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Rapid and efficient cell line development (CLD) process is essential to expedite therapeutic protein development. However, the performance of widely used glutamine‐based selection systems is limited by low selection efficiency, stringency, and the inability to select multiple genes. Therefore, an AND‐gate synthetic selection system is rationally designed using split intein‐mediated protein ligation of glutamine synthetase (GS) (SiMPl‐GS). Split sites of the GS are selected using a computational approach and validated with GS‐knockout Chinese hamster ovary cells for their potential to enable cell survival in a glutamine‐free medium. In CLD, SiMPl‐GS outperforms the wild‐type GS by selectively enriching high producers. Unlike wild‐type GS, SiMPl‐GS results in cell pools in which most cells produce high levels of therapeutic proteins. Harnessing orthogonal split intein pairs further enables the selection of four plasmids with a single selection, streamlining multispecific antibody‐producing CLD. Taken together, SiMPl‐GS is a simple yet effective means to expedite CLD for therapeutic protein production. [ABSTRACT FROM AUTHOR]

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes. [ABSTRACT FROM AUTHOR]

As the era of omics continues to expand with increasing ubiquity and success in both academia and industry, omics-based experiments are becoming commonplace in industrial biotechnology, including efforts to develop novel solutions in bioprocess optimization and cell line development. Omic technologies provide particularly valuable observational insights for discovery science, especially in academic research and industrial R&D; however, biomanufacturing requires a different paradigm to unlock actionable insights from omics. Here, we argue the value of omic experiments in biotechnology can be maximized with deliberate selection of omic approaches and forethought about analysis techniques. We describe important considerations when designing and implementing omic-based experiments and discuss how systems biology analysis strategies can enhance efforts to obtain actionable insights in mammalian-based biologics production.

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre‐transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG 1)-producing cells were generated, and the IgG 1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing. [ABSTRACT FROM AUTHOR]

Abstract Rapid and efficient cell line development (CLD) process is essential to expedite therapeutic protein development. However, the performance of widely used glutamine‐based selection systems is limited by low selection efficiency, stringency, and the inability to select multiple genes. Therefore, an AND‐gate synthetic selection system is rationally designed using split intein‐mediated protein ligation of glutamine synthetase (GS) (SiMPl‐GS). Split sites of the GS are selected using a computational approach and validated with GS‐knockout Chinese hamster ovary cells for their potential to enable cell survival in a glutamine‐free medium. In CLD, SiMPl‐GS outperforms the wild‐type GS by selectively enriching high producers. Unlike wild‐type GS, SiMPl‐GS results in cell pools in which most cells produce high levels of therapeutic proteins. Harnessing orthogonal split intein pairs further enables the selection of four plasmids with a single selection, streamlining multispecific antibody‐producing CLD. Taken together, SiMPl‐GS is a simple yet effective means to expedite CLD for therapeutic protein production.

Breast cancer, a formidable global health challenge, needs continuous translational research to understand the complexity of mechanisms and improve therapeutic and diagnostic strategies. Breast cancer cell lines are of paramount importance as they significantly contribute to the initial stage of research to understand cancer biology. This review provides insights into targeted therapies and immunotherapies that have emerged using in vitro models and microbiome analysis. It focuses on therapeutic development using cell lines and the limitations of tumor heterogeneity and microenvironment. We explore the evolving landscape of breast cancer cell lines from two‐dimensional (2‐D) cultures to patient‐derived xenograft (PDX) models advancing both fundamental and translational research. Patient‐derived xenografts, cell line‐derived xenografts (CDX), three‐dimensional (3‐D) cultures, organoids, and circulating tumor cells (CTC) models provide promising alternatives that capture the intricacies of the tumor microenvironment. This review bridges the gap between traditional cell lines and newer developments exploring the therapeutic and diagnostic advancements and needs for cell lines to expedite the progress in breast cancer research and treatment. [ABSTRACT FROM AUTHOR]

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines. [ABSTRACT FROM AUTHOR]

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever‐rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg‐based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state‐of‐the‐art technologies for cell culture‐based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single‐cell cloning strategy was established to generate monoclonal suspension Madin‐Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine‐relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell‐specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50‐fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high‐yield influenza virus production in chemically defined medium starting from a single cell. [ABSTRACT FROM AUTHOR]

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.

Molecular biological methods have emerged as inevitable tools to accompany the process of cell line development for the generation of stable and highly productive manufacturing cell lines in the biopharmaceutical industry. PCR-based methods are especially useful for screening and characterization of cell lines due to their low cost, scalability, precision and propensity for multidimensional read-outs. In this study, the diverse applications of droplet digital PCR (ddPCR) as a molecular biological tool for cell line development are demonstrated. Specifically, it is shown that ddPCR can be used to enable precise, sensitive and reproducible absolute quantification of genomically integrated transgene copies during cell line development and cell bank characterization. Additionally, an amplitude multiplexing approach is applied to simultaneously run multiple assays on different genetic targets in a single reaction and advance clonal screening by measuring gene expression profiles to predict the assembly and homogeneity of difficult-to-express (DTE) proteins. The implementation of ddPCR-based assays during cell line development allows for early screening at a transcriptional level, particularly for complex, multidomain proteins, where balanced polypeptide chain ratios are of primary importance. Moreover, it is demonstrated that ddPCR-based genomic characterization improves the robustness, efficiency and comparability of absolute transgene copy number quantification, an essential genetic parameter that must be demonstrated to regulatory authorities during clinical trial and market authorization application submissions to support genetic stability and consistency of the selected cell substrate. • Gene expression analysis by ddPCR can predict the assembly of bispecific antibodies. • Amplitude-multiplexing allows for a simultaneous quantification of several genes. • Precise and sensitive absolute quantification of transgene copy numbers. • High correlation between RNA sequencing, ddPCR and capillary gel electrophoresis. [ABSTRACT FROM AUTHOR]

