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PubMed2005

Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility.

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.

Academic Medical Centers↗

PubMed1994

Rapid and efficient purification of hepatitis A virus from cell culture.

Hepatitis A virus (HAV) characteristically remains strongly cell-associated when grown in culture, with only small yields in the culture supernatant. Cell factories (6000 cm2) of BS-C-1 cells infected with the cytopathic HM175A.Z strain of HAV for 3, 4 or 7 days were harvested using trypsin to disperse the infected cell monolayer, and cells were collected by low speed centrifugation. More than 70% of the yield of virus and viral antigen can thus be obtained in the packed cell pellet. Packed cell pellets were resuspended in 5 volumes of isotonic buffer and cell membranes lysed by the addition of a non-ionic detergent. After removal of nuclei by centrifugation, ionic detergent was added to the clarified cytoplasmic extract. Under these conditions, HAV particles (virions and empty capsids) are the only particulate material remaining in the sample, and were recovered in a single ultracentrifugation step through discontinuous sucrose/glycerol density gradients. In one day, this method yields viral antigen with minimal cellular contaminants, in a concentrated volume suitable for subsequent biochemical, vaccine or diagnostic uses. The yield of viral antigen over numerous batches varied from 200 to 1600 vaccine-equivalent doses per cell factory, with a titre of up to 1 x 10(10) infectious particles per ml.

Animals↗

PubMed2005

The growth of attenuated influenza vaccine donor strains in continuous cell lines.

The growth of the Russian live attenuated influenza vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and B/USSR/60/69 was studied in cells of the VERO and Madin-Darby canine kidney (MDCK) lines as six-well cultures and cell factories infected at different multiplicities of infection. Yields for A/Leningrad/134/17/57 and A/Leningrad/134/47/57 were comparable in either cell line over a range of multiplicities but were about 10-fold lower than in the allantoic fluids of infected chicken embryos. For both A/Leningrad/134/47/57 and B/USSR/60/69, yields from the MDCK line were about 10-fold higher than for the VERO line. For B/USSR/60/69, yields in eggs were approximately 100-fold higher than those obtained in the MDCK line. A feature of the growth of B/USSR/60/69 was its reduced capacity to produce infectious progeny in either cell line at multiplicities of infection of 2.0 or 1.0 pfu/cell. Inhibition was due probably due to the presence of defective-interfering particles and was not detected with A/Leningrad/134/17/57 or A/Leningrad/134/47/57 in cultures of either line infected at the same multiplicities. Yields for both A/Leningrad/134/47/57 and B/USSR/60/69 in cells of the MDCK line were comparable when grown in six-well cultures or cell factories.

Animals↗

PubMed2003

Cyanobacterial postgenomic research and systems biology.

The genomic era brought with it the capacity to unlock complex interactions in organisms and biological systems. Currently, by exploiting genomic and associated protein information through in silico analyses, postgenomic research is developing rapidly. This field, which encompasses functional genomics, structural genomics, transcriptomics, pharmacogenomics, proteomics and metabolomics, allows for a systems-wide approach to biological studies. To date, bacterial postgenomic research has focused mainly on a few representative pathogenic species, leaving the vast majority of the microbial community relatively overlooked. Amongst the under-represented microorganisms are the cyanobacteria, which are important for their beneficial natural product production, bioremediation and energy applications. Here, we highlight the current status of cyanobacterial postgenomic research and assess the potential for future metabolic engineering and "cell factory" or "microbial cell" development.

Biotechnology↗

PubMed2005

Upscaling of lentiviral vector production by tangential flow filtration.

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.

Animals↗

PubMed1995

Rabies veterinary virus vaccine produced in BHK-21 cells grown on microcarriers in a bioreactor.

BHK-21 cells were grown in microcarriers in the CELLIGEN CL 50 bioreactor to produce a stock of rabies veterinary virus vaccine PV (Pasteur virus) strain. Perfusion mode operation of this bioreactor produced between two- and fourfold larger yields (cells/ml) than traditional stationary cell culture systems (i.e., Blake, and Roller bottles or cell factory multitrays). The method employed harvested 281 of rabies virus in 200 h (infectivity titer 0.6 +/- 1.4 x 10(7) LD50 per ml) in a single operation. The risk of contamination is thus reduced when compared with traditional stationary methods which, in order to obtain the same amount of virus, would require the operation of 285 Blake bottles, or 143 Roller bottles, or 15 Cell Factory multitrays (10 trays). By perfusion mode operation of the bioreactor, 89% of the cell culture medium was recovered as vaccinal virus, which contrasts with the yield of only 50-59% using traditional cell culture systems. On the other hand, only 925 ml of fetal serum was required to obtain the 281 of rabies virus harvest as compared to the 3420 ml required by traditional methods.

