The “Push-to-Low” Approach for Optimization of High-Density Perfusion Cultures of Animal Cells

Konstantinov, Konstantin; Goudar, Chetan T.; Ng, Maria; Meneses, Renato; Thrift, John; Chuppa, Sandy; Matanguihan, Cary; Michaels, James; Naveh, D.

doi:10.1007/10_016

Cited by 79 publications

(92 citation statements)

References 16 publications

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“…The process space depends on several factors, which dictates the boundaries inside which this production optimization is taking place. These factors are (quoted in part from Konstantinov et al (2006)):…”

Section: Optimization Of the Process Parametersmentioning

confidence: 98%

“…Reducing the medium usage without compromising the process performance can be desirable by optimizing the medium composition and using concentrated media (Runstadler 1992;Hiller et al 1994;Ozturk 1996;Konstantinov et al 2006;Yang et al 2014).…”

Section: Medium Selectionmentioning

confidence: 99%

“…The cell specific perfusion rate is an established strategy where the perfusion rate is a linear function of the cell density (Ozturk 1996;Konstantinov et al 2006):…”

Section: Perfusion Rate Strategy Based On Csprmentioning

confidence: 99%

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Perfusion Processes

Chotteau

2014

Cell Engineering

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The interest for perfusion is increasing nowadays. This new focus has emerged from a synergy of a demand for disposable equipment and the availability of robust cell separation device, as well as the need for higher flexibility and lower investment cost. The cell separation devices mostly used today are based on filtration, i.e. alternating flow filtration, tangential flow filtration, spin-filter, or acceleration/gravity, i.e. inclined settler, centrifuge, acoustic settler. This paper gives an introduction to the basic concepts of perfusion and its practical implementation. It reviews the actual cell separation devices and describes the approaches used in the field to develop and optimize the perfusion processes.

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Section: Optimization Of the Process Parametersmentioning

confidence: 98%

Section: Medium Selectionmentioning

confidence: 99%

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Perfusion Processes

Chotteau

2014

Cell Engineering

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“…The system consists of a bioreactor for cell culture and an external inclined settler for cell retention ( Fig. 1; Batt et al, 1990;Konstantinov et al, 2006;Searles et al, 1994). Cell-depleted harvest was continuously removed from the bioreactor through the settler and fresh media was continuously supplied to the bioreactor to maintain the culture volume.…”

Section: Bioreactor Evaluationmentioning

confidence: 99%

Anti‐apoptotic genes Aven and E1B‐19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture

Nivitchanyong

Martinez

Ishaque

et al. 2007

Biotech & Bioengineering

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The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.

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“…On the other hand, perfusion has gained much interest, once again 36 . The productivity of newly developed perfusion processes is much greater than what those developed in the 80's achieved, and more importantly, volume exchanges are low (1-2 reactor volumes per day), which potentially drives cost of goods down 35,37,38 . Furthermore, the continuous mode can be extended to the purification platform, thus reducing the total processing time and the risks associated with degradation pathways 32 .…”

Section: Cell Culture Process Optimizationmentioning

confidence: 99%

Tailoring recombinant protein quality by rational media design

Brühlmann

Jordan

Hemberger³

et al. 2015

Biotechnology Progress

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Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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The “Push-to-Low” Approach for Optimization of High-Density Perfusion Cultures of Animal Cells

Cited by 79 publications

References 16 publications

Perfusion Processes

Perfusion Processes

Anti‐apoptotic genes Aven and E1B‐19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture

Tailoring recombinant protein quality by rational media design

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