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.

Chinese hamster ovary (CHO) cells are the predominant mammalian production host for complex therapeutic glycoproteins, yet they retain numerous functions that are superfluous in industrial bioprocesses. Given the intrinsic limitations in cellular resources such as ATP and ribosomal capacity, we hypothesized that targeted elimination of non-essential genomic regions could liberate these resources for bioproduction, ultimately enhancing cellular performance. Building on our previous demonstration of large-scale deletions up to 800 kilobase pairs, we expanded our approach to achieve megabase-scale deletions via a Cas9-mediated strategy. Non-essential genomic regions were identified by mapping all publicly available essential genes onto the CHO genome, while additional boundaries were defined by predicted essential metabolic genes derived from metabolic models tailored to growth and recombinant protein production data. Bioenergetic costs for replication, transcription, and translation of genes within these regions were calculated using cell-line-specific expression data, allowing us to prioritize targets with high cumulative costs. The deletion strategy involved an initial transfection with sgRNAs targeting selected boundaries, yielding monoallelic deletion clones, followed by a second transfection to obtain biallelic deletions in this diploid system. Tiling PCR and whole-genome nanopore sequencing confirmed precise excision, with observed reintegration events—ranging from complete reintegration to reassembled large fragments (up to multi million base pairs)—that did not result in detectable gene expression. Fed-batch cultivations of both the host and recombinant antibody-producing clones demonstrated that even megabase-scale deletions encompassing regions with highly expressed genes did not compromise key bioprocess parameters such as viable cell density, productivity, or cell viability compared to the parental cell line. These findings provide a promising blueprint for developing a streamlined, genome-reduced CHO cell chassis that reallocates cellular resources from energetically expensive, non-essential processes to enhanced bioproduction, setting the stage for the next generation of optimized CHO cell factories.

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.

Rapid and scalable production of biotherapeutics, including vaccine antigens, is critical for responding to public health emergencies. Typical workflows for developing production-ready clonal Chinese Hamster Ovary (CHO) cell lines are lengthy, requiring 8–10 months. To expedite early-stage clinical material generation, non-clonal CHO stable pools offer a promising alternative, reducing timeline from several months to a few weeks. However, the productivity of random-integration stable pools is lower than that of clones due to heterogeneous expression profiles, with some cells being high producers, some moderate and others non-producing. Identifying and fishing out high producers from a heterogenous pool would thus improve its overall productivity. Because assessing expression levels of cells in a stable pool is challenging as most of the recombinant protein product is secreted in the culture medium, we describe a surrogate surface marker-based approach to identify and enrich for high-producers from a heterogenous stable pool expressing a CD200-Fc fusion protein. We developed a bipartite vector system wherein the strength of the promoter driving the surface marker cassette can be modulated, such that its expression has minimal impact on target protein production. We tested two classes of surface markers- glycosylphosphatidylinositol (GPI)-anchored (GPI-mRFP) and transmembrane domain (TMD)-based (PD-L1, CD4) to compare their effectiveness in labeling and selectively enriching for high CD200-Fc-expressing cells. Furthermore, we characterized and compared this approach with an established methodology known as cold capture, wherein under cold conditions, the secreted product is transiently retained at the CHO cell surface. Our results show that cell surface expression of both GPI-anchored and TMD based surrogate marker correlates with target protein expression. This enabled us to identify and sort for high-expressing subpopulations, which showed higher transgene copy numbers, mRNA expression levels, and cell specific productivity, leading to a 2-fold increase in the volumetric titers. Overall, this strategy represents a significant advancement to shorten biomanufacturing timelines and support global health initiatives.

Chinese hamster ovary (CHO) cells have been the industry standard for biotherapeutics production for several decades. During this time, great efforts have been made to improve supporting infrastructure and environmental controls, while other advances came in the form of bespoke medias for optimized clone bioproduction. However, the CHO genome as an engineerable means of improving manufacturing outcomes has remained elusive, largely due to difficulties in target identification. Previously, our group developed and validated a first-in-class CHO CRISPR guide RNA library, built upon the de novo assembly and annotation of the CHO-K1 based CHOZN® GS-/- genome. As large-scale genomics studies become more financially accessible through cheaper sequencing costs, implementation of these unbiased, genome-scale screening tools becomes more practical for the CHO bioprocessing community. Here, we demonstrate the utility of our CHOZN®GS-/- CRISPR library in identifying new metabolic selection mechanisms via a genetic depletion screen and confirm the utility of these target genes via the development and testing of isogenic knockout clones which could ease the cell line development process for bispecific antibodies.

