Process parameter shifting: Part II. Biphasic cultivation—A tool for enhancing the volumetric productivity of batch processes using Epo‐Fc expressing CHO cells

Abstract

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

Proliferation control strategies to improve productivity and survival during CHO based production culture

“…Protein production is dependent on the phase of the cell-cycle and several genes such as those involved in ribosome biogenesis and protein translation are expressed highly in the G1 phase (Al-Rubeai and Emery 1990;Al-Rubeai et al 1992;Moore et al 1997;Fussenegger et al 1998Fussenegger et al , 2000Kaufman et al 1999Kaufman et al , 2001Carvalhal et al 2003;Ibarra et al 2003;Yoon et al 2003a, b;Fogolin et al 2004;Bi et al 2004;Trummer et al 2006). Cells arrested at the end of G1-phase of cell cycle are metabolically more active and bigger in size than non-arrested cells (Carvalhal et al 2003;Bi et al 2004).…”

“…Cells arrested at the end of G1-phase of cell cycle are metabolically more active and bigger in size than non-arrested cells (Carvalhal et al 2003;Bi et al 2004). For these reasons, the G1-phase of the cell cycle is considered the ideal time for increased production of recombinant proteins and G1 arrest has been used to increase the productivity in a number of commercially relevant cell lines such as hybridomas and CHO (Al-Rubeai and Emery 1990;Al-Rubeai et al 1992;Moore et al 1997;Fussenegger et al 1998Fussenegger et al , 2000Kaufman et al 1999Kaufman et al , 2001Ibarra et al 2003;Yoon et al 2003a, b;Fogolin et al 2004;Trummer et al 2006). Some studies have reported the S phase as the optimal production phase (Lloyd et al 2000;Fox et al 2005), an example being the increased production of human interferon-c (IFN-c) upon increasing the percentage of CHO in S phase (Fox et al 2005).…”

“…(I) Temperature shift to conditions of mild hypothermia Low culture temperature has been shown to reduce the growth rate, increase culture longevity and specific productivity in CHO cells expressing wide range of recombinant proteins Ohsuye 1998, 1999;Kaufmann et al 1999;Schatz et al 2003;Yoon et al 2003aYoon et al , b, 2006Fox et al 2004;Fogolin et al 2004, Bollati-Fogolin et al 2005Trummer et al 2006). In this laboratory reducing the temperature of suspension CHO-K1 cells from 37 to 31°C during the latter stages of exponential growth resulted in an immediate cessation of proliferation.…”

“…Cultures grown at 31°C achieved peak viable cell numbers that were almost 20% lower than in cells cultured for another 24 h at 37°C (Kumar et al in preparation). A shift in the proportion of cells from the S to the G1 phase of the cell cycle has been observed at reduced temperatures which results in a state close to growth arrest and has been shown to improve productivity (Moore et al 1997;Kaufamann et al 1999;Hendrick et al 2001;Yoon et al 2003a, b;Fogolin et al 2004;Trummer et al 2006). Specific examples include the finding that 80% of secreted alkaline phosphatase (SEAP) producing and 76% of erythropoietin (EPO) producing CHO line accumulate in G0/G1 phase of cell cycle following reduction of the culture temperature to 30°C with associated increases in productivity (Kaufmann et al 1999;Yoon et al 2003b).…”

“…The mechanism whereby cells at lower temperatures improve productivity is still poorly understood although recent studies have demonstrated that reduced culture temperature invokes a coordi- nated response involving the cell cycle, transcription and translational machinery, and the arrangement of the cytoskeleton (Chuppa et al 1997;Moore et al 1997;Ohsuye 1998, 1999;Jorjani and Ozturk 1999;Hendrick et al 2001;Yoon et al 2003b;Fox et al 2004Fox et al , 2005Baik et al 2006;Al-Fageeh et al 2006). Low culture temperature results in reduced metabolism (glucose/medium consumption, oxygen uptake, lactate production and ammonia production) and shear sensitivity which leads to delayed initiation of apoptosis and helps in extending the duration of the stationary/production phase (Moore et al 1997;Chuppa et al 1997;Ohsuye 1998, 1999;Jorjani and Ozturk 1999;Hendrick et al 2001;Yoon et al 2003a, b;Trummer et al 2006;Wong et al 2006). Reduced culture temperature has also been shown to increase levels of recombinant mRNA, either due to enhanced transcription of the recombinant gene of interest or increased mRNA stability (Hendrick et al 2001;Yoon et al 2003b;Fox et al 2004Fox et al , 2005.…”