Real-time monitoring of protein secretion in mammalian cell fermentation: measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM)

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. (c) 1995 John Wiley & Sons, Inc.     

A whole-cell biosensor as in vitro alternative to skin irritation tests

This study presents the time-resolved detection of chemically induced stress upon intracellular signaling cascades by using genetically modified sensor cells based on the human keratinocyte cell line HaCaT. The cells were stably transfected with a HSP72-GFP reporter gene construct to create an optical sensor cell line expressing a stress-inducible reporter protein. The time- and dose-dependent performance of the sensor cells is demonstrated and discussed in comparison to a label-free impedimetric monitoring approach (electric cell-substrate impedance sensing, ECIS). Moreover, a microfluidic platform was established based on μSlidesI(0,4)Luer to allow for a convenient, sterile and incubator-independent time-lapse microscopic observation of the sensor cells. Cell growth was successfully achieved in this microfluidic setup and the cellular response to a cytotoxic substance could be followed in real-time and in a non-invasive, sensitive manner. This study paves the way for the development of micro-total analysis systems that combine optical and impedimetric readouts to enable an overall quantitative characterization of changes in cell metabolism and morphology as a response to toxin exposure. By recording multiple parameters, a detailed discrimination between competing stress- or growth-related mechanisms is possible, thereby presenting an entirely new in vitro alternative to skin irritation tests.     

On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin

The secretions of molecules by cells are of tremendous interest for both fundamental insights studies and medical purposes. In this study, we propose a new biochip-based approach for the instantaneous monitoring of protein secretions, using antibody production by B lymphocytes cultured in vitro. This was possible thanks to the Surface Plasmon Resonance imaging (SPRi) of a protein biochip where antigen proteins (Hen Egg Lysozyme, HEL) were micro-arrayed along with series of control proteins. B cell hybridomas were cultured on the chip and the secretion of immunoglobulins (antibody) specific to HEL was monitored in real-time and detected within only few minutes rather than after a 30-60 min incubation with standard ELISA experiments. This fast and sensitive detection was possible thanks to the sedimentation of the cells on the biochip sensitive surface, where local antibody concentrations are much higher before dilution in the bulk medium. An other interesting feature of this approach for the secretion monitoring was the independence of the SPR response--after normalization--regarding to the density of the surface-immobilized probes. Such biosensor might thus pave the way to new tools capable of both qualitative and semi-quantitative analysis of proteins secreted by other immune cells.     

HLA-linked nonresponsiveness to Cryptomeria japonica pollen antigen. I. Nonresponsiveness is mediated by antigen-specific suppressor T cell

The objective of this study was to elucidate the cellular mechanism of IgE nonresponse to the Cryptomeria japonica (Japanese cedar) pollen antigen (CPAg), which was shown in our previous study to be HLA-linked (1). We established an assay system for the measurement of small amounts of anti-CPAg IgE antibody, both in an antigen-specific and isotype-specific manner, and a culture system to induce antigen-driven IgE antibody synthesis in vitro. By using these methods, we clarified that the function of the HLA-DR molecule in the CPAg-driven IgE response is similar to that of I-A or I-E molecule in mice, namely the product of immune response genes (Ir-genes), because anti-HLA-DR monoclonal antibody blocked the response, and the interaction between monocyte and monocyte-depleted peripheral blood lymphocytes (PBL) to respond to CPAg was restricted by HLA-DR. Furthermore, PBL from nonresponders revealed a specific IgE response to CPAg when the Leu-2+3- T cell fraction was depleted, thereby suggesting that even nonresponders have Leu-2-3+ T cell and B cell clones specific for CPAg, and they apparently show no response due to the presence of CPAg-specific Leu-2+3- suppressor T cells. This suppressor T cell fraction abolished the IgE response of the autologous B + monocyte + Leu-2-3+ T cell in a CPAg-specific manner. The current cellular analysis together with our previous genetic analysis strongly suggest that the HLA-linked IgE nonresponse to CPAg is mediated by CPAg-specific suppressor T cells. The HLA-linked gene controlling the nonresponsiveness to CPAg is thus designated as the immune suppression gene for CPAg (Is-CPAg). Mapping of Is-CPAg within HLA-DQ subregion is discussed.     

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

大鼠脑红蛋白(NGB)的原核表达、抗体制备及其细胞分布

脑红蛋白 (NGB)是新发现的与脑内氧供应密切相关的分子 .为了检测细胞内脑红蛋白的表达、亚细胞分布从而对该分子进行深入的功能研究 ,成功地将大鼠脑红蛋白基因编码区构建于原核表达载体pGEX 4T 2 ,转化大肠杆菌BL2 1(DE3) ,获得融合表达产物 .对含有融合蛋白的包含体进行溶解和复性 ,用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化 ,通过免疫家兔获得了兔源性抗NGB多克隆抗体 .采用Western印迹分析技术 ,用该抗体检测NGB基因的真核表达产物 ,证明该抗体有较好的针对NGB蛋白的专一性 ,可用于对NGB的结构和功能研究 .同时 ,用该抗体进行免疫组化分析发现 ,正常成年大鼠神经系统中有较多的NGB免疫反应阳性细胞分布 ,提示NGB是与神经系统功能密切相关的重要分子     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.     

