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.     

Exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody

Online monitoring of Chinese hamster ovary fed‐batch cell cultures via two‐dimensional fluorescence spectroscopy (2DFS) was evaluated in this work. Particular attention was directed toward different process strategies regarding the use of nutrient‐rich feed media and temperature shifts. These intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. Principal component analysis of the obtained fluorescence data showed that temperature shift and feeding strategy had a considerable impact on the fluorescence signals. Partial least square regression models were calculated for the prediction of glucose, lactate, monoclonal antibody (mAb), and viable cell concentrations (VCC). It was aimed to integrate all 2DFS datasets in the respective calibration models regardless of the process‐strategy‐dependent diversity. Contrary to the expectations, it was feasible to calibrate soft sensors for the online prediction of glucose (7 latent variables (LVs), = 0.97, rout mean squared error of prediction (RMSEP) = 1.1 g L^?1), lactate (5 LV; = 0.96; RMSEP = 0.5 g L^?1) and mAb concentrations (4 LV; = 0.99; RMSEP = 11.4 mg L^?1). Feeding and temperature shifts had the highest impact on the VCC model (3 LV; = 0.94; RMSEP 3.8 × 10⁵ mL^?1), nevertheless the prediction of VCC from the fed‐batch 2DFS data was feasible. The results strongly indicate that variances in the datasets due to the process strategy can be tolerated to some extent by the respective soft sensors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1592–1600, 2016     

Comparative study of non-invasive monitoring via infrared spectroscopy for mammalian cell cultivations

Process analytical technology (PAT) is a guide to improve process development in biotech industry. Optical sensors such as near and mid infrared spectrometers fulfill an essential part for PAT. NIRS and MIRS were investigated as non-invasive on line monitoring tools for animal cell cultivations in order to predict critical process parameters, like cell parameters as well as substrate and metabolite concentrations. Eight cultivations were performed with frequent sampling. Variances between cultivations were induced by spiking experiments with intent to break correlations between analytes; to keep causality of the models; and to increase model robustness.     

In‐line monitoring of amino acids in mammalian cell cultures using raman spectroscopy and multivariate chemometrics models

The application of PAT for in‐line monitoring of biopharmaceutical manufacturing operations has a central role in developing more robust and consistent processes. Various spectroscopic techniques have been applied for collecting real‐time data from cell culture processes. Among these, Raman spectroscopy has been shown to have advantages over other spectroscopic techniques, especially in aqueous culture solutions. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman‐based chemometrics models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. The chemometrics models based on Raman spectroscopy data demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. The model for methionine would have to be further refined to improve quantification.     

Generic Raman‐based calibration models enabling real‐time monitoring of cell culture bioreactors

Raman‐based multivariate calibration models have been developed for real‐time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO‐based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1004–1013, 2015     

Data reconciliation of concentration estimates from mid‐infrared and dielectric spectral measurements for improved on‐line monitoring of bioprocesses

Real‐time data reconciliation of concentration estimates of process analytes and biomass in microbial fermentations is investigated. A Fourier‐transform mid‐infrared spectrometer predicting the concentrations of process metabolites is used in parallel with a dielectric spectrometer predicting the biomass concentration during a batch fermentation of the yeast Saccharomyces cerevisiae. Calibration models developed off‐line for both spectrometers suffer from poor predictive capability due to instrumental and process drifts unseen during calibration. To address this problem, the predicted metabolite and biomass concentrations, along with off‐gas analysis and base addition measurements, are reconciled in real‐time based on the closure of mass and elemental balances. A statistical test is used to confirm the integrity of the balances, and a non‐negativity constraint is used to guide the data reconciliation algorithm toward positive concentrations. It is verified experimentally that the proposed approach reduces the standard error of prediction without the need for additional off‐line analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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     

Comparison of spectroscopy technologies for improved monitoring of cell culture processes in miniature bioreactors

Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the throughput of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in large‐scale bioreactors. The small volume of miniature bioreactor cultures (15 mL) is incompatible with the large sample volume (600 µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman, and 2D‐fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design‐of‐experiment approach is described that utilizes previously analyzed cell culture supernatant to assess metabolite concentrations under various conditions while providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D‐fluorescence could more accurately measure ammonium concentration (RMSE_CV 0.031 g L^?1) than Raman and NIR. Raman spectroscopy, however, was more robust at measuring lactate and glucose concentrations (RMSE_CV 1.11 and 0.92 g L^?1, respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D‐fluorescence. The implementation of Raman spectroscopy increases at‐line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:337–346, 2017     

A fluorescence lifetime spectroscopy study of matrix metalloproteinases‐2 and ‐9 in human atherosclerotic plaque

Matrix metalloproteinase (MMP)‐2 and ‐9 play important roles in the progression of atherosclerosis. This study aims to determine whether MMP‐2 and ‐9 content in the fibrotic caps of atherosclerotic plaque is correlated with plaque autofluorescence. A time‐resolved laser‐induced fluorescence spectroscopy (TR‐LIFS) system was used to measure the autofluorescence and assess the biochemical composition of human plaques obtained from carotid endarterectomy. Results presented here demonstrate for the first time the ability to characterize the biochemical composition as it relates to MMP‐2 and ‐9 content in the atherosclerotic plaque cap using a label‐free imaging technique implemented with a fiberoptic TR‐LIFS system. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line,FL‐SJC

About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated CD19⁺⁺, CD20⁺, CD22⁺⁺, HLA‐DR⁺, CD10⁺, CD38⁺, Lambda⁺ CD23^‐, CD5^‐ and Kappa^‐. The chromosome karyotypic analysis confirmed the co‐existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.     

Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic β‐cell activation

Nutrient secretagogues activate mitochondria of the pancreatic β‐cell through the provision of substrate, hyperpolarisation of the inner mitochondrial membrane and mitochondrial calcium rises. We report that mitochondrial matrix pH, a parameter not previously studied in the β‐cell, also exerts an important control function in mitochondrial metabolism. During nutrient stimulation matrix pH alkalinises, monitored by the mitochondrial targeted fluorescent pH‐sensitive protein mtAlpHi or ³¹P‐NMR inorganic phosphate chemical shifts following saturation transfer. Compared with other cell types, the resting mitochondrial pH was surprisingly low, rising from pH 7.25 to 7.7 during nutrient stimulation of rat β‐cells. As cytosolic alkalinisation to the nutrient was of much smaller amplitude, the matrix alkalinisation was accompanied by a pronounced increase of the ΔpH across the inner mitochondrial membrane. Furthermore, matrix alkalinisation closely correlates with the cytosolic ATP net increase, which is also associated with elevated ATP synthesis rates in mitochondria. Preventing ΔpH increases in permeabilised cells abrogated substrate‐driven ATP synthesis. We propose that the mitochondrial pH and ΔpH are key determinants of mitochondrial energy metabolism and metabolite transport important for cell activation.     

Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2‐DE and MS led to the generation of their 2‐DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2‐DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.     

A novel function for selenium in biological system: selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production

As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.     

Carbazole–azine based fluorescence ‘off–on’ sensor for selective detection of Cu2+ and its live cell imaging

A new carbazole–azine based fluorescent sensor was synthesized and characterized. The selectivity of the sensor for Cu²⁺ over other counter ions in a dimethyl sulfoxide/H₂O mixture was shown through enhancement in fluorescence – an off to on transformation. The specificity of the probe towards Cu²⁺ was evident in ultraviolet/visible, fluorescence, Fourier transform infrared and mass studies. Application of the probe in the cell imaging and cytotoxicity of living cells is illustrated.     

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd.