d(GC) - Association of Biotechnology and Pharmacy

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