Abstract Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever‐rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg‐based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state‐of‐the‐art technologies for cell culture‐based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single‐cell cloning strategy was established to generate monoclonal suspension Madin‐Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine‐relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell‐specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50‐fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high‐yield influenza virus production in chemically defined medium starting from a single cell.

ABSTRACTThe COVID-19 pandemic highlighted the urgent need for life-saving treatments, including vaccines, drugs, and therapeutic antibodies, delivered at unprecedented speed. During this period, recombinant antibody research and development cycle times were substantially shortened without compromising quality and safety, thanks to prior knowledge of Chemistry, Manufacturing and Controls (CMC) and integration of new acceleration concepts discussed below. Early product knowledge, selection of a parental cell line with appropriate characteristics, and the application of efficient approaches for generating manufacturing cell lines and manufacturing drug substance from non-clonal cells for preclinical and first-in-human studies are key elements for success. Prioritization of established manufacturing and analytical platforms, implementation of advanced analytical methods, consideration of new approaches for adventitious agent testing and viral clearance studies, and establishing stability claim with less real-time data are additional components that enable an accelerated successful gene to clinical-grade material development strategy.

Stable, highly productive mammalian cells are critical for manufacturing affordable and effective biological medicines. Establishing a rational design of optimal biotherapeutic expression systems requires understanding how cells support the high demand for efficient biologics production. To that end, we performed transcriptomics and high‐throughput imaging studies to identify putative genes and morphological features that underpin differences in antibody productivity among clones from a Chinese hamster ovary cell line. During log phase growth, we found that the expression of genes involved in biological processes related to cellular morphology varied significantly between clones with high specific productivity (qP > 35 pg/cell/day) and low specific productivity (qP < 20 pg/cell/day). At Day 10 of a fed‐batch production run, near peak viable cell density, differences in gene expression related to metabolism, epigenetic regulation, and proliferation became prominent. Furthermore, we identified a subset of genes whose expression predicted overall productivity, including glutathione synthetase (Gss) and lactate dehydrogenase A (LDHA). Finally, we demonstrated the feasibility of cell painting coupled with high‐throughput imaging to assess the morphological properties of intracellular organelles in relation to growth and productivity in fed‐batch production. Our efforts lay the groundwork for systematic elucidation of clone performance using a multiomics approach that can guide future process design strategies. [ABSTRACT FROM AUTHOR]

The majority of recombinant adeno‐associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale‐up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa‐based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3‐based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3‐4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity‐based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time‐efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines. [ABSTRACT FROM AUTHOR]

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all‐in‐one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site‐specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double‐cut and double‐nick donor formats significantly improved targeting accuracy by 2.3–8.3‐fold and 19–22‐fold, respectively, compared to standard circular donors. Notably, Cas9‐mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low‐fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR‐based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications. [ABSTRACT FROM AUTHOR]

The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ∼3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1–3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods. • Improved productivity and stability of product quality during cell line development using transposase. • Interrogation of integration sites and their effect on the genome in recombinant bioproduction cell lines. • Chromosome painting and transgene locus amplification (TLA) for genetic characterization of bioproduction cell lines. • Mechanisms and strategies to prevent transgenic silencing during cell line development. [ABSTRACT FROM AUTHOR]

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis. • Identification of transgene location is critical to assure monoclonality of CHO cells. • spPCR-based genome walking assay was developed and applied to recombinant CHO cells. • Python script-assisted selection of restriction enzyme can improve spPCR success rate. • spPCR saves required analysis time and cost substantially compared to current methods. • spPCR may aid cell line development by providing a simple and rapid analysis platform. [ABSTRACT FROM AUTHOR]

The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product. [ABSTRACT FROM AUTHOR]

For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high‐quality biologics with human‐like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long‐term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR‐Cas systems, the use of CRISPR‐Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast‐track product development process with consistent, improved product yield and quality. [ABSTRACT FROM AUTHOR]

Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MICL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD4 takes into account the available metadata and summaries all relevant statistics generated in steps 1–3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined. [ABSTRACT FROM AUTHOR]

