Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Abstract

Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell-specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell-specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady state. This perfusion process was then successfully scaled up to a 100 L single-use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891-901, 2017.

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

“…In this study, we investigated the benefit of a rapid and empirical DoE‐driven workflow to develop novel perfusion media by blending chemically defined basal media and established feed supplements. Using this empirical approach, a larger design space can be investigated when the exact media ingredients are unknown, compared to a more rational and targeted optimization approach . Rapid screening of a total of eight different cell supplement solutions in two different basal media was demonstrated.…”

“…High cell densities were supported at minimal CSPR as low as 10–30 pL/c/d and no changes of product quality were induced by the included feed supplements. The high‐performing perfusion media, developed from this empirical design approach, can subsequently be used as starting point for further rational and additional targeted optimization …”

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies

“…Despite differences among the various perfusion operations which are utilized for recombinant protein expression in CHO cell culture, including N − 1 perfusion, concentrated fed‐batch and continuous perfusion production, the starting point for perfusion medium development is typically the basal and feed media used for fed‐batch culture. One successful case of perfusion medium development, which resulted in a titer of 1.2 g/L per day, was achieved by optimizing the ratio between existing basal and feed media, removing unnecessary components, increasing medium depth, optimizing osmolality, and rebalancing amino acids and vitamins . However, more so than with fed‐batch media design, logistical issues such as simplicity of operation and compatibility with manufacturing facilities must be taken into account in the development of perfusion media.…”

“…Concentrated media formulated for precise delivery of nutrition can facilitate the manufacturing process and reduce cost . Although perfusion experiments and processes are typically executed in bench‐scale bioreactors, inexpensive and convenient scale‐down models for perfusion have been developed in deep well plates, spin tubes, and automated bioreactors, which can enable the large statistical DoE screens required to optimize perfusion media …”

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

“…A proprietary chemically defined medium for perfusion was used as the background for all experiments (Lin, Leighty, Godfrey, & Wang, ). Different levels of Na and K was achieved by interchanging Na forms of certain medium components with the respective K forms (e.g., potassium chloride for sodium chloride).…”