Work presented at this edition

• Posgrado en Ciencias Bioquímicas. Facultad de Química, Universidad Nacional Autónoma de México, 04510, Ciudad de México, México
• Houston Methodist Research Institute, Department of Radiology, 77030, Houston, Texas, U.S.
• Subdirección de Investigación Básica, Instituto Nacional de Cancerología, 14080, Ciudad de México, México.

* Presenting author

Outcomes for advanced gastric cancer remain poor. Emerging approaches, such as Chimeric Antigen Receptor (CAR) T-cell therapy, have generated promising results, but concerns about on-target, off-tumor toxicities persist. CD44 variant 6 (CD44v6), an alternative splicing isoform absent in most normal tissues, is upregulated in gastric cancer. Here, we engineered second-generation CAR T cells derived from the antibody BIWA 8 and evaluated their antitumor activity in two intraperitoneal gastric cancer models. Primary T cells were isolated from healthy-donor buffy coats, activated with human T-Activator CD3/CD28 Dynabeads, and transduced with lentiviral particles. The final product was harvested on day 10. CAR functionality was assessed in vitro with tumor cells expressing CD44v6. Six- to eight-week-old male NSG mice were engrafted intraperitoneally with either the gastric cancer cell line SNU638 or NCI-N87, both expressing GFP and Luciferase. T cells were administered intravenously. Tumor burden was determined by whole-body bioluminescence imaging. In vitro assays demonstrated CAR functionality based on binding and activation assays. CAR T cells also mediated specific antigen-dependent killing. Mice engrafted with either SNU638 or NCI-N87 that received CAR T cells showed significant suppression of tumor burden and a higher survival rate compared to control groups. Our results suggest that CD44v6 could be considered an antigen suitable for generating CAR-based therapies.

• Universidad de Costa Rica

* Presenting author

Organoids are three-dimensional (3D) in vitro cultures that recapitulate the architecture, cellular composition, functionality, and physiological characteristics of their tissue of origin. They are generated from stem cells, capable of self-renewal, proliferation, and differentiation under defined culture conditions. Organoids bridge reductionist two-dimensional cultures and animal models, but implementing them requires reproducible access to extracellular matrices, conditioned media, and tissue-processing workflows. The aim of this project was to establish the first murine colon organoid platform in Costa Rica, contributing to the development of advanced organoid-based research capacity in Central America and providing a novel resource for biomedical research, including studies of Clostridioides difficile infection and colorectal carcinogenesis. Colons were isolated from C57BL/6 mice and flushed with phosphate-buffered saline (PBS) containing antibiotics. The tissue was cut into small fragments and mechanically dissociated in 10 mM EDTA to isolate intestinal crypts. Crypt-enriched fractions were collected by centrifugation in 0.1% bovine serum albumin (BSA), filtered through a 70-µm cell strainer, and centrifuged to obtain a crypt pellet. The crypts were embedded in Geltrex and cultured in an in-house-produced L-WRN conditioned-medium formulation (Advanced DMEM/F12 + 1 X Glutamax + 1X Penicillin/Streptomycin + 20 % Fetal Bovine Serum, supplemented with 10 µM ROCK inhibitor). Cultures were passaged weekly, and morphology was assessed by microscopic observation and photographic documentation. Passage-5 organoids were harvested in TRIzol for downstream RNA isolation. During the first passage, cellular debris was observed within the organoid lumen, likely reflecting the initial tissue adaptation to the culture conditions. However, organoid numbers progressively increased with subsequent passages, demonstrating successful expansion and long-term maintenance. Furthermore, organoids remained morphologically stable, showing a rounded shape, double brighter membrane, homogeneous size, and a small lumen, and viable after two weeks without passaging, indicating the robustness of the culture conditions. We established a locally implemented murine colonic organoid workflow combining primary crypt isolation, in-house L-WRN medium production, sustained propagation, and molecular sample recovery. To our knowledge, this is the first murine colonic organoid platform established in Costa Rica, and one of the few installed in Latin America. This platform creates the technical foundation for controlled studies of intestinal host–pathogen interactions and carcinogenic processes while expanding regional access to advanced 3D culture technologies.

• Alternative Methods Section, Laboratory of Biological Testing, University of Costa Rica, 11501, Costa Rica
• Institute of Health Investigations, University of Costa Rica, 11501, Costa Rica
• Alternative Methods Section, Laboratory of Biological Testing, University of Costa Rica; Institute of Pharmaceutical Investigations, University of Costa Rica, 11501, Costa Rica

* Presenting author

Cell culture-based assays are valuable tools for product safety assessment and quality control because they provide reproducible and sensitive methods for detecting biological responses while supporting New Approach Methodologies (NAMs) that reduce animal use in toxicological testing. Despite the growing popularity of electronic cigarettes, information on the biological effects of commercially available e-liquids in Costa Rica remains limited. This study evaluated the cytotoxic effects of commercial banana-watermelon flavored e-liquids with and without nicotine using murine fibroblasts (L929 ATCC CCL-1) and macrophages (RAW 264.7 ATCC TIB-71). The objective was to assess the suitability of these in vitro models for product safety evaluation and quality control. L929 fibroblasts and RAW 264.7 macrophages (2 × 10⁵ cells/mL) were exposed for 48 hours to serial dilutions of commercial e-liquids containing or lacking nicotine. Cell viability was measured using the MTT assay according to internationally recognized in vitro cytotoxicity testing principles (OECD Guideline 129, 2010). All experiments were conducted in triplicate to ensure reproducibility. Both cell lines exhibited concentration-dependent responses, with cell viability increasing as e-liquid concentrations decreased. Exposure to both formulations reduced cell viability, although nicotine-free e-liquids consistently produced greater cytotoxicity than nicotine-containing formulations. Based on the dose-response curves, the estimated IC₅₀ for nicotine-free formulations was within the tested concentration range, whereas nicotine-containing formulations did not reduce viability below 50%, indicating an IC₅₀ above the highest concentration evaluated. L929 fibroblasts were more sensitive to the tested formulations than RAW 264.7 macrophages. In macrophages, viability values exceeded 100% at higher dilutions, suggesting increased metabolic activity associated with cellular activation rather than enhanced proliferation. The comparable response patterns observed for both nicotine-containing and nicotine-free formulations indicate that components other than nicotine, including propylene glycol, vegetable glycerin, or flavoring agents, may play a major role in the cytotoxic effects observed. Murine cell culture models proved to be reliable tools for the preliminary safety assessment and quality control of commercial e-liquids. The combined use of fibroblasts and macrophages provides complementary information by evaluating both cytotoxicity and metabolic responses. These findings support the application of standardized in vitro methodologies as effective screening tools and reinforce the importance of cell-based assays within NAMs for evaluating product safety before more complex biological testing.

