The Structural Characterization of Recombinant Human Interferon
The recombinant human interferon gamma (rhIFN-γ)
that is produced in E. coli by a method based on recombinant DNA
technology, increases macrophage cytotoxicity. In this study to evaluate
the structural characterization of rhIFN-γ, considered two conditions:
fresh sample (4°C) and old sample (6 months in 25°C) of rhIFN-γ.
Fresh sample (4°C) and old sample (6 months in 25°C) analyzed
by analytical methods for determination of biological activity and structural
analysis (deamidated and oxidized, heterodimers, monomer and aggregates,
covalent dimer and SDS-PAGE). The structural characterization of them
The human interferons (hIFNs) are a family of proteins. The initial discovery
of which was based on their ability to inhibit viral growth in target
cells (Friedland, 1996). Their biological effects in vivo include
antiviral activity, cell growth inhibitor and immunomodulator activity
(Pitha, 2007; Dheda et al., 2005; Perez et al., 1990; Chen
et al., 1992). IFN-γ is more potent than the other two IFNs
(α,β) in its immunomodulatory activities. The antiproliferative
effects make it a potentially useful against cancer (kidney cell carcinoma,
colon cancer and rheumatoid arthritis). The IFN-γ is secreted by
human antigen stimulated T-lymphocytes in response to viral infection
and various other inducer. The native human interferon gamma is composed
of 143 amino acid residues with a total molecular mass of 20-25 kD. It
is glycosylated and does not contain cysteine residues (Khalilzadeh et
al., 2004; Mohammadian-Mosaabadi et al., 2007). In 1986, hIFN-γ
cDNA was cloned and expressed in Escherichia coli. This rh-IFN-γ
produced in E. coli is not glycosylated and has methionine as its
N-terminal residue. The total molecular mass of rh-IFN-γ is 17 kD
that it is physiologically active (Pitha, 2007). The protein consists
of non covalent dimers of two identical monomers.
Recombinant proteins expressed in genetically transformed cells must
accurately represent the natural molecules that they are intended to replace
or complement. They should also fulfill the minimum requirements for biological
efficacy, safety and quality criteria as do other preparations intended
for pharmaceutical use (Zhang and Tong, 1992).
The deferent testing is done for evaluation of this recombinant product
so as biological activity and structural analysis.
This study describes the characterization of final product of rhIFN-γ
as structural analysis: covalent dimer and oligomer, deamidated, oxidized,
monomer and aggregate form of hIFN-γ in fresh and old sample that
described and comparison of the electrophoretic pattern and biological
activity was done.
MATERIALS AND METHODS
Analysis of covalent dimer: Size Exclusion Chromatography (SEC)
with HPLC method was used to check purity of rhIFN-γ and the presence
of undesired form including covalent dimer. The column (7.5x300 mm 10,000-300,000
Dalton), ultropak TSK G3000 SW LKB was equilibrating with sodium phosphate
buffer (0.2M, pH 6.8) at a flow rate of 1 mL min-1 and mixture
of molecular mass standards, BSA (66 kD), trypsin inhibitor (20.1 kD),
lysozyme (14.4 kD) as resolution solution, fresh sample and old sample
of rhIFN-γ as tests solution were used. The effluent was monitored
at 210 nm.
Analysis of deamidated and oxidized forms and heterodimers: Ion
Exchange Chromatography (IEC) with HPLC method was used to check these
forms of rhIFN-γ. The column (IEC sp-825 Shodex 8 mm ID x 75 mm L-1)
was equilibrated with ammonium acetate 0.05 M, pH 6.5 (buffer A) and developed
with a linear gradient (100-0%), (0-100%) ammonium acetate 1.2 M , pH
6.5 (buffer B) in a 57 min period at flow rate 1.2 mL min-1
in 35°C and tests solution (fresh and old) were injected and detected.
The effluents were monitored at 280 nm.
Analysis of monomer and aggregates: The column (7.5x300 mm 10,000-500,000
Dalton), ultropak TSK G3000 SW LKB was used to check monomer and aggregates
form of rhIFN-γ. After equilibrating of column with potassium chloride
(1.2 M) in solution A (0.59 g L-1 Succinic acid and 40 g L-1
mannitol pH 5) at flow rate 0.8 mL min-1, 20 μL of each,
resolution solution (BSA + Imukin), tests solution (fresh and old) were
injected and detected at 214 nm.
Analysis of purity of rhIFN-γ by SDS-PAGE: This analysis
was carried out by SDS-PAGE. The test was performed under reducing conditions
using resolving gels of 15% acryl amide and silver staining as the detection
purities fresh and old samples of rhIFN-γ.
