Biomedical subjects

A Kamen

Publications and source records attributed to A Kamen.

At least 19 recordsLinked to original sources

PubMed2005

Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors.

To meet increasing needs of adenovirus vectors for gene therapy programs, development of efficient and reproducible production processes is required. Perfusion cultures were employed to allow infection at greater cell concentrations. In an effort to define culture conditions resulting in enhanced productivities, experiments performed at different feed rates and infected at various cell densities were compared using metabolic flux analysis. The highest specific product yields were achieved in experiments performed at high perfusion rates and/or low cell concentrations. The intracellular flux analysis revealed that these experiments exhibited greater glycolytic fluxes, slightly higher TCA fluxes, and greater ATP production rates at the time of infection. In contrast, cultures infected at high cell density and/or low medium renewal rates were characterized by a more efficient utilization of glucose at the time of infection, but the specific product yields achieved were lower. The intracellular flux analysis provided a rational basis for the implementation of a feeding strategy that allowed successful infection at a density of 5x10(6)cells/ml.

Adenoviridae↗

PubMed2005

Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.

Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.

Cell Culture Techniques↗

PubMed2002

Low-protein medium affects the 293SF central metabolism during growth and infection with adenovirus.

In this study the metabolism of 293SF cells grown in serum-free and low-protein medium was analyzed. This cell line is known for its ability to replicate recombinant adenovirus, mainly used in gene therapy applications. A complete model composed of the main glycolytic, glutaminolytic, and amino acids pathways, as well as the internalization fluxes of certain compounds into the mitochondria, is used for metabolic flux calculations. The pentose-phosphate cycle is also added to the biochemical reactions set and was independently measured with labeled 14C-glucose. Different feeding strategies in two different media were analyzed with the model, and the theoretical ATP production was also calculated. The two media were similar in their glucose and amino acid composition, but one contained BSA at 1g/L whereas the other had a very low protein content. Use of low-protein medium resulted in up to fourfold higher adenoviral vector production. In this medium, glucose utilization was more efficient, as it entered the TCA cycle more efficiently. Also, lower glutamine and amino acids consumption were observed as well as lower lactate and ammonia production. This increased TCA activity led to a twofold higher ATP production in the low-protein medium.

Adenosine Triphosphate↗

PubMed2001

Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles.

An anion-exchange-high-performance liquid chromatography (AE-HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2 x 10(10) and 7 x 10(10) VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14 x 10(9) VP/ml and a slope of 3.04 x 10(5) VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.

Adenoviridae↗

PubMed2001

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.

Adenoviridae↗

PubMed2001

Improved high-performance liquid chromatographic method in the analysis of adenovirus particles.

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.

Adenoviridae↗

PubMed2001

Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor.

An emerging sensor technology referred to as electric cell-substrate impedance sensing (ECIS) has been extended for monitoring the behavior of insect cells including attachment, motility, and mortality. In ECIS, adherent cells were cultured on an array of eight small gold electrodes deposited on the bottom of tissue culture wells and immersed in a culture medium. Upon the attachment and spreading of cells on the gold electrode, the impedance increased because the cells acted as insulating particles to restrict the current flow. Experimental data revealed that insect cells interacted differently with various proteins used to precoat the gold electrode with concanavalin A as the best promoter to accelerate the rate of cell attachment. After the cells were fully spread, the measured impedance continued to fluctuate to reflect the constant motion and metabolic activity of the cells. As the cell behavior was sensitive to external chemicals, the applicability of ECIS for inhibition assays was demonstrated with HgCl2, trinitrotoluene, trinitrobenzene (TNB), and 2-amino-4,6-dinitrotoluene as model systems. Unlike conventional assays, the quantitative data obtained in this study are taken in real time and in a continuous fashion to depict cell motility and mortality.

Animals↗

PubMed2000

A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture.

