Biomass estimation using fluorescence measurements in <i>Pichia pastoris</i> bioprocess

Surribas, Anna; Montesinos, José Luis González; Valero, Francisco

doi:10.1002/jctb.1352

Cited by 24 publications

(17 citation statements)

References 22 publications

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“…Based on literature it was possible to associate some regions of this map to specific fluorophors, namely Phe, Tyr, Trp , pyridoxine, and riboflavin Marose et al, 1998;García et al, 2001). In bioreaction culture bulks, the maximum fluorescence emission for Trp normally reported corresponds to excitation at 290 nm Horvath et al, 1993;Surribas et al, 2006c). However, the maximum fluorescence signal for each fluorophor changes with the environmental conditions .…”

Section: Process and 2d Fluorescence Dynamicsmentioning

confidence: 97%

“…These compounds have maximum fluorescence signals at different excitation/emission wavelength pairs (l exc /l emi ). Therefore, scanning the culture bulk by 2D fluorometry over the course of a bioreaction provides dynamic information of all fluorophors, directly or indirectly reflecting changes in cell´s concentration, metabolome, and envirome Hisiger and Jolicoeur, 2005b;Surribas et al, 2006c).…”

Section: Introductionmentioning

confidence: 99%

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In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures

Teixeira

Portugal

Carinhas

et al. 2008

Biotech & Bioengineering

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The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1-IL2 cultured in batch and fed-batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r(2) = 0.95; 99.2% of variance captured in the training set; r(2) = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r(2) = 0.99 and 99.7% of variance captured in the training set; r(2) = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on-line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures.

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Section: Process and 2d Fluorescence Dynamicsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures

Teixeira

Portugal

Carinhas

et al. 2008

Biotech & Bioengineering

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show abstract

“…These methods are, thus, based on only partially valid assumptions and may lead to significant errors in the predictions. In the field of spectroscopy, indirect biomass monitoring has been reported using fluorescence measurements [4,7]. Direct determination of biomass concentration is done either by biological quantification methods (viable cell counting or petri plating) or by exploring the physical properties of the cells.…”

Section: Introductionmentioning

confidence: 99%

Cole–Cole, linear and multivariate modeling of capacitance data for on-line monitoring of biomass

Dabros

Dennewald

Currie

et al. 2008

Bioprocess Biosyst Eng

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This work evaluates three techniques of calibrating capacitance (dielectric) spectrometers used for on-line monitoring of biomass: modeling of cell properties using the theoretical Cole-Cole equation, linear regression of dual-frequency capacitance measurements on biomass concentration, and multivariate (PLS) modeling of scanning dielectric spectra. The performance and robustness of each technique is assessed during a sequence of validation batches in two experimental settings of differing signal noise. In more noisy conditions, the Cole-Cole model had significantly higher biomass concentration prediction errors than the linear and multivariate models. The PLS model was the most robust in handling signal noise. In less noisy conditions, the three models performed similarly. Estimates of the mean cell size were done additionally using the Cole-Cole and PLS models, the latter technique giving more satisfactory results.

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“…Taking advantage of fiber optics, fluorescence excitation-emission matrices (usually called fluorescence maps) have been collected inside bioreactors to get information on all culture bulk fluorophores and their change over culture time used to correlate with target bioprocess variables (e.g. Lindemann et al, 1998;Skibsted et al, 2001;Surribas et al, 2006;Wolf et al, 2007). In general, to establish those correlations multivariate 0168-1656/$ -see front matter © 2010 Elsevier B.V. All rights reserved.…”

Section: Introductionmentioning

confidence: 99%