• CONICET, UNL, FBCB, Centro Biotecnológico del Litoral (CBL), Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina
• UNL, FBCB, Centro Biotecnológico del Litoral (CBL), Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina

* Presenting author

Canine atopic dermatitis (CAD) is a multifactorial inflammatory skin disease characterized by chronic pruritus, skin barrier dysfunction, and reduced quality of life. Interleukin-31 (IL-31), a T helper 2-associated cytokine has been found to be one of the key pruritogens in several species, including dogs. The binding of IL-31 to its specific receptors, IL-31RA and OSMRβ, triggers the strong activation of signaling pathways involved in itch perception and inflammation. Consequently, IL-31 has emerged as an attractive therapeutic target for the treatment of CAD. Our research group is developing a recombinant Fusion Protein designed to sequester canine IL-31 and prevent its interaction with the IL-31 receptor complex. The preclinical development and functional validation of this biotherapeutic require a reliable source of biologically active recombinant canine IL-31 (rcIL-31) for analytical and binding assays. To support these studies, the aim of this work is to develop a production process for rcIL-31 and to establish its structural and functional characterization as a critical reagent for the evaluation of our novel Fusion Protein. A stable CHO-K1 producer cell line was generated by lentiviral transduction followed by puromycin selection and adapted to suspension culture to enable scalable protein production. Recombinant cIL-31 was purified by immobilized metal affinity chromatography (IMAC). Structural characterization was performed using SDS-PAGE, Western blot, thermal shift assays, and circular dichroism spectroscopy to assess purity, identity, conformational integrity, and thermal stability. Receptor-binding assays using DH82 cells and confocal fluorescence microscopy were performed to evaluate protein functionality. The interaction between rcIL-31 and the Fusion Protein was assessed by thermal shift assays. A cell-based reporter assay using DH82 cells is currently under development to validate the biological activity of rcIL-31. Recombinant cIL-31 was successfully purified and showed appropriate purity, identity, conformational integrity, and thermal stability. Receptor-binding assays confirmed the structural functionality of rcIL-31. Analysis of concentration-dependent thermal shifts (ΔTm) generated binding curves consistent with a non-cooperative interaction, confirming specific recognition of IL-31 by the Fusion Protein. Overall, this work successfully established a scalable production process and a comprehensive characterization strategy for biologically active rcIL-31 and demonstrated its structural integrity and functionality. This achievement provides an essential reagent for the analytical and preclinical evaluation of our IL-31-neutralizing Fusion Protein as a promising therapeutic approach for canine atopic dermatitis.

• a Department of Chemical Engineering, Biotechnology, and Materials, Universidad de Chile, Santiago, Chile. b Mammalian Cell Culture Lab, Universidad de Chile, Santiago, Chile.
• a Department of Chemical Engineering, Biotechnology, and Materials, Universidad de Chile, Santiago, Chile. b Mammalian Cell Culture Lab, Universidad de Chile, Santiago, Chile. c Institute of Cell Dynamics and Biotechnology (ICDB), Santiago, Chile.

* Presenting author

Mesenchymal stem cells (MSCs) hold great potential for biomedical and biotechnological applications due to their capacity to differentiate into multiple cell lineages and secrete bioactive molecules. However, their clinical and industrial translation is limited by the challenges of large-scale expansion, which requires culture conditions that support proliferation, preserve functionality, and accommodate their adherent nature. Spinner flask bioreactors combined with microcarriers represent a promising strategy to scale up MSC production for therapeutic use, which demands high cell doses, by enabling suspension-like growth while increasing the available surface area. Nevertheless, the costs associated with this approach remain high, requiring the optimization of culture conditions to improve process efficiency and reduce operational costs without compromising critical quality attributes of the cells. The objective of this study is to design and evaluate feeding strategies for MSC culture in spinner flasks with microcarriers, based on experimentally determined metabolic criteria. Metabolic characterization of MSCs was performed in plate cultures, at different glucose and lactate concentrations to identify growth-limiting thresholds. Kinetic parameters were determined from experimental data, and metabolic criteria were established for the design of feeding strategies. In addition, amino acid consumption and production profiles were characterized to identify critical amino acids in metabolism. The conventional culture scheme in spinner flask cultures, using a fixed microcarrier surface area and daily medium replacement, was compared with two alternative strategies: increasing microcarrier surface area in proportion to the effective cell growth rate, and an adaptive feeding strategy based on previously defined metabolic criteria. Scaling microcarrier surface area according to growth rate was a key factor driving increases of up to 54.3 percent in cell proliferation, compared with the conventional scheme, from approximately 103 million to 160 million cells. The adaptive feeding strategy based on lactate and glucose concentrations reduced fresh culture medium consumption by 21.1 percent relative to the conventional scheme. Amino acid profiling revealed differential consumption and production patterns among specific amino acids, pointing to additional metabolic targets relevant for future feeding strategies and process scale-up. Results show that surface area scaling based on growth rate improved MSC proliferation. In parallel, metabolic criteria for adaptive feeding reduced medium consumption in microcarrier-based cultures. These findings also highlight the potential operational advantages of spinner flask cultures for reducing manual handling and facilitating MSC expansion at larger scales. Overall, this work provides a foundation for the development of more efficient, cost-effective and scalable MSC expansion processes.

• Laboratorios de Especialidades Inmunológicas S.A. de C.V.

