Viral vectors such as AAVs or VLPs are still produced via transient gene expression (TGE) due to the toxicity of certain elements, which makes constitutive expression difficult for host cells. However, scaling up TGE-based bioprocess is limited by the cell density effect (CDE), which reduces cell-specific productivity as cell concentration increases. Changes in the physiological state of cells at high density are believed to be one of the explanations for CDE. Previous research has revealed significant alterations in molecular components and metabolic pathways following transfection. Among these, the downregulation of glycosphingolipid biosynthesis has been identified as a key factor impacting cell viability and homeostasis during TGE. The overexpression of UDP-glucose ceramide glucosyltransferase (UGCG), a key enzyme in glycosphingolipid biosynthesis, has demonstrated improvements in both transfection efficiency and VLP production. However, since UGCG was only transiently expressed in these studies, its full impact was limited by CDE constraints. In this work, we evaluated the effect of constitutive and inducible UGCG overexpression in HEK293SF-3F6 cell lines using a targeted integration platform to assess its influence on transfection efficiency and VLP production. Our findings indicate that constitutive UGCG overexpression leads to significant metabolic and signaling pathway reprogramming, resulting in reduced transfection efficiency and lower VLP yields at low cell densities. Proteomic analysis identified key pathways responsible for these metabolic alterations. Conversely, inducible UGCG overexpression demonstrated improved transfection efficiency and enhanced VLP production at high cell densities upon induction. These insights highlight the importance of controlled UGCG expression to optimize recombinant VLP production strategies at high cell densities.

Cell line development is a critical step in the production of biologics and plays a decisive role in determining the commercial viability of the final process. The method of transgene integration, in particular, significantly influences the efficiency of generating highly productive clones, while also impacting screening efforts and timelines. In recent years, several biopharmaceutical companies have transitioned from randomly integrating their gene of interest into host cells to using transposase-mediated transgene integration. Transposases, which are typically co-transfected as DNA or mRNA along with the transgene flanked by the requisite transposon recognition sites, operate via a cut-and-paste mechanism, efficiently integrating the entire transgene expression cassette into the host cell genome at multiple locations. As most transposases were silenced by their originating host during evolution, it is difficult to identify a highly active variant suitable for the generation of industrially relevant cell lines. In this study, we computationally mined and screened for previously unknown transposases and successfully identified two novel variants with a surprisingly high baseline activity. After highlighting the capability of these novel transposases by benchmarking them against internal and external controls, we applied computationally guided protein engineering to generate hyperactive variants. Additionally, we combined improvements on protein level with multi-objective codon optimization (MOCO) to synergistically enhance the efficiency for transgene integration of our novel transposase. In conclusion, this study highlights the deployment of protein and RNA optimization for maximum transposase efficiency leading to highly productive, recombinant protein expressing CHO cells.

Commercial production cell lines must express biopharmaceutical proteins at high yields with suitable product quality in minimal time. To address the bottleneck of cell growth in cell line development and biopharmaceutical manufacturing, we employed an optimized lentiviral whole-genome knockout (KO) screen targeting 17,761 expressed genes with six gRNAs each to investigate the genetic mechanisms driving a fast-growth phenotype in CHO cells. This screen integrates refined multi-dimensional hit prioritization from multiple sampling time points and a high-throughput system for functional validation of growth-enhancing genes. The library was cultivated under shake flask conditions and in a 30-day, 3 L perfusion bioprocess, representing typical upstream biopharmaceutical processes. The screen achieved 70–80% single-copy integrations and >5,000x library coverage, significantly improving data quality compared to typical lentiviral genome-wide KO screens. We identified 198 growth-enhancing genes clustering into 69 functional gene sets with common pathways and functions. We use the established high-throughput validation system combining arrayed CRISPR KO libraries from the 198 gene hits with robot-assisted cultivation, measuring growth rates and viability. These findings provide insights into the genetic basis of fast-growth phenotypes, enabling targeted engineering of CHO cell lines to enhance efficiency in biopharmaceutical manufacturing. With this study, we established a comprehensive framework for genetic screening in CHO cells, addressing growth bottlenecks and enabling the rapid development of robust, high-yield production platforms for biopharmaceuticals.

