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

Abstract

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.

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

“…One such undesirable metabolite is lactate. Quite interestingly, CHO cells made apoptosis resistant by over-expression of E1B-19K, Aven, and XIAP stop producing and actually begin consuming lactate (Dorai et al, 2009). Perhaps the increased lifespan of our BAX and BAK-deleted cells under starvation conditions is a result of a similarly altered metabolism.…”

Towards dynamic metabolic flux analysis in CHO cell cultures

“…For example, process parameters such as pH, temperature, CO 2 and osmolarity have been optimized [12][13][14], and complex feeding strategies have been devised using limited glucose and glutamine feeding, or feeding of other nutrients [15][16][17]. In addition, metabolism of CHO cells has been genetically engineered to enhance anaplerosis by overexpression of pyruvate carboxylase genes [18] and antiapoptotic genes such as Bcl-2 [19].…”

Proteomic Analysis of Bioreactor Cultures of an Antibody Expressing CHOGS Cell Line that Promotes High Productivity

“…In an attempt to increase overall productivity of these cell lines, a number of investigations have been undertaken in the past several years. These investigations, which follow closely those published in the literature, include 1) determining the optimal copy numbers of H-ch and L-ch of the therapeutic Ab in the transfectomas [17,40]; 2) utility of apoptotic resistant host cell lines [13,23,24]; 3) increased secretion of Abs via over-expression of chaperones [3] (unpublished observation); 4) prolongation of the G1 phase where protein synthesis is particularly more efficient [25] and 5) judicious use of expression systems and cell line selection strategies [2,41,42]. Recently, the "omics" approach for The effect of four different media components on multiple cell culture parameters.…”

“…A subset of cell lines that had increased Ab titers showed enhancement of H-ch and L-ch transcript levels, while another subset of cell lines showed minimal or no increase of these transcripts. Possible mechanisms by which the latter set of cell lines showed enhancement of Ab titers included: 1) increased stability of production cell lines [18][19][20], decreased apoptosis [21,22]; 2) increased secretion of Abs via chaperones [3] and 3) improved metabolic state of the cell line [23,24] that might lead to an increased proliferation of the cells and/or 4) prolongation of the G1 phase where protein synthesis is particularly more efficient [25].…”