* Presenting author

Regulatory restrictions on animal use for safety testing have driven the development of alternative in vitro methods. Among these, reconstructed human epidermis (RHE) models are widely used to assess skin irritation and corrosion. However, these models commonly use primary keratinocytes, requiring continuous donor supply and specialized reagents for cell isolation and expansion, thereby increasing production costs. In this study, an RHE model based on HaCaT cells was developed, offering continuous availability and ease of proliferation. The model was generated using HaCaT keratinocytes cultured under an air–liquid interface (ALI). Cell differentiation and formation of a stratified epidermal structure with barrier function were promoted using specific growth factors and supplements. Subsequently, the model was evaluated according to OECD Test Guidelines 431 and 439 for in vitro skin corrosion and irritation, respectively. Under the established conditions, a stratified structure with morphological and functional characteristics of human epidermis was obtained. The model met applicable acceptance criteria in OECD Test Guidelines 431 and 439. For skin irritation, two products previously evaluated using the EpiDerm® model referenced in the Guidelines showed the same skin irritation classification when tested with the developed model. For skin corrosion, 12 proficiency substances (four Category 1A, four non-corrosives and four Category 1B/1C) were evaluated according to OECD TG 431. Of these, 4/4 Category 1A substances were correctly classified, and 4/4 non-corrosive substances were also correctly classified, while only 1/4 Category 1B/1C substances were correctly subcategorized. Immunohistochemical staining suggested incomplete or heterogeneous epidermal differentiation, while hematoxylin and eosin staining indicated suboptimal stratum corneum formation. These findings are consistent with previous reports describing limitations in terminal differentiation and stratum corneum formation in HaCaT-based epidermal models. Despite this limitation, the current barrier function of the model was sufficient to distinguish corrosive from non-corrosive substances. Further optimization is required to achieve reliable subcategorization of corrosive substances. The results evidence the feasibility of using HaCaT cells to generate a reconstructed human epidermis model for the evaluation of skin corrosion and irritation. The model demonstrated barrier function and produced classification results consistent with validated models for the substances and products evaluated, supporting its potential application in quality control. The use of a continuously available cell line may reduce production costs and dependence on primary cell donors. Further optimization of stratum corneum formation and expansion of the validation dataset are required to improve model performance and enable reliable subcategorization of corrosive substances.

• Laboratory of Biotechnology and Biopharmaceuticals, Department of Physiopathology, Faculty of Biological Sciences, Universidad de Concepción, Edmundo Larenas 234, Concepción, Chile.

* Presenting author

Historically, the control of bovine viral diarrhea virus (BVDV) has relied on vaccination programs using inactivated or modified-live vaccines. However, the high genetic and antigenic diversity of the virus limits the cross-protection provided by some commercial formulations and may restrict the magnitude and duration of the immune response elicited. In this context, a pentavalent vaccine candidate based on five recombinant E2 protein variants from different BVDV subgenotypes was proposed. The aim of this study was to develop a reproducible laboratory-scale production platform using CHO-K1 cells adapted to suspension culture to produce the five antigens and to validate their immunological functionality. Five CHO-K1 cell lines, each producing a recombinant E2 antigen, were adapted to serum-free suspension culture through the progressive replacement of RPMI with EX-CELL® medium. Production was performed in batch mode using a 250 mL spinner bioreactor. The antigens were purified by immobilized metal affinity chromatography and anion exchange chromatography. Functional characterization was performed using indirect ELISA to assess antigen recognition by sera from naturally infected cattle, virus neutralization assays against BVDV subgenotypes 1b, 1c, 1d, and 1e in MDBK cells, and cross-reactivity assays among the five antigens using sera from immunized mice. All five cell lines were successfully adapted to suspension culture without compromising antigen expression. Spinner culture showed reproducible kinetics across three consecutive batches for each antigen, with maximum cell densities approaching 4 × 10⁶ cells/mL and viability ≥90% throughout the production process. Purified antigens exhibited purity exceeding 90%. Functional characterization showed that all five recombinant antigens were recognized by antibodies present in sera from naturally infected animals, indicating the preservation of native antigenic epitopes. Sera from immunized animals exhibited neutralizing activity against BVDV subgenotypes 1b, 1c, 1d, and 1e (P = 0.0095), and cross-reactivity among the five antigens was demonstrated, with both homologous and heterologous recognition. Overall, the established platform enables the reproducible production of recombinant E2 antigens with adequate quality. The subgenotypes and epitopes recognized by antibodies from naturally infected animals, elicited neutralizing activity against multiple BVDV subgenotypes and exhibited cross-reactivity among one another. These findings support their potential use as components of a pentavalent vaccine candidate designed to provide broad antigenic coverage against the genetic diversity of BVDV.

• Universidad de Concepción
• Servicios Médicos y Oftalmológicos MyS Limitada

* Presenting author

Nerve growth factor (NGF) is a neurotrophin essential for neuronal survival, differentiation, and regeneration. Its mature bioactive form, β-NGF, is a 26-kDa homodimer and is currently used as treatment for neurotrophic keratitis. However, commercial production in Escherichia coli is limited by inclusion body formation, requiring protein solubilization, in vitro refolding, enzymatic cleavage, and endotoxin (LPS) removal. In contrast, Chinese hamster ovary (CHO) cells provide an endotoxin-free expression platform capable of proteolytic processing and secretion of recombinant proteins in their biologically active form. This study evaluated the effect of the CHO host cell line on the maturation and biological activity of recombinant NGF. Stable CHO-K1 and CHO-S cell lines were generated using the PiggyBac transposon system. Adherent CHO-K1 cells were transfected with polyethyleneimine (PEI), whereas suspension CHO-K1 and CHO-S cells were electroporated. Following G418 selection and fluorescence-activated cell sorting (FACS) to enrich high-producing populations, cells were cultured under serum-free conditions. Culture supernatants were analyzed by Western blotting, recombinant NGF was purified by immobilized metal affinity chromatography (IMAC), and biological potency was determined using a TF-1 cell proliferation assay. Western blot analysis revealed marked differences in proteolytic processing between the two CHO cell lines. Both adherent and suspension CHO-K1 cells efficiently processed pro-NGF through endogenous furin activity, secreting exclusively mature NGF (~14 kDa per monomer). In contrast, CHO-S secreted a mixture of pro-NGF and mature NGF, indicating incomplete precursor processing. Accordingly, NGF produced in CHO-K1 exhibited biological potency comparable to the E. coli-derived reference standard (EC₅₀ = 0.84 and 0.87 ng/mL, respectively), whereas CHO-S-derived NGF showed reduced potency (EC₅₀ = 1.4 ng/mL). Because pro-NGF displays biological properties distinct from mature NGF and can activate apoptosis-associated signaling pathways, its presence represents an undesirable quality attribute for therapeutic applications. These findings demonstrate that the proteolytic processing capacity of the host cell is a critical quality attribute that determines both the molecular composition and biological activity of recombinant NGF. CHO-K1 emerges as a promising platform for the serum-free production of mature, functional, endotoxin-free NGF, providing a potentially more efficient strategy for the manufacturing of ophthalmic biotherapeutics.

