Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T‐75 flask

Vallejos, Jose R.; Brorson, Kurt; Moreira, A.; Rao, Govind

doi:10.1002/bit.22649

Cited by 20 publications

(21 citation statements)

References 43 publications

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“…This fact led to a decrease in the energy supply needed for cell development, consequently leading to a drop in the cell growth specific rate, as reported by Vallejos et al (2009). Maximum concentration of viable cells/mL at the second day of culture (Fig.…”

Section: Monoclonal Antibody Productionsupporting

confidence: 56%

“…As mentioned before, reductions in the mAb productivity (Li et al 2006) as well in cell growth (Vallejos et al 2009) show that different cell lines can behave differently. Data obtained for MRSA-antiPBP2a mAb producing hybridoma cells which showed high specific mAb antibody production rate until P10 (Fig.…”

Section: Monoclonal Antibody Productionmentioning

confidence: 69%

“…Lactate concentrations greater than 3 g/L can inhibit cell growth (Legazpi et al 2005). High-values for the yield of lactate formation (Y Lac/Gluc ) are usually an indication that the cells may be in a state of less energetic efficiency, and can adversely affect productivity and quality of the product of interest (Vallejos et al 2009;Acosta et al 2007). The maximum concentration of lactate obtained did not exceed 0.9015 g/L during cultures performed at passage P20, for example.…”

Section: Metabolismmentioning

confidence: 99%

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Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Corrêa

Senna²,

Sousa³

2015

Cytotechnology

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Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, fastergrowing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies ]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.

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Section: Monoclonal Antibody Productionsupporting

confidence: 56%

Section: Monoclonal Antibody Productionmentioning

confidence: 69%

Section: Metabolismmentioning

confidence: 99%

See 1 more Smart Citation

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Corrêa

Senna²,

Sousa³

2015

Cytotechnology

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“…13 Robust q O2 and q CO2 estimation is thus critical for bioprocess development and is also important for monitoring and diagnosing manufacturing bioreactors. While recent q O2 estimation efforts have focused on small scale cultures such as spinner flasks, 14 T-flasks, 8 and microtiter plates, 15 limited data on q O2 and q CO2 estimation in high density perfusion cultures are available.…”

Section: Introductionmentioning

confidence: 99%

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

Goudar

Piret

Konstantinov

2011

Biotechnology Progress

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We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.

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“…It not only affects the growth rate and protein synthesis (Miller et al 1987) but also regulates glucose and glutamine consumption and lactate and ammonia generation (Vallejos et al 2010). A DO concentration between 20 and 50% airsaturation is usually maintained in bioreactors for better cell growth and recombinant protein expression.…”

Section: Introductionmentioning

confidence: 99%

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

et al. 2011

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A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k(L)a value of the Tubespin at standard conditions was 24.3 h(-1), while that of a spinner flask was only 2.7 h(-1). The maximum cell density in the Tubespin bioreactor reached 6 × 10(6) cells mL(-1), which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.

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Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T‐75 flask

Cited by 20 publications

References 43 publications

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

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