Caractérisation du système inductible au cumate pour la production de protéines thérapeutiques en cellules CHO

Therapeutic proteins represent a class of therapeutics for the treatment of various diseases such as cancer or autoimmune diseases. Due to their ability to perform post-translational modifications, necessary to obtain biologically active proteins, most of these marketed proteins are produced in Chinese Hamster Ovary (CHO) cell lines. The development of large-scale highly productive CHO cell lines is a long (6 to 18 months) and very expensive and labor-intensive process. This starts with the transfection followed by the selection of cells having randomly integrated the gene coding for the protein of interest into their genome, resulting in a heterogeneous population (pool) in terms of production, product quality and growth. In recent years, the use of these pools has become a very popular approach for protein biochemical characterization and preclinical studies, requiring the rapid and cost-effective production of sufficient quantities of material. However, drug regulatory authorities require that cell lines be derived from a single progenitor cell (or clone). Obtaining a highly productive cell line requires a screening step to isolate a clone with both high  productivity and growth rate. This step is considered as the limiting step of the cell line development process because the very low frequency of high-producer clones requires the screening of thousands of cells. So far, no specific mechanism has explained the low occurrence of high-producer clones in selected pools. However, the combination of molecular and cellular mechanisms in response to stress due to overexpression of the gene of interest could be involved. In our laboratory, we have developed a CHOBRI/rcTA cell line containing the cumate gene switch inducible expression system. This system, unlike most of constitutive systems used in the industry, allows defining the time and the expression level of the protein of interest. Indeed, by the addition of the cumate inducer, the chimeric rcTA transactivator binds to the strong CR5 promoter and triggers transcription. In this research project, we hypothesised that the use of an inducible expression system could limit the potential stress induced by the overexpression of the recombinant protein during the selection process. The frequency of high-producer clones in the selected population would then be increased, improving the population productivity and facilitating the screening step. We first developed and optimized an efficient platform for the rapid production of recombinant proteins via the generation of pools, by combining plasmid expression vectors and the CHOBRI/rcTA cell line. We thus obtained populations which produced about 0.9 g/L of the fusion protein hCD200Fc and about 0.35 g/L of the antibody rituximab. For each population, the production stability after a one-month culture and after a freeze-thaw cycle was also demonstrated. Furthermore, for both proteins, the final product quality analysis showed no major differences between proteins produced either from a stable clone or from a stable pool. Finally, we showed the superiority of the CR5 inducible promoter compared to two constitutive promoters commonly used in the industry. We then studied the impact of recombinant protein expression on the productivity and clonal distribution of the selected pool. For this, two stable pools resulting from the same transfection and producing hCD200Fc were generated. For one of them, protein expression was triggered during the selection process by addition of cumate. Subsequently, for each pool, fed-batch production was compared and clonal distribution via micro-population screening was studied. We have thus demonstrated that a decrease in recombinant protein expression during the selection process leads to a significant increase in pool productivity, due to an enrichment of cells with increased specific productivity. Finally, during selection process, we observed an increase in cell mortality together with an increase in the expression of an endoplasmic reticulum stress marker in the population expressing high levels of hCD200Fc. These results suggest that during the selection process, overexpression of the recombinant protein results in activation of the endoplasmic reticulum stress response pathways by the cell, which could then limit its growth or induce apoptosis. This project contributed to the development of an efficient platform for the rapid production of recombinant proteins via the generation of stable pools. In addition, the cumate inducible system characterization in this context has demonstrated the real advantage of using an inducible system to reduce the development time and costs of stable cell lines with high production potential.

Keyphrases: Expression stable, Cumate-inducible expression system, CHO cell, Recombinant protein production, Stable expression