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Development of a rapid Polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth
Boateng, Bernard O. Elcoroaristizabal, Saioa Ryder, Alan G.
Boateng, Bernard O.
Elcoroaristizabal, Saioa
Ryder, Alan G.
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Date
2021-01-27
Keywords
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journal article
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Citation
Boateng, Bernard O., Elcoroaristizabal, Saioa, & Ryder, Alan G. (2021). Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth. Biotechnology and Bioengineering, 118(5), 1805-1817. doi:https://doi.org/10.1002/bit.27694
Abstract
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 (
), 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 (
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Science Foundation Ireland
European Regional Development Fund
European Regional Development Fund
Publisher
Wiley
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License
CC BY