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

This study was performed in order to evaluate a new LED‐based 2D‐fluorescence spectrometer for in‐line bioprocess monitoring of Chinese hamster ovary (CHO) cell culture processes. The new spectrometer used selected excitation wavelengths of 280, 365, and 455 nm to collect spectral data from six 10‐L fed‐batch processes. The technique provides data on various fluorescent compounds from the cultivation medium as well as from cell metabolism. In addition, scattered light offers information about the cultivation status. Multivariate data analysis tools were applied to analyze the large data sets of the collected fluorescence spectra. First, principal component analysis was used to accomplish an overview of all spectral data from all six CHO cultivations. Partial least square regression models were developed to correlate 2D‐fluorescence spectral data with selected critical process variables as offline reference values. A separate independent fed‐batch process was used for model validation and prediction. An almost continuous in‐line bioprocess monitoring was realized because 2D‐fluorescence spectra were collected every 10 min during the whole cultivation. The new 2D‐fluorescence device demonstrates the significant potential for accurate prediction of the total cell count, viable cell count, and the cell viability. The results strongly indicated that the technique is particularly capable to distinguish between different cell statuses inside the bioreactor. In addition, spectral data provided information about the lactate metabolism shift and cellular respiration during the cultivation process. Overall, the 2D‐fluorescence device is a highly sensitive tool for process analytical technology applications in mammalian cell cultures.     

Soft sensor based on 2D‐fluorescence and process data enabling real‐time estimation of biomass in Escherichia coli cultivations

In bioprocesses, specific process responses such as the biomass cannot typically be measured directly on‐line, since analytical sampling is associated with unavoidable time delays. Accessing those responses in real‐time is essential for Quality by Design and process analytical technology concepts. Soft sensors overcome these limitations by indirectly measuring the variables of interest using a previously derived model and actual process data in real time. In this study, a biomass soft sensor based on 2D‐fluorescence data and process data, was developed for a comprehensive study with a 20‐L experimental design, for Escherichia coli fed‐batch cultivations. A multivariate adaptive regression splines algorithm was applied to 2D‐fluorescence spectra and process data, to estimate the biomass concentration at any time during the process. Prediction errors of 4.9% (0.99 g/L) for validation and 3.8% (0.69 g/L) for new data (external validation), were obtained. Using principal component and parallel factor analyses on the 2D‐fluorescence data, two potential chemical compounds were identified and directly linked to cell metabolism. The same wavelength pairs were also important predictors for the regression‐model performance. Overall, the proposed soft sensor is a valuable tool for monitoring the process performance on‐line, enabling Quality by Design.     

The red‐edge effects: 30 years of exploration

In 1970, three laboratories independently made a discovery that, for aromatic fluorophores embedded into different rigid and highly viscous media, the spectroscopic properties do not conform to classical rules. The fluorescence spectra can depend on excitation wavelength, and the excited‐state energy transfer, if present, fails at the ‘red’ excitation edge. These red‐edge effects were related to the existence of excited‐state distribution of fluorophores on their interaction energy with the environment and the slow rate of dielectric relaxation of this environment. In these conditions the site‐selection can be provided by variation of the energy of illuminating light quanta, and the behaviour of selected species can be followed as a function of time and other variables. These observations found extensive application in different areas of research: colloid and polymer science, molecular biophysics, photochemistry and photobiology. In particular, they led to the development of very productive methods of studying the dynamics of dielectric relaxations in protein and membranes, using the tryptophan emission and the emission of a variety of probes. These studies were extended to the time domain with the observation of new site‐selective effects in emission intensity and anisotropy decays. They stimulated the emergence and development of cryogenic energy‐selective and single‐molecular techniques that became valuable tools in their own right in chemistry and biophysics research. Site‐selection effects were discovered for electron‐transfer and proton‐transfer reactions if they depended on the dynamics of the environment. This review is focused on the progress in the field of red‐edge effects, their applications and prospects. Copyright © 2002 John Wiley & Sons, Ltd.     

Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T‐75 flask

Routine cell culture is done in small‐scale disposable vessels (typically 0.1–100 mL volumes) in academia and industry. Despite their wide use in bioprocess development (i.e., process optimization and process validation), miniature process scouting devices (PSDs) are considered “black boxes” because they are generally not equipped with sensors. In this study, we show that on‐line monitoring of dissolved oxygen (DO) and pH in a T‐75 flask‐based PSD can be achieved during cell passaging and that this information can be linked to different cellular metabolic states. In this case, on‐line monitoring of DO and pH show three distinctive metabolic regions in passages 1–18, 19–28, 29–54 and in particular, the shift in the pH curve, the specific oxygen uptake rate (q_O2), and the lactate production rate to the oxygen consumption rate yield (Y_Lac/ox) confirm the existence of these distinctive metabolic regions. These findings are particularly useful because they show that sensor equipped PSDs can help to monitor cell culture behavior after thaw, in pre‐ and seed culture prior to scale‐up and in development/optimization studies. Such routine monitoring will help to develop more consistent cell culture techniques. Biotechnol. Bioeng. 2010;105: 1040–1047. © 2009 Wiley Periodicals, Inc.     