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

With the aim of increasing protein productivity of Chinese hamster ovary (CHO) cells, we sought to generate new CHO hosts with favorable biomanufacturing phenotypes and improved functionality. Here, we present an innovative approach of enriching the CHO host cells with a high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing difficult-to-express bispecific molecules generated from the MMP-enriched host outperformed the parental host by displaying (1) improved fed-batch productivity; (2) enhanced long-term cell viability of pools; (3) more favorable lactate metabolism; and (4) improved cell cloning efficiency during monoclonal cell line generation. Proteomic analysis together with Western blot validation were used to investigate the underlying mechanisms by which high MMP influenced production performance. The MMP-enriched host exhibited multifaceted protection against mitochondrial dysfunction and endoplasmic reticulum stress. Our findings indicate that the MMP-enriched host achieved an overall “fitter” phenotype that contributes to the significant improvement in biomanufacturing capability.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come. [ABSTRACT FROM AUTHOR]

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters. [ABSTRACT FROM AUTHOR]

For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology. [ABSTRACT FROM AUTHOR]

The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months. • Chinese hamster ovary cells. • Cell line development. • Transposase. • Acceleration. • Monoclonal antibody. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production. [ABSTRACT FROM AUTHOR]

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Abstract Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process.

Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources., (© 2024 American Institute of Chemical Engineers.)

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single‐cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production. [ABSTRACT FROM AUTHOR]

Cell line development (CLD) represents a critical, yet time‐consuming, step in the biomanufacturing process as significant resources are devoted to the scale‐up and screening of several hundreds to thousands of single‐cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single‐cell cloning (SCC) using limiting dilution or fluorescence‐activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single‐cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process‐derived clones demonstrated equivalent long‐term stability compared with standard process‐derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability. [ABSTRACT FROM AUTHOR]

Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot. [ABSTRACT FROM AUTHOR]

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein‐based biologic drugs. Here, we describe the Leap‐In Transposase® platform, a novel transposon‐based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high‐titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap‐In provides several opportunities to de‐risk programs early and reduce timelines and resources. [ABSTRACT FROM AUTHOR]

The rapid growth of bispecific antibodies and antibody fusion molecules in biopharmaceuticals necessitates precise process development. The molecular design, the biophysical properties, such as protein hydrophobicity and charge-charge interactions, and the process development workflows are crucial to manage the critical quality attribute (CQA) of development candidates at the development and manufacturing stage. This study focuses on aggregation mitigation for an immunocytokine, HLX101, comprised of an engineered cytokine fused to the knob chain of a Knob-into-Hole IgG. Aggregation and other CQAs were monitored and controlled throughout cell line development (CLD), expression, and purification. Our study effectively identified HLX101 clone #10 as optimal for large-scale manufacturing by demonstrating stable expression and by balancing high yield with low aggregation. This was achieved by using purification process results to guide the selection of the final clone in the cell line development, integrating both processes to optimize overall outcomes in immunocytokine production. Thus, clone #10, with its favorable profile, emerged as more efficient, enhancing overall yield and quality. This underscores the importance of considering both upstream and downstream processes in immunocytokine production. • Develop a stable high-yield asymmetric IgG-like immunocytokine cell line via transposon system. • Highlight the balancing between clones' yield and quality. • Establish a purification workflow customized for asymmetric IgG-like immunocytokine. • Factor in product aggregate level at cell line development for best post-purification yield. [ABSTRACT FROM AUTHOR]

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 10 7  TU mL -1  in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 10 7  TU mL -1  in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 10 7  TU mL -1  day -1  for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34 + cells, reducing the LV quantities required for gene and cell therapy applications., (© 2024 CSL Innovation GmbH and The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.)

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen., (© 2024 Wiley‐VCH GmbH.)

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

• UCOE element was associated with increased yield of antibody. • UCOE pools had greater number of positive clones and more stable expression. • Clones that stably co-express mAb and its specific SINEUP had more mAb titer. • Stably expressed SINEUP clones had stable long-term expression. • Combined UCOE and SINEUP provided more optimized expression conditions in CHO cells. The growing need for monoclonal antibodies (mAbs) necessitates the development of novel and efficient production approaches. Regulatory elements like ubiquitous chromatin-opening elements (UCOEs) have been employed for improvement of the mAb expression in the Chinese hamster ovary (CHO) cells. SINEUPs are a class of long non-coding RNAs, which can improve the translation of partly overlapping mRNAs. A combination of these two elements might lead to higher production of mAbs. Therefore, the current study was conducted to investigate the effects of SINEUPs and A2UCOE on the expression of an IgG1 in the CHO-K1 cells. Hence, after constructing the mAb, mAb-SINEUP, and mAb-UCOE vectors, four stable cell pools were generated through combining the above vectors. According to the expression analysis, antibody yields were higher in the mAb-SINEUP and mAb-UCOE cell pools compared to the mAb cells. In addition, the cells possessing both SINEUP and UCOE elements provided the best expression. Persistent mAb expression was observed for over 2 months in these cells, whilst the expression was decreased in the mAb pool. SINEUP and UCOE positively influenced the stable mAb expression. It can be concluded that the SINEUP and UCOE enhance the antibody stability and expression level separately and their combination improves the mAb production in the CHO cells. [ABSTRACT FROM AUTHOR]