نبذة مختصرة : Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early-stage development and optimization of a polarised Total Synchronous Fluorescence Spectroscopy (pTSFS) method for protein quantification in a hydrolysate-protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (¿), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarised) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of > 5000 was achieved for concentrations of BSA/YST 1.2/10 g L-1 with TSFS|| . Optimisation using genetic algorithm and interval Partial Least Squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast ( ; This publication emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant number (14/IA/2282, Advanced Analytics for Biological Therapeutic Manufacture, to AGR). We also thank Agilent Technologies (Mulgrave Victoria, Australia) for an instrument loan. The authors declare no financial or commercial conflict of interest. ; peer-reviewed
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