Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Abstract

Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547–559, 1997.

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…Whereas several research groups reported that the specific growth rate was inversely proportional to the level of recombinant protein expression (Gu et al, 1996;Jiang et al, 2006;Page and Sydenham, 1991;Pendse et al, 1992), others observed no significant influence of recombinant protein overexpression on the specific growth rate of recombinant CHO cells (Fann et al, 2000;Schröder and Friedl, 1997), or, in line with our observations, that recombinant CHO subclones were quite heterogeneous in regards to m and that there was no clear correlation between Figure 3. Specific productivities q mAb [pg cell À1 day À1 (pcd)] of high-, low-, and medium-producer CHO-mAb subclones isolated from (A) the 250 nM MTX amplified cell pool 2.11, and (B) the 500 nM MTX amplified cell pool 2.11. m and q mAb (Kim et al, 1998a).…”

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

“…The ATIII secreting cell lines CHO-A11-A2, CHO-A11-A27, CHO-A11-A279, and CHO-A11-A279-C7 were derived from CHO DUKXB11 cells and secrete increasing amounts of ATIII (Schröder and Friedl, 1997a;Zettlmeissl et al, 1987). The cell line CHO-SS3-A2 (Tebbe et al, 1995) was a kind gift of Prof. J. Lehmann (University of Bielefeld, Bielefeld, Germany).…”

“…A 6 x stock solution of the casein peptone soybean flour peptone broth was filter sterilized and than added to the final medium. Human fibronectin was isolated from human blood plasma (German Red Cross, Frankfurt/Main, Germany) as described before (Schröder and Friedl, 1997a). Animal-source derived components used in the serum-free formulation were fetuin from fetal calf serum (no.…”

“…The anchorage-dependent cell lines CHO DUKXB11, CHO-A11-A2, CHO-A11-A27, CHO-A11-A279, and CHO-A11-A279-C7 were cultivated as described previously (Schröder and Friedl, 1997a). The suspension cell line CHO-SS3-A2 was cultivated in 250 ml spinner flasks at 37°C, 10% CO 2 and stirring at Schröder,Matischak,and Friedl 5 30 rpm.…”

“…Colony forming efficiencies at seeding densities of 23 or 230 cells/cm 2 were determined as described before (Schröder and Friedl, 1997b). Conditioned media for clonal growth assays were collected 3 h after seeding cells at 2·10 4 cells/cm 2 into 75 cm 2 T-flasks as described by Schröder and Friedl (1997a).…”