| Popis: |
Industrial mammalian cell culture uses complex media, such as eRDF, to support cell growth and product formation as an integral part of the process; as a result the analysis of media variance/quality is of critical importance[1, 2]. Furthermore, there is a need to analyse the media in their prepared state. The use of fluorescence Excitation-Emission Matrix (EEM) spectroscopy as a potential tool for the rapid, in-situ, and routine quantitative and qualitative analysis of eRDF cell culture media was investigated. The presence of multiple chromophores in the sample leading to high absorbance values causes large Inner Filter Effects (IFE) introducing an intrinsic non-linearity into the EEM data. The first step of this research was to use PARAFAC2 on an EEM dataset collected from eRDF solutions of different concentrations to model sample matrix and concentration effects on the emission of individual fluorophores. Using a classical standard addition (cSA) method coupled with NPLS, it was then shown that quantification of tryptophan (Trp), tyrosine (Tyr), pyridoxine (Py) and riboflavin (RF) in the eRDF media could be achieved by fluorescence analysis on these types of samples; prediction errors of less than 5 % were obtained. However, due to its complex photo-physics a prediction error of 10 % for folic acid (FA) was achieved. By using cSA, only samples with analyte concentrations larger than their initial concentrations in the Test sample (c0) could be quantified, thus a modified standard addition (mSA) [3] was required to be able to predict concentrations less than c0. This method was refined through the selection of (1) sub-sections of the full EEMs and (2) specific samples and analytes to use in the calibration step. This led to prediction errors of less than 5% to be achieved for Trp, Tyr, Py and RF with a method that allowed for prediction in any sample with analyte concentrations above or below c0. The use of fluorescence was also investigated for the monitoring of cell culture media change during storage. The PARAFAC analysis of these EEMs suggested that fluorescence could be used to monitor photo-degradation of samples exposed to light. In order to observe changes that were not due to light exposure, Surface Enhanced Raman Scattering (SERS) was used in combination with PCA analysis and the results suggested that cysteine degradation could be monitored. The implementation of SERS (sample handling and preparation technique) was then optimized for application in quantitative analysis; in particular the importance of appropriate sample to colloid ratio was discussed. SERS was then shown to be a potential technique for the determination of FA concentration in eRDF solutions with an error of ~ 9 % using a cSA based method. |