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno-associated virus production by fluorescence spectroscopy

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.     

Advanced online monitoring of cell culture off‐gas using proton transfer reaction mass spectrometry

Mass spectrometry has been frequently applied to monitor the O₂ and CO₂ content in the off‐gas of animal cell culture fermentations. In contrast to classical mass spectrometry the proton transfer reaction mass spectrometry (PTR‐MS) provides additional information of volatile organic compounds by application of a soft ionization technology. Hence, the spectra show less fragments and can more accurately assigned to particular compounds. In order to discriminate between compounds of non‐metabolic and metabolic origin cell free experiments and fed‐batch cultivations with a recombinant CHO cell line were conducted. As a result, in total eight volatiles showing high relevance to individual cultivation or cultivation conditions could be identified. Among the detected compounds methanethiol, with a mass‐to‐charge ratio of 49, qualifies as a key candidate in process monitoring due to its strong connectivity to lactate formation. Moreover, the versatile and complex data sets acquired by PTR MS provide a valuable resource for statistical modeling to predict non direct measurable parameters. Hence, partial least square regression was applied to the complete spectra of volatiles measured and important cell culture parameters such as viable cell density estimated (R² = 0.86). As a whole, the results of this study clearly show that PTR‐MS provides a powerful tool to improve bioprocess‐monitoring for mammalian cell culture. Thus, specific volatiles emitted by cells and measured online by the PTR‐MS and complex variables gained through statistical modeling will contribute to a deeper process understanding in the future and open promising perspectives to bioprocess control. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:496–504, 2014     

In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures

The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived “pure concentration” profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.     

Laser tissue welding analyzed using fluorescence,Stokes shift spectroscopy,and Huang‐Rhys parameter

Near infrared (NIR) continuous wave laser radiation at the 1,450 nm wavelength was used to weld porcine aorta and skin samples via the absorption of combitional vibrational modes of native water in the tissues. The fluorescence spectra were measured from the key native molecules of welded and non‐welded tissues at specific excitation and emission wavelengths from collagen, elastin, and tryptophan. The changes in the fluorescence intensities and differences in Stokes shift (Δν_ss) of key native fluorophores were measured to differentiate the Huang‐Rhys parameter values (S) of the chromophores. The strength of coupling depends on the local electron‐vibration intra‐tissue molecular environment and the amount of polar solvent water surrounding the net charges on collagen, elastin, and tryptophan. The S values for both non‐welded and welded tissues were almost the same and less than 3, suggesting minimal changes in the local molecular environment as a result of welding. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In‐situ monitoring of Saccharomyces cerevisiae ITV01 bioethanol process using near‐infrared spectroscopy NIRS and chemometrics

The application feasibility of in‐situ or in‐line monitoring of S. cerevisiae ITV01 alcoholic fermentation process, employing Near‐Infrared Spectroscopy (NIRS) and Chemometrics, was investigated. During the process in a bioreactor, in the complex analytical matrix, biomass, glucose, ethanol and glycerol determinations were performed by a transflection fiber optic probe immersed in the culture broth and connected to a Near‐Infrared (NIR) process analyzer. The NIR spectra recorded between 800 and 2,200 nm were pretreated using Savitzky‐Golay smoothing and second derivative in order to perform a partial least squares regression (PLSR) and generate the calibration models. These calibration models were tested by external validation and then used to predict concentrations in batch alcoholic fermentations. The standard errors of calibration (SEC) for biomass, ethanol, glucose and glycerol were 0.212, 0.287, 0.532, and 0.296 g/L and standard errors of prediction (SEP) were 0.323, 0.369, 0.794, and 0.507 g/L, respectively. Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:510–517, 2016     

Two‐photon excited fluorescence lifetime measurements through a double‐clad photonic crystal fiber for tissue micro‐endoscopy

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two–photon excitation and time‐correlated single photon counting detection through a double‐clad photonic crystal fiber (DC‐PCF) without pre‐chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one‐photon‐based techniques, may be combined with morphology‐yielding techniques such as photoacoustic and ultrasound imaging. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Two‐dimensional fluorescence as soft sensor in the monitoring of biotransformation performed by yeast

Soft sensors are powerful tools for bioprocess monitoring due to their ability to perform online, noninvasive measurement, and possibility of detection of multiple components in cultivation media, which in turn can provide tools for the quantification of more than one metabolite/substrate/product in real time. In this work, soft sensor based on excitation‐emission fluorescence is for the first time applied for the monitoring of biotransformation production of 2‐phenylethanol (2‐PE) by yeast strains. Main process parameters—such as optical density, glucose, and 2‐PE concentrations—were determined with high accuracy and precision by fluorescence fingerprinting coupled with partial least squares regression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:299–307, 2017     

