The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

Coco-Martin, J.M.; Oberink, Jan W.; Groot, Tiny A. M. van der Velden-de; Beuvery, E. C.

doi:10.1007/bf02540031

Cited by 13 publications

(5 citation statements)

References 33 publications

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“…The data also supports the hypothesis that cells in the G1 phase are most productive. These findings are in agreement with several previous studies demonstrating a high specific productivity of hybridoma cell lines during the G1 phase [36][37][38][39][40][41][42] and may be considered for further optimization of the process, for instance with appropriate feeding strategies or cultivation modes. Analysis of the IgG expression on the single-cell level via flow cytometry was performed in order to evaluate the homogeneity of the cell population.…”

Section: Cell Sortingsupporting

confidence: 92%

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Moretti¹,

Behr²,

Walter³

et al. 2010

Engineering in Life Sciences

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The improvement of specific productivity is a continuous challenge for bioprocesses involving mammalian cells, and hence, high‐throughput methods and low‐cost strategies are needed for the selection of high producers. The aim of this study was the productivity improvement of the hybridoma cell line IV F 19.23. For this purpose, a cell surface affinity matrix assay was established to identify and select high producers. This assay is based on the binding of secreted monoclonal antibodies to an affinity matrix assembled on the outer cell membrane. A protein microarray approach was used to investigate and optimize the functionality of the affinity matrix. The protein microarray was particularly useful to identify critical steps of the staining method, such as unspecific binding, before it was applied to the hybridoma cell line. Secreting hybridomas were treated with the affinity matrix and then selected via flow‐cytometric cell sorting in four consecutive bulk sort rounds. The applied bulk strategy, allowing low screening costs, resulted in a 125% increase in specific productivity of the cell line in comparison to the initial population.

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Section: Cell Sortingsupporting

confidence: 92%

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Moretti¹,

Behr²,

Walter³

et al. 2010

Engineering in Life Sciences

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“…There was a bigger increase in the mAb concentration in BM basal medium after the cultivation of 96 h, when there was a big drop in the specific cell growth rate (data not shown). It was also reported that, for a hybridoma cell line, IgG is mainly synthesized during the S phase but secreted into the culture fluid as the cells go through the G 1 phase (Coco-Martin et al, 1992). The maximal mAb concentration (i.e., 182.4 mg/L) in the BM basal medium was 10-fold higher than that (i.e., 16.8 mg/L) in the DMEM basal medium.…”

Section: Resultsmentioning

confidence: 95%

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG_2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System

Tang

Ohashi

Hamel

2007

Biotechnology Progress

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A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.

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“…The hybridoma cells started to secrete antibody after cultivation for 5 days during that time, lactic acid and ammonia was also produced. It have been reported that hybridoma cells mainly synthesized monoclonal antibodies during the S phase but secreted into the culture fluid as cells go through the G 1 phase of the cell cycle 51. Combining the results with 2D batch cultivation, it is assumed that with decreasing growth rate (during days 23–30), cells remain in G 1 phase for a longer time and therefore there was an increase of the antibody productivity with increasing death rate.…”

Section: Resultsmentioning

confidence: 95%

Three‐dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles

Nilsang

Nehru

Plieva

et al. 2008

Biotechnology Progress

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in Wiley InterScience (www.interscience.wiley.com).Cell proliferation and long-term production of monoclonal antibody IgG 2b by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 Â 9 mm 2 ). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine þ 2 mM a-ketoglutarate or L-alanyl-glutamine (GlutaMAX TM ) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine þ a-ketoglutarate was similar to cells grown on Gluta- MAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 Â 10 6 cells mL À1) and highest specific mAb production rate (3.9 lg mL À1 10 À4 viable cell day À1), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production.

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The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

Cited by 13 publications

References 33 publications

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG_2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System

Three‐dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles

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The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

Cited by 13 publications

References 33 publications

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Characterization and improvement of cell line performance via flow cytometry and cell sorting

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System

Three‐dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles

Contact Info

Product

Resources

About

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG_2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System