Introducing therapeutic protein genes into mammalian cells can be done in several ways. Transposon systems consisting of a transposon encoding the genetic cargo and a transposase enzyme releasing the cargo from the transposon and inserting it into the genome have become a leading method for developing stable cell lines, effectively addressing issues with older random integration techniques. We used orthologous transposase/transposon pairs to insert genes for increased protein production while reducing the activity of other genes. This allowed us to change cell behavior and improve the quality of the produced proteins. We sequentially introduced three orthogonal transposons into CHO cells using three corresponding transposases, creating a new CHO-K1 cell line. We then used this line to generate high-producing cell clones with desirable protein characteristics. The first transposon introduced a selection marker. The second inserted a gene for an IgG therapeutic antibody, resulting in stable, high-producing clones yielding over 5 g/L. The third transposon limited the formation of glycans on the IgG, significantly reducing fucose levels to below 10%. Throughout this process, we used advanced genetic analysis (TLA and NGS) to confirm accurate transposon integration, enzyme specificity, and the inserted genes' stability over time. Glycan modification did not affect cell growth or protein production, and we observed no loss or rearrangement of the transposons. The produced IgG was functional and showed improved immune cell-killing activity in lab tests. The protein production and quality of the final engineered clone remained consistent for more than 60 cell generations.

Metabolic selection systems, glutamine synthetase (GS) and dihydrofolate reductase (DHFR), have played a key role in establishing CHO cell processes capable of routinely generating high monoclonal antibody yields. In this study, we introduce nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in cellular NAD+ biosynthesis, as a novel metabolic selection marker for CHO cells and compare it with industry standard GS selection. NAMPT and GS were used to drive the expression of green fluorescence protein (GFP) and a trastuzumab biosimilar in NAMPT-knockout and GS-knockout host cells, respectively. Compared to the GS system, NAMPT selection demonstrated stronger selection stringency for GFP. Gene amplification, using small molecule inhibitors further enhanced the expression in both systems. In the production of trastuzumab, an industrially relevant recombinant protein, NAMPT-selected pools achieved comparable yields to those generated using the GS system. These data demonstrate the significant potential of NAMPT as a selection marker, offering a promising alternative to the industry-standard GS selection system for generating CHO cell populations for biopharmaceutical manufacturing.

Generating hetero-oligomers from a mixture of recombinantly expressed and purified proteins has always been arduous. It becomes even more challenging for large multimeric proteins. Using a series of ingenious modifications to existing techniques and tools, we have artificially generated variants of a trimeric proapoptotic serine protease, HtrA2, that is associated with several diseases, including neurodegeneration and cancer. With a sequential chemical denaturation/renaturation strategy followed by the introduction of variable and additional tags at the N- and C-termini of the macromolecules, we successfully purified these proteins using a modified affinity chromatography technique. This protocol generated hetero-oligomeric HtrA2 variants in a bacterial system by engineering and reorganizing the protomers obtained from the mixture of purified homo-oligomers differing in the number of a C-terminal protein-protein interaction domain - PDZ or active-site mutations (replacing the active serine with alanine). This effort was taken to explicate the contributions of each monomer and/or their domains in modulating the activity of the large multimeric HtrA2 ensemble. This will not only facilitate obtaining a homogenous population of difficult-to-purify hetero-oligomers with nominal differences in their physico-chemical properties from a set of recombinant proteins, but also help understand the contribution of each protomer through further biochemical and/or biophysical characterizations. This study, therefore, paves the way toward understanding the structural and functional intricacies of various proteins with biomedical and biotechnological importance.

Efficient expression of complex biopharmaceutical antibodies requires robust, high-titre cell lines and rapid manufacturing processes. Many conventional cell line development workflows involve generating mini-pools to enhance productivity and maintain stable expression over extended periods. Here, we present a novel synthetic production-phase promoter that enables high-level expression of complex and difficult-to-express molecules in bulk-pools, while significantly reducing recovery times. This promoter enhances the scalability of cell line development, improves consistency across production batches, and accelerates timelines, offering a promising strategy to meet the demands of rapid and cost-effective biopharmaceutical production.