Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells

Abstract

To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10-9-27) were cultivated at 30, 33, and 37 degrees C. At a culture temperature lower than 37 degrees C cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37 degrees C to 33 degrees C, more than a 2.5-fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33 degrees C was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced q(EPO). The q(EPO) at 33 degrees C was 0.35 +/- 0.08 microg/10(6) cells/h, which was approximately 4-fold higher than that at 37 degrees C. Although the highest q(EPO) of 0.49 +/- 0.14 micro/10(6) cells/h was obtained at 30 degrees C, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on q(EPO). Like q(EPO), the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced q(EPO) at low culture temperature. The quality of EPO produced at 33 degrees C in regard to isoform pattern, sialic acid content, and in vivo biological activity was comparable to or even better than that produced at 37 degrees C. Taken together, the results obtained demonstrate the potential of the application of low culture temperature to the commercial EPO production in rCHO cells.

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

“…negatively growth-associated) in the CHO1-15 500 cell line. This finding is in agreement with several investigators who have correlated reduced specific growth rate to increased recombinant protein productivity for CHO cell lines utilizing the DHFR amplification system (Fox et al 2005;Fussenegger et al 1997;Hayduk and Lee 2005;Hendrick et al 2001;Kaufmann et al 1999;Yoon et al 2003Yoon et al , 2005. Although not conclusive, this negative growth-association suggests that the protein may be preferentially or exclusively produced during the G1 cell cycle phase.…”

Proliferation control strategies to improve productivity and survival during CHO based production culture

“…A shift in the proportion of cells from the S to the G1 phase of the cell cycle has been observed at reduced temperatures which results in a state close to growth arrest and has been shown to improve productivity (Moore et al 1997;Kaufamann et al 1999;Hendrick et al 2001;Yoon et al 2003a, b;Fogolin et al 2004;Trummer et al 2006). Specific examples include the finding that 80% of secreted alkaline phosphatase (SEAP) producing and 76% of erythropoietin (EPO) producing CHO line accumulate in G0/G1 phase of cell cycle following reduction of the culture temperature to 30°C with associated increases in productivity (Kaufmann et al 1999;Yoon et al 2003b). More examples are given in Table 1.…”

Enhanced Production of hCTLA4Ig by Adding Sodium Butyrate and Sodium Pyruvate

“…식물세포의 낮은 생산성을 향상 시키고 정확한 발현수준을 조절하기 위해서 당 고갈에 의하 여 발현되는 벼 유래의 α-amylase RAmy3D promoter와 같이 특정 조건에 의해 발현이 유도되는 inducible promoter에 대 한 연구가 진행되고 있다 [5]. 또한 고발현을 제공하는 숙주 세포나 발현 벡터를 개발하는 방법 [6], 배지를 최적화 하 는 방법 [5], 낮은 온도에서 배양하는 방법 [7] 등 세포주나 공정개발을 통한 생산성 증진 연구도 활발하다. 동물세포배 양을 이용한 재조합단백질 생산의 경우 small molecule enhancer (SME)를 첨가하여 단백질 생산량을 높이는 연구 도 다수 수행되어 생산 현장에 적용되고 있다 [8].…”