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.