From cancer-beating Rituximab to arthritis-relieving Humira, antibody drugs today have become wildly successful in treating a staggering variety of ailments. Unfortunately, these blockbuster therapeutics can also be prohibitively expensive. Currently, antibody drugs are commonly mass-produced in Chinese hamster ovary (CHO) cells because the latter grow rapidly under lab conditions. However, the media used to culture them is expensive, driving up the cost of production for drug manufacturers.
To improve the efficiency of antibody production in CHO cells, a team from A*STAR’s Bioprocessing Technology Institute (BTI) worked with researchers from Merck & Co. to identify specific CHO cell genes that would improve antibody production when silenced. As current small interfering RNA (siRNA) screens are not specific to CHO cells, the researchers created a CHO-specific siRNA library by narrowing down siRNAs homologous to CHO sequences from a Merck proprietary library.
Screening more than 2,900 CHO-specific siRNAs that each could silence a particular gene, the researchers identified four gene targets that affected antibody production across multiple recombinant CHO cell lines. The team then used CRISPR-Cas9 technology to permanently remove those genes and validate their impact on antibody production.
“siRNA screening can give you hints as to which genes to target. But you still have to knock the gene out to confirm its impact. If you want to proceed to large-scale production, you cannot possibly keep using siRNAs in your solution—you have to do a permanent gene knockout,” Zhiwei Song, Senior Principal Scientist at A*STAR’s BTI and the corresponding author of the study, explained.
The genes identified in the siRNA screen were then knocked out with CRISPR-Cas9 in an antibody-producing CHO cell line. Three of the four gene target knockouts showed promising results. Most notably, the Cyp1a2 gene target showed the highest increase in antibody titer after knockout, at 1.7-fold from the baseline, with the least impact on cell growth.
Researchers can explore larger siRNA libraries if they want to look for more gene targets, said Song. “There are 24,000 genes in the CHO cell line, we only used 3,000. If you use a larger siRNA library, you’ll probably identify more,” he said.
While Song noted that combinations of knockouts did not further improve productivity, biotherapeutic manufacturers can look into removing the identified Cyp1a2 to potentially increase antibody production efficiency. “Pao Chun Lin, the first author of the paper, made a significant contribution to this work, as did our collaborators at Merck,” he added.
The A*STAR-affiliated researchers contributing to this research are from the Bioprocessing Technology Institute (BTI).