Multi-Dimensional Fluorescence Spectroscopy for Quantitative Cell Culture Media Analysis

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

Multi-Dimensional Fluorescence Spectroscopy for Quantitative Cell Culture Media Analysis

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

Chemometric approaches to low-content quantification (LCQ) in solid-state mixtures using Raman mapping spectroscopy.

The low-content quantification (LCQ) of active pharmaceutical ingredients or impurities in solid mixtures is important in pharmaceutical manufacturing and analysis. We previously demonstrated the feasibility of using Raman mapping of micro-scale heterogeneity of solid-state samples combined with partial least squares (PLS) regression for LCQ in a binary system.1 However, PLS is limited by the need for relatively high calibration sample numbers to attain high accuracy, and a rather significant computational time requirement for the large Raman maps. Here we evaluated alternative chemometric methods which might overcome these issues. The methods were: net analyte signal coupled with classical least squares (NAS-CLS), multivariate curve resolution (MCR), principal component analysis with CLS (PCA-CLS), and the ratio of characteristic analyte/matrix bands combined with shape-preserving piecewise cubic polynomial interpolation curve fitting (BR-PCHIP). For high (>1.0%) piracetam analyte content, all methods were accurate with relative errors of prediction (REP) of:peer-reviewed2018-10-3

Monitoring cell culture media degradation using surface enhanced Raman scattering (SERS) spectroscopy

Journal articleThe quality of the cell culture media used in biopharmaceutical manufacturing is a crucial factor affecting bioprocess performance and the quality of the final product. Due to their complex composition these media are inherently unstable, and significant compositional variations can occur particularly when in the prepared liquid state. For example photo-degradation of cell culture media can have adverse effects on cell viability and thus process performance. There is therefore, from quality control, quality assurance and process management view points, an urgent demand for the development of rapid and inexpensive tools for the stability monitoring of these complex mixtures. Spectroscopic methods, based on fluorescence or Raman measurements, have now become viable alternatives to more time-consuming and expensive (on a unit analysis cost) chromatographic and/or mass spectrometry based methods for routine analysis of media. Here we demonstrate the application of surface enhanced Raman scattering (SERS) spectroscopy for the simple, fast, analysis of cell culture media degradation. Once stringent reproducibility controls are implemented, chemometric data analysis methods can then be used to rapidly monitor the compositional changes in chemically defined media. SERS shows clearly that even when media are stored at low temperature (2-8 degrees C) and in the dark, significant chemical changes occur, particularly with regard to cysteine/cystine concentration

Comprehensive, quantitative bioprocess productivity monitoring using fluorescence EEM spectroscopy and chemometrics

Journal articleThis study demonstrates the application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative predictive analysis of recombinant glycoprotein production cultured in a Chinese hamster ovary (CHO) cell fed-batch process. The method relies on the fact that EEM spectra of complex solutions are very sensitive to compositional change. As the cultivation progressed, changes in the emission properties of various key fluorophores (e. g., tyrosine, tryptophan, and the glycoprotein product) showed significant differences, and this was used to follow culture progress via multiple curve resolution alternating least squares (MCR-ALS). MCR-ALS clearly showed the increase in the unique dityrosine emission from the product glycoprotein as the process progressed, thus provided a qualitative tool for process monitoring. For the quantitative predictive modelling of process performance, the EEM data was first subjected to variable selection and then using the most informative variables, partial least-squares (PLS) regression was implemented for glycoprotein yield prediction. Accurate predictions with relative errors of between 2.3 and 4.6% were obtained for samples extracted from the 100 to 5000 L scale bioreactors. This study shows that the combination of EEM spectroscopy and chemometric methods of evaluation provides a convenient method for monitoring at-line or off-line the productivity of industrial fed-batch mammalian cell culture processes from the small to large scale. This method has applicability to the advancement of process consistency, early problem detection, and quality-by-design (QbD) practices.IRCSE

Rapid quantification of tryptophan and tyrosine in chemically defined cell culture media using fluorescence spectroscopy

The rapid and inexpensive analysis of the complex cell culture media used in industrial mammalian cell culture is required for quality and variance monitoring. Excitation-emission matrix (EEM) spectroscopy combined with multi-way chemometrics is a robust methodology applicable for the analysis of raw materials, media, and bioprocess broths. We have shown that the methodology can identify compositional changes and predict the efficacy of media in terms of downstream titer [1]. Here we describe how to extend the measurement methodology for the quantification of tryptophan (Trp), tyrosine (Tyr) in complex chemically defined media. The sample type is an enriched basal RDF medium in which five significant fluorophores were identified: Trp, Tyr, pyridoxine, folic acid, and riboflavin. The relatively high chromophore concentrations and compositional complexity lead to very significant matrix effects which were assessed using PARAllel FACtor analysis2 (PARAFAC2). Taking these effects into account. N-way partial least squares (NPLS) combined with a modified standard addition method was used to build calibration models capable of quantifying Trp and Tyr with errors of similar to 4.5 and 5.5% respectively. This demonstrates the feasibility of using the EEM method for the rapid, quantitative analysis of Trp and Tyr in complex cell culture media with minimal sample handling as an alternative to chromatographic based methods.IRCSE