Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios

Analysis of the four bicistronic vectors for mAb and mRNA expression in established stable cell lines.

<p>Stably transfected pools were generated by transfection of CHO-S cells with various bicistronic vectors containing either IRES or F2A sequences and different promoters (CHEF or CMV). Levels of mAb and mRNA expression were measured by ELISA and qRT-PCR respectively. Black bars represent the mAb titer and gray bars represent mRNA fold induction. The error bars represent the standard deviation of three independent measurements.</p

Analysis of the stability of antibody expression over time by stable cell pools transfected with two vectors containing F2A sequences.

<p>Both stable cells were cultivated for 18 weeks upon removal of puromycin as a selection marker. Every 2 weeks, antibody expression was monitored and measured by ELISA. The other pools exhibited the same expression pattern; the data from one of them was represented.</p

mAb expression assessment in the clonal cell lines.

<p>Dual promoter cell pools underwent clonal selection using limiting dilution method. <b>A</b>, In1/2.5-pBLPCH cells, 120 clones, in 1/5-pBLPCH cells, 130 clones, and in N-pBLPCH cells, 125 clones were recovered. Transposition based cells showed more productivity in comparison to conventional ones. <b>B</b>, More than 60% of clones in 1/2.5-pBLPCH cells had more than 1000μg/L expression while a similar percent of N-pBLPCH derivative cells had less than 100μg/L expression level. Around 21% of clones derived from 1/2.5-pBLPCH parental cells produced mAb more than 2500μg/L whereas two other groups had a comparable number of clones in this area and more than 10% of their clones expressed mAb in this level. <b>C</b>, Four clones of each pool were assessed for specific productivity and growth rates. As it is demonstrated, specific productivities of conventional clones were the lowest and transposition based clones were more productive. Clones growth rate were comparable in all evaluated clones. Error bars indicated SD of triplicate measurements.</p

mRNA levels in established pools were assessed by quantitative real-time PCR.

<p><b>A</b>, In dual promoter bearing cells both light and heavy chains, are expressed via an independent expression cassette. Transposition effect was assessed at the level of transcription of each chain and as it is demonstrated in the picture both chains expression rate increase in a similar way. HC to LC ratio analysis showed that HC in all dual promoter cells was produced in a greater amount. <b>B</b>, Light chain mRNA levels in single ORF mAb expressing cells were evaluated by real-time PCR using N-pBLPCH-LC mRNA as the control gene. Error bars indicate SD of triplicate measurements. ANOVA was used to detect statistically significant differences between the generated pools (P< 0.05).</p