The CHOZN® GS-/- CHO cell line is a robust production platform for monoclonal antibodies and other recombinant protein therapeutics. With the increasing use for bispecific antibodies in antibody therapies, we aim to demonstrate the platform's performance in bispecific cell line development and manufacturing. Utilizing various 3-chain bispecific designs such as SEEDbodies and either common light chains or scFv; our resulting bispecific clones exhibit high productivity, stability, and product quality following our standard cell line development workflows and templated fed-batch and perfusion bioreactor processes. We employed a two-plasmid system, with each vector housing a unique heavy chain and its corresponding light chain. Two selection strategies were tested: either glutamine selection for both plasmids, or sequential puromycin and glutamine selection. Both selection strategies yielded productive, stable, and high quality clones. Our top clones maintain a minimum of 80% heterodimer; and have similar glycosylation and charge variant profiles across clones and assays. These clone attributes extend from spin tube studies to 3L Mobius® single use fed-batch bioreactors and Mobius® Breez 2mL dynamic perfusion microbioreactors. Spin tube fed-batch titers reach 2-3.5 g/L, while 3L Mobius® fed-batch exceeds 3.5 g/L, and Mobius® Breez perfusion systems reach 3.5 g/L/day in volumetric productivity. Our performance metrics highlight the CHOZN® GS-/- CHO platform's potential in bispecific cell line development and manufacturing.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the leading platform in gene therapy due to their favourable safety profile and high transduction efficiency. However, biomanufacturing constraints, particularly low production yields in HEK293-based systems, limit their scalability and drive the search for alternative hosts. Chinese Hamster Ovary (CHO) cells, the gold standard for biopharmaceutical production, present a promising alternative, yet no CHO line has been established for efficient rAAV production, and the cellular response to AAV and adenoviral (AdV) gene expression remains largely unexplored. This study represents the first comprehensive analysis of CHO cell responses to individual AAV and AdV proteins at both the transcriptomic and proteomic levels. We engineered expression constructs for isolated AAV and AdV gene products and systematically evaluated their impact on CHO cellular pathways. Surprisingly, functionally redundant AAV proteins elicited distinct cellular responses, with limited overlap in differentially expressed transcripts. Both transcriptomic and proteomic data suggest novel roles for AAV capsid and accessory proteins beyond their canonical functions observed in HEK293 systems. Additionally, AdV proteins displayed unexpected regulatory activities. Notably, expression of viral proteins resulted in the downregulation of immune and energy metabolism pathways in CHO cells. Our findings uncovered key host-virus interactions leading to potential candidate targets for host cell engineering. This work contributes to a better understanding of the bottlenecks associated with rAAV production in CHO cells and highlights potential directions for future optimization strategies.

Chinese Hamster Ovary (CHO) cells remain the dominant host system for the production of therapeutic monoclonal antibodies (mAbs), routinely achieving titers exceeding 10 g/L. However, the growing demand for more complex biologics, such as bispecific antibodies (bsAbs) capable of engaging multiple antigens simultaneously, presents significant challenges in cell line development. These engineered, multi-chain formats often suffer from imbalanced chain expression, inefficient assembly, and substantially reduced productivity—posing a major bottleneck to their commercialization. To address this, we developed a novel signal peptide (SP) cleavage prediction algorithm that surpasses current state-of-the-art models in both precision and recall. Applying this predictor to the CHO-K1 proteome, we identified over 800 previously uncharacterized CHO-derived signal peptides. These were combined with existing human SP sequences to construct a diverse library of approximately 4,000 signal peptides. We systematically applied this SP library to the heavy chains of various complex bsAb formats—including scFv-Fc and CrossMab—enabling chain-specific fine-tuning of molecule expression. This approach led to as much as a threefold increase in antibody titers, demonstrating the critical role of signal peptide selection in optimizing the expression of structurally complex antibodies. In conclusion, our work highlights the utility of chain-specific signal peptide engineering—particularly leveraging CHO-derived sequences—as a powerful and generalizable strategy to enhance bispecific antibody production in CHO cells.

The utilization of diverse cell expression platforms, including insect cells, HEK (Human Embryonic Kidney) cells, CHO (Chinese Hamster Ovary) cells, and Escherichia coli (E. coli), has become a pivotal strategy in addressing challenges associated with “difficult-to-express" biological targets. Each cell expression platform offers unique advantages tailored to the specific requirements of the target proteins. E. coli systems are favoured for their rapid growth and high yield, though they may struggle with complex post-translational modifications (PTMs). Insect cell systems, utilizing baculovirus vectors, excel in producing proteins with intricate PTMs, crucial for functional studies. HEK cells are widely used for their human-like PTMs and ease of genetic manipulation, making them ideal for producing proteins in their native conformation. CHO cells are indispensable for producing therapeutic proteins with human-like modifications and are extensively used in biopharmaceutical production. HEK and CHO cells also offer the advantage of secreting proteins into the surrounding medium making downstream processing much easier and cost effective. As the Department of Protein Science and Structural Biology within Sygnature Discovery, we are a contract research organization (CRO) that provides custom made, soluble and membrane proteins for use in drug discovery, research applications as well as X-ray crystallography, NMR and Cryo-EM structures of both novel and precedented proteins. Here we show real examples of how we’ve utilized each of our different expression platforms (Insect, HEK, CHO, E. coli) to successfully express membrane proteins (for structural work), intrinsically disordered transcription factors, large multi subunit protein complexes (the largest being a 12 subunit protein complex) and labelled proteins for NMR studies. This was achieved by leveraging the strengths of each individual expression platform to overcome the limitations posed by “difficult-to-express" targets; thereby, facilitating advancements in drug discovery, structural biology and therapeutic development.

