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)
and Biotechnology (NIGEB, Tehran, Iran). mPEG
(20 kDa) and SPA derivatives of mPEG (5 and
40 kDa) were purchased from SUNBIO (Anyang
City, South Korea).
mPEG activation by SPA: mPEG-SPA was
prepared and characterized according to Perry
and Kwang (27). Briefly, dried mPEG (1 mmol)
was dissolved in 20 mL of dry toluene. Then,
0.365 g of potassium tert butoxide was added to
the reaction mixture, which was then stirred for
6 h at 80 °C under nitrogen atmosphere. Then, 5
mL of methyl 3- bromo propionate was added
slowly to the reaction mixture, and was stirred
for another 20 h at room temperature under
nitrogen atmosphere. Then, the reaction mixture
was filtered and precipitated by adding dry diethyl
ether. The dried precipitated product was
dissolved in 25 mL of 1 N NaOH solution and
stirred for 2 h at room temperature under nitrogen
environment. HCl (6 N) was then slowly added
to the solution, until obtaining pH 3. Chloroform
was further added to the solution in order to
extract the reacted mPEG. Finally, the reacted
mPEG was reprecipitated by cold ethyl ether. Ten
grams of dried precipitated product and 0.52 g
of N-hydroxy succinimide were dissolved in 50
mL of methylene chloride under nitrogen
atmosphere. Then, 0.74 g of N, Ndicyclohexylcarbodiimide
was added to the
solution which was stirred in ice bath for 20 h.
After removing the precipitated dicyclohexyl urea,
the product was precipitated by adding dry diethyl
ether and dried in vacuum, overnight. The final
product (mPEG-SPA) was characterized by
Fourier Transform Infrared (FT-IR) Spectrometer
and stored in vacuum at -20°C until using (28,
29).
IFN PEGylation: IFN was conjugated to mPEG-
SPA derivatives of different molecular weights (5,
20 and 40 kDa) at the defined pH and protein/
mPEG, according to the designed experiment,
introducing in the following sections. The reaction
conditions were optimized to obtain high
conjugation yields in a reproducible manner.
Briefly, IFN solution (0.3 mg/mL) in phosphate
buffered saline (PBS, 20 mM, pH 7.2) was filtered
Interferon Beta-1a PEGylation
231
through a 0.45 µm pore size syringe filter. mPEG–
SPA was dissolved in 100 µL of dimethylsulfoxide
and added dropwise to the cold IFN solution (1.5
mL). Conjugation was allowed to proceed at 4°C
with an end-to-end rotation and quenched with
the addition of glycine (final concentration of 15
mM). The IFN–mPEG conjugate solution was
then stored at 4°C (19, 30).
Size-exclusion-HPLC (SE-HPLC): The
composition of IFN–mPEG conjugates was
analyzed using an HPLC system (Younglin
SDV30 PLUS, South Korea) consisting of a
Waters SP930P pump and M730D UV detector
equipped with an Autochro data module. The
samples were loaded on to a 8 mm × 300 mm
Shodex protein column KW-802.5 (Showa Denko
KK, Japan) through a guard column and eluted
with PBS (20 mM, pH 7.4) as a mobile phase.
The flow rate was adjusted to 0.8 mL/min and
the elutes were monitored by UV detection at 280
nm. The unmodified protein fraction as HPLC
response was calculated from the difference
between the unmodified protein peak area before
and after the reaction (30).
Ninhydrin test: Ninhydrin method was used to
determine the degree of modification by
measuring the number of free amino groups on
the surface of the modified and unmodified
proteins. Fifty microliters of the protein solution
was mixed with 100 µL of distilled water, 50 µL
of 4 M acetate buffer (pH 5.1) and 200 µL of
Ninhydrin reagent. The mixture was heated at
100 °C for 15 min, cooled at 10 °C for 10 min,
and diluted with 1 mL of 50% ethanol. After
centrifuging at 14000 rpm, the optical absorbance
was measured at 570 nm. The degree of
modification was calculated from the difference
between the free amino groups before and after
the reaction (31).
Design of experiments, full factorial design
at two levels: The first step (using Yates table
(32)) identified the most effective variables on
the PEGylation reaction using 5 and 20 kDa linear
mPEG-SPAs. The selection of these factors was
according to the literature on IFN PEGylation (25,