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

Abstract

11The rapid and inexpensive analysis of the complex cell culture media used in industrial 12 mammalian cell culture is required for quality and variance monitoring. Excitation-emission 13 matrix (EEM) spectroscopy combined with multi-way chemometrics is a robust methodology 14 applicable for the analysis of raw materials, media, and bioprocess broths. We have shown 15 that the methodology can identify compositional changes and predict the efficacy of media in 16 terms of downstream titre [1]. Here we describe how to extend the measurement 17 methodology for the quantification of specific tryptophan (Trp), tyrosine (Tyr) in complex 18 chemically defined media. The sample type is an enriched basal RDF medium in which five 19 significant fluorophores were identified: Trp, Tyr, pyridoxine, folic acid, and riboflavin. The 20 relatively high chromophore concentrations and compositional complexity lead to very 21 significant matrix effects which were assessed using PARAllel FACtor analysis2 22 (PARAFAC2). Taking these effects into account, N-way Partial Least Squares (NPLS) 23 combined with a modified standard addition method was used to build calibration models 24 capable of quantifying Trp and Tyr with errors of ~4.5 and 5.5% respectively. This 25 demonstrates the feasibility of using the EEM method for the rapid, quantitative analysis of 26Trp and Tyr in complex cell culture media with minimal sample handling as an alternative to 27 chromatographic based methods. 28 29

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

“…This gives fluorescence-based techniques high sensitivity and good signal-to-noise (S/N) ratios; routine assays that use conventional spectrometers can have limit of detection (LoD) down in the nanomolar concentration range. 28,29 This sensitivity has been exploited to produce methods with single molecule detection such as fluorescence correlation spectroscopy (FCS) for the study of proteins. 30 For complex biological molecules and samples there can be multiple fluorophores and other photophysically active molecules present, and all are involved in fluorescence emission.…”

“…This is not a trivial undertaking as many of the biogenic samples generated by BioPharma are biologically, chemically, and physically unstable and even apparently minor differences in sample handling (e.g., stirred not shaken) can, and will, cause major differences in measurements for all techniques. 28,32,[34][35][36]58,59,73,118,131 Sample Handling A very important issue in BioPharma concerns the physical nature of the sample and whether one is testing solids or liquids. Solid-state cell culture media, for example, are complex powders (e.g., media, hydrolysates, etc.)…”

Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures

“…Excitation/emission matrix (EEM) spectroscopy is a powerful method to rapidly assess qualitative and quantitative changes in complex mixtures of biological material, such as cell culture media 5, 12. Due to the measurement principle, EEM spectroscopy is intrinsically focused on photophysically active molecules, and offers high sensitivity and throughput capability.…”

“…These properties render it ideally suited for detecting and monitoring light‐induced changes in media composition, and also allow for easy method transfer to routine quality control applications in industrial biotechnological production. In order to best possibly avoid inner filter effects and work above the cutoff for high absorbance of interfering components, the EEM wavelength region investigated in this study was limited to the range 300–500 nm (λ ex ) and 400–600 nm (λ em ) 12, 13…”

“…Fortunately, the same molecular characteristics that render a compound susceptible to photo‐induced damage also render it an attractive target for spectroscopic analysis. Using its fluorescence properties, subtle changes in the configuration of a molecule can be monitored with high sensitivity for the typical concentration ranges found in media 5, 12…”