The Role of Media Development in Process Optimization: An Historical Perspective - The development of culture media continues to improve biopharmaceutical manufacturing processes. - BioPharm

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The Role of Media Development in Process Optimization: An Historical Perspective
The development of culture media continues to improve biopharmaceutical manufacturing processes.


BioPharm International Supplements


Growth Supplements

Although cells may be adapted to grow in media devoid of growth factors, growth factor supplements still are essential for the growth of many cells in culture. Insulin traditionally has been used as a mitogen and also is involved in glucose amino acid uptake, lipid metabolism, and DNA synthesis.18 Recombinant insulin has been available since 1982 (Genentech, Eli Lilly) and is the most universal supplement in SFM. Although insulin is the growth factor of choice, it is required at supra-physiological concentrations (2–10 mg/mL) to support cell growth and viability under culture conditions.19,20 It is widely accepted that insulin action is primarily through the activation of the IGF-I receptor (IGF-IR) rather than its own insulin receptor (IR).21

An insulin-like growth factor analog, LONG R3 IGF-I (Novozymes) has been developed that acts directly at a much higher potency on the IGF-IR and has been shown to be equivalent to or out perform insulin and IGF-I in supporting CHO cell growth and productivity.22,23 LONG R3 IGF-I, an animal-free recombinant supplement typically is used at 200-fold lower concentrations than insulin because of its increased IGF-IR affinity. It has a distinct biological advantage over other growth factor supplements because of a 100-fold reduced affinity for inhibitory IGF-binding proteins. Stimulation of the IGF-IR results in the activation of a number of signalling pathways, some of which are known to have key mitogenic and anti-apoptotic effects. LONG R3 IGF-I results in greater activation of these signalling molecules, thereby increasing culture longevity and productivity. LONG R3 IGF-I has been shown to be an effective alternative to insulin as a growth factor supplement for sustaining cell growth and viability in serum-free culture at industrial scale.24

Transferrin

Transferrin is required to transport iron into cells, which is essential for cell growth and the regulation of key metabolic processes, such as DNA synthesis and oxygen transport.25 Transferrin has also been shown to play an important role in binding heavy metals in culture. Industrial cell lines such as CHO and NS0 require transferrin to attain optimal cell growth and productivity. Transferrin has been available in the form of serum-derived purified human transferrin (hTf) or bovine transferrin (bTf). Alternatively, inorganic iron salts have been used to supply iron to mammalian cells. To supply high-density cell cultures with sufficient iron, elevated concentrations of iron salts are required that use low affinity non-transferrin receptor pathways. This can have a negative effect on cell growth because of the formation of free radicals and oxidative stress from the unbound ferric or ferrous irons. Precipitation of iron hydroxide in the culture medium also can lead to limited bioavailability of iron to the cell.26

Attempts to provide an efficient supply of iron by chemical chelators such as aurintricarboxylic acid or 2-hydroxy-2,4,6-cycloheptatrein-1-one (tropolone) has had limited application across a variety of cell lines because of unpredictability in controlling the intracellular redox cycle and cell oxidation processes.


Figure 1
A recombinant analogue of human transferrin, CellPrime rTransferrin AF (rTransferrin), (Novozymes) has shown equivalence to hTf and superiority to bTf in stimulating cell growth and productivity across a number of cell lines (Figure 1).27,28 rTransferrin binds specifically to the transferrin receptor, facilitating iron uptake into the cell.

Supplements in Combination

The demand for a universal and robust cell culture media has led to the need to identify cell culture components that substitute for the growth-promoting effects of serum. Nutrient requirements for individual cell lines have been found to differ considerably and, therefore, it has been difficult to design a single serum-free media for the growth of cell lines of commercial interest. In response to this demand, combinations of essential serum proteins have been examined for their ability to stimulate cell growth and productivity in a variety of industrially relevant cell lines.


Figure 2
Studies in CHO cells have shown that growth and viability were adequately maintained in the absence of serum only when both IGF-I and transferrin were overexpressed in genetically engineered CHO cells.29 The combined action of two recombinant forms of these serum proteins, IGF-I (LONG R3 IGF-I) and transferrin (CellPrime rTransferrin AF) on CHO cell growth and productivity in SFM has been investigated (Figure 2). Results from this study show that a combination of these two recombinant proteins promote a synergistic increase in the levels of cell growth and productivity above those obtained from a standard SFM or each protein on its own.

Conclusion

Biopharmaceutical companies require cell-culture media to be animal free, serum-free, defined, and cost-effective. In addition, there is a need to maintain or enhance process productivity while satisfying regulatory requirements for the elimination of serum components. A major driver for this has been the concern over contamination of the final drug product with adventitious agents derived from animal components. The industry has seen various attempts to produce robust animal-free, chemically defined media, and more recently, a protein-free media that is acceptable to regulatory agencies. However, the time involved in adapting cells to defined media, which often have resulted in reduced growth rates and product titers, has shown media development to be an important factor in the increasing cost of manufacture of the final drug product.

With a variety of recombinant, animal-free, defined protein supplements such as growth factors, transferrin, and albumin entering the market, the biopharmaceutical industry now has innovative and safer alternatives to serum and other animal-derived supplements. This situation allows manufacturers to rethink their media development to achieve greater process performance in a more regulatory compliant way.

Sally Grosvenor is a senior scientist and scientific communications manager at Novozymes, Ltd., Thebarton, Australia, +61 883547787,


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