dc.description.abstract | Interferon (IFN) with important immunomodulatory function. is one of the members of cytokines They are produced in response to viruses and they inhibit virus replication. Besides antiviral activities, they also show antiproliferative and immunomodulatory activities. The pleiotropic properties of IFNs have been successfully exploited in therapeutic application in several types of cancer and viral infection diseases, including leukemia, lymphoma, hepatitis B, hepatitis C…etc. However, most of the products currently on the market are recombinant IFNs expressed from Escherichia coli (E. coli) or Chinese Hamster Ovary (CHO) cell. Due to the absent or different glycosylation of such recombinant proteins, they exhibit certain shortage, including shorter half-life, less potency and almost 100% side effect. The need of natural human IFNs for clinical use is on the rise in the last several years.
The human interferon-a (IFN-a) family is comprised of 13 homologous subtypes with 78-95% identity at amino acid sequence level. Only IFN-?2 and IFN-?14 are glycosylated subtypes. Previous studies have demonstrated that each of the IFN-? subtypes showed quantitatively distinct patterns in the antiviral, growth inhibitory, and other biologic activities. And IFN-a with natural structural conformation and similar composition of subtypes has higher efficacy and is less immunogenic. Until now, the minute quantities and the complexity of subtypes composition of native IFN-a produced from human cells still hindered the development of naturally derived products. In this study, the patented human lymphoblastoid cell line was cultured in bioreactor. Our goal is to establish an analytic methodology by proteomics approach to analyze a group of proteins at low concentration (?g/ml) with high identity in amino acid sequence. The purified IFN-a by a 3-step column chromatography was prepared by CytoPharm Inc. Taiwan. The IFN-? mixture was further separated into 11-15 major peaks by reversed-phase high performance liquid chromatography. The molecular weight of this preparation is 19-24 kD with 50-80% higher than 20 kD, which indicating the glycosylated subtypes. From the analysis of SDS-PAGE and stained by glyco-staining, the glycosylated subtypes were located in the peaks with earlier retention time than recombinant IFN-?2a when separated by RP-HPLC and contained 57-60% of total peak area. And there are no detectable N-linked subtypes from the deglycosylation analysis indicating there were no IFN-?14 in purified IFN-? that also confirmed by MS/MS analysis. Based on the peptide sequences analysis by electrospray quadrupole-time-of-flight tandem mass spectrometry, confirmed the present of 5 subtypes (?1(13)、?2、?5、?8、?21) and several subtypes (?4/10/14/17、?4/7/16/17/21) were not completely identified. Therefore, there were at least 6 and most 11 subtypes in purified IFN-?. For the sample pretreatment for MS analysis, changing the temperature of reduction from 37?C to 56?C raised the peptide coverage from 10% to 47% when analyzed by tandem mass spectrometry. And 58-66% amino acid sequence coverage for the identified subtypes was obtained when cooking the sample for 5 minutes before reduction. Our study provides an effective and sensitive approach to characterize the subtype composition of an IFN mixture that may efficiently accelerate the development of natural IFN drug. | en_US |