Radio Labeling Of Polyethylene Glycol Recombinant Human Interleukin-6 (PEG-rhIL-6), The Purification And Identification Of The Labeled PEG-rhIL-6

Interleukin 6 (IL-6) is a multi-functional cytokine that is produced by T lymphocyte hybridoma cells and other cells (such as monocytes, fibroblasts, endothelial cells, etc.). It plays a central role in both host defense and hematopoietic activities. It is also one of the major mediators of the acute phase inflammatory response. The registration studies about IL-6 have come to stage of phase Ⅰ-Ⅱ in USA and Japan. However, cytokines have generally poor stability in vivo, and a high dose is needed to achieve sufficient clinical effect. In addition, they would also cause unfavourable side-effects due to the pleiotropic functions.In order to circumvent the poor stability and pleiotropic activities of cytokines, some studies have been performed by the modification of cytokines molecule using water-soluble polymers. Conjugation of the polymer polyethylene glycol (PEG) to proteins can significantly decrease their clearance from plasma, and thus decrease renal excretion rate of proteins and prevent the degradation of proteases. This would result in prolonging the half-lives of bioactive proteins and potentiating their clinical effects. Polyethylene glycol-modified interleukin-6 (PEG-IL-6), were able to selectively increase their function of promoting platelet production, but not other unfavourable functions. A new biological drug - PEG-rhIL-6 developed by a graduate school of China is also in the clinical stage of registration study. 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 labeled product is very important to study the pharmacokinetics of the newly developed biological product when using isotope tracing technique. The present study is designed to investigate the labeling of PEG-rhIL-6, purification of labeled product and therefore, to prepare the labeled PEG-rhIL-6 with high purity and biological activity.In this study Iodine-125 was used as labeling nuclide, and the PEG-rhIL-6 was labeled by the common used chloramines-T and the two-phase chloramines-T, respectively. The influence of those two labeling methods on incorporation of iodine to PEG-rhIL-6, specific activity and biological activity of labeled PEG-rhIL-6 were investigated. The labeled compound was purified by both gel filtration and ultrafiltration method, respectively. The purity of the labeled PEG-rhIL-6 was determined by both trichloroacetic acid (TCA) and SDS-PAGE, and the biological activity was determined by the MTT. Inaddition, the influence of the temperature and protective protein on the stability of labeled PEG-rhIL-6 was also investigated. The results are as follows:1. The iodine incorporation and specific radioactivity were 74.5% and 5.513 X 105Bq/ug of PEG-rhIL-6 labeled by the two-phase used chloramines-T. The purity of labeled PEG-rhIL-6 purified by both gel filtration and ultrafiltration were over 99% with TCA. The labeled PEG-rhIL-6 showed two bands with Rf of 0.289 and 0.542, respectively, Which is identical to that of standard PEG-rhIL-6 with SDS-PAGE.2.The iodine incorporation and specific radioactivity were 62.3% and 4.625 X 105Bq/ug of PEG-rhIL-6 labeled by the common used chloramines-T, respectively. The purity of labeled PEG-rhIL-6 purified by both gel filtration and ultrafiltration were over 99% with TCA method. The labeled PEG-rhIL-6 showed three bands. Among them, one band has a heavy molecule with Rf of 0.214, the other two bands were identical to that of standard PEG-rhIL-6. This indicated that part of PEG-rhIL-6 might be damaged in labeling when using common chloramines-T.3. The labeled PEG-rhIL-6 can be purified using ultrafiltration, ultrafiltration purified products have higher protein recovery (92%) and concentration (920ug/ml) than that of gel filtration purified products.4. The biological activity of labeled PEG-rhIL-6 was determined by MTT method. It showed the biological activity of labeled PEG-rhIL-6 by the common used chloramines-T was lower than that of PEG-rhIL-6 labeled by the two-phase chloramines-T method.5. The conditions of storage (temperature and protective protein) influenced the stability of labeled PEG-rhIL-6. The radiochemical purity of labeled PEG-rhIL-6 was determined by TCA at certain time. The purity of labeled PEG-rhIL-6 without BSA in storage buffer decreased to 81% at 15, 30, 45 days, respectively, when it is stored at room temperature, -4°C and -20 °C, respectively. After adding protective protein (BSA) in storage buffer, it took 10-15 days more for the purity of labeled PEG-rhIL-6 decreased to 81% This indicated that low temperature and protective protein would be help for the storage of the labbelled compound.In conclusion, two-phase chloramine-T method has the following advantages: are easier to handle, with higher iodine incorporation, and lower loss of biological activity for the labeling of PEG-rhIL-6 compared to common used chloramines-T. In addition, the ultrafiltration method, which makes the purification procedure faster, simpler and the results in high protein recovery, is suitable for the purification of the labeled protein. The labeled protein with protective protein was preserved in -20 °C can maintain a good stability, which can fulfil the requirement of pharmacokinetics.