• Universidad Tecnológica de La Habana José Antonio Echeverría,Centro de Inmunología Molecular

* Presenting author

The expiration of the pembrolizumab patent in 2028, after reaching sales exceeding USD 29 billion in 2024, represents a strategic opportunity for the development of high-impact biosimilars in oncology. In this context, the anti PD1 biosimilar 2C12 was developed at the Center for Molecular Immunology, aimed at expanding access to immuno-oncology therapies and strengthening national production capacity. As part of its clinical development, technology transfer from the pilot plant to the production plant was carried out in 2025, with the purpose of ensuring the supply required for a Phase III clinical trial.The objective was not limited to reproducing the process at a larger scale, but rather to generate experimental evidence that would allow predicting and controlling culture behavior up to the industrial scale. To this end, cell growth and production were initially characterized through batch culture, determining a maximum cell concentration of 4.06 × 10⁶ cells/mL, a maximum specific growth rate of 0.026 h⁻¹, a specific productivity of 5.73 mg/cell/h, and a maximum productivity of 143.73 mg/L/d. Based on this characterization, the culture behavior was evaluated in a continuous process at 80 L scale under two dilution rates (0.008 h⁻¹ and 0.006 h⁻¹). Although the values obtained were lower than those reported for other cell lines, the culture maintained a viability above 97% and stability over multiple generations, providing the experimental basis for defining industrial operating conditions. A validated mathematical model also made it possible to predict cell behavior in perfusion and reduce the risks associated with scale-up. The implementation of the process in a 1500 L bioreactor made it possible to reach a steady state of 2.17 × 10⁷ cells/mL, with viability above 90% and a CSPR of 53.26 pL/cell/d. Specific productivity reached 11.42 mg/cell/h, approximately double that obtained in batch, while volumetric productivity was 96.59 mg/L/d, 84% higher than previously recorded for biosimilars at the center. The fermentation process accounted for 55% of the total production cost, within the range reported internationally for biosimilars. These results demonstrated the feasibility of the scale-up and lay the foundations for robust and competitive industrial production of the anti PD1 biosimilar 2C12.

• Mammalian Cell Culture Lab MCCL, Department of Chemical Engineering, Biotechnology and Materials, University of Chile. Av. Beauchef 851, 8370458, Santiago, Región Metropolitana, Chile.
• Oncobiomed Advanced Cell Technology. Málaga 85, 7550144, Las Condes, Región Metropolitana, Chile.

* Presenting author

Cutaneous melanoma is a highly aggressive neoplasm. To generate effective antitumoral responses, Oncobiomed's TAPCells® technology employs antigen-presenting cells stimulated with tumor lysates from the MCL 1, MCL 2, and MCL 3 cell lines. Given that their adherent growth limits scalability, the transition to suspension cultures using microcarriers was evaluated. This work aims to design a suspension culture strategy on Cytodex 1 in repeated-batch mode and characterize its metabolic behavior to generate lysates with immunogenic properties. Human melanoma cell lines MCL 1, MCL 2, and MCL 3 were cultured in spinner flasks with RPMI 1640 medium and Cytodex 1 for 8, 10, and 14 days, replacing the medium every 60 h, 24–48 h, and 48–120 h, respectively. Cell concentration and metabolic profiles were measured daily, aiming to maintain glucose above 5 mM and lactate and ammonium values below 10 mM and 5 mM, respectively. Microcarrier-adhered cells were subjected to heat shock, nitrogen lysis, filtration, and cryopreservation to produce the lysate. To evaluate the lysate’s immunogenicity in dendritic cells, the assessment of markers CD83, CD86, CCR7, MHC I, and MHC II is currently being established. Although growth rates did not exceed those of adherent culture, MCL 1 and MCL 2 reached a higher maximum surface cell density in suspension culture. Medium exchange frequencies in MCL 1 and MCL 2 caused glucose and glutamine depletion along with an excess of lactate (>10 mM), restricting proliferation. MCL 3 exhibited slow initial growth that recovered upon maintaining glucose between 5 and 8 mM. Regarding medium requirements, MCL 1 is estimated to require a supplementation of 18% cysteine and 12% serine, whereas MCL 2 requires 25% cysteine. Glutamine was the most consumed amino acid, exhibiting the highest specific consumption rate at 0.052 mmol/10⁹ cells/h in MCL 3, followed by arginine with a peak consumption rate of 0.011 mmol/10⁹ cells/h in MCL 2. Preliminary results from the lysate's efficacy evaluation demonstrated a morphological shift toward an extended phenotype characteristic of mature dendritic cells upon exposure to the lysate. Culture on Cytodex 1 increased the surface cell density for MCL 1 and MCL 2 compared to adherent culture. Adjusting the medium exchange frequency, supplementing critical amino acids (cysteine and serine), and increasing the seeding density for MCL 3 constitute the next steps to advance toward scalable TAPCells® production, moving this therapeutic platform closer to clinical application in melanoma treatment.

