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".

Keywords: 2D‐fluorescence spectroscopy; CHO cell cultivation; MVDA; PAT; in‐line bioprocess monitoring; metabolism monitoring.

Figure 1

New fluorescence device set‐up scheme

Figure 2

Fiber arrangement in newly designed probe

Figure 3

Observed versus. predicted plots of each analyte tested in the DoE‐Study: (A) BSA, (B) FAD, (C) NADH, (D) NAD, (E) Pyridoxine, (F) Riboflavin

Figure 4

(A) Linear fused spectra from cultivation K1, colored according to batch age [h] from Blue (0 h) to Red (250 h); Saturated Raleigh‐Wavelengths were excluded for BEM; (B) Time‐course of several important excitation‐emission wavelength combinations chosen from Figure 4, that can be associated to different Fluorophores; Feeds: F1‐F7

Figure 5

(A) Score Plot of PCA‐model from cultivations K1‐K3 with score t[1] plotted against score t[2]; dotted‐arrow indicates course of the batch maturity; (B) loading plot of pc 1 and (C) pc 2 with peaks from the rayleigh‐backscattering as well as those stemming from different intrinsic fluorophores

Figure 6

(A) Batch Control Chart of Batch Evolution Model made from Cultivations K1‐K3 where Principal Component t[1] is plotted against the Batch Age; Dashed green line is the average time‐course of the Scores from K1‐3; Feeds: F1‐8; (B) Prediction Batch Control Chart of K4 and K5 where the Predicted Score tPS[1] is plotted against the Batch Age; Average and ±3 h. Deviation Limits correspond to those from the BEM (Figure 6 )

Figure 7

Comparison of the NADH‐signal courses over time measured with the new 2DF‐System brown) and the state‐of‐the‐art BioView^® sensor (grey); Feed addition: F1‐F7