Decreased chimeric antibody productivity of KR12H-1 transfectoma during long-term culture results from decreased antibody gene copy number

The stability of KR12H-1 transfectoma in regard to chimeric antibody production was examined during long-term, repeated batch culture without selection pressure using antibiotics. Both serum-supplemented and serum-free media were used. Regardless of the medium used, the specific antibody productivity (q(Ab)) of transfectoma decreased by 60% to 88% during 70-day culture. This loss of antibody productivity was not due mainly to the appearance of a nonproducing population (NP) of transfectoma. The percentage of a producing population (P), which was monitored by the limiting dilution method, remained over 90% until the end of culture, indicating that the q(Ab) of P decreased during the culture. Flow cytometric data also showed the increase of cell population with low fluorescence intensity during culture, indicating that the intracellular antibody content of P decreased. The subclones of P obtained at the end of long-term culture were further characterized. Compared with the q(Ab) of P at the beginning of long-term culture, the q(Ab) of most P subclones was significantly low, confirming that the loss of antibody productivity was due mainly to the decreased q(Ab) of P during long-term culture. The decreased antibody gene copy number of P subclones was found to be partly responsible for the decreased q(Ab) of P during long-term culture. (c) 1996 John Wiley & Sons, Inc.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implications for proliferation and productivity

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.     

SSGF-I,a potent growth-promoting substance for mammalian cells from swine serum

A small amount of swine serum markedly stimulated cell growth for high productivity subclones derived from a mouse human-human heterohybridoma, N12-16.63, secreting an anti-tetanus toxoid human monoclonal antibody in a polyethylene glycol (PEG)-containing serum-free medium, PEG-86-1. A growth promoting substance, SSGF-I, was isolated from the serum by ammonium sulfate fractionation, Cibacron blue F3A-G affinity chromatography, DEAE-agarose ion exchange chromatography, and gel filtrations on Trisacryl GF 2000 and Sephacryl S-300. SSGF-I was characterized as a low density lipoprotein (LDL) of swine serum by its physico-chemical properties. It promoted cell growth synergistically with PEG and its optimum concentration was 1 to 100g/ml. Human LDL was less active, and human or swine high density lipoprotein (HDL) and very low density lipoprotein (VLDL) were inactive. Based on these results, we propose an improved serum-free medium, PEG-86-3, which contains all the ingredients of PEG-86-1 and 10g/ml SSGF-I. This medium is useful for not only high productivity heterohybridomas but also for a variety of lymphoid cell lines.     

Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure

Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.     

Immobilization can improve the stability of hybridoma antibody productivity in serum-free media

Hybridoma cells (S3H5/gamma2bA2) were cultivated in spinner flasks with 1% serum media and serum-free media. Monoclonal antibody productivity was maintained in 1% serum media. However, cells in serum-free media showed a decrease in antibody productivity, and it completely disappeared in IMDM-based low protein medium. This loss of antibody productivity was not observed when the cells were immobilized in alginate beads. In fact, immobilization enhanced the specific MAb productivity.     

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.     

Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions

Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date. One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal. In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures. Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity. This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media. These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions. To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80. At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement. Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions. After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale.     

Effect of biomass concentration on the specific solvent productivity ofClostridium acetobutylicum in chemostat culture

Summary Using a defined medium in chemostat culture, an inverse relationship between the biomass concentration and the specific butanol productivity has been observed. It is suggested that this is due to the cell population not being homogeneous, and that a change in the nutrient balance leads to a cha in the relative proportions of acidogenic, solventogenic and inert cells (spores).     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Comparison of cell line maintenance procedures on insect cells used for producing baculoviruses

Summary A gypsy moth cell line, IPLB-LdEIta, maintained under various conditions was tested for susceptibility to and productivity of two baculoviruses, the Autographa californica nucleopolyhedrovirus (AcMNPV) and Lymantria dispar nucleopolyhedrovirus (LdMNPV). The results suggest that cells maintained in serum-containing medium (modified TC100) were more susceptible (on the basis of titers in an endpoint assay) to LdMNPV than cells maintained in a serum-free medium (ExCell™ 400). Such a difference was not apparent with AcMNPV. Similarly, little difference existed in the proportion of cells containing occlusion bodies (OBs) a wk after inoculation with AcMNPV (i.e., the percent infected) in any LdEIta strains, although one combination of cells and medium (cells maintained in ExCell 400 but infected in TC100) showed a lower percent infection with LdMNPV. Even though the percentage of cells infected varied little, the number of OBs produced varied by 3 logs with AcMNPV and 11/2 logs with LdMNPV. In each case, cells normally grown in ExCell 400 and infected in the same medium produced the lowest number of OBs. However, productivity was improved when cells normally grown in ExCell 400 were infected in TC100. Even more interesting was that cells normally grown in TC100 produced more AcMNPV OBs when infected in ExCell 400 medium. This suggests that changing culture medium (regardless of the normal maintenance medium) can stimulate virus production. In addition to examining virus productivity in LdEIta cells in both serum-containing and serum-free media, I also tested a strain maintained at low temperature (17° C) for over a yr. This maintenance protocol was not detrimental for LdMNPV productivity and was slightly stimulatory for production of AcMNPV.     

High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells

Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O₂ permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactae production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements. Correspondence to: M. Ogata     

Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.     

High-Density culture of mouse-human hybridoma in serum-free defined medium

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.     

Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy

Immunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines.     

Corticosteroid effects on lipid lateral diffusion in CEM-C1 and CEM-C7 acute lymphoblastic leukemia cells

We have examined glucocorticoid effects on CEM-C7 and CEM-C1 subclones of a leukemic human T-cell line using fluorescence photobleaching recovery techniques. Incubation with 10(-5) M triamcinolone acetonide (TA) increased lipid lateral diffusion on steroid-sensitive CEM-C7 cells but had no effect on steroid-resistant CEM-C1 cells. CEM-C7 cells incubated in serum-free medium responded only to TA but, when fetal calf serum was added to the incubation medium, would also respond to 10(-5) M dexamethasone and hydrocortisone. Thus, glucocorticoids can cause increased lipid lateral diffusion in CEM-C7 cells, while having no effect on steroid-resistant CEM-C1 cells.     

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.     

Growth factors and hyperthermia. I. The relationship between hyperthermic cell killing and the mitogenic response to serum and growth factors

Chinese hamster ovary HA-1 cells were tested for their ability to respond to mitogenic stimulation after hyperthermia at 45 degrees C. Cells were arrested by 24 h incubation in serum-free Eagle's MEM. Heating of arrested cells in serum-free medium did not alter heat sensitivity compared to exponentially growing cells heated in serum-containing medium. After hyperthermia cells exhibited a delay in the ability to undergo mitogenesis. Recovery of the capacity for mitogenesis occurred during the 24 h following heating and was able to take place in the absence of serum. After recovery in serum-free medium, cells were simultaneously assayed for survival and mitogenesis as measured by [3H]Thy uptake. With increasing heating time, surviving fraction and mitogenesis decreased. The reduction in survival was similar to the reduction in [3H]Thy incorporation. The relationship between mitogenesis and cell death was studied in more detail with flow cytometry. At a relatively mild heat dose of 30 min at 45 degrees C (survival = 30%), a small population of cells (9%) was found to be clonogenically dead yet capable of being stimulated to progress from G1 to G2-M. At a more severe heat dose of 40 min at 45 degrees C (survival = 3%), stimulation of dead cells could not be detected. Therefore, hyperthermia impairs mitogenic ability, but at low heat doses, a subpopulation of killed cells can still be stimulated to progress through the cell cycle.