• Programa de Doctorado en Farmacología, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, México · Instituto de Biología Molecular en Medicina y Terapia Génica, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, México
• Instituto de Biología Molecular en Medicina y Terapia Génica, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, México
• Instituto de Investigación en Enfermedades Crónico Degenerativas, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, México
• Instituto de Biología Molecular en Medicina y Terapia Génica, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, México · Tecnológico de Monterrey, EMCS, Zapopan, Jalisco, México

* Presenting author

Pulmonary fibrosis is a progressive disease characterized by excessive deposition of extracellular matrix and disruption of normal lung architecture. Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, has emerged as a potential therapeutic target due to its role in regulating inflammatory and profibrotic signaling pathways. This study evaluated the antifibrotic effect of the adenoviral vector AdSIRT1 in a murine model of bleomycin-induced pulmonary fibrosis. Pulmonary fibrosis was induced in mice by a single transoral administration of 0.17 mg bleomycin in 60 μL of injectable water. The experimental design included 24 mice distributed among six groups. AdSIRT1 was produced as a first-generation recombinant adenoviral vector and obtained through amplification in HEK-293 cells, followed by viral particle recovery and purification using cesium chloride density-gradient ultracentrifugation, based on a previously established methodology. Viral preparations were evaluated by particle and infectious-unit titration. Lung tissue was subsequently analyzed macroscopically and histologically. Masson's trichrome and Picrosirius Red staining were used to evaluate extracellular matrix and collagen deposition, respectively, while immunohistochemistry for α-SMA was performed to assess myofibroblast activation. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test or Kruskal-Wallis followed by Dunn's post hoc test, with p<0.05 considered statistically significant. Bleomycin administration produced marked pulmonary alterations, characterized by increased extracellular matrix deposition, collagen accumulation, and enhanced α-SMA immunoreactivity. Treatment with AdSIRT1 significantly reduced fibrotic changes compared with the bleomycin group. Masson's trichrome staining demonstrated a significant reduction in extracellular matrix deposition following AdSIRT1 treatment, while Picrosirius Red staining showed a significant decrease in collagen deposition. In addition, AdSIRT1 markedly reduced α-SMA immunoreactivity, indicating decreased activation of collagen-producing myofibroblasts. Macroscopic examination also showed an improvement in pulmonary morphology after AdSIRT1 treatment. These findings demonstrate that AdSIRT1 exerts a significant antifibrotic effect in bleomycin-induced pulmonary fibrosis, characterized by reduced collagen and extracellular matrix deposition, decreased myofibroblast activation, and partial restoration of lung architecture. The results support SIRT1-targeted gene therapy as a promising strategy for the development of novel approaches to pulmonary fibrosis.

• UNL, CONICET, FBCB, Centro Biotecnológico del Litoral, Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina.
• UNL, FBCB, Centro Biotecnológico del Litoral, Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina.

* Presenting author

Long-term stability of transgene expression represents one of the greatest challenges in developing mammalian cell lines for recombinant therapeutic protein production. Viral promoters such as CMV are still the most widely used regulatory elements, although maintaining stable expression during prolonged culture remains difficult. Endogenous promoters, which respond to the cell's own transcriptional machinery and culture conditions, offer an attractive alternative. Building on our previous characterization of PromA and PromB, which supported stable fluorescent reporter expression and efficient production of a difficult-to-express recombinant protein, this study aimed to expand the repertoire of endogenous CHO-K1 promoters and evaluate enhancer fusion as an additional strategy to modulate promoter activity. Three novel endogenous promoters, PromD, PromE and PromF, were isolated from highly expressed CHO-K1 genes in bioreactor cultures and characterized in silico for putative transcription factor binding sites. Their activity was evaluated using ZsGreen1 in transiently transfected CHO-K1, HEK293T and BHK-21 cells and in stable CHO-K1 cell lines. Then, PromD, PromE and PromF, together with PromA and PromB, were fused to the cytomegalovirus enhancer (ECMV) and re-evaluated following the same approach. CMV+ECMV and EF1α served as controls. In both stages, transient promoter activity was assessed by fluorescence microscopy and flow cytometry. Two sets of stable CHO-K1 lines were generated for each construct and monitored for 60 generations. Transcript levels were quantified by RT-qPCR at representative generations. Initially, native PromD exhibited the highest activity across CHO-K1, HEK293T and BHK-21 cells, reaching levels similar to the controls. PromE displayed intermediate activity, whereas PromF consistently yielded the lowest expression. In two sets of stable CHO-K1 lines, PromD and PromE sustained expression comparable to PromA, PromB and EF1α, while PromF and CMV+ECMV showed similarly low expression. In the second stage, fusion to ECMV did not significantly improve transient promoter activity for any of the five endogenous sequences. Both sets of stable CHO-K1 lines yielded consistent results, with flow cytometry and RT-qPCR in excellent agreement. In some cases, ECMV-fused constructs showed a trend toward higher expression than their native counterparts, although these differences were not statistically significant. This study substantially expands the repertoire of endogenous CHO-K1 promoters available for mammalian expression. PromA, PromB, PromD and PromE represent a competitive set of endogenous regulatory elements supporting robust, sustained transgene expression. Beyond the trends observed with ECMV-fused variants, the strong activity of the native promoters highlights their potential for developing high-performing CHO platforms for therapeutic protein production.

• Programa de Maestría en Ciencias Bioquímicas, Universidad Nacional Autónoma de México, Circuito de Posgrado, Edificio B, Primer Piso, Ciudad Universitaria, Alcaldía Coyoacán, C.P. 04510, Ciudad de México, México.
• Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, The Netherlands
• São Carlos Institute of Physics, University of São Paulo, São Carlos, Brazil
• Departamento de Farmacia, Universidad Nacional Autónoma de México, Ciudad de México, México
• Instituto de Investigación en Materiales, Universidad Nacional Autónoma de México, Ciudad de México, México.
• Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Mexico.
• Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, Alcaldía Coyoacán C.P. 04510, Ciudad de México, México.

