d(GC) - Association of Biotechnology and Pharmacy

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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
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Table 1. The selected variables and their corresponding levels for full factorial design. Variable Low level High level (1) (2) A: Polymer molecular weight (KDa) 5 20 B: pH 7.4 8.0 C: The ratio of protein to mPEG fractions 1:10 1:40 Table 2. Yates table design (full factorial design) for screening the factors. Treatment Polymer pH Protein/mPEG HPLC Ninhydrin Combination a MW (B) fractions (C) Response Response (kDa) (A) 1 5 7.4 1:10 0.879 0.128 a 20 7.4 1:10 0.868 0.135 b 5 8.0 1:10 0.860 0.153 ab 20 8.0 1:10 0.809 0.208 c 5 7.4 1:40 0.655 0.352 ac 20 7.4 1:40 0.588 0.415 bc 5 8.0 1:40 0.612 0.405 abc 20 8.0 1:40 0.545 0.468 a The low level of any variable is denoted by l, and the high level of any variable is denoted by its lower-case letter. Table 3. The selected variables and their corresponding levels for Taguchi design. Variable First level (1) Second level (2) Third level (3) A:Polymer molecular weight (KDa) 5.0 12.5 20.0 B: pH 7.4 7.7 8.0 C: The ratio of protein to mPEG fractions 1:10 1:25 1:40 Protein PEGylation: The degree of polymer attachment to IFN-β was assessed by HPLC for PEGylated proteins using linear polymers with molecular weights of 5 and 20 kDa, and also the branched polymer of 40 kDa. The samples were loaded on a SE-HPLC column to separate the PEGylated and non-PEGylated proteins on the basis of their molecular size, as evaluated by the Autochro 3000 software. For each test, the ratio of the non-PEGylated protein peak to the total Interferon Beta-1a PEGylation 233 peaks represented the response. Therefore, in this procedure, a lowers ratio leads to higher levels of polymer attachment to the protein. Furthermore, Ninhydrin test was also done to obtain the total amount of polymer attached to the protein. In this test, the reaction of Ninhydrin with free amino groups on the protein surface showed that the PEGylated protein reacts with lower levels of Ninhydrin in comparison to the non-PEGylated protein. Consequently, in this test,
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