Multi-wavelength (2D) fluorescence spectroscopy represents an important step towards exploiting the monitoring potential of microtiter plates (MTPs) during early-stage bioprocess development. In combination with multivariate data analysis (MVDA), important process information can be obtained, while repetitive, cost-intensive sample analytics can be reduced. This study provides a comprehensive experimental dataset of online and offline measurements for batch cultures of Hansenula polymorpha. In the first step, principal component analysis (PCA) was used to assess spectral data quality. Secondly, partial least-squares (PLS) regression models were generated, based on spectral data of two cultivation conditions and offline samples for glycerol, cell dry weight, and pH value. Thereby, the time-wise resolution increased 12-fold compared to the offline sampling interval of 6 h. The PLS models were validated using offline samples of a shorter sampling interval. Very good model transferability was shown during the PLS model application to the spectral data of cultures with six varying initial cultivation conditions. For all the predicted variables, a relative root-mean-square error (RMSE) below 6% was obtained. Based on the findings, the initial experimental strategy was re-evaluated and a more practical approach with minimised sampling effort and elevated experimental throughput was proposed. In conclusion, the study underlines the high potential of multi-wavelength (2D) fluorescence spectroscopy and provides an evaluation workflow for PLS modelling in microtiter plates.
Keywords: 2D fluorescence spectroscopy; high-throughput; microtiter plate; multivariate data analysis; online monitoring.