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Gains in Cell Growth and Productivity through the Supplementation and Design of Cell Culture Media
Notes from Cell Culture Engineering Conference (CCE XIII) Apr 22-26, 2012 – A guest blog by Steve Pettit, Director Cell Culture, InVitria For the bioproduction of protein therapeutics from CHO cells it is important to maximize cell growth and productivity in order to lower the overall cost of producing the therapeutic protein. There were several presentations at the CCE XIII conference that described novel approaches to achieve increased cell density and productivity through medium supplementation and design.
Christopher Shen (Keck Graduate Institute) presented data that demonstrated enhanced growth and productivity of CHO in bioreactors using medium supplemented Cellastim™ recombinant human albumin (rHSA). The data showed that supplementation of the medium with Cellastim™ increased cumulative cell density and the production of an antibody product. The beneficial effect of supplementing medium with Cellastim™ was additive to the positive effects achieved with nutrient feeds. Moreover, rHSA did not interfere with the purification of antibody via protein A chromatography and may sometimes increase the recovery of antibody in low load scenarios.
Several presentations showed that the optimization of medium components in order to balance metabolic flow can result in substantial gains in cell growth and productivity. For background, cells need to generate sufficient ATP to maintain cell growth. During glycolysis, one glucose is converted into 2 molecules of pyruvate. Pyruvate is converted to acetyl CoA which is used for the generation of ATP via the Kreb cycle. In a balanced metabolic scenario, nearly all the pyruvate produced from glycolysis enters the kreb cycle, resulting in the efficient generation of ATP.
Cells grown in culture often have an unbalanced cell metabolism that result in increased accumulation of toxic products that inhibit cell growth and productivity. For example, physiological levels of glucose are ~ 1 g/L. In cultures where cells are grown to high density, physiological levels of glucose can be consumed rapidly. Glucose is typically used in media at concentrations higher than physiological concentration to circumvent the need for frequent medium changes, In this example, the high concentration of glucose results in excess flow though glycolysis, which in turn, results in the generation of excess pyruvate. Excess pyruvate is then converted into lactic acid, as a waste product. Lactic acid (lactate) is a potent inhibitor of many cell types that can reduce cell growth and productivity. High lactate production by cells also indicates that glucose was utilized inefficiently to generate cellular ATP.
During cell metabolism, amino acids can be consumed via entry into the Kreb cycle, or amino acids can be synthesized for use by the cell via Kreb cycle. Excess amino acids in media formulations can also result in metabolic unbalancing. For example, excess glutamine can enter the Kreb cycle which results in the generation of toxic ammonia. Thus, the favored approach for the development of cell culture medium capable of supporting high density growth is to balance the amino acid and glucose levels to minimize the generation of toxic lactate and ammonia. Lower levels of lactate and ammonia in the medium translate into increased cell density and increased productivity.
Alan Dickson (Univ. of Manchester) used the levels of glycine, citrate, lactate, and sorbitol produced by CHO cells as an indicator of metabolic inefficiency. Using these analytes, they developed a metabolic model to optimize energetic transfer. Using this model, they were able to compose a simple 4 –component nutrient feed that doubled CHO cell density and product yield. Thus, simple component changes that positively affect metabolic balancing can produce dramatic cell density and yield increases.
Nate Freund (Keck Graduate Institute) showed that CHO cells adapted to growth in high (toxic) levels of lactate undergo profound cell growth increases when returned to medium with reduced lactate levels. Cell densities as high as 35 million viable cells/ml-among the highest in the industry-were achieved in batch culture using lactate-adapted CHO cells. Lactate production was reduced 8-fold and fewer pH adjustments of the medium were required due to the reduced production of lactate.
Yen-Tung Luan (Pfizer) approached medium component balancing by analyzing the total amino acid content of CHO cells and the products generated (the mass balance of each amino acid). Through this approach a model was generated to estimate the optimum levels of nutrients to maximize metabolic efficiency and reduce lactate accumulation. The model indicated that as much as a doubling of cell density is achievable though these methods.
Bhanu Chandra Mulukulta (University of Minnesota) showed that CHO cells could shift from lactate production to lactate consumption (and higher productivity) by limiting glucose levels while the cells are growing slowly. The shift to lactate consumption was accompanied by an increase in the production of antibody. Cell engineering techniques that regulated the rate of glycolysis such as manipulating AKT-mTOR expression levels also promoted lactate consumption and higher productivity.
Ziomara Gerdtzen (Univ. of Chile) showed that CHO cells could be engineered to produce less lactate by introducing multiple changes in the galactose metabolic pathway in order for galactose to be utilized as an alternative to glucose. CHO cells transfected with GALK1(galactose utilization) and Slc2a8 (galactose transport) were able to grow to high density in medium supplemented with galactose rather than glucose. The engineered cells produced much less lactate compared to control cells grown in glucose supplemented medium.
In summary, these presentations demonstrate the benefit of supplementing cell culture media with recombinant albumin and of medium design to maximize the growth and productivity of CHO cells for bioproduction.