* Presenting author

In vitro blood–brain barrier (BBB) models are valuable tools for evaluating drug delivery strategies and their interactions with brain endothelial cells. Curcumin is a hydrophobic compound with potential applications in the central nervous system, although its physicochemical properties limit its delivery. Menthol has been proposed as a BBB permeation enhancer. This study used a human brain endothelial model to evaluate the effects of free and nanoparticle-encapsulated menthol on barrier integrity and the transport of curcumin-loaded polymeric nanoparticles. hCMEC/D3 human brain microvascular endothelial cells were cultured for five days on collagen-coated Transwell® inserts. Barrier formation was monitored by microscopy and transendothelial electrical resistance (TEER). Curcumin-loaded PVM/MA nanoparticles were compared with curcumin–menthol-loaded nanoparticles and curcumin-loaded nanoparticles co-administered with free menthol (200 µg/mL). Transport was evaluated by curcumin-associated fluorescence in the basolateral compartment, while endothelial-associated fluorescence was examined by optical and confocal microscopy. hCMEC/D3 cells formed a confluent monolayer, with TEER increasing from approximately 20–25 Ω·cm² to >50 Ω·cm² by day five. Encapsulated menthol did not enhance basolateral recovery, after 72 h, cumulative recovery was 39.7 ± 3.7% for curcumin-loaded nanoparticles and 27.9 ± 0.4% for curcumin–menthol-loaded nanoparticles. Free menthol significantly decreased TEER but did not increase basolateral recovery. Release studies showed that the nanoparticles released a substantial fraction of their curcumin cargo, which may have contributed to the progressive loss of nanoparticle-associated fluorescence over time. However, curcumin remained associated with the nanoparticles after 24 h, supporting their continued fluorescence-based tracking during the transport experiment. Persistent fluorescence was also detected in the endothelial layer after extensive washing, suggesting endothelial cargo retention. Menthol-induced barrier modulation did not predict enhanced nanoparticle transport. Curcumin release from the nanocarriers may partially explain the progressive loss of nanoparticle-associated fluorescence, although the persistence of curcumin cargo after 24 h indicates that fluorescence remained useful for nanoparticle tracking. These findings highlight the importance of considering drug release and endothelial retention alongside TEER and direct transport measurements when evaluating nanocarrier interactions with in vitro BBB models.

• Universidad Técnologica de La Habana, Centro de Inmunología Molecular
• Centro de Inmunología Molecular

* Presenting author

Recombinant human erythropoietin (rhu-EPO) and its hyposialylated variant NeuroEPO are biopharmaceuticals of great relevance produced at the Center for Molecular Immunology. rhu-EPO is used to treat anemia in chronic kidney disease, while NeuroEPO has shown neuroprotective properties in diseases such as Alzheimer's and Parkinson's. The current process scheme is oriented towards obtaining highly sialylated isoforms, which limits the proportion of hyposialylated isoforms required for NeuroEPO. The aim of this work was to determine the influence of modifications in the operating conditions during fermentation on NeuroEPO production, seeking to increase its productivity without compromising culture stability. A control run was compared with an experimental run in an industrial-scale bioreactor operated in perfusion mode. Four scenarios were defined in pseudo-steady states (coefficient of variation of viable cells <10% for at least 3 days). Progressive changes included temperature (36.5-37.0 °C), dissolved oxygen (DO) (50-10%), agitation (110-140 min⁻¹) and purge rate (180-120 L/d). Kinetic parameters, volumetric productivities (mg/L/d) and isoform profiles were determined for the control run and the scenarios evaluated in the experimental run. The results showed that the modifications, under all conditions evaluated, did not affect culture stability (cell concentrations ranged from 1.21 to 1.45×10⁷ cells/mL and average cell viabilities exceeded 95.0%). In the experimental run, the total mass of NeuroEPO increased by 55.4% and a maximum percentage of hyposialylated isoforms of 57.0% was achieved. NeuroEPO volumetric productivity increased by 23.6%, while total rhu-EPO productivity remained at comparable levels. Scenario 4 proved to be the most favorable condition for NeuroEPO production. Total production costs increased by 26.1%, while the unit cost of NeuroEPO decreased by 24.3%, confirming the technical and economic efficiency of the implemented modifications. The operational modifications of Scenario 4 (temperature = 37.0 °C, DO = 10%, agitation = 140 min⁻¹, purge = 120 L/d) allowed a significant increase in NeuroEPO productivity and the proportion of hyposialylated isoforms, while maintaining cell stability. The economic analysis confirms the technical and economic viability of this strategy, reducing the unit cost of the product of greatest therapeutic interest. These results provide a solid foundation for the industrial optimization of the NeuroEPO production process.

• Pontificia Universidad Católica de Valparaíso

* Presenting author

The continuously increasing prevalence of Inflammatory Bowel Diseases (IBD) highlights the limited effectiveness of current conventional treatments in addressing the highly multifactorial causes of this chronic condition. Within this clinical context, advanced therapies based on mesenchymal stem cells (MSCs) are emerging as a promising alternative, primarily due to their well documented immunomodulatory capacity. Specifically, the targeted activation of MSCs via Toll-like receptor 3 (TLR3) using Poly(I:C) has been identified as a strategic method to enhance their anti-inflammatory phenotype. Consequently, this study aimed to address the production of conditioned medium (CM) derived from TLR3-activated MSCs, comprehensively evaluating the impact of distinct culture configurations namely 2D monolayers and 3D spheroids on their glycolytic profile and the secretion of critical soluble anti-inflammatory factors like interleukin-10 (IL-10). Umbilical cord derived MSCs (passage 6) were incubated under standard conditions (5% CO2, 21% O2) in high glucose complete DMEM (10% FBS, 1% antibiotics). For the 2D configuration, cells in T-25 flasks were stimulated for one hour with Poly(I:C) upon reaching 80% confluence. For the 3D system, spheroids comprising 20,000 or 40,000 cells were generated using the hanging drop method and inoculated into ULA plates at densities of 4 and 16 spheroids per well. The CM collected from both systems at 24 and 48 hours revealed insightful results. Spheroid diameters averaged 554±31 µm for the 20,000 cell group and 608±37 µm for the 40,000 cell group. While the metabolic index (ΔLactate/ΔGlucose) for 2D cultures sat slightly above 1, the 3D spheroids consistently maintained indices between 0.4 and 1.2. Crucially, regarding the secretory profile, IL-10 remained undetectable in all 2D cultures and low density (4 spheroid) wells. However, within the high density 16 spheroid configuration at 48 hours, smaller spheroids demonstrated vastly superior performance, secreting 0.83 pg/mL with Poly(I:C) and 0.7 pg/mL in controls, compared to just 0.09 pg/mL for larger spheroids. This represented a remarkable 21 fold increase in IL-10 secretion between the 24 and 48 hour windows for the smaller spheroids. Ultimately, while Poly(I:C) preconditioning safely maintains structural and metabolic integrity, achieving effective immunomodulatory IL-10 secretion strictly requires high density 3D configurations, proving that optimizing spheroid size and packing is key to maximizing MSC therapeutic potential.