Recent advancements in biotechnology, coupled with the increasing recognition of the unique properties of recombinant immunoglobulin M (IgM), have generated growing interest in these antibodies for therapeutic and diagnostic applications. A major driver of this progress is the widespread adoption of phage display technology, a valuable tool in antibody discovery and engineering that allows the complete synthetic generation of antibodies. The combination of phage display methodology and CHO expression system provides a powerful platform to produce recombinant IgMs addressing several challenges arising from the complex nature of these antibodies. The recombinant IgMs offer a promising alternative to traditional reagents in immunodiagnostic kits, with many advantages including the reduced reliance on human sera. In this work, we employed phage display technology to identify single-chain variable fragment (scFv) sequences with high affinity for the Rubella viral particle, a key reagent in the Diasorin IgM Rubella LIAISON® assay. This strategy allowed the isolation of scFv in only 18 days. Six pairs of sequences were then assembled on IgM scaffolds and produced as full-length antibodies in transient expression. The six antibodies were characterized in term of productivity and immunochemical performance, leading to the selection of the best IgM to proceed with stable expression. Finally, a hyper-producing IgM anti-Rubella stable clone was generated by our improved CHO cell line development process. Fed-batch production yielded 1.2 g/L of recombinant IgM, while preserving its multimeric structure. The recombinant IgM excellently replaces human positive sera for IgM anti Rubella, currently used in Diasorin IgM Rubella LIAISON® assay as calibrators and positive control. Signal emission and consistency were the parameters used to evaluate the comparison. Additionally, the longterm stability of the recombinant IgM was assessed. Along with optimal reactivity performance, its high production titer enables the expansion of the kit calibration range. This significant achievement highlights the potential of this integrated platform and marks a significant step toward leveraging recombinant IgM in diagnostic and therapeutic applications.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of mAbs and other protein biologics. Selection of high-producing cell lines is a key step in the process of manufacturing a novel biologic and requires an extensive and lengthy screening campaign of several hundreds of clonally-derived cell lines. We have previously reported the development of an efficient cumate-inducible expression system. Here, we present a new GMP-banked parental cell line, CHO2353, amenable to both constitutive or cumate-inducible expression. We first present our process for selecting CHO pools and then cell lines using a semi-automated approach, where imaging analysis provides >99% probability that selected cell lines are single-cell derived. Following stable pool selection with MSX, fluorescently labeled cells are deposited at one cell per well in 384-well plates using FACS and pictures of each well are taken to assess monoclonality. Hundreds of cell lines are then screened in 96-well plate format to identify top producers, which then a subset of clones enter expression stability study in a 6-deepwell plate format (~20 mL). High-producing, stable cell lines are then tested in 1-5 L bioreactors. We found that ~75% of selected cell lines show stable expression after > 60 generations in culture. We also present how we have engineered our platform for the production of antibodies with reduced fucosylation, and recent development of a minipools selection approach allowing to select more productive CHO clones. Antibody productivity for pools reached 2-4 g/L while that of clones reached 6 g/L. Finally, we present recent data with a newly engineered CHO cell line (CHONRC4), where expression from CHO pools reached titers of 4.0 g/L and 6.5 g/L for two model proteins.

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.

The baculovirus expression system is recognised as one of the main platform technologies for the production of proteins in insect cells. For many years, the Sf9 cell line has been popular for both the amplification of recombinant viruses and the production of proteins. It was produced by clonal selection from Sf21 cells, which were originally derived from the pupal ovarian cells of Spodoptera frugiperda (Fall armyworm). In this study, we aimed to produce a rhabdovirus-free insect cell line for production of proteins for use as human vaccines. We used a frozen vial of early passage Sf21 cells (Oxford, 1981) as our starting material. A number of cell lines were derived by rounds of single-cell cloning and testing for the presence or absence of the insect rhabdovirus by RT-PCR. Putative rhabdovirus-free cell lines were expanded in ES-AF (animal-free) medium and were also tested for the ability to support baculovirus amplification and protein production. One cell line, SfC1B5, was selected for banking and further study. The SfC1B5 cell line has since been adapted to grow in chemically-defined medium and extensive testing has demonstrated the cell line supports recombinant baculovirus production, amplification and protein yields similar to that obtained with Sf9 cells. We report use of the cell line to make Gc and Gn surface glycoproteins of Crimean Congo Hemorrhagic Fever virus as a candidate vaccine. However, for a range of secreted proteins, including viral antigens, higher yields were obtained in the SfC1B5 cell line. Where the secreted protein is to be used as a vaccine for human or animal health, the increase in yield significantly reduces the cost of vaccine production per unit dose.

Hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used effectively in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, GigaGen established a platform for development and manufacturing of recombinant polyclonal antibodies (pAbs), which comprise a mixture of >1,000 individual antibodies produced en masse. Two drugs of this class have now entered clinical development: GIGA-2050, for treatment of SARS-CoV-2, and GIGA-2339, for treatment of chronic Hepatitis B virus. To achieve these milestones GigaGen overcame several key challenges, including development of a single site targeted integration cell line, optimization of the upstream process using that cell line, as well as development of novel methods to monitor the upstream and downstream processes to ensure lot to lot consistency.

Recombinant adeno-associated viral vectors (rAAV) have emerged as a transformative platform in treating rare genetic diseases, evidenced by seven recent FDA approvals. These vectors are distinguished by their long-term expression capabilities, favorable safety profiles, and diverse capsid types that enable targeting of different tissues. While initially successful in treating rare monogenic disorders, rAAV applications have expanded to include more common conditions in ophthalmology and CNS disorders. Beyond gene replacement and regulation, rAAV vectors are now being explored as delivery vehicles for antibodies, cytokines in immunotherapy, and vaccine development. Despite their versatile potential, recombinant Adeno-Associated Virus (rAAV) treatments have experienced suboptimal adoption rates. A key challenge lies in the cost-effective manufacturing of high-quality AAV vectors at scale compared to traditional biologic products. Streamlining manufacturing processes and reducing costs while maintaining vector quality could help overcome the barriers to wider adoption. Currently, transient transfection-based production in HEK293 cells represents the most widely used platform, while stable producer cell lines emerge as the preferred future production system. Although recent years have seen significant improvements through process understanding and optimization, comprehensive knowledge of these production systems remains limited. This study presents an overview, progress, and outlook of stable producer cell line-based AAV production systems, focusing on helper virus mechanisms, host cell pathway analysis, and digital tools advancing AAV production excellence.

H5N1 is a highly pathogenic avian influenza virus that is responsible of the highly infectious respiratory illness called “bird flu”. Since 1996, the virus has expanded across the globe spilling into different animal groups such as poultry, wild and domestic mammals, as well as over 900 humans including one case in BC, Canada (November 2024). Since 2022 an increase of the outbreak has been reported, causing the death of millions of birds and leading to the death of approximately 20 million hens as of January 2025, and one human death in the USA. The virus is constantly evolving, and it has diversified and accumulated mutations throughout the years. Despite no transmission between humans having been reported yet, there are concerns that the virus is only a few mutations away from being able to transmit between humans, leading to a new potential pandemic. Most of the current available H5N1 vaccines are manufactured through a lengthy egg-based production process and the protection they provide is often based on outdated H5N1 strains. An alternative option for flu vaccines is the use of recombinantly produced antigens, such as hemagglutinin. Since CHO is the industry’s most used cell factory to produce r-proteins with a glycosylation similar to humans, their use is more and more considered for vaccine manufacturing. In my presentation, I will detail our strategy to produce a H5N1 recombinant hemagglutinin (H5) antigen in CHO cells that is able to generate immunogenicity in vivo, as well as improving its manufacturability.

Antibodies are the best-selling class of drugs in a fast-growing pharmaceutical market. To ensure the efficacy of an antibody therapeutic, it is recognized that in addition to target binding, multiple functional and developability properties need to be addressed early and concurrently in the drug development process. ATUM has created a platform, AntibodyGPS®, that enables assessing and improving multiple antibody properties simultaneously while minimizing the number of samples and assays required. The combination of multi-dimensional generative machine learning (ML) and high-quality analytics enables the holistic engineering of lead molecules into drugs.

