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