Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α.

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

Comment on "Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor".

In the original paper, Li and co-workers [ACS Appl. Mater. Interfaces 2022, 14, 17128-17141] described their approach to select specific hybridoma cells from a polyclonal hybridoma pool by using a cell surface anchor to catch the secreted antibody. The antigen-specific detection was performed with streptavidin-labeled antigen and a PE-labeled anti-F(ab')2 antibody. The present comment offers a clearer description of the selection system originally published by Listek et al. in 2020 and provides further information about the importance of controls and recent adaptations made by our lab.

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line.

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.