The last two decades have seen a growing interest in the role of D-amino acids within the nervous system. In particular, D-serine is now recognized as a key neuromodulator in its role as a more potent co-agonist than glycine for N-methyl-D-aspartate receptors (NMDARs), which are widely recognized as the key receptor responsible for synaptic plasticity. Accordingly, aberrations in D-serine signalling have been consistently associated with several pathological conditions. However, despite our understanding of D-serine’s role in the nervous system, the molecular mechanisms that govern its dynamics remain unclear, with recent works challenging its role as a gliotransmitter. To address these gaps in knowledge, tools with the requisite spatiotemporal resolution, such as genetically encoded fluorescent protein-based indicators, are necessary to monitor D-serine dynamics. To date, the only genetically encoded indicator for D-serine is a FRET-based indicator, called DserFS, based on a bacterial periplasmic binding protein (PBP). PBPs are ideal scaffolds for sensor engineering because they are orthogonal to neurons, offer large changes in fluorescence in response to ligand binding and can be targeted to arbitrary cellular compartments. However, DserFS shows a limited dynamic range relative to single fluorescent protein-based indicators and requires exogenous addition for imaging in brain slices. Here we present our work on engineering a genetically encoded single fluorescent protein-based indicator for D-serine from DserFS. Indeed, preliminary results indicate that our D-serine sensor shows large fluorescence changes with micromolar affinities and good membrane localization. We anticipate that our new D-serine indicator will open new avenues for investigating D-serine dynamics within the nervous system.

This work introduces a spouted variant of the Thomson 7L Optimum Growth flask, currently the largest working volume flask available in the market. The design features a basal spout with integrated tubing, engineered as a closed system for cellular transfer operations. The system's low retention volume architecture optimizes culture transfer efficiency for seeding several liters or serving as an inoculation vessel for large-scale bioreactors. This configuration reduces both culture exposure risks and the total number of vessels required for large-volume cell culture operations.

Chinese Hamster Ovary (CHO) cells are widely used for the manufacturing of antibodies and, more recently, subunit vaccines. To this end, transient transfection is a rapid and cost-effective means of expressing lead candidates in CHO cells and is particularly useful when needing to produce novel subunit vaccine variants within a short time frame. In a conventional process, the timing of transfection is generally decided by a predetermined viable cell density (VCD) trigger. However, this strategy may not translate well when culturing CHO cells using continuous cultures or N-1 perfusion, due to the cultures not following conventional growth curves. To determine an ideal time to carry out transient transfection in such cultures, an oxidation-reduction potential (ORP) probe may aid in this decision-making process. ORP is an indirect measure of the presence of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, which in higher concentrations can negatively impact cellular productivity and quality attributes of the products such as post-translational modifications. To determine whether ORP levels at the time of CHO cell transfection have any impact upon production yield of the therapeutic marker, namely SARS-CoV-2 spike protein, transient transfection was performed in 1 L bioreactors while monitoring readings from an online ORP probe. The discovery of an ideal ORP range and VCD combination for transient transfection may provide a useful parameter for CHO cell readiness for transfection. Furthermore, monitoring of the ORP signal post-transfection can serve as an indicator of the general health and productivity of the culture. Changes in redox potential captured with the ORP probe may be correlated with production rate that generally decline after a few days post-transfection. This would in turn help us further improve cellular productivity by adding antioxidants, for instance, at the appropriate time to reduce cellular oxidative stress.

Perfusion bioreactors are emerging as a powerful platform for continuous monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells with advantages in productivity, product quality, and operational efficiency. However, conventional perfusion processes typically rely on offline sampling and delayed analytics to guide process decisions. This piecewise approach often leads to reactive rather than proactive control, where flow rates, feed concentration and downstream adjustments are only made after metabolite imbalances or performance issues arise. In this study, we present the design of an advanced perfusion bioreactor platform built around the Sartorius Biostat® B-DCU 2L bioreactor system coupled with a Repligen ATF (alternating tangential flow) controller. Enhanced with integrated online sensors, the system enables real-time process awareness and more responsive control. Alongside standard sensors (pH, dissolved oxygen, temperature) the system incorporates a multi-spectrum UV sensor and a capacitance probe to provide continuous data on metabolic state, nutrient consumption, cell density, and product yield. The central aim of this work is to demonstrate how a real-time sensing strategy can overcome the limitations of traditional offline workflows. Leveraging the Sartorius Cellca 2 CHO strain for maximum protein production, the system uses continuous monitoring to enable early detection of metabolic shifts to support dynamic control of perfusion rates, nutrient supplementation, and process parameter shifts including temperature and dissolved oxygen. This level of process visibility has the potential to boost productivity over extended culture periods, improve overall titre, and maximize space-time yield while simultaneously informing downstream operations. By transitioning from manual adjustments to adaptive control, this platform reflects a broader push toward integrated and data-driven biomanufacturing. Future work will focus on leveraging these sensor outputs to develop predictive control algorithms and fully closed-loop systems for robust, scalable mAb production.

