Radio-labelling Of Long-Lasting Erythropoietin(LL-EPO) And Its Purification

LL-EPO is a newly developed biological product for treating anemia with long-lasting effect as compared to the currently used EPO in the clinic. As far as we know, pharmacokinetic study is needed for any new biological product to meet the requirements of registration. Several techniques can be used to study the pharmacokinetics of a new biological product. Among them, isotope tracing technique is most widely used. However, the purity and biological activity of the labelled product is very important when using isotope tracing technique to study the pharmacokinetics of the newly developed biological product. The study is designed to investigate the labelling of LL-EPO, purification of labelled compound, and therefore, to prepare the labelled LL-EPO with high purity and biological activity.In this study Iodine-125 was used as labelling nuclide, and the LL-EPO was labelled by the common used chloramine-T and the modified two-phase chloramine-T method, respectively. The labelled compound was purified by both gel filtration and ultrafiltration method, respectively. The purity of the labelled LL-EPO was determined by both trichloroacetic acid (TCA) and SDS-PAGE method, and the biological activity was determined by the reticulocyte counting method . In addition, the influence of the temperature on the purity of the labelled LL-EPO was also investigated. The results are as follows:1. The present modified two-phase chloramine-T method has several advantages over common used two-phase chloramine-T method, including a small bottle with cover was used as reaction container and the reaction system was better airproofed; the absorption of radio-labelled protein was prevented by silanization of the bottle; the filter paper , as thesource of Ck, was hung in the bottle, the concentration of the oxidant can be adjusted, therefore, result in a higher iodine incorporation.2. The iodine incorporation and specific radioactivities were 89% and 5.82xl05Bq ug'1 for LL-EPO labelled by the modified two-phase chloramine-T method and were 20.65% and 3.62*105 Bq ug"1 for LL-EPO labelled by the common used chloramine-T method, respectively. The purity of labelled LL-EPO purified by both gel filtration and ultrafiltration were over 96% with TCA method purification. The labelled LL-EPO showed two bands with Rf of 0.28 0.49, respectively, which is identical to that of standard LL-EPO through SDS-PAGE. There was no loss of biological activity of LL-EPO after labelling as determined by reticulocyte counting method.3. The ultrafiltration can be used to purify the radio-labelled LL-EPO , with the higher protein recovery and concentration than with gel filtration method.4. Temperature takes an important role in the storage of the labelled LL-EPO. The purity decreased faster and earlier at room temperature than at 4C.In conclusion, the advantages of the modified two-phase chloramine-T method are easy handle, high iodine incorporation , and no loss of biological activity of labelled LL-EPO. In addition, the ultrafiltration method, which makes the purification procedure faster, simpler, and results in high protein recovery, is suitable for the purification of the labelled protein.