Program

The increasing emergence of new gene therapies, advanced personalized therapies and vaccine new formats using transient transfection of mammalian cells as production platform is evidencing the need to develop more efficient production processes allowing to decrease the manufacturing costs and facilitate access of patients to these new therapeutics. This presentation will focus on bioprocess improvement for the production of viral particles, such as Virus Like Particles and Adeno Associated Viruses, including a) the so-called extended gene expression (EGE), b) the cell engineering to increase AAV release out of the cells and c) functionalization of VLPs for a tuberculosis vaccine generation. EGE is a technology to increase the production of viral particles by transient transfection in HEK293 cells and is based in continuous medium exchange by perfusion combined with multiple transfections. The process has been demonstrated both for VLPs (HIV-1 Gag VLPs) and AAVs (AAV1 and AAV9) at bioreactor scale, using continuous perfusion with the ATF system, with substantial extension of the production span from the conventional 48-72 hours to 120-150, with a 5-12-fold increase in total harvested VLPs or AAV yield. The EGE produced viral particles have also been integrated with two-step chromatography purification process. Cell engineering of HEK293 cells has also been applied to improve the capacities of AAV secretion, and therefore further increase the yields of harvested AAVs. Quantitative multiplexed proteomics of cells in EGE conditions revealed coordinated remodeling of host-cell pathways involved in vesicle trafficking, lipid biosynthesis, protein homeostasis, and nuclear transport. This proteomic analysis allowed to identify key host regulators of exo-rAAV biogenesis and secretion, including CD63, VPS37B, SMPD3, and SNAP47. Targeted modulation of these pathways enabled rational enhancement of extracellular rAAV recovery. Using a DoE-based design, a 50% viral egress into the supernatant was achieved, demonstrating the capacity to further improve AAV production bioprocess. Finally, an example of functionalization of HIV-1 Gag virus-like particles and the concomitant extracellular vesicles produced in HEK293 cell culture, displaying short antigenic peptides on their surface through an engineered CD81 protein will be discussed. Both particle types were functionalized with three peptide antigens and characterized for particle yield, morphology and antigen presentation. Immunogenicity and protective efficacy were evaluated in mice using a progressive tuberculosis model by measuring antigen-specific antibody responses, bacterial burden in lung and spleen, and histopathological damage following challenge with M. tuberculosis. Engineered VLPs and EVs efficiently displayed antigenic peptides without altering particle size or morphology. All vaccine candidates were produced at comparable levels and showed similar surface presentation of the inserted peptides. In vivo, only one of the virus-like particle–based vaccines elicited a detectable antigen-specific antibody response.

Biopharmaceuticals have transformed the treatment of life-limiting diseases, offering tremendous therapeutic success and unique commercial potential. However, the growing demand for new therapeutic modalities requires innovation in biopharmaceutical manufacturing to improve access to and the cost-effectiveness of these transformative medicines. The transition from traditional batch production to continuous manufacturing represents a critical challenge in this field, requiring the development of novel technologies to make this transition possible. This presentation focuses on findings related to the engineering of Chinese hamster ovary (CHO) cell factories with enhanced capabilities for continuous biopharmaceutical production. Although CHO cells have been widely used in batch and fed-batch processes, their performance in continuous production remains limited. Over the past 15 years, our research has sought to understand CHO cell biology under continuous culture (chemostat) conditions, identifying key aspects for optimization at the cellular level, such as the control of cell proliferation and apoptosis, to improve CHO cell performance in continuous manufacturing.

Lactate accumulation remains a major limitation in CHO cell culture, affecting process robustness, productivity, and product quality. The transcription factor HIF-1α regulates glycolytic gene expression and drives metabolic rewiring toward lactate formation. Here, we evaluated whether CRISPR-Cas9 disruption of HIF-1α reduces lactate overflow and shifts glucose metabolism in CHO-S cells. CHO-S cells were edited via CRISPR-Cas9 targeting the HIF-1α locus and isolated by single-cell cloning. Partial disruption and heterogeneous editing across the locus were confirmed by PCR amplification of a fluorescent reporter integrated through homology-directed repair, while protein-level disruption was verified by Western blot of nuclear extracts. From 28 edited clones, five were randomly selected for batch and fed-batch characterization against parental CHO-S cells. Glucose and lactate concentrations were monitored over time to estimate kinetic and metabolic parameters. In suspension batch cultures, top-performing clones showed a marked reduction in lactate accumulation during exponential growth, decreasing specific lactate production rates by 63% to 74% compared to parental cells. These clones also reduced specific glucose uptake by 34%, indicating lower glycolytic flux and more efficient carbon utilization. However, metabolic responses and kinetic profiles varied across clones, reflecting inherent clonal variability after genome editing. Performance improvements were more pronounced under glutamine-limited fed-batch cultures. Overall, HIF-1α disruption induces a clear metabolic shift in CHO-S cells, offering a practical strategy to reduce acidification, simplify pH control, and mitigate lactate-related constraints in biomanufacturing.

