Raman spectroscopy and chemometrics for on‐line control of glucose fermentation by Saccharomyces cerevisiae

This work presents the use of Raman spectroscopy and chemometrics for on‐line control of the fermentation process of glucose by Saccharomyces cerevisiae. In a first approach, an on‐line determination of glucose, ethanol, glycerol, and cells was accomplished using multivariate calibration based on partial least squares (PLS). The PLS models presented values of root mean square error of prediction (RMSEP) of 0.53, 0.25, and 0.02% for glucose, ethanol and glycerol, respectively, and RMSEP of 1.02 g L^?1 for cells. In a second approach, multivariate control charts based on multiway principal component analysis (MPCA) were developed for detection of fermentation fault‐batch. Two multivariate control charts were developed, based on the squared prediction error (Q) and Hotelling's T². The use of the Q control chart in on‐line monitoring was efficient for detection of the faults caused by temperature, type of substrate and contamination, but the T² control chart was not able to monitor these faults. On‐line monitoring by Raman spectroscopy in conjunction with chemometric procedures allows control of the fermentative process with advantages in relation to reference methods, which require pretreatment, manipulation of samples and are time consuming. Also, the use of multivariate control charts made possible the detection of faults in a simple way, based only on the spectra of the system. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

Lab and pilot scale batch cultivations of a CHO K1/dhfr^? host cell line were conducted to evaluate on‐line multifrequency permittivity measurements as a process monitoring tool. The β‐dispersion parameters such as the characteristic frequency (f_C) and the permittivity increment (Δε_max) were calculated on‐line from the permittivity spectra. The dual‐frequency permittivity signal correlated well with the off‐line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off‐line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C_M), and effects caused by medium conductivity (σ_m) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f_C as a result of a fall in intracellular conductivity (σ_i) was identified as responsible for the observed changes in the dual‐frequency permittivity signal. It is hypothesized that the β‐dispersion parameter f_C is indicative of changes in nutrient availability that have an impact on intracellular conductivity σ_i. On‐line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

In situ near infrared spectroscopy for analyte‐specific monitoring of glucose and ammonium in streptomyces coelicolor fermentations

There are many challenges associated with in situ collection of near infrared (NIR) spectra in a fermentation broth, particularly for highly aerated and agitated fermentations with filamentous organisms. In this study, antibiotic fermentation by the filamentous bacterium Streptomyces coelicolor was used as a model process. Partial least squares (PLS) regression models were calibrated for glucose and ammonium based on NIR spectra collected in situ. To ensure that the models were calibrated based on analyte‐specific information, semisynthetic samples were used for model calibration in addition to data from standard batches. Thereby, part of the inherent correlation between the analytes could be eliminated. The set of semisynthetic samples were generated from fermentation broth from five separate fermentations to which different amounts of glucose, ammonium, and biomass were added. This method has previously been used off line but never before in situ. The use of semisynthetic samples along with validation on an independent batch provided a critical and realistic evaluation of analyte‐specific models based on in situ NIR spectroscopy. The prediction of glucose was highly satisfactory resulting in a RMSEP of 1.1 g/L. The prediction of ammonium based on NIR spectra collected in situ was not satisfactory. A comparison with models calibrated based on NIR spectra collected off line suggested that this is caused by signal attenuation in the optical fibers in the region above 2,000 nm; a region which contains important absorption bands for ammonium. For improved predictions of ammonium in situ, it is suggested to focus efforts on enhancing the signal in that particular region. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Application of (S)‐TBMB carboxylic acid‐based on‐line HPLC‐exciton CD analysis to acyclic vicinal alcohols and amino alcohols

Applications of the on‐line HPLC‐exciton CD analysis using (S)‐2‐tert‐butyl‐2‐methyl‐1,3‐benzodioxole‐4‐carboxylic acid [(S)‐TBMBC‐OH] that can simultaneously determine the enantiomeric compositions and the absolute configuration of cyclohexane‐1,2‐diols and diamines as well as acyclic vicinal diols and amino alcohols were studied. Di‐O‐ or di‐N,O‐(S)‐TBMBC derivatives of acyclic terminal vicinal diols, 2‐hydroxy‐1‐amines, and nonterminal vicinal diols gave symmetrical exciton CD spectra between enantiomers, indicating their absolute configurations. However, Di‐N,O‐(S)‐TBMBC derivatives of 2‐amino‐1‐ols did not always give symmetrical exciton CD spectra between enantiomers, but their 2‐phthalimido‐1‐O‐(S)‐TBMBC derivatives gave symmetrical exciton CD spectra, indicating their absolute configurations. All these (S)‐TBMBC derivatives were separated by normal‐phase HPLC and unequivocally determined by the on‐line HPLC‐exciton CD analysis without recourse to reference samples. Chirality 11:149–159, 1999. © 1999 Wiley‐Liss, Inc.     

A multivariate calibration procedure for UV/VIS spectrometric monitoring of BHK‐21 cell metabolism and growth

Monitoring mammalian cell culture with UV–vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off‐line UV–vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK‐21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.50 ± 0.10 mM and 2.21 ± 0.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UV–vis at‐line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed‐batch feeding schemes. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:241–248, 2014