Chinese Hamster Ovary (CHO) cells are the industry gold standard for expressing therapeutic monoclonal antibodies (mAbs), and selecting high-producing clones early in the production pipeline is essential for efficient patient delivery. This can be achieved by identifying biomarkers that accurately predict cell productivity. However, current approaches for biomarker discovery rely heavily on low-throughput, labour-intensive methods that limit scalability and slow development timelines. These limitations hinder comprehensive assessment of large clone panels and obscure the full extent of transcriptional heterogeneity at early stages. To address these challenges, we present a novel high-throughput, multiplexed approach for early clonal characterization using single-cell RNA sequencing (scRNA-seq). By co-culturing and barcoding multiple CHO clones in a pooled format, we enable efficient, parallelized transcriptomic profiling of thousands of individual cells across multiple clones in a single experiment. This strategy reduces batch effects and accelerates screening, providing a more holistic view of clonal behaviour at the single-cell level. Our analysis revealed consistent transcriptomic signatures associated with high-productivity clones and identified productive subpopulations within clones. Beyond biomarker discovery, this platform offers a scalable, data-rich framework for characterizing transcriptional heterogeneity and performance across clones — delivering valuable insights for early decision-making in cell line development.

The development of cell lines that reliably express large titers of biologics such as monoclonal antibodies (MAbs) is an important step of bioprocess development. After transfection of CHO cells, stable pools are obtained, and clones are isolated. As each clone can behave distinctly when cultivated, clone selection is crucial. This study proposes an adaptable multifactorial data analysis method for clone selection that takes multiple process parameters into account, in addition to the traditionally considered titer and cell growth. We applied our innovative approach on existing in-house data corresponding to the generation of CHO clones producing Omalizumab, an IgG1 monoclonal antibody. Twenty-four clones were chosen for expression stability screening tests conducted in extra deep well plates (18 mL). Among them, eight stable clones were selected for scalability evaluation performed in benchtop stirred-tank bioreactors (0.75-1 L). Considering parameters related to productivity, cell growth and expression stability led to the selection of different clones for bioreactor scalability assessment experiments compared to those originally selected based on a conventional method. As efficient CO2 stripping is challenging in large-scale bioreactors, experiments were conducted with and without addition of air in the bioreactor headspace to modulate the concentration of CO2 in the media. We found that cultures without overlay air addition reached a pCO2 of up to 190 mmHg. Of note, we showed the increased concentration of CO2 to be beneficial. Indeed, on average, we measured 1.31-fold higher final titer, 1.14-fold higher cell specific productivity, and 1.13-fold greater peak viable cell density for cultures exposed to a greater pCO2. In addition, these cultures benefitted from 3 to 5 more days above 80% viability, and their titer kept increasing until the end of the culture (17-21 days). We integrated statistical tools to reliably analyze datasets relating to productivity, cell growth, and key cellular metabolites. This new approach helped selection of a robust clone which performed similarly for low and high pCO2, offering a better potential for subsequent scale-up. These findings underscore the great potential of MVDA to improve bioprocess development.

Suspension MDCK cells are a highly relevant cell substrate for scalable and efficient production of influenza A virus (IAV). Considering the high heterogeneity within conventional cell populations, the development of clonal cell lines has resulted in candidates with superior growth characteristics and high IAV yields (Zinnecker et al., 2024). However, proteomic analysis could help to further understand specific properties of such clonal cell lines and help to identify best producers for vaccine manufacturing. In the present study, we compare proteome alterations between two IAV-infected suspension MDCK cell clones (C59 & C113, Sartorius, Germany) to elucidate differences in cell growth, size, metabolism and IAV productivity. Using advanced mass spectrometry, a total of 5177 host cell proteins were detected in both cell clones. Protein network analysis of the differentially expressed proteins with respect to cell growth revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in the highly productive cells, whereas steroid biosynthesis and DNA replication were more active in the faster growing cells. After infection, 122 proteins were significantly upregulated (log2 fold change ≥1) in the high-producing cell line, including proteins associated with membrane trafficking. In addition, proteins that have cross-links to the IAV-NS1 protein and proteins that support virus production were identified. In addition, 98 proteins associated with antiviral signaling pathways such as Met and TNF signaling were downregulated (log2 fold change ≤1). In the less producing cell line, 77 proteins were downregulated and 57 upregulated after infection. Here, RNA metabolism seemed to be downregulated, whereas the TCA cycle and stress response were upregulated. Overall, we were able to identify important differences between a fast-growing and a high-producing clonal MDCK cell line, revealing potential bottlenecks and providing further insights into the efficient production of IAV in cell cultures.