Human erythropoietin (hEPO) is a pleiotropic glycoprotein placed at the intersection of two distinct biological programs: erythropoiesis, mediated by its homodimeric receptor, and cytoprotection/neuroplasticity in the central nervous system, mediated by a heterodimeric receptor complex. This dual identity makes hEPO a hinge molecule: a single scaffold from which divergent therapeutic outcomes can be engineered depending on how its glycosylation is manipulated. Glycoengineering by hyperglycosylation, understood as the introduction of N- or O-glycosylation sites, was used as an operative tool to steer hEPO along two opposite trajectories: potentiating its native erythropoietic activity, or blocking this action by dissociating its two biological functions. To potentiate erythropoiesis, multiple copies of a novel peptide tag (mGMOP) were fused to hEPO termini, generating the variant mGMOP3-EPO-mGMOP. This hGM-CSF-derived tag, which carries the APAR epitope recognized by mAb CC1H7 (generated in our lab), was modified to provide 6 potential O-glycosylation sites. The variant was purified by immunoaffinity chromatography, yielding a 99% pure product with 100%-recovery. The resulting increase in sialic acid content markedly improved pharmacokinetics relative to commercial rhEPO, extending half-life 3.4-fold and reducing plasma clearance 4-fold, without altering protein conformation. In vivo, the novel variant displayed a hematopoietic potency 2.5-fold higher than rhEPO, confirming that hyper-O-glycosylation amplified the molecule's original function. To dissociate hEPO's activities, single amino acid substitutions were introduced at defined positions of the native sequence to create an additional N-glycosylation site (muteins Mut 45_47 and Mut 104, expressed in CHO.K1 and HEK-293 cells). This site- specific modification abolished erythropoietic activity in vitro while preserving, and in several assays enhancing, neurobiological activity: neuritogenesis, filopodia formation, and synaptogenesis. More importantly, a significant in vivo increase in dendritic arborization of hippocampal pyramidal neurons was observed regardless of the producing cell line. Together, these two engineering strategies converge on the same hinge molecule to illustrate opposite design principles: glycosylation added extrinsically, as a fusion tag, amplifies the erythropoietic program, whereas glycosylation inserted intrinsically, at a single defined site, uncouples receptor-specific functions. This dual perspective positions hEPO as a paradigmatic model for rational glycoengineering in the design of next-generation biopharmaceuticals with tailored, receptor-selective activity profiles.

The study of cell physiology and the mechanisms associated with communicable and noncommunicable diseases is undergoing a paradigm shift driven by the development of three-dimensional (3D) cell models. Organoids, derived from stem cells, are complex structures capable of recapitulating relevant aspects of the architecture, cellular diversity, and functionality of in vivo organs, overcoming some of the major limitations of traditional two-dimensional cell cultures. This presentation will address the fundamentals of organoid technology, including their generation, characterization, and main methodological approaches, with particular emphasis on their application as models of the intestinal tract. Examples will be presented illustrating their use to address different microbiological challenges, including the analysis of the infectivity and invasion of Salmonella strains, the modeling of critical biological stages in the life cycle of Toxoplasma gondii, and the study of host–pathogen interactions during Trypanosoma cruzi infection. The potential of these systems for studying chronic inflammatory processes, noncommunicable diseases, and their applications in personalized medicine will also be discussed. Overall, these 3D models enable the study of infection and host responses within a context of greater tissue complexity and with higher physiological relevance than conventional cell-based systems. Their ability to reproduce aspects of tissue organization and function makes them highly predictive tools for investigating the cellular and molecular events that contribute to pathogenesis. In addition to reducing reliance on in vivo animal models, organoids offer new opportunities to investigate disease mechanisms and evaluate host responses in a more integrated manner. The incorporation of models derived from species of veterinary and zoonotic relevance further expands these approaches toward a One Health perspective, integrating human, animal, and environmental health in the study of infectious diseases.

Organ-on-a-chip systems are not merely miniaturized cell cultures, but rather experimental platforms designed to reproduce, under controlled conditions, components of the microenvironment that determine cellular function. This talk will present LaNSBioDyT’s experience in the interdisciplinary development of these platforms, from defining the biological question to microfluidic design, microfabrication, integration of cell culture, and functional validation. Drawing on representative cases and projects—including models of the blood–brain barrier, axonal growth, and neural tube, among others—the presentation will illustrate how geometry, materials, flow, co-culture interfaces, and observation and measurement strategies must be selected according to the phenomenon under investigation. The aim is to demonstrate that innovation arises from the iterative interaction between biology, physics, and engineering, rather than from miniaturization alone.

