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