As a crucial surfactant, polysorbate (PS) is deployed in the final formulation buffer to stabilize biologics – often monoclonal antibodies – and thus ensuring the safety and efficacy of the drug product. However, in recent years it was shown that Chinese hamster ovary (CHO) host cell proteins (HCPs) possessing hydrolytic activity could persist the purification process of the biotherapeutic, and were found at trace levels in the final drug product. Over time, even trace amounts of hydrolases can possibly cleave PS ultimately compromising stability of the drug product and might even lead to the occurrence of (sub)-visible particles. Tackling strategies encompass the entire bioprocess, including cell line engineering, downstream optimization, or alternative formulation approaches. However, mitigation strategies during upstream processing are scarce and usually only focus on the harvest procedure to avoid leakage of intracellular HCPs. Here, we present a novel strategy to reduce PS degradation via choice of cultivation media. During head-to-head cultivation of CHO cell lines in various cultivation media, we revealed strongly differing PS degradation properties when assaying the harvested cell culture supernatant. This phenomenon seemed to be lipoprotein lipase (LPL) related, since PS degradation was substantially reduced in LPL knockout cells even in media that showed pronounced PS degradation. Consequently, PS degradation clearly correlated with LPL protein abundance in the supernatant. In order to elucidate the connection between media and varying LPL levels, we analyzed media dependent cellular LPL expression and could rule out differences in transcription as the root cause. Consequently, we focused on media dependent effects on LPL protein expression, secretion and stability. In addition, media components were correlated to LPL expression to identify possible stabilizing or destabilizing agents in the media composition. The understanding of the influence of cell culture media components on hydrolytic HCP expression and hence varying PS degradation offers a novel solution to mitigate PS degradation in CHO cell bioprocesses. Our results suggest that by rational media design we might be able to reduce the activity of critical PS-degrading hydrolases in the future.

Antibody-producing cells like CHO are typically cultivated in shake flasks, limiting miniaturization and throughput due to volume and high-velocity shaking requirements. We propose a high-throughput microfluidic bioprocess in a 384-well plate format, where CHO cells are encapsulated in a hydrogel and receive nutrients and oxygen via rocking induced media flow through hydrogel channels, enabling efficient mass transfer with minimal infrastructure and energy needs. We identified alginate as the optimal hydrogel for CHO cell encapsulation, preserving their rounded morphology. Alginate enabled easy cell retrieval via EDTA-mediated Ca² chelation, achieving a 97 ± 16% yield, facilitating downstream analysis. We established CHO cell cultivation in alginate within a perfusable 384-well plate, using a microfabricated platform (OrganoBiotech) where encapsulated cells occupy the middle well, and gravity-driven flow is maintained by differential media levels and rocking-induced perfusion. To enhance mass transfer, 20 µm-scale granules of gelatin were incorporated at the cell encapsulation stage. Gelatin was removed upon alginate crosslinking and temperature elevation, leaving behind a porous path. To fabricate the slab, 25 μL of the alginate solution with CHO cells (1x106 mL) were deposited into the middle well, allowing the solution to evenly spread across the well, followed by a rapid crosslinking using a CaCl2 solution yielding an even distribution of live cells in 3-dimensions. After 10 days, CHO cells cultivated in alginate with rocking induced flow, exhibited higher density and viability compared to those cultivated in a static alginate slab, or a standard 384-well plate with cells growing in 2-dimensions at the bottom of the wells . Quantification of the IgG concentration using the Octet platform further highlighted the superiority of the microfluidic platform, with significantly higher total mAb amount under flow compared to a static cultivation in alginate or a standard 384-well plate (Fig. 1). Acknowledgements: The authors thank Amgen for funding, Rene Hubert, Tracy Lee, Brett Eyford and Eric Gislason for helpful discussion.

In mammalian cell-based bioprocesses, glycosylation of recombinant therapeutic proteins requires stringent control and monitoring as it may affect therapeutic efficacy and safety. The sialylation of glycosylated moieties plays an important role in determining the in vivo clearance of therapeutic proteins by extending their serum half-life. In this study, six enzymatically synthesized oligosaccharides (3’-sialyllactose, 6′-sialyllactose, sialyllacto-N-tetraose a, disialyllacto-N-neotetraose, lacto-N-tetraose, and lacto-N-neotetraose) and lactose were evaluated for their effectiveness on the N- and O-glycan profiles of etanercept, a therapeutic Fc-fusion glycoprotein consisting of 3 N- and 13 O-glycosylation sites, produced in recombinant Chinese hamster ovary (rCHO) cells. Of these seven compounds, only sialylated oligosaccharide supplementation increased the proportion of the acidic isoforms, di-sialylated N-glycan and di-sialylated O-glycan, as well as the total sialic acid content of etanercept, although the degree of the effect varied. Increased sialylation resulted from increased concentrations of intracellular CMP-sialic acid, with the highest increase observed for disialyllacto-N-neotetraose, which contains the most di-sialic acid residues. In contrast, four sialylated oligosaccharides did not enhance the sialylation of etanercept produced by human embryonic kidney 293 (HEK293) cells. Taken together, enzymatically synthesized sialylated oligosaccharides represent novel supplements to enhance the sialylation of therapeutic Fc-fusion glycoproteins in rCHO cell culture.

