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https://doi.org/10.1002/dta.2691
Copy DOIJournal: Drug Testing and Analysis | Publication Date: Sep 12, 2019 |
Citations: 6 |
Thermal sensitivity, as a practical measure of thermostability, is an interesting quality attribute that can be used in the quality control (QC) release of biopharmaceuticals. This article investigates circular dichroism (CD) spectroscopy and nano-differential scanning fluorimetry (nano-DSF) to evaluate the thermal stability of E.coli L-asparaginase (L-ASNase) for QC purposes. In CD, molar ellipticity as a function of temperature (from 20 to 80°C) was measured at 222nm. Different L-ASNase samples dissolved in different diluents were investigated by determining the melting temperature (Tm ) from the first derivative curve as well as the slope of the fitted sigmoidal function of the temperature gradient CD data. The obtained Tm values could be correlated with the L-ASNase sample origin as well as with the pH of the diluent. The Tm values obtained from the CD data were moreover consistent with the Tm values determined by nano-DSF, confirming their reliability. Next to the Tm value, also the slope of the fitted sigmoidal CD-function was able to differentiate different L-ASNase samples, including unstressed from stressed protein. By using both the Tm and the curve slope, the thermal stability of L-ASNase was investigated, demonstrating and recommending the use of this heat-stress characteristic as a QC quality attribute of proteins, which can be applied to detect substandard and falsified proteins.
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