Loss of Antibody Productivity During Long-Term Cultivation of a Hybridoma Cell Line in Low Serum and Serum-Free Media (original) (raw)
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The loss of antibody productivity in continuous culture of hybridoma cells
Biotechnology and Bioengineering, 1990
Two hybridoma lines, HB8178 and AFP-27, were grown in continuous culture. The concentrations of viable cells as well as those of various nutrients and metabolites reached steady-state values. The concentrations of either total IgG or antigen-specific antibody, however, failed to reach steady-state values but rather continuously decreased over the course of the cultures. The fraction of antibody-producing cells in the total cellular population also continuously decreased in the AFP-27 cultures. Comparison of the specific antibody productivity based on either the entire population or the antibodyproducing fraction of the population over time suggests that the decrease in productivity was at least partly due to the occurrence of a nonproducing subpopulation of cells.
Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media
Biotechnology and Bioengineering, 1991
Two murine hybridoma cell lines (167.465.3 and S3H5/ y2bA2) were adapted to grow in low-serum and serumfree media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/y2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/y2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.465.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.
Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production
Journal of Biotechnology, 1990
A murine hybridoma cell line (167.4(35.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 102 to 105 cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. (31utamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 106 cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 103 cells per ml grew 30% slower than those at 10 4 or 10 5. This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.
Cell growth and monoclonal antibody production in the presence of antigen and serum
Biotechnology Progress, 1995
The impact that the continuous presence in the fermentation broth of the cognate antigen has on the serum-supplemented hybridoma cell cultures was investigated. Both soluble and immobilized antigen a t various concentrations was applied. The cell line (ATCC TIB191) was cultured in a serum-supplemented PFHM-I1 medium in T-flasks. Sepharose gel beads provided the immobilization matrix, and bovine y-globulin was the carrier protein upon which the antigen, picric acid, was conjugated. Produced antibody, after elution from the beads by displacement with free picric acid, was measured with a n ELISA. Soluble antigen-carrier protein conjugates showed no effect on the cultures, but the immobilized antigen had a strong influence on them.
Biotechnology Progress, 1991
The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/72bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monodtype model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest a t 20-50 % air saturation. Glucose and glutamine uptake rates increased at DO above 10 % and below 5 % air saturation. Cell growth rate was optimal a t pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased a t low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed a t pH values below 7.2. Higher concentrations of antibody were obtained a t high serum levels, between 20 % and 40% DO, and a t p H 7.20 due to higher viable cell numbers obtained.
Biotechnology Progress, 1991
The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/72bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monodtype model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest a t 20-50 % air saturation. Glucose and glutamine uptake rates increased at DO above 10 % and below 5 % air saturation. Cell growth rate was optimal a t pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased a t low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed a t pH values below 7.2. Higher concentrations of antibody were obtained a t high serum levels, between 20 % and 40% DO, and a t p H 7.20 due to higher viable cell numbers obtained.
Serum‐free media in hybridoma culture and monoclonal antibody production
Biotechnology and …, 1988
The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum-free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum-free formulations and selection rules for the serum-free ingredients. In general, serum-free media which are "lipid rich" can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum-free media, irrespective of the lipid content. 2. Improves reproducibility of cell culture growth and product yield.
Journal of Biotechnology
The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across ...
Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures
Cytotechnology, 1994
It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle GI phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.
Regulation of antibody production by hybridoma cultures
Cellular Immunology, 1987
A series of anti-DNA antibody-producing hybridomas were obtained by fusing spleen cells from 6-month-old MRL/lpr. autoimmune prone. mice with P3X63-Ag8 myeloma cells. Rabbit anti-idiotype (id) antibodies specific for several of the hybridoma proteins were prepared. It was shown that the anti-id antibody inhibited immunogiobulinin secretion by the hybridoma cells in an id-specific manner. Inhibition of antibody production was not due to a cytotoxic effect. since the anti-id, in fact, stimulated proliferation of the hybridoma cells. L IYX7 4cadcmic