• Upstream Process Manager, Center for Molecular Immunology, Havana, Cuba

* Presenting author

Longtime continuous perfusion for mammalian cells is an engineering challenge but introduce a technological improvement in a validated pharma platform is a real nightmare for any process engineer. Our company based the industrial EPO production in perfusion cultivation by Spinfilters technology. This was old fashioned and poor efficient system compared with the state-of-the-art Hollow Fibers based devices. As part of the company expansion and market growth we looked forward to improving our perfusion technology to a hollow fibers cartridges system. We faced a challenge not only technical but regulatory. The introduction of the new technology switched a hard to control almost empirical operation by a well defined TFF process. Engineering calculations for design a new recirculation system and pump were made as well as automation improvements. Kinetics and Physiological parameter were determined and compare with historical production data. Downscale of downstream process were performed to assess the quality attributes of the molecule an ensure the process validation. Introduction of the new perfusion technology leaded to an improvement in bioreactors automation and feasibility as well as operational cost reductions and process intensification. For process validation three stable reproducible runtimes were performed and compared with historical production data attending not only to upstream parameters but also to molecule quality attributes. Increments in productivities and cost reductions derived from this technology update allow us to spread out the coverage of EPO and ensure it affordability to new emergent markets. Reduction in mechanical complexity diminish the contamination risks and lowered the maintenance costs. Stable controlled operation allows to increase the productivities and yields as well as ensure the quality of the product. Introduction of this technology from the seeding bioreactor shortened the process gaps from upstream steps.

• Escuela de Ingeniería Bioquímica. Laboratorio de Tecnologías en cultivos Celulares. Pontificia Universidad Católica de Valparaíso
• Centro Regional en Estudios de Alimentos Saludables CREAS
• Escuela de Alimentos. Pontificia Universidad Católica de Valparaíso
• scuela de Ingeniería Bioquímica. Laboratorio de Tecnologías en cultivos Celulares. Pontificia Universidad Católica de Valparaíso

* Presenting author

Alzheimer's disease (AD) is characterized by the interaction of oxidative, metabolic, and proteostatic stress that compromises neuronal function. The cholesterol-derived metabolite 27-hydroxycholesterol (27-OHC) and L-kynurenine, a metabolite of the kynurenine pathway, have been identified as relevant cellular stressors capable of increasing reactive oxygen species (ROS) and activating stressresponsive signaling pathways. It has been demonstrated that persistent activation of the protein kinase R-like endoplasmic reticulum kinase (PERK)-integrated stress response (ISR) axis is associated with increased expression of the β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and subsequent amyloid-β accumulation. In this study, we investigated the potential of a bioprocessed coffee-derived phenolic fraction to modulate redox homeostasis, PERK-dependent signaling, and amyloidogenic processing in SH-SY5Y neuronal cells exposed to distinct AD-related stressors. SH-SY5Y cell lines were exposed to 27-OHC, L-kynurenine, or hydrogen peroxide (H₂O₂). Intracellular ROS levels were quantified at the single-cell level using spectral flow cytometry. Functional redox status was assessed by measuring the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR). PERK-dependent ISR activation was evaluated by western blotting for phosphorylated PERK (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and C/EBP homologous protein (CHOP). Downstream effects were assessed by measuring BACE1 expression and intracellular amyloid-β1–40 accumulation. Human fibroblasts were included as a non-neuronal control. Each of the 3 stressors increased intracellular ROS levels and altered antioxidant enzyme activity in SH-SY5Y cells. Treatment with the coffee-derived phenolic fraction at 300 µg/mL reduced ROS levels and restored, at least in part, the activities of SOD, GPx, and GR, with the magnitude of the response varying according to the stress condition. These changes in REDOX status coincided with attenuation of PERK-dependent ISR signaling. In parallel, treatment with the phenolic fraction was associated with reduced BACE1 expression and lower intracellular amyloid-β1–40 accumulation. Taken together, these findings suggest that the bioprocessed coffee-derived phenolic fraction exerts effects beyond a simple reduction in ROS levels, involving both antioxidant enzyme activity and PERK-dependent stress signaling in a neuronal cell model. Its modulation of the redox–PERK axis was accompanied by a reduction in amyloidogenic processing under different AD-related stress conditions. These results support the further evaluation of coffee-derived phenolics as neuroprotective bioactives and potential active ingredients for nutraceutical strategies aimed at promoting healthy aging. Funding: This work was supported by FONDECYT Postdoctoral Project No. 3250833, funded by the National Agency for Research and Development (ANID), Chile.

• Laboratorio de Cultivo de Células Mamíferas MCCL, DIQBM, Universidad de Chile; Center of Interventional Medicine for Precision and Advanced Cellular Therapy IMPACT.
• Center of Interventional Medicine for Precision and Advanced Cellular Therapy IMPACT; Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso.
• Center of Interventional Medicine for Precision and Advanced Cellular Therapy IMPACT; Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso; CREAS, Centro Regional de Estudios en Alimentos Saludables.
• Laboratorio de Cultivo de Células Mamíferas MCCL, DIQBM, Universidad de Chile; Center of Interventional Medicine for Precision and Advanced Cellular Therapy IMPACT; Núcleo Milenio Agronomía Marina de Holobiontes Algales MASH.

* Presenting author

The positive impact on the expansion of mesenchymal stem cells (MSC) derived from human umbilical cord, of antioxidant supplementation of culture medium was verified. A mathematical model was proposed to capture the dynamics of the medium's antioxidant capacity, as determined by FRAP, and to predict its reduction over time. This model, together with nutrient consumption rates, was used to propose an antioxidant replenishment and feeding strategy that helps sustain a favorable redox environment. The results indicate that the oxidative state of the medium is a relevant parameter to optimize culture operation strategies.

