الفهرس 
Hepatitis C in EgyptOnly 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).