Animals↗

PubMed2005

Focusing in bioproduction science.

As in other Biotechnological fields, the microbial production of recombinant proteins and other biomolecules can be approached from multiple angles through the help of diverse technologies of increasing complexity. To better reach all the specialized niches in bioproduction, Microbial Cell Factories is now inviting authors to prepare concise Reviews (eventually miniReviews), covering relevant areas that deserve specific and highly focused attention. By the publication of such contributions, the journal will promote the revision of new insights around the Cell Factory concept in a highly comprehensive way, in molecular, cellular and environmental contexts.

Journal Article↗

PubMed2005

Microbial isoprenoid production: an example of green chemistry through metabolic engineering.

Saving energy, cost efficiency, producing less waste, improving the biodegradability of products, potential for producing novel and complex molecules with improved properties, and reducing the dependency on fossil fuels as raw materials are the main advantages of using biotechnological processes to produce chemicals. Such processes are often referred to as green chemistry or white biotechnology. Metabolic engineering, which permits the rational design of cell factories using directed genetic modifications, is an indispensable strategy for expanding green chemistry. In this chapter, the benefits of using metabolic engineering approaches for the development of green chemistry are illustrated by the recent advances in microbial production of isoprenoids, a diverse and important group of natural compounds with numerous existing and potential commercial applications. Accumulated knowledge on the metabolic pathways leading to the synthesis of the principal precursors of isoprenoids is reviewed, and recent investigations into isoprenoid production using engineered cell factories are described.

Bioreactors↗

PubMed2025

Evolution and applications of genome-scale metabolic models in yeast systems biology studies.

Genome-scale metabolic models (GEMs) can be used to simulate the metabolic network of an organism in a systematic and holistic way. Different yeast species, including Saccharomyces cerevisiae, have emerged as powerful cell factories for bioproduction. Recently, with the dedicated efforts from the scientific community, significant progress has been made in the development of yeast GEMs. Numerous versions of yeast GEMs and the derived multiscale models have been released, facilitating integrative omics analysis and rational strain design for different types of yeast cell factories. These advancements reflected the evolution and maturation of yeast GEMs together with a model ecosystem around them. This review will summarize the development and expansion of yeast GEMs and discuss their applications in yeast systems biology studies. It is anticipated that yeast GEMs will continue to play an increasingly important role in pioneering yeast physiological and metabolic studies in coming years.

Systems Biology↗

PubMed2026

Engineering Bacillus Subtilis for Efficient Biosynthesis of Riboflavin: Current Knowledge and Future Perspectives.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Bacillus subtilis↗

PubMed1993

Agarose gel electrophoresis system for the separation of antibiotics used in animal agriculture.

A novel electrophoresis system using agarose gel has been developed for the separation and as an aid in the classification of antibiotics. This system utilizes Nunc cell factory disposable tissue culture dishes, which serve as bioassay dish and cooling chamber for agarose gel, in a custom designed electrophoresis unit. Tris(hydroxymethyl) methylamine-succinate buffer at pH 6.0 and 8.0 are employed as the electrolyte for electrophoresis. Bioautography was used as the indicator of mobility. Any agar diffusion assay can be modified to use this system. A suggested name for this system is Nunc cell factory agarose gel electrophoresis (NUAGE). Selected antibiotics, representative of the aminoglycoside, beta-lactam, macrolide, moenocinol, peptide, polyene, polyether, quinone and tetracycline classes, were separated with this system.

Animal Feed↗

PubMed1974

Vaccinia virus replication in enucleate BSC-1 cells: particle production and synthesis of viral DNA and proteins.

