Human Thrombopoietin Hormone Recombinant Cell Culture Technology Research

In the current study, a method of high-density cell culture for producing rhTPO was developed. These research included construction of a CHO cell line stably transfected with rhTPO eDNA, selection of the cell line that highly expressed rhTPO protein, development of an assay for measuring the in-vitro activity of rhTPO, development of a method of cell culture using bioreactor, and finally, the purification and characterization of rhTPO.In brief, a mammalian expression plasmid containing TPO eDNA and a selectable marker was first introduced into CHO cells. A cell line, which was named STP0-l, was obtained following the standard selection and amplification procedures. This STP0-l cell line could stably express rhTPO at a high level.The optimization of cell culture was then performed and the resulting level of expression reached 2,000 units/mi (7 mglml) of rhTPO when the ST1抩-l cells were cultured in flasks. The high-density cell culture was studied using method of packed-bed bioreactor with continuous perfusion culture. Following optimization of the bioreaction condition, such as medium, pH, dissolved oxygen (DO2), agitation speed, glucose concentration, and perfusion rate, the cell density could reach 6 X 106/ml and level of TPO expression could reach 12,000 units/mI (40 mg/mi) by using a specific protein-free medium. As a result, an average of 100 liters of conditioned medium could be harvested using a bioreactor with a 5-liter vessel. A total of 650 mg of rhTPO protein could be obtained following the purification process, which included a series of filtration and chromatography steps.The characterization of purified rhTPO showed 99% purity by High-Performance-Liquid Chromatography (HPLC) and SDS-Polyacrylamide GelElectrophoresis (PAGE), 70 -120 kD of the apparent molecular weight, multiple Iso-Electric Focusing (IEF) bands with p1 <5, and a single smeared band on a WesternBlot analysis. The N-terminal amino acid analysis showed correct sequence as3deduced from TPO genetic code.In summary, a cell line that could highly express rhTPO was constructed, an efficient in-vitro activity assay of rhTPO and a complete process for production and purification of rhTPO was developed. These results provided a solid ground for large-scale manufacture process development as well as eventual clinical application of rhTPO.