Studies demonstrate structural and functional similarity between recombinant human transferrin derived from rice and native human transferrin
A guest blog by Steve Pettit, Ph.D., Director, Cell Culture Development, InVitria
Transferrin in Cell Culture
Cells need the right amount of iron at the right time to maintain health and to continue to grow and divide. Too little or too much iron leads to poor growth and productivity. In nature, iron is delivered to cells through the iron binding protein transferrin, present in mammalian blood. Transferrin is a universal iron carrier designed to deliver the appropriate amount of iron to cells via a receptor-mediated transfer of iron from transferrin to the cell. Specific transferrin receptors on the surface are up regulated or down regulated based on the cell’s iron need. Due to this ability of cells to regulate iron uptake, transferrin is uniquely able to deliver the exact amount of iron needed by the cells. This is important because it eliminates the problem of cells taking in too much iron and causing cell damage, which can happen with other iron sources.
Due to its important iron delivery function, transferrin is a critical component of animal- free culture media formulations and has been successfully used to supplement many culture media for mammalian and stem cell lines. Fetal Bovine Serum (FBS) contains transferrin, but in serum-free systems, the lack of transferrin can lead to poor performance. Bovine blood transferrin and human donor blood derived transferrin can be used to supplement when serum is absent, but the use of animal products or blood donor derived products raises the risk of pathogen contamination and can lead to variability due to the nature of the raw material that is used to make these products.
In an effort to achieve completely animal-free cell culture many have chosen to utilize iron compounds instead of transferrin. Iron compounds are animal-free, provides iron to cells, is very inexpensive, and easy to obtain. The challenge to using iron compounds is that they may provide too much iron. Iron not bound to transferrin can cause Fenton oxidation reactions that can damage cells . It is relatively tricky to establish the right balance of iron within the cell when using these alternatives. In addition, iron can cause difficulties in downstream processing by fouling expensive chromatography resins. Another problem is that direct iron supplementation is not as effective as transferrin in many of the leading cell lines used in bioscience research including hybridoma, stem cells, and vero cells.
Today, cell culture scientists have an alternative to human serum derived transferrin or bovine transferrin thanks to recombinant DNA technology. Recombinant Transferrin is now available as a way to ensure successful iron delivery and maintain animal-free culture.
Recombinant vs. Native Transferrin
To ensure the quality and comparability of recombinant transferrin when compared with native human transferrin (nhTF), recombinant human transferrin (rhTF) derived from rice, was examined in two studies to evaluate structural and functional similarity to native transferrin.
The first study, “Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.),” published in Protein Expression and Purification, Zhang, et. al. demonstrates structural and functional similarity between rhTF from rice and nhTF. The authors describe the method for manufacturing the recombinant transferrin in rice and also the purification methods.
The rhTF was compared with nhTF in several areas including:
- Amino (N)-terminal-sequence analysis
- Molecular weight (MALDI) and PNGase F digestion
- Isoelectric point
- RP-HPLC analysis
The data demonstrated that the rhTF is structurally similar to the nhTF.
In order to access biological equivalence, the authors conducted an iron binding assay and a cell growth and productivity assay. The iron-binding assay measured the rhTF’s ability to bind and release iron reversibly compared to nhTF and results confirmed equivalent function.
To compare cell growth and productivity, the authors tested both recombinant and human native transferrin in the serum-free culture of hybridoma cells. They found the dose response to be the same for proliferation and stimulation of cell growth and antibody production.
The latest study, “Characterization of transferrin receptor mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.),” published in BMC Biotechnology, Zhang, et. al. went one step further and confirmed functional similarity between recombinant human transferrin (rhTF) derived from rice and human derived transferrin (nhTF) in cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support.
In the study, the TFR binding capacity and equilibrium dissociation constant were compared between rhTF and nhTF. In order to compare TFR binding, a competitive binding affinity assay was used. Results showed the same competitive binding and dose-dependent inhibition in both rhTF and nhTF. For the equilibrium dissociation constant the values were different but still within the normal range and both rhTF and nhTF had a similar dose-dependent binding profile.
TFR-Mediated endocytosis was also compared. After endocytosis kinetics were assayed in HeLa and Caco-2 cells and pulse chase assays were conducted, the data demonstrate that retention and recycling of both in Caco-2 and HeLa cells is similar.
Lastly, authors compared the ability of both rhTF and nhTF to support cell growth and function in two cell lines, HL-60 and Sp2/0 hybridoma cells. In addition, antibody production was also measured in hybridoma cells to further test whether normal cellular function is occurring. In the HL-60 cell culture proliferation was compared using a dose response. It was found that both rhTF and nhTF increased cell proliferation in serum-free media at the same dose concentrations.
In the hybridoma culture, hybridoma cells were first grown in serum-free media that contained the iron compounds ferric nitrate and ferric sulfate (DMEM/F12 medium). Even though these iron compounds were present in the medium the cells failed to grow beyond two days without transferrin. For the next study, hybridoma cells were cultured in serum-free DMEM/F12 media with either rhTF in one of three levels of iron saturation – iron-free, partially iron saturated and iron saturated or nhTF in one of three levels of iron saturation. Results showed that all six variations of either rhTF or nhTF had similar growth kinetics regardless of the iron saturation levels. Next a dose response study was conducted to compare rhTF to nhTF using the following cell culture performance parameters:
- Log – phase cell proliferation after 3 days of culture
- Sustained cell growth measured by cumulative cell density through 6 days of culture
- Antibody productivity at the end of batch culture on day 6
- Six day cumulative cell density or integral of the viable cell concentration
The data show that both rhTF and nhTF elicited production of a similar antibody yield at equivalent concentrations.
Recombinant Human Transferrin is an Attractive Alternative to Native Transferrin
These studies demonstrate that rhTF from rice is a safe and effective alternative to nhTF, as both the rice derived and native transferrin is structurally similar and show equivalent performance. The rice-derived rhTF has the additional advantage that it is animal-free and cost effective. The rhTF from rice that is used in the study is offered by InVitria under the name Optiferrin and is manufactured in a dedicated, commercial-scale facility with strict standard operating procedures for cGMP compliance.