We describe in detail a robust, sensitive, and versatile functional assay for G-protein-coupled receptors (GPCRs) expressed in human embryonic kidney (HEK) 293-EBNA (Epstein-Barr virus nuclear antigen) (designated 293E) cells. The ability to grow these cells in suspension, in conjunction with the use of the secreted form of the human placental alkaline phosphatase (SEAP) as the reporter enzyme transcriptionally regulated by 5-cyclic AMP (cAMP) response elements (CREs) (Chen et al., Anal. Biochem. 226, 349-354 (1995)), makes this CRE-SEAP assay potentially attractive for high-throughput screening (HTS). A 293E clonal cell line, stably transfected with the CRE-SEAP plasmid, was initially characterized with compounds known to activate intracellular signal transduction pathways similar to those activated by GPCRs. Forskolin and cAMP analogues were potent at inducing SEAP expression but calcium ionophores (A23187 and ionomycin) were without effect. The forskolin response was also potentiated by the protein kinase C activator phorbol myristate acetate as well as the phosphodiesterase inhibitor isobutylmethylxanthine. Previously established cell lines expressing the G(alphas)-coupled DP or the G(alphaq)-coupled-EP(1) prostanoid receptors were stably transfected with the reporter gene construct and clones were selected based on their ability to secrete SEAP upon agonist challenge. Pharmacological characterization of the DP and EP(1) receptors displayed a similar rank order of potency for several known prostanoids and related compounds to that previously reported using classical binding assays or other functional assays. The CRE-SEAP assay was also used to characterize the EP(1) receptor antagonists SC-51322, SC-51089, and AH6809. In summary, we have established a reporter gene assay for GPCRs that couple to both G(alphas) and G(alphaq) and is amenable to HTS of both agonists and antagonists.

Base Sequence↗

PubMed2000

Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis.

Metabolic flux analysis is a useful tool to analyze cell metabolism. In this study, we report the use of a metabolic model with 34 fluxes to study the 293 cell, in order to improve its growth capacity in a DMEM/F12 medium. A batch, fed-batch with glutamine feeding, fed-batch with essential amino acids, and finally a fed-batch experiment with both essential and nonessential amino acids were compared. The fed-batch with glutamine led to a maximum cell density of 2.4x10(6) cells/ml compared to 1.8x10(6) cells/ml achieved in a batch mode. In this fed-batch with glutamine, it was also found that 2.5 mM ammonia was produced compared to the batch which had a final ammonia concentration of 1 mM. Ammonia was found to be growth inhibiting for this cell line at a concentration starting at 1 mM. During the fed-batch with glutamine, the flux analysis shows that a majority of amino acid fluxes and Kreb's cycle fluxes, except for glutamine flux, are decreased. This observation led to the conclusion that the main nutrient used is glutamine and that during the batch there is an overflow in the Kreb's cycle. Thus, a fed-batch with glutamine permits a better utilization of this nutrient. A fed-batch with essential amino acid without glutamine was also assayed in order to reduce ammonia production. The maximum cell density was increased further to 3x10(6) cells/ml and ammonia production was reduced below 1 mM. Flux analysis shows that the cells could adapt to a medium with low glutamine by increasing the amino acid fluxes toward the Kreb's cycle. Adding nonessential amino acids during this feeding strategy did not improve growth further and the nonessential amino acids accumulated in the medium.

Amino Acids↗

PubMed1999

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.

Adenoviridae↗

PubMed1998

A TrkA-selective, fast internalizing nerve growth factor-antibody complex induces trophic but not neuritogenic signals.

Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF.mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF. mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF.mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF. mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF.mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF.mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

Antibodies, Monoclonal↗

PubMed1998

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells.

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.

Adenoviridae↗

PubMed1994

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures.

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing beta-galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific beta-galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (< or = 3 x 10(6) cells mL-1). In cultures infected at later growth stages, beta-galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific beta-galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.

Alanine↗

PubMed1994

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells.

Human 293S cells, a cell line adapted to suspension culture, were grown to 5 x 10(6) cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5 x 10(5) cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C, how product yield can be maintained at high cell densities of 2 x 10(6) cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.

Adenoviruses, Human↗

PubMed1980