الفهرس | Only 14 pages are availabe for public view |
Abstract C viral infection in Egypt has one of the highest prevalence rates in the world. The rhIFN-α2b protein was approved by FDA as antiviral for HBV and HCV treatment and anti-tumor drug. Due to their complex nature and the nature of production process, recombinant pharmaceutical proteins often contain micro heterogeneity represented in presence of different forms (isoforms) of the produced protein. We have previously developed limited pilot scale method for enriched production of properly refolded rhIFN-α2b isoform in E. coli as intracellular insoluble aggregates. In this study, a detailed molecular and biological characterization of the properly refolded rhIFN-α2b isoform produced using the previously developed method was carried out to validate it as a therapeutic drug and establish its quality assessment criteria. To prepare the rhIFN-α2b isoform, refolding reaction was cross filtered and this isoform has been purified from the cross filtered refolding reaction by means of high resolution anion ion exchange chromatography using resource Q column. Target peak harboring this isoform of rhIFN-α2b has the proper MW (19 KDa) and high purity as proved by silver stained SDSPAGE. The linear epitopes of purified rhIFN-α2b isoform was immunologically confirmed with western blot using Rabbit monoclonal anti human interferon-α2b IgG. Evaluation of endotoxins level in the purified rhIFN-α2b isoform using LAL assay revealed mean endotoxin concentration of 0.03 EU/mg which is far below the internationally accepted safe level (1 EU/mg protein) which with purity results proved the efficiency of the previously developed purification method and the safety of the produced rhIFN-α2b isoform. Using RP-HPLC, the rhIFN-α2b isoforms gave only one sharp symmetrical peak at retention time (RT) 116 min in comparison to run buffer as a proof for purity and as a quality parameter for the process and product. Mass spectrometry assessment of its intact mass showed average mass of 19337 Da equivalent to that of the expressed rhIFN-α2b protein without any chemical modification and without the first methionine. These results along with that of HPLC ensured the homogeneity and purity of the prepared isoform. As quality control measure, peptide mapping for the prepared rhIFN-α2b isoform after reduction/alkylation using urea as a denaturing agent showed complete digestion and superior stable peptide map pattern compared to that without urea as it gave many more sharp high RP-HPLC peaks with lower molecular weight peptides (majority less than 13kDa) in silver stained SDS-PAGE. Indirect sandwich ELISA for human IFN-α2 using structural epitopes specific monoclonal antibodies has successfully detected the prepared protein at concentrations as low as 626 pg/ml protein which implies the conservation of these structural epitopes. The in vitro antiviral activity evaluated for the prepared rhIFN-α2b isoform through assessment of 50% cytopathic effect (CPE) protection for monkey kidney epithelial vero cells challenged with VSV compared with that of standard rhIFN-α2 showed high activity (2.5x108±1.1x108IU/mg) comparable to the commercially produced brands. The in-vivo clearance study of the prepared rhIFN-α2b intramuscularly administered to SD female rats as a single dose of 500 μg/kg (approximately 2 MIU/rat) measured using ELISA for the prepared human interferon alpha 2b isoform proved biological stability as it gave a maximum plasma concentration of 33,792 pg/ml after 0.25 h (Tmax) of administration then the concentration started to decline till complete clearance within three hours. The period required for reduction of plasma concentration to one half (t1/2) was 0.54 h. The previously prepared commercial interferons alpha 2b were heterogeneous mixtures of isoforms which enhance the development of binding and neutralizing antibodies. The prepared rhIFN-α2b isoform is highly purified nonheterogeneous preparation with chemical, structural and immunological properties of native protein. This should strongly reduce the development of binding and neutralizing antibodies especially in long term treatment schemes as in HCV therapy with enhancement of therapeutic efficiency and increased safety. Additionally, several quality control assays have been established for the prepared IFN-α2b isoform and preparation process. For the purity and integrity (SDS-PAGE, RP-HPLC, LAL assay and mass spectrometry) for homogeneity (RP-HPLC and Mass spectrometry), for stability, chemical and functional properties (RP-HPLC, mass spectrometry and antiviral activity assay). |