d(GC) - Association of Biotechnology and Pharmacy

More documents

Recommendations

Info

Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Table 4. An L 9 Taguchi array for IFNs PEGylation. Trial Polymer Protein/mPEG HPLC Ninhydrin Number MW (kDa) pH fractions Response Response 1 5.0 7.4 1:10 0.879 0.128 2 12.5 7.7 1:10 0.838 0.179 3 20.0 8.0 1:10 0.805 0.218 4 5.0 7.7 1:25 0.774 0.256 5 12.5 8.0 1:25 0.712 0.271 6 20.0 7.4 1:25 0.621 0.280 7 5.0 8.0 1:40 0.605 0.412 8 12.5 7.4 1:40 0.611 0.390 9 20.0 7.7 1:40 0.579 0.431 the result is represented as the ratio of the absorption difference between the non- PEGylated and PEGylated to the absorption of non-PEGylated protein. Therefore, in Ninhydrin test, a higher ratio shows higher levels of polymer attachment to the protein. Full factoriel design: By considering the obtained results by HPLC and Ninhydrin tests, full factorial design was employed to screen the main effective factors on PEGylation, using linear polymers with molecular weights of 5 and 20 kDa. Table 2 shows data regarding the full factorial design using linear PEGs of 5 and 20 kDa. According to Table 2, by increasing the level of pH, polymer molecular weight, and the ratio of protein to mPEG fractions, HPLC responses have decreased and Ninhydrin responses increased, indicating that the degree of PEG attachment to the protein has increased. Diwan and Park reported that by increasing the polymer molecular weight, pH, and the ratio of protein to mPEG fractions during PEGylation of IFN-β with mPEG-SPA, the degree of PEGylation increased (28). By considering the analysis of variance (ANOVA) and p-values (Table 5) less than 0.01, the most important factors were determined. These results show that PEG molecular weight (A), pH (B), and the ratio of protein to mPEG fractions (C) are important but the effect of their interactions are not significant. Ahmad Abolhasani et al 234 Taguchi statistical design: By considering the important factors, Taguchi method (L 9 array) was used to obtain the optimum conditions using the linear PEGs. Table 4, shows the obtained results by Taguchi design using linear PEGs of 5 and 20 kDa. According to Table 4, by increasing the levels of pH, polymer molecular weight and the ratio of protein to mPEG fractions, HPLC responses have decreased and Ninhydrin responses increased, indicating an increase in attachment of PEG to the protein. This shows that the amount of protein PEGylation has been improved by increasing the level of the factors. Hence the optimum conditions for PEGylation that were obtained by software are as follows: pH 8; the ratio of protein to mPEG fractions 1:40; PEG molecular weight 20 kDa. One factor at a time design: Figure 2 shows the obtained results for one factor at a time design using 40 kDa branched PEG. PEG attachment to the protein increased following increases in pH and the ratio of mPEG to protein, as HPLC responses decreased and Ninhydrin responses increased. It shows the optimum condition for PEGylation reaction using the branched 40 kDa PEG, as pH 8 and the ratio of protein to mPEG fractions 1:40. At this point, the non PEGylated protein component is at its minimum and the numbers of the reacted amino groups are at their maximum. Table 6 shows the optimum conditions
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) and the predicted and experimental responses obtained by HPLC and Ninhydrin tests from Taguchi and one factor at a time designs. It is observed that under similar conditions, the branched PEG could produce more PEGylated protein. Determination of antiviral activity: In this study, results of the biological activity of PEGylated IFNβ are reported as qualitative and quantitative responses. The qualitative results are presented in Figure 3, obtained by an optical microscope from the infected HeLa cells. These images show the cells’ health conditions and biological activity, indicating that cells with distinct shapes, sizes and cell walls were still viable. 235 Also, the optical absorption by the HeLa cells’ growth medium was measured and compared. A change in the color of HeLa cells’ growth medium indicates the viability of cells and the amount of cell density. Changing the yellow tetrazole to the purple formazan occurs just when the reductase enzymes are active, because tetrazol reacts with the reductase enzymes (MTT Test). Therefore, such changes in the color of the medium are used to measure the quantity of viable cells. The levels of optical absorption by the purple formazan solution was measured and compared with the two states: the solution containing the relevant cells and the control cells. Figure 3a shows the cell growth medium used as control. Fig. 2. The effect of different ratios of protein to mPEG at two different pHs on the extent of PEGylation progress; (a) HPLC results and (b) Ninhydrin results obtained from one factor at a time design for 40 kDa mPEG. Fig. 3. Biological activities in the hela cells. (a) Control cell (b) Control cells+interferon before the PEGylation reaction, (c) control cells+interferon before PEGylation reaction+virus, (d) control cells+PEGylated interferon, (e) control cells+PEGylated interferon+ virus, (f) control cells+virus. Interferon Beta-1a PEGylation
Page 3 and 4:
Current Trends in Biotechnology and
Page 5 and 6:
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72: Current Trends in Biotechnology and
Page 121: Current Trends in Biotechnology and
Page 147 and 148: 6th Annual Convention of Associatio
show all

d(GC) - Association of Biotechnology and Pharmacy

Create successful ePaper yourself

Delete template?

Save as template?