The relevance of cell size in a CHO fed batch process: Metabolic and transcriptomic characterization

Normally the growth profile of a CHO cell fed-batch process can be divided into a growth phase followed by a stationary (non-growth) phase and a cell death phase. In this study, using a commercially available media system in a 10 liter fed-batch reactor, the growth phase is followed by a phase where cell division stops but cell growth continues in the form of an increase in cell size (Figure 1). During the cell size increase (SI) phase, the average volume and dry weight per cell increase linearly with time up to nearly threefold. Cell cycle and transcriptome analysis show that the cell size increase is related to an arrest of cells in the G1 and G2 phase of the cell cycle in combination with a continued biomass formation. The SI phase is characterized by accumulation of fatty acids and formation of lipid droplets in the cells. Furthermore, the mAb specific productivity per cell increases linearly with the cell volume, while the specific essential amino acids consumption rates per cell remain fairly constant and comparable to that in the NI phase. As a consequence the yield of product on nutrients increases in the SI phase. Metabolic flux balancing shows that also the yield of product on oxygen consumed and CO2 produced is increased in the SI phase, which means lower gas-flow rates can be used to reach the same volumetric productivity. In summary, cell size is an important parameter to consider in CHO cell processes. A better mechanistic understanding on how the cell size is influenced by process conditions can be used to further optimize these processes

Metabolic characterization of a CHO cell size increase phase in fed-batch cultures

Normally, the growth profile of a CHO cell fed-batch process can be divided into two main phases based on changes in cell concentration, being an exponential growth phase and a stationary (non-growth) phase. In this study, an additional phase is observed during which the cell division comes to a halt but the cell growth continues in the form of an increase in cell size. The cell size increase (SI) phase occurs between the exponential proliferation phase (also called the number increase or NI phase) and the stationary phase. During the SI phase, the average volume and dry weight per cell increase threefold linearly with time. The average mAb specific productivity per cell increases linearly with the cell volume and therefore is on average two times higher in the SI phase than in the NI phase. The specific essential amino acids consumption rates per cell remain fairly constant between the NI and the SI phase, which agrees with the similar biomass production rate per cell between these two phases. Accumulation of fatty acids and formation of lipid droplets in the cells are observed during the SI phase, indicating that the fatty acids synthesis rate exceeds the demand for the synthesis of membrane lipids. A metabolic comparison between NI and SI phase shows that the cells with a larger size produce more mAb per unit of O2 and nutrient consumed, which can be used for further process optimization.</p

Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

In a Chinese Hamster Ovary (CHO) cell fed-batch process, arrest of cell proliferation and an almost threefold increase in cell size occurred, which is associated with an increase in cell-specific productivity. In this study, transcriptome analysis is performed to identify the molecular mechanisms associated with this. Cell cycle analysis reveals that the cells are arrested mainly in the G0/G1 phase. The cell cycle arrest is associated with significant up-regulation of cyclin-dependent kinases inhibitors (CDKNs) and down-regulation of cyclin-dependent kinases (CDKs) and cyclins. During the cell size increase phase, the gene expression of the upstream pathways of mechanistic target of rapamycin (mTOR), which is related to the extracellular growth factor, cytokine, and amino acid conditions, shows a strongly synchronized pattern to promote the mTOR activity. The downstream genes of mTOR also show a synchronized pattern to stimulate protein translation and lipid synthesis. The results demonstrate that cell cycle inhibition and stimulated mTOR activity at the transcriptome level are related to CHO cell size increase. The cell size increase is related to the extracellular nutrient conditions through a number of cascade pathways, indicating that by rational design of media and feeds, CHO cell size can be manipulated during culture processes, which may further improve cell growth and specific productivity

Transcriptome analysis for the scale-down of a CHO cell fed-batch process

Transcriptome and metabolism analysis were performed to evaluate the scale-down of a CHO cell fed-batch process from a 10 L bioreactor to an ambr 15® (ambr) system. Two different agitation scale-down principles were applied, resulting in two different agitation rates in the ambr system: 1300 RPM based on the agitator tip speed, and 800 rpm based on the volumetric power input (P/V). Culture performance including cell growth, product titer, glycosylation, and specific consumption/production rates of metabolites was the same for both agitation rates in the ambr and was comparable to that of the 10 L system. The initial variation in gene expression between the inocula for the ambr and 10 L system was no longer present after three days of culture, indicating comparable culture conditions in both systems. Based on principal component analysis, changes in gene expression over time were similar between both scales with less than 6% variation. 2455 genes were uniquely regulated in the ambr system compared to 1604 genes in the 10 L system. Functional analysis of these genes did not reveal their relations with scale or cellular function. This study further strengthens that the ambr system gives representative culture performance for the 10 L bench-scale bioreactor.</p