Chinese hamster ovary (CHO) cells are the preferred host for biopharmaceutical production. However, a major limitation is that they undergo apoptosis (programmed cell death) triggered by various stress factors restrigting culture lifespan and final titre. Though genetic engineering has been used to curb apoptosis, further improvements are needed. In this study, two novel anti-apoptotic genes (AAg) with broad protective functions were identified and overexpressed independently in an IgG-producing CHO cell line, as well as the reference anti-apoptotic gene bcl (BCL2) for comparison. Each modified line was compared to the parental IgG CHO cell line (control). After 48 h apoptosis-induction with camptothecin, the AAg2 cell line had 57% and 75% less cells in early and late apoptosis stages respectively, than the control. In batch cultures, both novel AAg cell lines achieved a peak viable cell density (VCD) similar to the control, while the Bcl-2 cell line reached 40% lower VCD. No extension in culture duration was observed in the AAg cell lines. However, the AAg2 cell line showed 31% higher IgG production and a 65% increase in cell-specific productivity compared to the control. In fed-batch culture, with sodium butyrate treatment, the AAg2 cell line extended culture duration by at least four days and increased IgG titer by 68% (Fig. 1). These findings demonstrate that the AAg2-modified line offers enhanced resistance to apoptosis and highlights their potential to enhance CHO cell viability and productivity.

Chinese hamster ovary (CHO) cell lines are widely used in biopharmaceutical production and are continuously optimized for efficiency. Under bioreactor conditions, many endogenous cellular functions become redundant, imposing unnecessary transcriptional and translational burden to the cells. To address this, genome reduction via precise genome editing offers a powerful solution by removing non-essential DNA and reducing host cell protein levels to simplify downstream purification. Previously, large-scale genomic deletions of up to 1 megabase pair (Mbp) were achieved, laying the foundation for a minimal CHO genome. This study builds on this achievement by investigating the molecular mechanisms driving large-scale deletions in CHO cells. To facilitate the generation of large-scale genomic deletions, various transfection methods of CRISPR/Cas9/sgRNA ribonucleoprotein (RNP) complexes were tested. Using the most effective method, large-scale model deletions of several Mbps in size were generated, and the cellular repair dynamics under physiological conditions were monitored over time via a deletion-specific quantitative PCR (qPCR) assay. The results indicated a very fast onset of large scale deletion repair mechanisms and that time-dependent formation of deletions is influenced by their size. To elucidate the repair mechanisms enabling these large-scale genome deletions, specific DNA double-strand break (DSB) repair pathways were selectively modulated using small molecule inhibitors. Establishing non-toxic modulator concentrations for CHO cells allowed for the application of a qPCR-based deletion assay to monitor early double-strand break repair events, revealing the involved key molecular mechanisms. Furthermore, the combined use of small molecules targeting several different DNA repair pathways led to a significant increase in deletion efficiency at later time points following RNP delivery, facilitating an efficient clone selection. These findings provide insights into the currently unexplored repair mechanisms and dynamics of large-scale genomic deletions. In addition, the finding that small molecule treatments improve CRISPR/Cas9-mediated deletion efficiency may has the potential to advance CHO cell engineering for biopharmaceutical applications in the future.

Efficient recombinant protein production often depends on stable cell lines, especially for multisubunit complexes like virus-like particles (VLPs) and adeno-associated viruses (AAVs) used in vaccines and gene therapy. Nevertheless, generating stable cell lines is time-consuming and challenging, especially for products requiring multiple and large transgenes. Current biopharmaceutical production processes are based on stably transfected Chinese hamster ovary (CHO) cells, however for various products, human-like post-translational modifications are required. Thus, there is a need for a versatile, cell type-independent platform for fast stable cell line development. Our system addresses this need by using baculoviral transduction of mammalian cells (BacMam), which is cost-effective, scalable, and efficient. BacMam has several key advantages: (i) it doesn’t require high-biosafety laboratories, (ii) it efficiently transduces various cell types, and (iii) it is suitable to deliver large DNA fragments into the cellular nuclei. Hence, we developed the REMBAC platform (Rapid Efficient Manifold Baculovirus Transduction), enabling site-specific genome integration of large transgenes with customizable expression levels based on BacMam. Our expression cassettes have been optimized by including several beneficial elements such as insulators to protect against host-cell silencing. Our system ensures efficient gene delivery across different cell types and is designed to integrate the transgenes without leaving viral footprints. By combining BacMam’s versatility with homologous recombination for site-specific integration, and using a homing endonuclease for precise transgene excision, REMBAC allows the co-expression of multiple transgenes at controlled levels. Thus, REMBAC facilitates stable cell line development for a wide range of biopharmaceutical applications, including biologics like monoclonal antibodies or bionanoparticles such as VLP vaccines or AAV gene therapy vectors.