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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) PEG conjugation (PEGylation) is an established technology that donates many beneficial effects to the proteins, including increased circulation half-life, reduced immunogenicity and antigenicity and decreased toxicity (7). Since the initial demonstration of PEGylated proteins as therapeutic agents, several proteins have been PEGylated and shown useful properties in clinical applications (8). In the PEGylation technology, the size and structure of the PEG moiety, play important roles in the pharmacokinetic and pharmacodynamic properties of the resulting protein conjugates. Increasing the molecular size by PEGylation in particular, slows the renal ultrafiltration and prolongs the residence time of the drugs in blood circulation (9). Therefore, the current PEGylation technology needs high-molecular-weight PEG reagents to obtain favorable pharmacokinetic profiles (9-11). In fact, branch-structured PEGs allow for a higher molecular weight of up to 60 kDa as compared to linear PEGs with less than 30 kDa molecular weights. In addition, the branched PEGs act as if they are much larger than linear PEGs of the same molecular weight and show more effectiveness in protecting the proteins from proteolytic degradation and in reducing immunogenicity (7, 9). A linear 5-kDa PEG was conjugated to interferon alpha (IFN-α), in an early attempt (12); however, this conjugate did not make any significant improvements in increasing the circulation half-life, compared to the unmodified IFN (13). To improve the pharmacokinetic properties, a linear 12-kDa PEG was conjugated to IFN and the resulting conjugate (PEG-Intron, Schering–Plough), showed a significant increasing in the circulation half-life, when compared to the unmodified IFN, with measurable serum concentrations detected after single weekly administrations (14). The next generation of PEGylated IFNs was obtained by conjugating a branched 40-kDa PEG structure to IFN via an amide linkage. This Ahmad Abolhasani et al 230 product, Pegasys; Roche, showed superior efficacy over the unmodified IFN, with a significant increasing in the circulating half-life and reduced renal clearance, resulting in a strong antiviral response throughout a once-weekly dosing schedule (15-17). Similar success was recently achieved using a trimer-structured 43kDa PEG, which is the slightly modified form of the branched PEG prepared by attachment a 3kDa PEG to the branched 40-kDa PEG (18). This mono-PEG43K-IFN was absorbed slowly and had markedly reduced clearance in rats, thereby increased the half-life, approximately 40-fold compared to the native IFN (19). Initial progress on PEG-IFN-β-1a has already been reported (9, 20, 21), but a general strategy for creating tailored PEGylated IFN-β- 1a has not been developed since 1990 (22). Similar success was recently achieved by conjugating a branched PEG structure to IFN-ß via an amide linkage, as mentioned above. Its product showed superior efficacy over unmodified IFN-β, with a significant increasing in the circulating half-life and reduced renal clearance, resulting in a strong antiviral response throughout a once-weekly dosing schedule (1, 23-26). For protein PEGylation, PEG must be initially activated in order to be able to react with the functional groups on the protein surface, mostly ε-amino group of lysine. In previous studies, methoxy-PEG (mPEG) has been activated by succinimidyl propionic acid (SPA) as an useful activator for antibodies and pancreatic islet PEGylation (19, 25, 28). In the present study, we prepared and characterized IFN-β-1a modified with 5 and 20 kDa linear mPEG-SPAs and also, 40 kDa branched mPEG-SPA. We also reported the optimum conditions for PEGylation and biological activity of the PEGylated protein relative to the unmodified one. Materials and Methods Materials: Recombinant IFN-β-1a was obtained from National Institute of Genetic Engineering
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,
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