d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
30, 33). According to our experiments, an<br />
appropriate time for IFN PEGylation was 2 h (data<br />
not shown). The evaluated variables <strong>and</strong> their<br />
levels are showed in Table 1. Yates table design<br />
is presented in Table 2. All <strong>of</strong> the experiments<br />
were performed in duplicate, unless stated<br />
otherwise.<br />
Design <strong>of</strong> experiments, Taguchi statistical<br />
design: Taguchi statistical design was selected<br />
to determine the optimum conditions for IFN<br />
PEGylation, using 5 <strong>and</strong> 20 kDa linear mPEG-<br />
SPAs (34). An L 9 Taguchi array (34) was designed<br />
based on three variables (determined from the<br />
previous design) at three levels (Table 3) (30).<br />
The design (L 9 array) is shown in Table 4. All <strong>of</strong><br />
the experiments were performed in duplicate<br />
unless stated otherwise.<br />
Design <strong>of</strong> experiments, one factor at a time<br />
design: One factor at a time design was selected<br />
to study the impact <strong>of</strong> one variable (the ratio <strong>of</strong><br />
protein to mPEG fractions) on single response<br />
(HPLC or Ninhydrin test) for PEGylated IFN using<br />
40 kDa branched mPEG-SPA (32). To obtain the<br />
optimum conditions for PEGylation <strong>of</strong> IFN with<br />
40 kDa branched mPEG-SPA, the effect <strong>of</strong> the<br />
ratio <strong>of</strong> protein to mPEG fractions (at values <strong>of</strong><br />
1:10, 1:20, 1:30 <strong>and</strong> 1:40) at two different pHs <strong>of</strong><br />
7.4 <strong>and</strong> 8.0 was investigated.<br />
Determination <strong>of</strong> antiviral activity: The specific<br />
antiviral activity <strong>of</strong> IFN-ß- 1a was determined<br />
using a cytopathic effect (CPE) bioassay that<br />
232<br />
measures the ability <strong>of</strong> proteins to protect HeLa<br />
cells (grown at 37 °C/5% CO 2 ) challenged with<br />
the Vesicular Stomatitis Virus (VSV). HeLa cells<br />
suspended in 10 mL <strong>of</strong> Dulbecco´s modified<br />
eagles medium (DMEM) containing 5% fetal<br />
bovine serum, 9.53 g/L DMEM medium, 0.1 g/L<br />
<strong>of</strong> sodium pyruvate, 0.3 g/L <strong>of</strong> L-glutamine, 2.5-<br />
3.5 g/L <strong>of</strong> sodium bicarbonate <strong>and</strong> 50 µg/mL <strong>of</strong><br />
kanamycin-neomycin, were added to a 96-well<br />
microtiter plate <strong>and</strong> incubated for 24 h at 37° C.<br />
Serial dilutions <strong>of</strong> duplicate IFN-β-1a st<strong>and</strong>ards<br />
<strong>and</strong> test samples were then added, <strong>and</strong> the cells<br />
were incubated for further 24 h. The medium was<br />
removed, <strong>and</strong> VSV virus was added. The cells<br />
were incubated for 48 h, then 50 µL <strong>of</strong> 5 mg/mL<br />
<strong>of</strong> 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl-2Htetrazolium<br />
bromide (MTT) in PBS was added.<br />
After 1 h <strong>of</strong> incubation, the medium was removed<br />
<strong>and</strong> the wells were washed with 100 µL <strong>of</strong> PBS.<br />
The cells <strong>and</strong> dye were then solubilized with 100<br />
µL <strong>of</strong> 1.2 N HCl in 2-propanol, <strong>and</strong> the optical<br />
absorbance <strong>of</strong> medium was then measured at<br />
570 nm (1).<br />
Results <strong>and</strong> discussion<br />
Activation <strong>of</strong> mPEG: The synthesized mPEG-<br />
SPA was analyzed by FT-IR spectroscopy (FT-<br />
IR system: NICOLT, Lexus 670, USA). The FTIR<br />
spectrum <strong>of</strong> mPEG-SPA showed a peak at 1,725<br />
cm-1 , indicating the presence <strong>of</strong> a carbonyl bond.<br />
These spectra indicated that the mPEG-SPA was<br />
successfully synthesized (Figure 1).<br />
Fig. 1. FTIR spectrum <strong>of</strong> (a) 20 kDa mPEG <strong>and</strong> (b) 20 kDa mPEG-SPA.<br />
Ahmad Abolhasani et al