A novel LED‐based 2D‐fluorescence spectroscopy system for in‐line monitoring of Chinese hamster ovary cell cultivations – Part I

Abstract

A new two‐dimensional fluorescence sensor system was developed for in‐line monitoring of mammalian cell cultures. Fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra‐ and extracellular fluorophores in the cell broth. The fluorescence signals correlate to the cells’ current redox state and other relevant process parameters. Cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majority of spectral variation. Accordingly, the newly developed device utilizes three high‐power LEDs as excitation sources in combination with a back‐thinned CCD‐spectrometer for fluorescence detection. This setup was first tested in a lab design of experiments study with process relevant fluorophores proving its suitability for cell culture monitoring with LOD in the μg/L range. The sensor was then integrated into a CHO‐K1 cell culture process. The acquired fluorescence spectra of several batches were evaluated using multivariate methods. The resulting batch evolution models were challenged in deviating and “golden batch” validation runs. These first tests showed that the new sensor can trace the cells’ metabolic state in a fast and reliable manner. Cellular distress is quickly detected as a deviation from the “golden batch”.

Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth

“…Intrinsic yeastolate emission (and most cell culture media) is more complex because of the large number of small molecule fluorophores present. The emission can be split into three main regions (Figures 1 and S4, SI): R1 ( λ ex = 240–300 nm) mostly related to emission from aromatic amino acids (B. Y. Li et al, 2012); R2 ( λ ex = 320–360 nm) corresponding to larger fluorophores such as vitamins (Faassen & Hitzmann, 2015), and R3 ( λ ex > 360 nm) corresponding to co‐factors and flavins (Graf et al, 2019). The strong yeastolate and BSA spectral overlap (Figure 1, R1) coupled with the presence of multiple chemical constituents from yeastolate, like paramagnetic ions, histidine (Lakowicz, 2006), and also non‐emitting chromophores (B. Li et al, 2010), significantly reduces BSA emission intensity through quenching and IFE processes.…”

Realization of user‐friendly bioanalytical tools to quantify and monitor critical components in bio‐industrial processes through conceptual design

“…Limits in the figure depend on analyte and process. As the arrows and positions of the citations [14–52] illuminate, many of the methodologies have undergone gradual improvements in performance. These achievements have moved their placement of the circular diagrams towards the intersected areas.…”

“…The strength of these methods is the simplicity of transduction of the signal, compared to methods with biorecognition or other reaction schemes (through antibodies, enzymes, samples treatments, etc.) [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29].…”

“…with biosensors based on enzyme electrodes, microgravity sensors, and thermal biosensors) but few are used in industrial practice and if so, only in process R&D. Successful commercially examples could be noted, F I G U R E 5 Analytical performance with critical limits for response time, sensitivity, selectivity, and cost effectivity. Selected methodologies are cited in the diagram: near-infrared- [15][16][17], 2D fluorescence- [18][19][20], and Raman spectroscopies [21][22][23], in-situ microscopy [24][25][26], holography [27,28], calorimetry [29,30], enzyme electrodes [31], enzyme thermistors [32][33][34], localized surface plasmon resonance [35], immuno-capacitive sensors [36][37][38][39], electrodes [40], aptamers [45], molecular imprinted polymers [47], electronic noses and tongues [49], and lateral flow sensors [52]. Optical and spectroscopic methods labelled blue, calorimetric red, methods based on biorecognition green, other methods with grey.…”

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

“…ex275, 280 nm/em300, 330-350 nm), and phenylalanine (max. ex260 nm/em280 nm) [22][23][24]. The strong difference in the 2D FL data most probably derived from the different trace element concentrations of the media batches.…”