Catalan-Tatjer, D. | bioRxiv Science

bioRxiv2024

Evaluating apoptotic gene efficiency for CHO culture performance using targeted integration

Chinese hamster ovary (CHO) cells have long been the favoured platform for producing complex biopharmaceuticals such as monoclonal antibodies (mAbs). Cell death is a critical factor in all CHO cultures, dictating duration until harvest in batch cultures and viable cell density in perfusion. The programmed cell death, or apoptosis, pathway has been widely studied due to its relevance in affecting cell culture performance and the extensive knowledge about its protein-to-protein interaction network. However, clonal variation seen with random integration has confounded results and it remains unclear which effector genes should be overexpressed. Here, we employed the recombinase-mediated cassette exchange (RMCE) strategy to develop isogenic cell lines expressing one copy of erythropoietin, as model protein product, and various anti-apoptotic genes: bcl-2 from CHO and human origin, bcl-xL from CHO and human origin, mcl-1 and bhrf-1. We tested the generated isogenic cell lines in the presence of sodium butyrate, a well-known apoptotic initiator, in batch culture. The most promising candidates were cultured in fed-batch in the microbioreactor ambr(R)15 system. The observed phenotype varied significantly depending on the overexpressed gene, therefore the metabolic differences were further characterized using multiplexed quantitative proteomics. We showed that overexpressing bcl-2 from CHO origin significantly improved productivity and established a methodology to successfully test candidate genes via targeted integration. This will enable future metabolic engineering strategies to be more comparable and overcome the challenges faced thus far.

synthetic biology↗

bioRxiv2024

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

The production of recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is a highly intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids--pHelper, pRepCap, and pGOI-- play a distinct role and affect different cellular pathways when producing rAAVs. The expression balance of these different elements emphasizes the critical need to fine-tune the concentration of all three plasmids and transfection reagents effectively. The use of design of experiments (DOE) to find optimal plasmid and transfection reagent ratios is a powerful method to streamline the process. However, the choice of the DOE method and the design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: a rotatable central composite design (RCCD), a Box-Behnken design (BBD), a face-centered central composite design (FCCD), and a mixture design (MD). We compared the ability of the different models to predict optimal ratios, interactions among the three plasmids and transfection reagent, and the essentiality of studying the variability caused by uncontrolled random effects using blocking. Our findings revealed that MD, when coupled with FCCD, outperformed all other tested models. This outcome underscores the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.

bioengineering↗