosphate and sodium (as sodium chloride) were restored to the same molar ratio as in version 1 of the powder which resulted in a complete restoration of the biocapacitance profile driving historically comparable feed totals, titer, and osmolality (Figure ).…”