Abstract
Chinese hamster ovary cells (CHO) and dihydrofolate reductase (dhfr)/methotrexate (MTX) gene amplification system are commonly used to generate stable high-producer CHO cell clones in biopharmaceutical industries, such as antibodies, at the present time. Most biopharmaceutical antibody-based products display favorable safety profiles such as chimeric or humanized antibodies. Simulation of bio-pharmaceutical industrial process for antibody production, the expression system using CHO/dhfr- cell as a host cell to express anti-Japanese encephalitis virus (JEV) neutralizing chimeric antibody IgG1-2H2 and IgG1-E3.3. The silencing shRNA vector psd2, which was previously demonstrated the most effective in silencing dhfr RNA transcripts, improved antibody production in dhfr-deficient CHO (CHO/dhfr-¬) cells through dhfr/MTX gene amplification. Initially, a chimeric mouse-human antibody expression vector was used to express JEV neutralizing chimeric antibodies, IgG1-2H2 and IgG1-E3.3. Then, in addition to psd2, other silencing vectors that were mir-30 based sd2 silencing vector (pmir-30 sd2) and nonspecific of dhfr gene silencing vector (pscramble) were adopted to improve IgG1-2H2 expression in CHO/dhfr- cells. Compare to the average expression level of IgG1-2H2 of CHO/dhfr stable clones, the clones with the silencing vectors pMir-30 sd2 and psd2 were about 100% higher than that with pscramble silencing vector and without silencing vector. Higher level of IgG expression and more stable productivity in MTX-free medium was achieved in amplified stable clones containing psd2 or pMir30-sd2. The method proposed here can be applied to screen high producer cell clones for recombinant antibody or other biologics expression in CHO/dhfr- cells with efficient stable transfection. It can be beneficial for the production of recombinant protein therapeutics in bio-industry.