High cell density perfusion culture of hybridoma cells recycling high molecular weight components

Takazawa, Yoshiharu; Tokashiki, Michiyuki; Hamamoto, Kimihiko; Murakami, Hiroki

doi:10.1007/bf00146818

Cited by 40 publications

(6 citation statements)

References 13 publications

(8 reference statements)

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“…As can be seen in eq 2, the overall productivity, per bioreactor volume used, would be increased correspondingly. It has been previously reported that some hybridoma cell lines are growth phase-dependent with respect to the specific antibody production, while others are growth phase-independent (Suzuki and Olis, 1990;Takazawa et al, 1988); our work here indicates that the presence of antigen can shifi a cell line from one type to the other. This antigen-induced shift was also observed in proteinfree media (Dandulakis et al, 1994).…”

Section: Resultssupporting

confidence: 62%

Cell Growth and Monoclonal Antibody Production in the Presence of Antigen and Serum

Dandulakis

Herr

Kirwan

1995

Biotechnology Progress

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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 at various concentrations was applied. The cell line (ATCC TIB191) was cultured in a serum-supplemented PFHM-II medium in T-flasks. Sepharose gel beads provided the immobilization matrix, and bovine gamma-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 an ELISA. Soluble antigen--carrier protein conjugates showed no effect on the cultures, but the immobilized antigen had a strong influence on them. Cell growth rate and total antibody production decreased as the amount of immobilized antigen increased from 16:10 (units are in (mol of Ag/mol of carrier):(mg of carrier/mL of beads) to 36:40. Most importantly, the specific antibody production rate switched from growth phase-independent behavior in the antigen-free cultures to growth phase-dependent behavior in the immobilized antigen cultures. Furthermore, during the early stationary phase of the cultures with immobilized antigen, the specific antibody production was 20-100% higher relative to the production rate in antigen-free cultures. This increased specific antibody production rate would be particularly useful in increasing the volumetric productivity of perfusion-type bioreactors for hybridoma cell cultures.

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Section: Resultssupporting

confidence: 62%

Cell Growth and Monoclonal Antibody Production in the Presence of Antigen and Serum

Dandulakis

Herr

Kirwan

1995

Biotechnology Progress

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“…These include encapsulation, hollow fibers, cells entrapped on polymer beads, cells attached to ceramic supports, and cells in dialysis reactors.',4,5,7.12,14T25. 32 Such systems maintain cells for long periods at very low growth rates and high cell density, conditions which increase specific immunoglobulin secretion rates, to produce gram quantities of MCAB. However, the amount of MCAB produced is generally monitored by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants, but the quality of MCAB produced as judged by its molecular integrity is rarely tested.…”

Section: Ntrod Uctlonmentioning

confidence: 99%

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous‐flow culture with integrated product recovery

Mohan

Chohan

Eade

et al. 1993

Biotech & Bioengineering

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The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth.

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“…(a) Determination of the molecular weights of the 9.2.27 yza heavy and K light chains; 9.2.27 antibody was purified from cell culture supernatants with protein-A affinity chromatography, 10% reducing SDS gel. The molecular weight markers are myosin (H chain, 200,000), phosphorylase b (97,400), bovine serum albumin (68,000), ovalbumin (43,000), carbonic anhydrase (29,000) and P-lactoglobulin (18,400). (b) Fluorograph of a 10% reducing SDS gel of the MAb accumulated in the supernatant during the labeling period, for the cells collected in the stationary growth phase.…”

Section: Continuous Labeling Experimentsmentioning

confidence: 99%

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells

Bibila¹,

Flickinger²

1991

Biotech & Bioengineering

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A three compartment model (ER --> Golgi --> extracellular medium) is used here to describe the interorganelle transport and final secretion of an IgG(2a) monoclonal antibody (MAb) in 9.2.27 murine hybridoma cells. Model simulations of pulse-chase and continuous labeling experiments are used to gain a better understanding of the kinetics of MAb interorganelle traffic. Simulation results for the continuous labeling case compare well with experimental data obtained during continuous labeling of 9.2.27 hybridoma cells. Incorporation of this compartmental transport model into our previously developed model of MAb synthesis and assembly can provide a useful tool for analyzing the dynamics and regulation of the complete antibody secretory pathway under different growth and/or nutritional conditions.

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High cell density perfusion culture of hybridoma cells recycling high molecular weight components

Cited by 40 publications

References 13 publications

Cell Growth and Monoclonal Antibody Production in the Presence of Antigen and Serum

Cell Growth and Monoclonal Antibody Production in the Presence of Antigen and Serum

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous‐flow culture with integrated product recovery

A model of interorganelle monoclonal antibody transport and secretion in mouse hybridoma cells

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