|Send to printer »|
Tutorials : Dec 1, 2008 ( )
Optimizing Mammalian Cell Culture Performance
Iron Supplement Eliminates Issues Associated with Animal-Derived Options!--h2>
Mammalian cells, both in vivo and in vitro, require iron for cell growth, DNA replication, cellular respiration, and metabolism. Transferrin is the natural physiological method by which iron is transported into the cell.
Traditionally, transferrin has been supplied in the form of serum-derived human transferrin (hTf), bovine transferrin (bTf), or, more recently, with the move toward serum-free medium (SFM), inorganic iron salts have been used to supply iron to mammalian cells. To provide high-density cell cultures with sufficient iron, elevated concentrations of iron salts are required that utilize low-affinity nontransferrin receptor pathways.
An increase in iron concentration can have a toxic effect on the cell due to the accumulation of unbound ferric or ferrous ions. This toxicity can be caused by the formation of ferric hydroxides that precipitate in culture, altering media composition. Fe3+ can also be decreased by reducing agents in the medium to form Fe2+, promoting free-radical formation and causing oxidative damage to the cells.
Transferrin is an extracellular antioxidant, binding iron so tightly that little unbound iron is available to catalyze the production of free radicals. Iron-bound transferrin has an increased affinity for the transferrin receptor, which is then transported into the cell via receptor-mediated endocytosis.
Moreover, the transferrin-receptor pathway is more efficient than free inorganic iron in the targeting of iron toward the mitochondria. Transferrin is also thought to be involved in cellular growth and differentiation activities. These effects are thought to be at least partially independent of its iron-binding function as apo transferrin has been shown to have growth-promoting effects.
CellPrime™ rTransferrin AF (rTransferrin), manufactured by Novozymes and distributed through an exclusive partnership by Millipore, is expressed in a Saccharomyces cerevisiae system. It is a recombinant analog of human transferrin that has shown equivalence to serum-derived human transferrin (hTf) and superiority to bovine transferrin (bTf) in stimulating cell growth and productivity for a number of cell lines. Supplied as a human holo-transferrin analog, rTransferrin binds specifically to the transferrin receptor thereby facilitating the bioavailability of iron to the cell.
CHODUKXB11 clone expressing a human mAb against human interleukin (IL)-8 (referred to as DP-12) was cultured in DMEM/F12, 10% FBS, 4.5 g/L glucose, 2 µg/mL recombinant human insulin, 200 nM MTX and 2mM Glutamax™ in T25 flasks. Mouse myeloma cell line, Sp2/0 expressing anti TGFb-3, was cultured in DMEM/F12 (SAFC Biosciences), and 2 mM Glutamax™, ITS, 0.5% Probumin.
Prior to assay set-up, CHO cells were grown to between 70–90% confluence in T75 tissue culture flasks in 10–20 mL sterile culture media, and spun and detached using trypsin. Cells were then washed in 10 mL Dulbecco’s PBS, spun and resuspended in serum-free, growth factor-free medium. Cell viability was determined by the trypan blue exclusion method. Sp2/0 cells were grown to 70–90% confluence, spun, and resuspended in 10 mL fresh SFM media (as above minus Probumin®). The wash step was repeated, and viability determined by trypan blue exclusion method.
One thousand CHO cells and 1,500 Sp2/0 cells in fresh SFM were added to each well of a 96-well plate. All supplements and controls were tested in six replicate wells. Plates were incubated at 37ºC/5% CO2 and harvested at either day six or ten.
On the day of harvest, 100 µL of conditioned media was removed to a separate plate for IgG analysis. The metabolic dye MTS was added to the remaining media for analysis of viable cell numbers and absorbance was measured from each of the six replicate wells to determine growth. The conditioned media removed in the previous step was pooled for each sample to create three replicates for IgG analysis (ELISA or HPLC).
Results and Discussion
To examine the effect of rTransferrin on CHO cell growth and productivity, two concentrations (1 and 5 µg/mL) of the supplement were tested. A greater than twofold increase in growth for both concentrations of rTransferrin was observed above that of SFM by day 10 of the culture (Figure 1). Productivity results, analyzed in parallel with growth, also displayed an increase (>threefold) in IgG production when compared to SFM by day 10.
To determine whether rTransferrin
(5 µg/mL) displayed similar growth and productivity effects in CHO and Sp2/0 cells to iron supplements in common use within the industry, rTransferrin was compared with serum-derived hTf or bTf (each at 5 µg/mL) and three different inorganic iron salts.
The results in Figure 2 demonstrate that rTransferrin and hTf were equipotent in stimulating cell growth in both cell types studied. Cells treated with bTf, however, showed little increase in cell growth when compared to SFM. CHO cells treated with rTransferrin displayed an equal or better growth profile than the treatments with iron salts, while growth of Sp2/0 cells above that of SFM was observed only for cells treated with rTransferrin or hTf.
Productivity increases were obtained for both CHO and Sp2/0 cells when treated with rTransferrin and hTf compared to SFM. rTransferrin enhanced CHO cell productivity compared to the other iron supplements tested, displaying a 20% increase above hTf, a 30% increase above FeSO4 and Fe citrate, and a 10-fold increase above bTf and Fe EDTA. Again, in the Sp2/0 cell line, bTf and iron salts showed minimal productivity increase compared to SFM.
The biopharmaceutical industry is constantly striving for optimal cell growth and productivity in their upstream manufacturing process, while maintaining product/ process consistency and compliance. Historically, the number of animal-free defined protein supplements available for use as industrial cell culture supplements has been limited.
The data presented in this article describes a recombinant transferrin product that displays all the characteristics of serum-derived hTf without any of the regulatory issues.
In all treatments, rTransferrin was shown to be equivalent to or better than the alternative iron supplements examined. rTransferrin performed equally well when compared to hTf and significantly better than bTf in stimulating cell growth and productivity. These results would indicate an unexplained species specificity of transferrin and an apparent lack of affinity of bTf for the CHO and Sp2/0 transferrin receptor.
When compared to inorganic forms of iron, rTransferrin was again shown to deliver superior performance in growth and productivity even though the number of moles of iron added was ten times that for the iron salts compared to holo transferrin. Unbound iron is transported via nontransferrin receptor-mediated pathways that have shown to be less effective in driving cell growth and recombinant protein production in our experimental model when compared to the natural physiological method of transferrin.
CellPrime rTransferrin AF provides an alternative to other iron supplements commonly used in the cell culture industry by eliminating the use of animal-derived components while enhancing cell performance.
© 2016 Genetic Engineering & Biotechnology News, All Rights Reserved