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M Al-Rubeai

Publications and source records attributed to M Al-Rubeai.

At least 19 recordsLinked to original sources
PubMed2007

Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2.

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.

Animals↗
PubMed2004

Measuring the heterogeneity of protein loading in PLG microspheres using flow cytometry.

Poly (DL-lactide co-glycolide) (PLG) microspheres with mean sizes up to 1 microm containing Fluorescein Isothiocyanate labelled Bovine Serum Albumin (FITC-BSA) were prepared by the water-in oil-in water (w/o/w) emulsion solvent evaporation technique. Protein loading and loading efficiency determined by the BCA total protein assay increased with microsphere size as measured by laser diffractometry. Protein loaded microspheres were analysed using flow cytometry (FC) to provide fast and reproducible measurements of the size and protein loading of individual microspheres within a sample thereby quantifying in detail the batch heterogeneity. The FC analysis demonstrated that as the size of individual microspheres within a batch increased, so the protein loading tended to increase. For example, the protein loading of microspheres increased from 2.7 to 8.9 wt.% as the size of microspheres increased from 0.42 to 1.45 microm, respectively. Measurements taken during a subsequent protein release experiment indicated that smaller microspheres within a sample released their protein more quickly than larger sizes. Flow cytometry has been shown to provide detailed information, at the level of individual microspheres, about the heterogeneity in size and protein loading of a microsphere sample and could thus lead to improvement of the release characteristics of microsphere-based delivery systems for biopharmaceuticals.

Animals↗
PubMed2004

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity.

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.

Antibodies, Monoclonal↗
PubMed2002

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture.

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.

Animals↗
PubMed2001

Relationship between cell proliferation, cell-cycle phase, and retroviral vector production in FLYRD18 human packaging cells.

The relatively low concentrations of retroviral vectors produced by most packaging cells requires the optimization and intensification of their production to make a commercially viable product for gene therapy. While a number of reports exist concerning target cell-cycle effects on retroviral vector infection efficiency, no studies have been reported on the effects of packaging cell cycle on vector production. We have studied the effect of proliferation of the human packaging cell line, FLYRD18, on vector production. In addition, the titer levels of vector produced by cells in each phase of the cell cycle were compared. Numerous studies suggested progression of the cells through the cell cycle to be essential for vector production. However, vector release was found not to be predominant in any particular phase of the cell cycle. These findings indicate that packaging cell proliferation is important for optimal virus production and that arrest of the cells in any particular phase of the cell cycle affords no benefits in retroviral vector production. In contrast to previous reports (using other cell lines), we observed no temporary inhibition of cell cycle progression after detachment of cells from their substratum and that virus production occurred immediately after re-plating of the cells. The findings in this report are important for determining the optimal culture conditions for vector production by packaging cells in vitro.

Cell Count↗
PubMed2000

Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures.

The influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre.

Ammonia↗
PubMed2000

Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody.

The influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 microgram/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines.

Ammonia↗
PubMed1999

Improved cell line development by a high throughput affinity capture surface display technique to select for high secretors.

A novel process is described which permits rapid and objective selection of rare cells from a heterogeneous population based on quantity of secreted target protein. The process involves construction of an immobilised affinity surface display matrix that specifically binds secreted target product which is then detected using a fluorescent labelled ligand. Cells with the highest fluorescence can then be sorted using conventional flow cytometric technology. Overall, the whole process can be completed in less than 4 h during which time in the region of five million cells can be analysed. Cells are rapidly selected for in a quantitative manner compared to traditional methods which can take several months and have a reduced probability of finding low abundance high secretors due to practical limitations imposed on the number of cells which can be screened.

Affinity Labels↗
PubMed1999

Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay.

Apoptosis is a form of cell death in which the dying cell plays an active part in its demise. At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death. In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules. Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed. We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay. The results of this assay were compared with data generated by flow cytometry and manual scoring. All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture. Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface.

Animals↗
PubMed1999

Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta.

Brochothrix thermosphacta is a common meat spoilage bacterium. The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques. Methods for the quantification of this bacterium have been compared. Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth. The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml. Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml. The sensitivity of analysis, growth rates and lag time attained using these methods were also compared. As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate. The plate count method proved unable to detect the difference between bacteria existing as chains or single cells. The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts. This suggests that flow cytometry is capable of counting individual cells in a chain. Further investigation showed that passage of B. thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells. The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B. thermosphacta, a bacterium which exhibits complex morphologies.

Animals↗
PubMed1999

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Animals↗
PubMed1999

Bcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells.

Recent studies have suggested that Bcl-2 can affect cell cycle re-entry by inhibiting the transition from G0/G1 to S phase. In this study, we have taken a novel route to the study of the relationship between Bcl-2 expression and cell cycle progression. Continuous cultures of pEF (control) and Bcl-2 transfected murine hybridoma cells were operated at a range of dilution rates from 0.8 day-1 down to 0.2 day-1. The specific growth rate of the pEF cell line was the same as the dilution rate down to a value of 0.6 day-1. However, as the dilution rate was reduced stepwise to 0.2 day-1, the growth rate levelled-off at approximately 0.55 day-1 and this coincided with a fall in culture viability. By contrast, the specific growth rate of the Bcl-2 transfected cell line followed the dilution rate down to a value of 0.3 day-1 with high levels of cell survival. At high dilution rates, the cell cycle distributions were very similar for both cell lines. However, the distributions diverged as the dilution rate was reduced and, at a rate of 0.2 day-1, the percentage of G1 cells in the Bcl-2 culture was 80%, compared to only 56% in the pEF cell population. This corresponded with a greater extension in the duration of the G1 phase in the Bcl-2 cells, which was 1.7 days at the lowest dilution rate tested, compared to only 0.6 day for the pEF cell line. The durations of the G2/M and S phases remained constant throughout the culture. The maximum doubling time was 1.2 days in the pEF culture compared to 2.3 days in the Bcl-2 culture. Analysis of amino acids, ammonia and lactate concentrations indicated that the observed effects on cell cycle dynamics were probably not due to differences in the culture environment. It is suggested that the expression of Bcl-2 can effect G1 to S phase transition in continuously cycling cells, but this is only apparent at suboptimal growth rates.