The growth of vaccinia virus in monolayers of BSC-1 cells enucleated by centrifugation in the presence of cytochalasin B has been studied. No evidence for the production of infectious virus in these cells was obtained, and the production of virus particles was reduced to 8.3% compared with the yield from cytochalasin-treated, uncentrifuged cells. Virus DNA and early and late polypeptides were synthesized with normal timing in enucleate cells, but in reduced amounts; cleavage of structural polypeptide precursors P4a and Px also occurred in enucleate cells. Factories containing immature virus particles were demonstrated in enucleate cells by electron microscopy; these factories were reduced in number and size compared with those found in cytochalasin-treated, uncentrifuged cells.

Amino Acids↗

PubMed2025

Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability.

BACKGROUND: Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality. METHODS: We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness. RESULTS: Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use. CONCLUSIONS: Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

Humans↗

PubMedDate not supplied

Optimization of retroviral vector generation for clinical application.

BACKGROUND: For many inherited and acquired diseases of the blood system, gene transfer into hematopoietic cells is a promising strategy to alleviate disease-related symptoms or even correct genetic alterations. In clinical gene therapy applications, low transduction efficiencies have been a major limitation mainly because of insufficient effective titers of the retroviral supernatants used. Thus, optimization of clinical-grade vector production under current 'Good Manufacturing Practice' (GMP) conditions is a prerequisite for successful gene therapy trials. METHODS: We established stable retroviral producer clones with single integrations of a retroviral vector encoding for the multidrug-resistance gene 1 (MDR1). Optimization of vector production in multi-tray cell factories (MTCFs) was studied with particular regard to harvest medium, cell density and harvest time point. RESULTS: We demonstrated that high-titer vector stocks could be produced in serum-free medium. By reducing the volume of harvest medium, titers could be increased up to four-fold. Plating optimal cell densities of 1 x 10(4) cells/cm2, repetitive harvests of vector supernatant were feasible over four consecutive days. Combining the most advantageous culture and harvest parameters tested, we were able to produce large quantities of serum-free vector supernatant in 40-tray MTCFs. Highly efficient gene transfer into primary human CD34+ progenitor cells demonstrated the quality of these vector stocks. CONCLUSION: The large-scale vector-production protocol in MTCFs described here is easy to handle, is applicable to a wide range of adherent producer cell lines and, most importantly, complies with current GMP guidelines.

Antigens, CD34↗

PubMed2002

Enhanced bystander cytotoxicity of P450 gene-directed enzyme prodrug therapy by expression of the antiapoptotic factor p35.

Cytochrome P450 gene-directed enzyme prodrug therapy substantially augments intratumoral activation of anticancer prodrugs, such as cyclophosphamide (CPA), leading to a strong increase in antitumor effect without a corresponding increase in host toxicity. Attempts to additionally increase tumor cell kill by enhancing the intrinsic chemosensitivity of P450-expressing tumor cells by chemical means (depletion of cellular glutathione) or by coexpression of proapoptotic factors was shown to result in the desired increase in chemosensitivity, but with a decrease in net production of bystander cytotoxic drug metabolites because of accelerated death of the prodrug-activating tumor cells. Moreover, tumor cell P450 activity declined during the course of apoptosis induced by P450-activated CPA, limiting the potential of the tumor cell for continued production of activated drug metabolites. This limitation could be overcome by retroviral delivery of the baculovirus-encoded caspase inhibitor p35 to P450-expressing tumor cells. p35 substantially prolonged the activation of CPA by P450 "factory cells," leading to an increase in their bystander cytotoxicity toward P450-deficient tumor cells. This effect was greatest in tumor cells treated with CPA for an 8-h period, a schedule designed to model the effective time period of drug exposure in bolus CPA-treated patients in vivo. Notably, retroviral transduction of tumor cells with p35 did not induce drug resistance, as shown by the absence of long-term tumor cell survival or detectable colony formation activity after CPA treatment. These findings demonstrate that antiapoptotic factors, such as p35, can be used in a novel manner to enhance prodrug activation gene therapy by delaying tumor cell death, thereby increasing the net production of bystander cytotoxic metabolites and, hence, the overall effectiveness of the anticancer strategy.

Animals↗

PubMed2000

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Journal Article↗

PubMed2000

Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1).

Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells. As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines. This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system. The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo. Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m. Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line. Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells. Results from the production runs and the applied biosafety concept are described.

Animals↗

PubMed2005

Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus.

The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.

Base Sequence↗