Highly luminescent and biocompatible,l‐citrulline‐capped ZnS:Mn nanocrystals for rapid screening of metal accumulating Lysinibacillus fusiformis bacteria

Biocompatible and highly luminescent manganese doped zinc sulfide (ZnS:Mn) nanocrystals of average particle size 10 nm have been synthesized by capping with a novel amino acid ligand, l ‐citrulline. Though there are many reports on the bioimaging applications of nanostructured semiconductors, the present study focused on the detection of a special type of metal accumulating bacteria, Lysinibacillus fusiformis. This bacterium has significant applications in the disposal of metal components from industrial effluents. In this context, the detection of this bacterium is quite important and the present work demonstrates a novel technique for this bacterial detection. The synthesized nanocrystals were attached to Lysinibacillus fusiformis and characteristics of the bioconjugated system were studied. The blue shift observed in the ultraviolet‐visible absorption and photoluminescence spectra of the bioconjugated system, confirms conjugation of the Lysinibacillus fusiformis with l ‐citrulline‐capped ZnS:Mn. When the bioconjugated system (capped ZnS:Mn + bacteria) was observed using a fluorescent microscope under excitation wavelengths 365.4 nm (ultraviolet), 435.8 nm (blue) and 546.1 nm (green), fluorescence emissions were obtained in yellow, green and red regions respectively. The study of relative growth of Lysinibacillus fusiformis in the presence of l ‐citrulline‐capped ZnS:Mn proves biocompatible property of these nanocrystals and their tunable color properties under different excitation wavelengths make them ideal for biolabeling applications. Copyright © 2013 John Wiley & Sons, Ltd.     

The potential of random forest and neural networks for biomass and recombinant protein modeling in Escherichia coli fed‐batch fermentations

Product quality assurance strategies in production of biopharmaceuticals currently undergo a transformation from empirical “quality by testing” to rational, knowledge‐based “quality by design” approaches. The major challenges in this context are the fragmentary understanding of bioprocesses and the severely limited real‐time access to process variables related to product quality and quantity. Data driven modeling of process variables in combination with model predictive process control concepts represent a potential solution to these problems. The selection of statistical techniques best qualified for bioprocess data analysis and modeling is a key criterion. In this work a series of recombinant Escherichia coli fed‐batch production processes with varying cultivation conditions employing a comprehensive on‐ and offline process monitoring platform was conducted. The applicability of two machine learning methods, random forest and neural networks, for the prediction of cell dry mass and recombinant protein based on online available process parameters and two‐dimensional multi‐wavelength fluorescence spectroscopy is investigated. Models solely based on routinely measured process variables give a satisfying prediction accuracy of about ± 4% for the cell dry mass, while additional spectroscopic information allows for an estimation of the protein concentration within ± 12%. The results clearly argue for a combined approach: neural networks as modeling technique and random forest as variable selection tool.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

A new Schiff base as a turn‐off fluorescent sensor for Cu2+ and its photophysical properties

A new Schiff base receptor 1 was synthesized and its photophysical properties were investigated by absorption, emission and excitation techniques. Furthermore, its chromogenic and fluorogenic sensing abilities towards various metal ions were examined. Receptor 1 selectively detects Cu²⁺ ion through fluorescence quenching and detection was not inhibited in the presence of other metal ions. From fluorescence titration, the limit of detection of receptor 1 as a fluorescent ‘turn‐off’ sensor for the analysis of Cu²⁺ was estimated to be 0.35 μM.     

A completely noninvasive method of dissolved oxygen monitoring in disposable small‐scale cell culture vessels based on diffusion through permeable vessel walls

Disposable cell culture vessels are extensively used at small scales for process optimization and validation, but they lack monitoring capabilities. Optical sensors that can be easily adapted for use in small‐scale vessels are commercially available for pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO₂). However, their use has been limited due to the contamination and compatibility issues. We have developed a novel solution to these problems for DO monitoring. Oxygen diffusion through permeable vessel wall can be exploited for noninvasive monitoring. An optical oxygen sensor can be placed outside the oxygen permeable vessel wall thereby allowing oxygen diffusing through the vessel wall to be detected by the sensor. This way the sensor stays separate from the cell culture and there are no concerns about contaminants or leachants. Here we implement this method for two cell culture devices: polystyrene‐made T‐75 tissue culture flask and fluorinated ethylene propylene (FEP)‐made Vuelife^® cell culture bag. Additionally, mammalian and microbial cell cultures were performed in Vuelife^® cell culture bags, proving that a sensor placed outside can be used to track changes in cell cultures. This approach toward noninvasive monitoring will help in integrating cell culture vessels with sensors in a seamless manner. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:172–177, 2014