Combining high‐throughput screening of caspase activity with anti‐apoptosis genes for development of robust CHO production cell lines

Abstract

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

“…The diverse signals transmitted by each pathway converge upon the caspase proteases, which cleave protein targets and lead to cell death 28 . As a strategy to increase CHO cell longevity, caspase activation has been targeted with chemical inhibitors 30 and caspase-inhibiting proteins 24,[31][32][33] . We found that several cell lines contained extra copies of various caspases (Fig.…”

Progesterone Regulation of AVEN Protects Bovine Oocytes from Apoptosis During Meiotic Maturation1

“…AVEN is known to play a role in male and female germ cell development; downregulation of AVEN was associated with incomplete spermatogenesis in mice [18] and Vasa-homolog-deficient mice, which display abnormal spermatogenesis, fail to express AVEN [19], while over expression of human AVEN mRNA in Xenopus laevis oocytes delays P4 induced meiotic maturation [20]. AVEN protein has also been shown to inhibit the mitochondrial apoptosis pathway by binding to and inhibiting the self-association of pro-apoptotic APAF-1 [21,22], as well as binding to and enhancing anti-apoptotic BCL-x L activity [21,[23][24][25]. However, AVEN can induce apoptosis in cells with high levels of DNA damage, while promoting the recovery of cells with lower levels of DNA damage.…”

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].…”