Assay of biological activity: The method relies on viral cytopathic
effect (CPE) inhibition using semi-micro dyebinding technique in IFN-treated
cells and comparing activity of rhIFN-γ with standard samples of
The assay was performed by incubating a fixed count of vero (African
green monkey kidney) cell line with serial dilutions of rhIFN-γ followed
by challenging the cells with defined plaque-forming units of Vesicular
Stomatitis Virus (VSV), all according to standardized procedure (Tsanev
and Ivanov, 2001; Mohammadian-Mosaabadi et al., 2005).
The interferon activity was calculated as the reciprocal of the dilution
in the well of the titre plate where 50% of the vero cell monolayer is
protected from the CPE of challenging virus. The standard interferon gamma
was used in this test is WHO standard as: Interferon gamma, human 88/606
(National Institute of Biological Standard and Control Blanche Lane, England).
RESULTS AND DISCUSSION
The rhIFN-γ is a single chain protein has a molecular mass of 17
kD, The active form of it is considered to a homo dimmer in which two
monomers are none covalently bound in anti parallel Orientation (Tsanev
and Ivanov, 2001; Walsh Gary, 2003). So, in characterization of rhIFN-γ
is necessary to determine possible covalent dimer forms as undesired product
that form in the assessment of covalent dimers was done by gel filtration
Figure 1 shows the fresh sample in 4°C of rhIFN
lacks the covalent dimer forms but HPLC analysis of a rhIFN-γ stored
for 6 months in 25°C, shows the peaks with approximately 35 kD and
higher in comparison to molecular weight standards (BSA, Trypsin inhibitor,
Lysozyme) that seems belongs to covalent dimmer forms. Since, the rh IFN-γ
has no cysteine residue; the forms are not disulfide linked dimmer.
Since, one source of protein variability comes from oxidative protein
modification to analysis of rhIFN-γ The examination of oxidized and
deamidated forms of Protein was considered. Generally, these modified
forms are produced during manufacturing process or storage of proteins.
In order to determination of deamidated and oxidized forms, the fresh
sample in 4°C of rhIFN and the old sample stored 6 months in 25°C
was examined by strong cation exchange chromatography. The data are shown
in Fig. 2, in old sample principal peak (rhIFN-gamma)
is about 27.4 min and heterodimers 25.4 min, deamidated and oxidized forms
in 26.2 min were eluted at shorter retention time, relative to principal
peak. Fresh sample contains any unmodified (deamidated, oxidized, heterodimers)
||Analysis of covalent dimer, comparison of graphs from
(—), standard proteins which contains Bovine Serum Albumin (66
kD) peak No 1, Soybean Trypsin Inhibitor (20.1 kD) peak No 2 and lysozyme
(14.4 kD) peak No 3 and (……), fresh gamma interferon with
any peak and shoulder before main peak, (—), old sample
(6 mounts in 25°C) gamma interferon
In the purification process of rhIFNγ during unfolding and refolding
reactions, Some portion of monomers doesn`t assemble in to active dimer
forms and remain as monomers forms. Protein aggregation is a common issue
encountered during manufacture of bio pharmaceutical drug. Although the
aggregated forms are removed during purification process by SEC chromatography
and so on but it seems, some these forms because of stresses can lead
to protein aggregation, are observed in final product. So, the rhIFNγ
was analyzed by analytical SEC based on difference in size of monomer
and aggregates that the peaks of monomer, native dimer and aggregated
are isolated. Figure 3 shows that fresh sample is followed
the pattern of commercial rhIFNγ in standard sample that contains
native dimer form without any monomer and aggregate form. The old sample
has a little aggregate form and no monomer.
||Analysis of deamidated and oxidized forms and heterodimers,
comparison of graphs from old sample (6 mounts in 25°C) gamma
interferon (—) which contains principal peak (rhIFN-gamma) is
about 27.4 min and Heterodimers (25.4 min), Deamidated and oxidized
forms (26.2 min) were eluted at shorter retention time, relative principal
peak, with (—) fresh gamma interferon without unmodified
||Analysis of monomer and aggregates, comparison of graphs
from standard proteins (—), which contains Bovine Serum Albumin
(66 kD) with retention time equal to 8.85 min, commercial gamma interferon
with retention time 10.13 min, with (……) rhIFN gamma and
(—) old sample (6 months in 25°C) rhIFN gamma
In Fig. 4, the comparison of electerephoretic pattern
is shown that the old sample has a band between 35 and 25 kD bands of
standard sample that it may be is related to covalent dimer and a band
between 14.4 and 18.8 kD region is belonged to monomer form of IFN in
while the fresh sample has a one band in monomer region.
