Development of rapid spectroscopic methods for the analysis of cell culture media

Industrial scale cell culture is used for the production of many therapeutic agents such as protein and vaccines. Cell culture medium is a vital raw material used in these production processes. Formulation analysis of the medium is thus an essential task of any bioprocess. The medium is a critical aspect of the process because it has to supply all of the necessary nutrients and other factors to ensure growth and productivity. Small variations in medium composition can alter cell metabolism, thereby changing process efficiency and productivity. There is an ongoing need for analytical methods to ensure reproducible medium formulations; therefore, real¿time qualitative and quantitative analysis of medium components by spectroscopic methods in combination with chemometrics has the potential to be adapted as a PAT tool in bioprocesses. This thesis investigates the spectroscopic analysis and quantification of three medium components - D-glucose, eRDF and yeastolate - in model medium formulations by Raman, Surface Enhanced Raman Scattering (SERS) and two fluorescence approaches (Excitation Emission Matrix (EEM) and Total Synchronous Fluorescence Scan (TSFS)). These methods were used in conjunction with chemometrics to provide a wealth of information about medium composition: qualitative assessment and outlier detection through principal component analysis and robust principal component analysis, fluorophore detection and identification using parallel factor analysis and multivariate curve resolution, and quantitative analysis achieved with partial least squares. These studies complement previous studies in this laboratory where specific component quantification [1, 2] and variance analysis were used for characterising, screening [3-5] and quantifying the performances of cell culture media by spectroscopic methods [6-8]. The advantages of spectroscopic methods are that they require little to no sample preparation and they give spectra with rich information content suitable for the discrimination of subtle chemical and physical effects. The goal of this work was to see if these spectroscopic methods could be used to accurately quantify medium components, both simple (glucose) and complex (yeastolate and eRDF). The end-use application was to develop a quality assurance method for correct medium preparation/formulation. Quantitative accuracy varied with the methods due to various experimental factors. Various different pre-processing techniques were used to minimise unwanted spectral effects such as noise, intensity and baseline differences. With Raman, quantification of D-glucose, eRDF and yeastolate was achieved with an error of ~5%, ~16% and ~38% respectively. The SERS model gave error percentages of 16% for the eRDF and 12% error for yeastolate, while the best fluorescence model gave error figures of 5.4% for yeastolate and 7.2% for eRDF. These models show the potential of these spectroscopic methods for the measurement/identification of individual medium components within complex cell culture medium. However, the error level obtained suggests that improvement could be achieved through modification of the current experimental setup which would then lead to more accurate prediction of component concentrations.2017-02-1