Fig. 4.3.17 FTIR plots of native protein (black line i.e. reference at room temp.) protein with out excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 65°C for 5 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance).
Observing the FTIR plots for formulated protein using maltodextrin as an excipient (Fig- 4.3.17) it is clearly visible that, the plots of thermal denatured native protein goes side by side with that of the formulated protein. In the formulated protein the basic peak that corresponds to the -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure.
56 Fig.4.3.18 FTIR plots of native protein (black line i.e. reference at room temp.) protein with out excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 65°C for 10 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance).
In Fig. 4.3.18 all the three plots i.e. of control, native and formulated samples have similar kind of peaks which is clearly visible. In the formulated protein the basic peak that corresponds to the -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure.
57 Fig. 4.3.19 FTIR plots of native protein (black line i.e. reference at room temp.) protein with out excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 65°C for 40 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance).
Analyzing the result for Fig. 4.3.19, we observed the similar type of situation as that of the above two cases. The plots of thermal denatured native protein goes side by side with that of the formulated protein, whereas control shows different peaks than that of the other two. In the formulated protein the basic peak that corresponds to the -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure.
58 Fig. 4.3.20 FTIR plots of native protein (black line i.e. reference at room temp.) protein without excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 65°C for 60 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance).
For formulated sample in place of a peak corresponding to -helix at 1655 cm-1 we got a trough over here, which is a clear cut indication that -helix is not protected. Also the plots of thermal denatured native protein goes side by side with that of the formulated protein. So the excipient is not working in the specified temperature for the specific time of exposure.
59 Fig. 4.3.21 FTIR plots of native protein (black line i.e. reference at room temp.) protein without excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 75°C for 5 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance).
Formulated and heat treated protein lacks peaks at the region of 1655 cm‾1 (Fig.4.3.21) which is related to the -helix and also peaks are shifted from its original position that can be marked. The peak found in the region of 1687 cm‾1 is shifted to 1693 cm‾1, 1676 cm‾1 to 1679 cm‾1 and 1641 cm‾1 to 1644 cm‾1. In case of formulated ones, it does not protect the band at 1655 cm‾1 and there is shift of peaks and the peak numbers and hence the excipient fails to protect the protein in specified conditions.
60 Fig 4.3.22 FTIR plots of native protein (black line i.e. reference at room temp.) protein without excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 75°C for 10 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance)
As per Fig. 4.3.22, we observed the similar type of situation as that of the above two cases. The plots of thermal denatured native protein goes side by side with that of the formulated protein, whereas control shows different peaks than that of the other two. In the formulated protein the basic peak that corresponds to the -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure
61 Fig. 4.3.23 FTIR plots of native protein (black line i.e. reference at room temp.) protein without excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 75°C for 40 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance)
In the above figure we got totally irregular results. For the higher wave numbers native denatured sample showed similar peaks with that of control one, where as for lower wave number native denatured sample shows similar peaks as that of formulated one, but the very basic for stability of formulated protein is the peak that corresponds to the -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure
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Fig. 4.3.24 FTIR plots of native protein (black line i.e. reference at room temp.) protein without excipient heat treated (red line) and formulated sample (green line- spray dried with maltodextrin at 75°C for 60 min treatment). The graph shows second derivative form of the original spectrum of Amide-I region (X-axis: wave number 1700-1600 cm‾1 and Y-axis: absorbance
For this particular plot we observed at higher wave number all the three plots showing similar type of peaks but for lower wave numbers native denatured sample shows similar peaks as that of formulated one. And again the vital peak is missing i.e. -helix at 1655 cm-1 is not conserved. So the excipient is not working in the specified temperature for the specific time of exposure.
Now coming to the overall result analysis of the formulated sample using maltodextrin as excipient for the specific temperature and specified time of exposure to heat we can concluded that, maltodextrin can not protect the basic α-helix at 1655 cm- 1. Neither it shows protection for higher time of exposure nor for lower time of exposure. It neither protects at 75°C nor at 65°C. So it is not a better option to be used as an excipient.
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