Evaluating the expression profile and stability of different UCOE containing vector combinations in mAb-producing CHO cells

Abstract

Background: As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations.

Results: Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools.

Conclusions: In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.

Keywords: Cell line development; Chinese hamster ovary (CHO); Monoclonal antibody (mAb); Ubiquitous chromatin opening elements (UCOE).

Fig. 1

The schematic structure of plasmid vectors constructed and used in the studies. a Heavy chain coding plasmid vectors; pTracer-CMV2-HC (pH) and pTracer-CMV2-UCOE-HC (pUH). b Light chain coding plasmid vectors; pIRES2-DsRed2-LC (pL) and pIRES2-DsRed2-UCOE-LC (pUL)

Fig. 2

Antibody concentrations of different stable cell pools. a Antibody expression levels from generated pools were measured by ELISA. The error bars represent standard deviation of triplicate ELISA measurements. Data was statistically analyzed using ANOVA to detect significant differences between the generated cell pools (p < 0.05). b Western blot analysis of reduced supernatants from four pools using HRP conjugated goat anti-human IgG. The appearance of bands with the expected size of 50 KD for heavy chain and 25 KD for light chain verified the presence of antibody. Negative control (untransfected cell culture medium) (lane 1), Positive control (human IgG) (lane 2), protein molecular weight marker (lane 3), sample supernatants of CHO-HL (lane 4), CHO-HUL (lane 5), CHO-UHUL (lane 6) and CHO-UHL (lane 7)

Fig. 3

Antibody mRNA levels and gene copy numbers of the stable cell pools. a Relative HC, LC mRNA levels were determined by qRT-PCR and compared with antibody expression levels. b Relative HC, LC gene copy numbers were measured by qRT-PCR. The error bars represent standard deviation of three independent qRT-PCR assays. Data was statistically analyzed using ANOVA to detect significant differences between the generated cell pools (p < 0.05)

Fig. 4

Antibody expression levels of clonal cell lines from CHO-HL a CHO-HUL (b), CHO-UHUL (c), CHO-UHL (d) pools are shown and ranked from highest to lowest. Data was statistically analyzed using ANOVA to detect significant differences between the generated cell pools (p < 0.05)

Fig. 5

Comparison of specific antibody productivity (qmAb) [pg/cell/day] of three isolated clones from each cell pools. The error bars represent standard deviation of triplicate measurements. Data was statistically analyzed using ANOVA to detect significant differences between the generated cell pools (p < 0.05)

Fig. 6

Long-term antibody expression levels of the stable cell pools. The error bars represent standard deviation of three experiment replicates Data was statistically analyzed using ANOVA to detect significant differences between the generated cell pools (p < 0.05)