Adeno-associated viruses (AAVs) are indispensable viral vectors for fundamental and clinical research. In fact, AAVs are composed of a single-stranded DNA contained in a 25 nm capsid. These two components can be modified to create recombinant AAVs (rAAVs), which can then be designed for a specific use. For example, some rAAVs allow the delivery of optogenetic tools and render the study of in vivo mechanisms possible. Other rAAVs can cross the blood brain barrier and represent an interesting avenue for central nervous system gene therapy. However, while rAAVs enable a panoply of applications, the transduction of the viral particle itself remains very intricate and underknown. There is a critical need to further our understanding of this tool to optimize its use and therefore lead to the development of highly efficient viral vectors. Thus, the goal of this project is to develop a bio-imaging and analysis method to follow the journey of rAAVs throughout cells in real time. First, AAV capsids are labelled using dibenzocyclooctyne (DBCO) molecule paired with a fluorophore via click chemistry. This unique AAV labelling strategy was optimized to minimize the impacts on the viral capsid integrity and its transduction properties. Second, subcellular regions like the cell membrane and the nuclear envelope are tagged to create checkpoints during the rAAV transduction. Neurons will be the main cells of interests considering AAVs’ central nervous system gene therapy perspective. Third, the monitoring of the fluorescently labelled rAAVs infection process is made using real-time videorate fluorescence microscopy. Fourth, analysis and quantification of the AAV transduction will then be realized to obtain unprecedented data on this pathway. Furthermore, a wide variety of cellular models and AAV serotypes are compatible with the developed labelling methods, which will open the door to further studies on this viral vector and contribute to its widespread and optimized use.

Influenza vaccine production in cell culture offers a scalable and efficient alternative to egg-based manufacturing. Process intensification strategies can be used to further increase yields, reduce facility footprint, and shorten production timelines. This study evaluates different approches for intensified influenza A virus (IAV) production using two clonally-derived suspension MDCK cell lines, C59 and C113 (Sartorius, Germany), with distinct characteristics [1]. To drive cells to high cell density (HCD), we implemented a semi-perfusion strategy in shake flasks, achieving maximum densities of 42×10 C59 cells/mL and 17×10 C113 cells/mL. Upon infection, high hemagglutinin titers of 3.5 and 3.6 log10(HAU/100 µL) were reached for C59 and C113, respectively. Based on the small-scale results, the process was transferred to a 3 L bioreactor coupled to a KrosFlo tangential flow depth filtration (TFDF) perfusion lab scale system (Repligen, USA) with perfusion rates tailored to the specific needs of each cell line. The filter allowed for cell retention with continuous virus transmission enabling continuous clarification and final harvest, resulting in a 10-fold increase in space-time yield for C59 and a 4-fold increase for C113 over batch operation. In addition, we evaluated seed train intensification strategies, including N-1 perfusion and HCD cryopreservation for direct inoculation of the production bioreactor. Cells from N-1 perfusion showed reduced doubling times, but virus titers were unaffected compared to control infections. Direct inoculation from HCD cryovials enabled accelerated process initiation without additional precultures, demonstrating its applicability for streamlining manufacturing workflows. Overall, our study provides important insights into process intensification strategies for improved IAV production. We demonstrate scalable, high-yield solutions that increase productivity, reduce production times, and support the transition from batch to intensified cell culture-based influenza vaccine manufacturing.

Traditionally, Herpes Simplex Virus-1 is produced in adherent cells, typically Vero. The use of adherent cells, despite the availability of roller bottle systems, cell factories, or fixed bed/microcarrier bioreactors, poses significant challenges for handling, upscaling, and production. We, therefore, aimed to set up a suspension-cell process that would enable the production of high titers of HSV-1. Moreover, the process should allow for certain flexibility in production, suitable for both research-scale production and supply for potential clinical studies. First, we aimed to identify a suitable suspension cell line to propagate HSV-1. Different cell lines commonly used for virus production were evaluated. To our surprise, HEK293 cells proved to be the most promising for HSV-1 production. Following the identification of HEK293 as the most productive cell line, three different HEK-derived cell lines were tested in combination with various media. The best producing cell line, HEK-LTV cultured in Dynamis medium (Thermo Fisher Scientific), produced approximately 2×10^8 TCID50/ml of HSV-1. To further optimize the batch process, the influence of temperature during the infection and production phases was analyzed. Lowering the temperature to 35°C or 33°C did not improve viral production compared to culture at 37°C. Furthermore, cell density effects in both batch and semi-perfusion settings were studied. Finally, the batch process was transferred to a stirred tank bioreactor, which resulted in a comparable HSV-1 infectious titer of 1.3 x 10^8 TCID50/ml. In this setting, different agitation speeds were tested to assess the impact of shear stress on HSV-1 production. Current commercial HSV-1 production utilizes adherent cells, highlighting the need for the development of a suspension cell process that yields high viral titers. Here, we demonstrate a scalable HSV-1 production process in suspension HEK293 cells, yielding high viral titers.