Subpopulations of rat lung fibroblasts with different amounts of type I and type III collagen mRNAs

A fluorescence-based method using the cell sorter has been devised to separate rat lung fibroblasts into subpopulations. Type I or type III collagen antiserum was used as the primary antibody to react with parent rat lung fibroblasts. This was followed by a fluorescein-conjugated secondary antibody. Specificity of the primary collagen antibody was determined using a monoclonal beta-actin antibody and purified IgG as the primary antibodies. The fluorescent shift of parent rat lung fibroblasts was optimized for the amount of primary collagen antibody and secondary fluorescein-conjugated antibody. An increase in slot blot intensity was observed for pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) mRNAs with increasing amounts of cellular RNA. When precipitating with type I collagen antibodies, the total cellular steady-state levels of type I procollagen mRNAs were increased in the high intensity cells as compared with the low intensity cells. Alternately, when the type III collagen antibodies were used to precipitate the rat lung fibroblasts, the low intensity cells had increased type I procollagen mRNAs while the high intensity cells had increased type III procollagen mRNA. The subpopulations of rat lung fibroblasts after isolation using the fluorescent cell sorter were readily propagated for at least four passages.     

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.     

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.     

Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.     

Imaging of proteolytic activity using a conditional cell surface receptor

Programmed cell death (apoptosis) is a ubiquitous means utilized by multicellular organisms for elimination of unwanted cells during development and homeostasis. Dysregulated apoptosis is implicated in an array of clinical disorders including cancer, autoimmune diseases, neurodegenerative disorders, and ischemia. During programmed cell death, a series of proteases, known as caspases, with different specificities play crucial roles in the apoptotic process. Caspase-3, a group II cysteine aspartate protease, recognizes and cleaves substrates harboring the amino acid sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD), and it plays an important role in the terminal phase of apoptosis. Here we report the development of a novel imaging platform for sensing the activation of cellular proteases. A recombinant chimeric protein was constructed, composed of a cell-surface-targeted single-chain antibody (sFv) fused to a Golgi retention signal. The DEVD tetrapeptide sequence was included between the single-chain antibody and the Golgi retention signal as a caspase-3 protease cleavage site. When expressed in cultured cells this fusion protein was localized to Golgi bodies and was not detected on the cell surface. Induction of apoptosis resulted in cleavage of the fusion protein releasing the single-chain antibody from the Golgi retention signal in a caspase-dependent manner. As a result, in cells undergoing apoptosis the single-chain antibody was visualized at the cell surface by immunofluorescence microscopy. The expression of sFv on the surface of cells in a protease-dependent manner provides a unique opportunity for real-time imaging through the use of targeted nanoparticles. This methodology may provide for a multimodal noninvasive real-time imaging of apoptosis and a new opportunity for high-throughput screening of cell-death-modulating therapeutic agents.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

Actin-associated proteins in human neutrophils: identification and reorganization upon cell activation

The neutrophil cytoskeleton, especially the actin network, is thought to play a crucial role in neutrophil migration. However, little is known on the modulation of this network by actin-associated proteins. We have demonstrated the presence of immuno-reactive forms of alpha-actinin (an actin cross-linking and bundling protein) and vinculin (a putative actin-membrane linker) in human neutrophils using specific antibodies to chicken gizzard vinculin and bovine epithelial alpha-actinin. In contrast, talin, another putative actin-membrane linker protein, could not be detected in significant amounts in human neutrophils using a polyclonal antibody raised against chicken gizzard talin, which reacted with human platelet and lymphocyte talin. We have also analyzed the vinculin and alpha-actinin content of Triton X-100 insoluble cytoskeletons, isolated from resting and activated neutrophils. A small amount of alpha-actinin was already associated with the cytoskeleton of resting cells. Addition of chemotactic peptide to the cells rapidly increased the alpha-actinin content of the cytoskeletons 1.6 to 7-fold. This rapid increase was followed by a slower decrease to a lower level which, after 30 min of stimulation, was still significantly higher than that of control cells. The time-course of the association of alpha-actinin with the cytoskeleton paralleled that of actin association. This stimulus-induced rearrangement of cellular alpha-actin may thus play an important role in determining the structure of actin networks in motile neutrophils. Vinculin in contrast could not be detected in significant amounts in the Triton X-100-insoluble neutrophil cytoskeleton, not even after prolonged stimulation of the cells by chemotactic peptide.     

Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: a population analysis

Single-cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G(1), S(1) and G(2) + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single-cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G(1) phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G(1) phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G(2) + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.     

Development and application of a GFP-FRET intracellular caspase assay for drug screening

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.     

Protein-dependent ribozymes report molecular interactions in real time

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.