The most convenient method for interferon assay is quantitating its antiviral
activity or inhibition of cytopathic effect (CPE). In this research, we
looked for a fast and sensitive method to quantitative antiviral activity
of this cytokin based on cytopathic effect/dye uptake. After standardization
of method, activity of fresh and old IFN-γ was compared to standard
sample (WHO standard) (Fig. 5). In regard to biological
activity of standard sample, 20x106 IU mg-1, the
biological activity of fresh sample is near the WHO standard, but biological
activity of old sample approximately two times decreased. Antiviral activity
of rhIFN gamma in vero cell line shown in Fig. 6.
According to obtained results, we can observe that rhIFN-γ is a
sensitive protein. Modifications of rhIFN-γ such as deamidated and
oxidized, monomer, covalent dimer and aggregate happened in during life
time (0 and 6 months) and temperature change (4 and 25°C). These modifications
change the structure of rhIFN-γ and decrease approximately two times
biological activity. It may be these assessments are so necessary for
this protein using as a recombinant pharmaceutical drug.
||SDS-PAGE analysis of rhIFN-gamma. Lane 1 is MW standards
from MBI Fermentes Company which contain β-galactosidase 116.0
kD, BSA 66.2 kD, ovalbumin 45.0 kD, LDH 35 kD, Bsp981 restriction
endonuclease 25 kD, β-lactoglobulin 18.4 kD and lysozyme 14.4
kD. IFN-gamma band should stand between the last two bands. Lane 2
is old sample (6 mounts in 25°C) rhIFN-gamma and Lane 3 fresh
||Determination of anti-viral activity of IFN-gamma
Antiviral activity of rhIFN gamma in vero cell line.
Normal Vero cell line without any treatment (a), Vero cell line
that treated with 500 ng mL-1 of IFN-γ(WHO) and
challenged with 1/50,000 dilution of VSV stock after 48 h (b), Vero
cell line that treated with 500 ng mL-1 fresh sample
of IFN-γ (4°C) and challenged with 1/50,000 dilution of
our VSV stock after 48 h (c), Vero cell line that treated with 500
ng mL-1 old sample of IFN-γ (6 months in 25°C)
and challenged with 1/50,000 dilution of this VSV stock after 48
Chen, A.B., A.A. Championsmith, J. Blanchard, J. Gorrell and B.A. Niepelt et al., 1992. Quantitation of E. coli protein impurities in recombinant human interferon gamma. Applied Biochem. Biotechnol., 36: 137-152.
Dheda, K., Z.F. Udwadia, J.F. Huggett, M.A. Johnson and G.A. Rook, 2005. Utility of the antigen-specific interferon-gamma assay for the management of tuberculosis. Curr. Opin. Pulm. Med., 11: 195-202.
PubMed | Direct Link |
Friedland, J.S., 1996. Chemokines in viral disease. Res. Virol., 147: 131-138.
Khalilzadeh, R., S.A. Shojaosadati and N. Maleksabet, 2004. Process development for production of rh IFNγ expressed in E. coli. J. Ind. Microbiol. Biotechnol., 31: 63-69.
Mohammadian-Mosaabadi, J., H. Naderi-Manesh, N. Maghsoudi, M.A. Nassiri-Khalili, M.R. Masoumian and N. Malek-Sabet, 2007. Improving purification of recombinant human interferon γ expressed in Escherichia coli; effect of removal of impurity on the process yield. Protein Express. Purif., 51: 147-156.
Mohammadian-Mosaabadi, J., H. Naderi-Manesh, N. Maghsoudi, R. Khalilzadeh, S.A. Shojaosadati and M. Ebrahimi, 2005. Effect of oxidative stress on the production of recombinant human interferon-gamma in Escherichia coli. Biotechnol. Applied Biochem., 41: 37-42.
Perez, L., J. Vega and C. Chuay, 1990. Production and characterization of human gamma interferon from E. coli. Applied Microbiol. Biotechnol., 33: 429-434.
Pitha, P.M., 2007. Interferon: The 50th Anniversary. 1st Edn., Springer, New York, USA., ISBN: 978-3-540-71328-9.
Tsanev, R.G. and I.G. Ivanov, 2001 2001. Immune Interferon: Properties and Clinical Application. 1st Edn. CRC Press LLC, USA., ISBN: 0849311489.
Walsh Gary, 2003. Biopharmaceuticals: Biochemistry and Biotechnology. 1st Edn., John Wiley and Sons Ltd., New York, USA., ISBN:0470843268, pp: 150-167.
Zhang, Z. and K.T. Tong, 1992. Production, purification and characterization of recombinant human interferon. J. Chromatogr., 604: 143-155.
2019 Science Alert. All Rights Reserved