An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

Banik, Gautam G.; Heath, Carole A.

doi:10.1007/bf00387697

Cited by 17 publications

(10 citation statements)

References 36 publications

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“…A strategy designed to reduce and k d in perfusion could contribute to a reduction in both q S and q O2 , allowing to support a higher X v for the same amount of oxygen transferred to the system. Indeed, metabolic studies in batch (Doverskog et al, 1997;Fitzpatrick et al, 1993;Ramirez and Mutharasan, 1990); chemostat (Hiller et al, 1993;Robinson and Memmert, 1991) and perfusion culture (Banik and Heath, 1994;1995;Park and Ryu, 1994;Schmid et al, 1992;Seamans and Hu, 1990; present study) have demonstrated that a correlation can be established between and q glc or q gln . Further studies focusing on lymphoid cell metabolism also presented direct correlations between q O2 and q S (Kyung et al, 1994;Ramirez and Mutharasan, 1990;Zeng et al, 1998) and between q O2 and (Banik and Heath, 1995;Boraston et al, 1984, Hiller et al, 1993Ramirez and Mutharasan, 1990).…”

Section: Strategies Aimed At Reducing Sparging Requirementssupporting

confidence: 48%

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

Mercille¹,

Johnson²,

Lanthier³

et al. 2000

Biotechnol. Bioeng.

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One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL). Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc). Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed.

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Section: Strategies Aimed At Reducing Sparging Requirementssupporting

confidence: 48%

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

Mercille¹,

Johnson²,

Lanthier³

et al. 2000

Biotechnol. Bioeng.

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“…Perfusion presents a number of advantages over other modes of cultivation including increased volumetric productivity, reduced labor and costs, and rapid removal of easily inactivated products from the culture environment (Prior et al, 1989). However, although high density cultures of hybridoma or recombinant myeloma cells can be achieved, decreasing viabilities are observed in a number of different perfusion systems (Al-Rubeai et al, 1992;Avgerinos et al, 1990;Banik and Heath, 1994;de la Broise et al, 1991de la Broise et al, , 1992Hansen et al, 1993;Hiller et al, 1993;Johnson et al, 1996;Kitano et al, 1986;Mercille et al, 1994c;Reuveny et al, 1986;Schmid et al, 1992;Tokashiki and Takamatsu, 1993;Trampler et al, 1994;Van Erp et al, 1991). Under these conditions, an equilibrium is established in which a significant loss of cell viability is matched by a high level of proliferation, leading to a significant waste of metabolic energy that could be more appropriately diverted to recombinant protein production (de la Broise et al, 1991).…”

Section: Introductionmentioning

confidence: 95%

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

Mercille¹,

Massie²

1999

Biotechnol. Bioeng.

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“…Perfusion processes can eliminate the nutrient‐limitation and inhibitor‐accumulation problems inherent to animal cell‐suspension cultures. High viable‐cell densities and product yields have been observed using perfusion for the production of monoclonal antibodies (MAbs) (Banik and Heath, 1994; de la Broise et al, 1991; Flickinger et al, 1990; Hiller et al, 1993; Mercille et al, 1994; Mohan et al, 1993; Munster et al, 1991; Smith et al, 1991), and perfusion offers the added advantage of rapid removal from cultures of easily inactivated products (Prior et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Use of the Centritech Lab Centrifuge for Perfusion Culture of Hybridoma Cells in Protein‐Free Medium

Johnson¹,

Lanthier²,

Massie³

et al. 1996

Biotechnology Progress

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As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell-separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium. Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor. The centrifuge efficiently separated viable cells from dead ones. Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80%. The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed. This understanding of the separation process inside the centrifuge is important and may apply to other similar devices. Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion. However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case. Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge. This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L-1, equal to the performance of filter-based perfusion systems with the same cell line. It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 x 10(6) cells mL-1) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations.

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An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

Cited by 17 publications

References 36 publications

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

Use of the Centritech Lab Centrifuge for Perfusion Culture of Hybridoma Cells in Protein‐Free Medium

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