A complex system is composed of multiple subsystems whose interconnected components interact in a nonlinear manner. This is an inherent feature of biological systems and poses challenges to the design of cell engineering strategies aimed at improving biopharmaceutical production processes. Dynamic modeling is an in silico approach that provides a mechanistic perspective on cellular processes by incorporating enzyme kinetics, the influence of metabolite concentrations on reaction rates, and feedback regulatory mechanisms. This enables a deeper understanding of intracellular dynamics. The aim of our research is to develop models of intracellular processes as tools for generating key information that can contribute to the rational design of cell engineering strategies. We are particularly interested in processes occurring along the secretory pathway of CHO cells, which has been identified as a bottleneck limiting the achievement of high recombinant protein productivities in this cell type.

CHO cells are the preferred host for industrial recombinant biotherapeutic production, yet limited specific productivity and loss of transgene expression during prolonged culture remain major challenges. This work explores endogenous CHO promoters as alternatives to strong viral promoters, which can induce cellular stress and undergo epigenetic silencing. Using transcriptomic analysis of CHO-K1 cells grown in bioreactors, highly active endogenous promoters were identified and characterized in transient and stable expression systems. Selected promoters supported robust and sustained transgene expression over multiple generations and under reduced-temperature production conditions. PromA and PromB maintained stable production of the SARS-CoV-2 receptor-binding domain (RBD), while CMV+E-driven productivity declined over time. Additional promoters and rationally engineered variants further improved expression, with some combinations exceeding viral CMV promoter activity. Overall, these endogenous regulatory elements provide a compact, non-viral toolbox for developing stable, high-yielding CHO cell platforms for the production of human and veterinary biotherapeutics.

This talk outlines the biopharmaceutical journey, from cell line and process development through regulatory submission, paving the way for clinical trials and the desired commercial approval. Initially, basic concepts guiding biopharmaceutical research and development to established highly productive recombinant cell lines and efficient upstream/downstream processes, will be explored. Then, through the lens of the UFRJvac COVID-19 vaccine candidate, we will discuss key aspects required to reach regulatory submission — cell bank development and certification, adventitious agents evaluation, quality control and stability studies of drug substance and drug product, pre-clinical testing and regulatory filing, based on real experiences with regulatory agencies of different countries.

Monoclonal antibodies targeting MICA represent a promising strategy for modulating antitumor immune responses. However, in addition to biological activity, the development of therapeutic antibody candidates requires consideration of manufacturability attributes such as expression, productivity, and molecular stability. The human anti-MICA antibody c65, which targets the α1 domain of MICA, has previously characterized functional activity; however, experimental observations during its development indicated limited stability upon storage at 4 °C. In this study, we evaluated whether rational engineering of its variable regions could improve properties associated with antibody production and stability. A variant designated H+K was generated by engineering the framework regions (FRs) of the heavy and light chains. The parental (WT) and H+K variants were initially evaluated in scFv format through recombinant expression in Escherichia coli. H+K showed an approximately 72-fold increase in soluble expression and an approximately 13 °C increase in melting temperature (Tm) compared with WT, while retaining MICA recognition. The WT and H+K variable regions were subsequently transferred to an IgG1 format and transiently produced in CHO cells to determine whether the improvements observed in the scFv format translated to the full-length antibody produced in mammalian cells. Three independent transient CHO cell productions were performed for each condition by co-transfection of the heavy- and light-chain constructs. Viable cell concentration, cell viability, antibody concentration in the culture supernatant, and specific productivity (qP) were evaluated during culture. H+K cultures showed higher antibody production than WT cultures. When antibody production was normalized to cell biomass, H+K exhibited a 2.6-fold higher qP than WT (n = 3; p < 0.0001). The antibodies were subsequently purified and characterized. H+K exhibited a melting temperature of 69.0 °C, compared with 65.9 °C for WT, corresponding to a 3.1 °C increase in Tm. ELISA characterization showed that the introduced modifications preserved MICA-binding activity. Taken together, these results demonstrate that framework region engineering of the anti-MICA c65 antibody improves key attributes associated with manufacturability. Modifications that initially enhanced soluble expression and stability of the scFv in a bacterial system translated, in the IgG1 format, into significantly increased specific productivity in CHO cells and improved thermal stability, while preserving antigen recognition. These findings highlight the importance of considering variable-region properties during the early stages of engineering and selection of antibodies intended for production in mammalian cells. Keywords: CHO cells; monoclonal antibodies; MICA; antibody engineering; framework regions; specific productivity; thermal stability.