Amino Acids↗
PubMed1999

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture.

We have addressed the possibility that Ca2+, Mg2+ and K+ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca2+, Mg2+ or K+ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K+ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg2+ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca2+ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppressor gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg2+ ion deprivation and apoptosis inducing effects of Ca2+ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K+ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca2+ Mg2+ or K+ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.

Journal Article↗
PubMed1998

Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture.

Accurate identification and quantitation of apoptosis is essential for developing efficient strategies for optimisation of culture viability and productivity in cell lines of industrial significance. We have examined the possibility of using carboxy-seminaphthorhodafluor-1-acetoxymethylester (carboxy SNARF-1-AM), a pH sensitive fluoroprobe and FITC-labelled annexin V (AV), a probe specific to phosphatidylserine exposed on the surface of apoptotic cells, to monitor apoptosis and to determine the relationship between intracellular pH (pHi), apoptosis and cell cycle in hybridoma cells. Temporal changes in the distribution of proliferative capacity (S phase), metabolic activity (pHi), and cell death population dynamics were effectively and reliably determined using flow cytometry. Intracellular acidification was shown to precede the occurrence of apoptosis during batch culture and after treatment with campothecin, staurosporine and under adverse bioreactor conditions such as glutamine deprivation and oxygen deficiency. These results showed that the decrease in pHi can be used as an indicator of cellular deterioration and cell death. AV in combination with propidium iodide permitted the identification of viable, transient apoptotic and necrotic cells in heterogeneous cultures of control (PEF) cells. Hybridoma cells over-expressing bcl-2 were protected from intracellular acidification and phosphatidylserine exposure, which was associated with the suppression of apoptosis in these cells. A decrease in pHi was apparent even before the accumulation of the normally acidic G1 phase and the development of a sub-G1 region, characteristic of apoptotic cell behaviour. The pHi assay can therefore be used as a tool to predict future cell culture performance. reserved.

Annexin A5↗
PubMed1998

In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene.

The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture).

Amino Acids↗
PubMed1997

G2 cell cycle arrest and apoptosis are induced in Burkitt's lymphoma cells by the anticancer agent oracin.

The cytotoxic effect of the new potential intercalating anticancer drug oracin was studied on Burkitt's lymphoma cell line that overexpressed bcl-2 (BL bcl-2) and a control transfectant without the bcl-2 gene (BL SV2). Oracin showed a marked cytostatic effect on both BL SV2 and BL bcl-2 cells. IC50, as measured by the MTT assay, was approx. 5-times greater for BL bcl-2 cells (5.0 micromol/l) than for BL SV2 cells (1.0 micromol/l). There was no significant increase in apoptosis after 24 h of treatment with oracin (1.0 micromol/l) in both cell lines. However, after 48 h from the removal of oracin in BL SV2 culture the levels of apoptotic and secondary necrotic cells increased to 20 and 37%, respectively. In contrast, BL bcl-2 cells treated in a similar manner showed only basal levels of apoptotic and secondary necrotic cells. Analysis of the cell cycle profiles showed a significant increase of S and G2/M phases of the cell cycle in both cell lines after 6 h of drug treatment (1.0 micromol/l). The cells were arrested in G2/M phase of the cell cycle after 24 h, with no significant changes in cell viability. After 72 h, the viable BL SV2 cells were still in G2/M, however, the viability of this culture had fallen to approx. 5%. Flow cytometry analysis of the DNA content revealed the presence of a 'sub-G2' region, which represented the apoptotic cells. The BL SV2 cells died after 72 h while they were in the G2/M phase. Although the treated BL bcl-2 cells were similarly arrested in the G2/M phase, they nevertheless remained with a relatively high viability (68%).

Antineoplastic Agents↗
PubMedDate not supplied

Effects of culture parameters on the production of retroviral vectors by a human packaging cell line.

The use of retroviral vectors for human gene therapy requires the production of large quantities of high titer vector stocks. Maintaining high titers during the prolonged culture of packaging cells will require that critical parameters be controlled. The aim of this study was to determine which culture parameters critically affect the production/decay of retroviral vectors produced by the human packaging cell line FLYRD18/LNC-hB7. The stability of retroviral vectors released by this cell line was found to be temperature dependent (half-life of 6.9, 11.0, and 64.3 h when incubated at 37, 32, and 0 degrees C, respectively). Titers increased up to 10-fold when the packaging cells were cultured at 32 degrees C, compared to 37 degrees C, despite a decrease in cell yield (cell-specific titers were 20-fold higher). Virus titers were also over 10-fold higher when the packaging cells were cultured in a reduced serum concentration (1%) compared to 5%. Retrovirus production at a range of pH levels revealed a significant decrease in virus titer at pH levels below 6.8 and above 7.2, optimum titers being achieved in cultures at pH 7.2. Dissolved oxygen levels in the range 20-80% did not significantly affect titers under the conditions tested. Finally, a packed bed system containing the packaging cells immobilized on porous microcarriers was shown to sustain the production of active retroviral vectors for over 1 month, in relatively large volumes.

Blood↗