Fed‐batch culture of hybridoma cells in serum‐free medium using an optimized feeding strategy

Zhang, Li; Shen, Hong; Zhang, Yuanxing

doi:10.1002/jctb.940

Cited by 33 publications

(26 citation statements)

References 26 publications

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“…Numerous strategies have been devised to address the accumulation of excessive lactate build-up including (1) maintaining low medium glucose concentrations (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995), (2) feeding alternative sugars, including fructose (Martinelle et al, 1998, Altamirano et al, 2004Wlaschin and Hu, 2007), (3) partially knocking out lactate dehydrogenase (LDH) expression by homologous recombination or siRNA technology (Chen et al, 2001;Kim and Lee, 2007a); (4) over-expression of pyruvate carboxylase (Kim and Lee, 2007b); (5) use of dichloracetate (DCA), a pyruvate dehydrogenase (PDH) activator (via PDH kinase inhibition) (Stacpoole et al, 2003) and (6) oxamic acid, an LDH competitive inhibitor (Mothersill and Seymour, 1986). While the above strategies have been partially successful in reducing the lactate concentration, an alternative approach to consider is stimulating mitochondrial respiration in order to enhance culture performance.…”

Section: Discussionmentioning

confidence: 99%

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Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Dorai

Kyung

Ellis

et al. 2009

Biotech & Bioengineering

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ABSTRACT:In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a ''high'' glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in ''high'' glucose medium reached 690 mg/ L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

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Section: Discussionmentioning

confidence: 99%

“…4). For this reason, a number of researchers have developed process methodologies such as restricting glucose feed in order to minimize lactate accumulation in mammalian cell cultures (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995).…”

Section: Comparison Of Metabolic Profiles Of Apoptotic R and Control mentioning

confidence: 99%

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Dorai

Kyung

Ellis

et al. 2009

Biotech & Bioengineering

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“…Many researchers have touted the importance of reducing glucose concentrations in order to help minimize lactate accumulation in mammalian cell cultures (Kurokawa et al, 1994;Ljunggren and Haeggstroem, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995). A distinguishing feature of the standardized FBP-2 process platform is the use of an initial glucose concentration of $15 g/L which, counter to conventional wisdom, results in significant lactate consumption and very low overall lactate/glucose yield coefficients.…”

Section: Lactate Consumption Is Process-dependentmentioning

confidence: 97%

Protein‐free fed‐batch culture of non‐GS NS0 cell lines for production of recombinant antibodies

Burky

Wesson

Young

et al. 2006

Biotech & Bioengineering

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Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.

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“…In order to avoid limited nutrient concentration or excessive metabolic by-product accumulation (such as ammonia and lactate), most attempts to enhance culture productivity have focused on development of feed formulations and feed strategies (Hu et al 2011;Meghrous et al 2009;Spens and Häggström, 2007;Zhang et al 2004). Fed-batch process has become the dominant production technology for therapeutic and recombinant proteins due to its ease of operation, flexibility to be implemented and compatibility with large scale manufacturing (Huang et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

et al. 2012

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A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett-Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL TM 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.

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Fed‐batch culture of hybridoma cells in serum‐free medium using an optimized feeding strategy

Cited by 33 publications

References 26 publications

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

Protein‐free fed‐batch culture of non‐GS NS0 cell lines for production of recombinant antibodies

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

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