The recent launch of our CHOZN® Elite cell line provides a high-performing and versatile platform. To enhance its performance further, we have developed innovative media and feed solutions that ensure optimal growth and productivity throughout the entire, streamlined cell line development (CLD) and production workflow. This innovation addresses the challenges posed by reliance on multiple media types at different stages of the process, underscoring the necessity for a unified media and feed solution that can simplify workflows and boost productivity. Our engineered medium and feed prototypes are designed by leveraging digital tools, including smart experimental design and multivariate data analysis for screening and optimization, coupled with extensive cross-testing in scale-down models. Our data demonstrate that the prototype medium supports clonal selection during the cell line development process. Notably, it yields superior results in fed-batch cultures, achieving maximum titers that are 30-40% higher than those of commercially available platform products. Additionally, our prototype medium shows compatibility across multiple CHOZN® cell lines during adaptation and passaging, with an average doubling time of 17-25 hours. Importantly, our prototype exhibits significantly reduced galactosylation compared to other vendors’ products. Our prototype compacted medium samples, in combination with a single-part feed, are currently available for customer testing and evaluation.

Three-dimensional cell culture systems represent an alternative approach for generating in vitro models with an enhanced ability to reproduce the characteristics of the tissue microenvironment. However, the development and maturation of 3D constructs require strategies that improve nutrient and oxygen transport while providing appropriate physical stimuli to promote cellular behavior and function. This presentation will describe our group’s experience in the development and application of biomaterials and bioreactors for the dynamic culture of animal and human cells in three-dimensional systems, using alginate–chitosan scaffolds to support the formation of cellular constructs. The effects of conditions such as perfusion and electrical stimulation on cell viability, proliferation, organization, and maturation will be discussed. Using models developed for bone and cardiac tissue engineering, the advantages of dynamic culture over static systems will be examined. Finally, the potential of these platforms to develop more reproducible and functional 3D cell models will be discussed, with applications in tissue engineering, regenerative medicine, and the evaluation of novel therapeutic strategies.

Cutaneous melanoma is the most aggressive form of skin cancer, accounting for a disproportionate share of skin-cancer-related deaths relative to its incidence. Oncobiomed, a University of Chile spin-off, has developed an immune-based therapeutic alternative, TAPCells, which uses antigen-presenting cells trained with lysates from specific melanoma cell lines to trigger an immune response against the tumor. Lysate production currently relies on batch adherent culture, a format whose surface-limited scalability is the central bottleneck for clinical-scale manufacturing. This bottleneck was addressed as a process design problem, assessing two three-dimensional cell culture strategies, microcarrier-anchored and self-assembled spheroids, across three melanoma cell lines. In both cases, quantitative characterization of metabolite concentrations and cell-specific growth kinetics was used to define operating conditions and determine nutrient requirements for process design. For the microcarrier strategy, repeated fed-batch cultures on microcarriers in spinner flasks were run over 8–14 days, depending on the cell line. Although growth rates did not exceed those of the adherent system, maximum cell density in suspension surpassed the adherent baseline for two of the three lines. For the self-assembled spheroid strategy, adherent-culture data on growth rate, doubling time, and specific metabolic consumption/production rates informed two design decisions: an enriched medium targeting the nutrients most depleted during growth, and a fed-batch schedule triggered by measured nutrient thresholds. This data-driven design enabled cultures to reach a maximum cell density triple that of adherent culture, extend culture duration up to 150 h while maintaining viability above 80%, and keep doubling times within the adherent baseline range while preserving the cells' metabolic state. The resulting lysates showed higher levels of dendritic cell maturation markers than the adherent-derived reference, confirming the preservation and enhancement of their immunostimulatory properties. In both cases, systematic quantitative characterization, together with the suspension format itself, enables turning these cell line cultures into a manufacturable process. This characterization links the study of each line's growth requirements and response, to the operating conditions that make scale-up feasible, paving the way toward a manufacturable melanoma vaccine.

The application of engineering biology to prokaryote cell types is promoted as enabling the generation of new processes/products in a revolutionary and sustainable manner. Whilst the same experimental principles and technologies should be equally applicable to mammalian cell biofactories, the complexities and redundancies in mammalian cells have limited progress towards radical engineered enhancements in manufacturing efficiency. This presentation will develop the principles by which cell engineering strategies are developed for enhancement of the biomanufacturing efficiency to mammalian cells, exemplified by specific outcomes of studies on CHO cells in our laboratory.

Biotechnology innovation in Latin America faces the challenge of translating scientific findings into products that can advance to clinical trials, technology transfer, and commercialization. In this context, regulation should no longer be perceived as a barrier, but rather as a strategic tool that can facilitate development and reduce risks. This presentation will explore key global trends in cell culture, advanced therapies, and biological products, highlighting how an early regulatory strategy can accelerate technological maturation and market entry. Through real-world examples and the analysis of regulatory cases, mechanisms to strengthen the competitiveness of researchers, startups, and biotechnology companies in Latin America will be discussed.