Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures (original) (raw)
Related papers
Cytotechnology, 1992
A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G I phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of GI phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.
Journal of Biotechnology, 1991
During batch cultivation of the 1.13.17. hybridoma cell line, there is a 15 to 20-fold decrease in the levels of cytoplasmic and membrane-bound mAb, and a 7 to 10-fold decrease in the cellular levels of kappa and gamma chain mRNAs, as the cells pass from the exponential into the decline phase of growth. The profile of the specific mAb production rate does not correlate with the kinetics of either the cytoplasmic mAb or the specific mRNAs throughout the culture. Flow cytometry analyses have revealed that dead cells, which account for 40 to 70% of total cells during the decline phase, might significantly interfere with the determination of cytoplasmic mAb levels of cell-lysates ELISA and with the calculation of the specific mAb production rate. Possible influences of these parameters on mAb synthesis and secretion during hybridoma batch culture are discussed.
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.
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.
Biotechnology Progress, 1994
AB2-143.2 cells exhibit non-growth-associated antibody production (constant specific antibody production rate (qAb) throughout batch culture), while IND1 cells exhibit growth-associated production (decrease in qAb during stationary phase). AB2-143.2 cells increase qAb following abrupt batch osmotic stress. Enhanced antibody production in IND1 cells after batch osmotic shock is evidenced as maintenance of the higher exponential phase QAb into the stationary phase. AB2-143.2 cells also increase qAb in response to gradual osmotic stress in continuous culture. In contrast, IND1 cells decrease 9Ab during gradual osmotic stress. Although the two cell lines differ in antibody production, they have very similar intracellular antibody content profiles. Both show (1) constant antibody content during the exponential phase in control culture with a decrease as cells enter stationary phase; (2) maintenance of exponential-phase antibody content into the stationary phase after batch osmotic shock; and (3) no change in antibody content in response to gradual osmotic stress.
Kinetics of monoclonal antibody production in low serum growth medium
Journal of Immunological Methods, 1986
Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series 'of studies. The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media.
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.
Hybridoma, 1990
A murine hybridoma cell line (167.4G5.3) was adapted to grow in different serum concentrations over a six month period of time. Adaptation to low serum and to serum-free media improved growth rates, but at'low serum (1.25%) the antibody productivity was diminished. Flow cytometric analysis showed the presence of two distinct cell populations with respect to intracellular and surface antibody concentrations. The loss in antibody productivity during adaptation could be attributed to the appearance of a low antibody-containing cell population. Cultures maintained at high serum concentrations did not loose the original high antibody productivity. In a separate experiment the kinetics of growth improvement and loss of antibody production were studied for adaptation from 5% to 1.25% serum-containing media. Over a time period of about four months, the population shifted completely from high-producing cells to low-producing ones in response to the 1.25% environment. A shift-up from 1.25% to 20% serum resulted in the elimination of the low producing population. These results suggest that, for the cell line used, serum-containing factors prevent the loss of antibody productivity.
Biotechnology Letters, 1992
Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For tt:e murine hybridoma cell line $3It5/72bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 106 cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10 3 cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of l0 s cells/ml was necessary for the cells to grow in RPMI 1640 medium without, serum supplement, and an inoculnm density of t cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoeulum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma celt line used. Since the specific MAb production rate was independent of cultivation method and inoculum density~ the existence of autocriue growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.
Biotechnology Progress, 1999
The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowthassociated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate-and serum-depleted batch cultures.