• Centro Biotecnológico del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral

* Presenting author

Therapeutic proteins have revolutionized the treatment of diseases; however, their efficacy may be limited by rapid elimination from circulation. The fusion of glycosylation-inducing peptide tags represents a promising glycoengineering strategy to modulate their pharmacological properties. GMOP and mGMOP are peptide tags derived from the N-terminal region of granulocyte-macrophage colony-stimulating factor (GM-CSF), containing four and six potential O-glycosylation sites, respectively. Previous studies using a single copy of either tag fused to recombinant human interferon alpha-2b (rhIFN-α2b) showed increased molecular mass, sialic acid content, and plasma half-life compared with IFN-wt, but no significant pharmacokinetic differences between variants. These findings raised the question of whether increasing the number of fused copies could enhance the pharmacokinetic properties conferred by mGMOP compared with the same number of GMOP copies, as a consequence of the higher O-glycosylation and sialylation. Two rhIFN-α2b variants were generated by fusing three copies of GMOP or mGMOP to the N-terminus and one copy to the C-terminus. CHO producer cell lines were generated and adapted to suspension culture. Recombinant proteins were produced, purified and characterized for glycosylation, structural, and stability properties in comparison with IFN-wt. Biological activity was assessed by antiviral and antiproliferative assays, and plasma stability, pharmacokinetics, and antitumor activity were evaluated. GMOP3-IFN-GMOP and mGMOP3-IFN-mGMOP exhibited increased molecular masses and a shift toward more acidic isoforms compared with IFN-wt. This shift was more pronounced for the mGMOP variant and reflected its higher Neu5Ac content. Both variants preserved the secondary and tertiary structure of rhIFN-α2b while showing increased thermal and plasma stability. However, both hyper-O-glycosylated variants exhibited reduced in vitro biological activity. mGMOP3-IFN-mGMOP displayed a more favorable pharmacokinetic profile than GMOP3-IFN-GMOP, with lower apparent clearance and prolonged elimination half-life. In vivo, both variants reduced tumor growth compared with IFN-wt and PBS controls. Although no significant differences in final tumor volume or weight were observed between the variants, mGMOP3-IFN-mGMOP showed slower tumor growth, lower vascularization, and longer tumor doubling time. Multiple-copy fusion of GMOP and mGMOP represents an effective O-glycoengineering strategy to enhance the stability and pharmacokinetic properties of rhIFN-α2b while preserving its structural integrity and antitumor activity. The higher O-glycosylation and sialylation achieved with mGMOP resulted in a more favorable pharmacokinetic profile and slower tumor growth in vivo, suggesting that prolonged systemic exposure may compensate for the reduced in vitro activity. These findings support GMOP and mGMOP as promising tools for improving the pharmacological properties of therapeutic proteins.

• Centro Biotecnológico del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral.

* Presenting author

Biotherapeutics are complex proteins whose performance may be limited by their short half-life. Glycoengineering is a strategy to optimize these properties through the incorporation of glycans that can increase stability and reduce clearance. In this context, our laboratory developed the peptide tags GMOP and mGMOP, derived from the N terminus of hGM-CSF, which provide 4 and 6 potential O-glycosylation sites, respectively, and share the APAR epitope recognized by mAb CC1H7. This combination confers a bifunctional nature: they modify the pharmacokinetic properties of fusion proteins through O-glycosylation and serve as epitopes for detection, quantification, and purification through epitope-antibody interaction. This bifunctionality was previously demonstrated using interferon-α2b as a model protein. Here, their performance was evaluated in a second therapeutic protein, recombinant human erythropoietin (rhEPO), to establish a versatile platform applicable to other biotherapeutics. Two hyper-O-glycosylated variants, GMOP3-EPO-GMOP and mGMOP3 EPO mGMOP, were designed by incorporating three copies of each peptide at the N-terminus and one at the C-terminus of rhEPO. The variants were produced in CHO cells and purified by immunoaffinity using mAb CC1H7. Characterization included molecular mass, glycosylation profile, circular dichroism, thermal stability and plasma stability. Erythropoietic activity was evaluated in vitro, followed by pharmacokinetic and in vivo potency assessments based on hematological response in mice. The variants were purified by immunoaffinity chromatography using mAb CC1H7, achieving 96–99% purity and 100% recovery. They were detected by western blot and quantified by ELISA. Both exhibited increased apparent molecular mass and enrichment in acidic isoforms while retaining their structural characteristics. Increased melting temperatures and greater resistance to plasma degradation were also observed compared with rhEPO. Although in vitro erythropoietic activity decreased for the modified variants, marked pharmacokinetic improvements were observed. GMOP3-EPO-GMOP and mGMOP3-EPO-mGMOP increased elimination half-life 2.6- and 3.4-fold and reduced apparent clearance 2.5- and 4-fold, respectively. In vivo, both variants showed approximately 2.5-fold greater potency than reference rhEPO, with no significant differences between them. However, the single-dose regimen may have masked differences associated with their distinct pharmacokinetic profiles. Multiple incorporation of GMOP and mGMOP improved rhEPO stability and pharmacokinetic properties, with mGMOP showing a greater impact on half-life and clearance. Validation in a second therapeutic protein, together with the analytical utility demonstrated through mAb CC1H7, reinforces the bifunctional nature of these tags. Our findings support GMOP and mGMOP as versatile platforms for the development, characterization, and production of improved biotherapeutics, with potential applicability across different therapeutic proteins.

• Centro Biotecnológico del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral.
• Biotecnofe S.A.
• Centro de Medicina Comparada, Instituto de Ciencias Veterinarias del Litoral (ICiVet Litoral), UNL, CONICET
• Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral
• Instituto AP de Ciencias Básicas y Aplicadas, Universidad Nacional de Villa María; Instituto de Reproducción Animal Córdoba (IRAC)
• Centro Biotecnológico del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral. Cátedra de Biotecnología Animal. FBCB-UNL

* Presenting author

Recombinant bovine follicle-stimulating hormone (rbFSH) represents a promising alternative to pituitary-derived FSH for bovine superovulation, overcoming limitations associated with batch variability, LH contamination, and short circulating half-life. In previous work, we developed a hyperglycosylated long-acting rbFSH variant (rbFSH LD) by fusing three copies of the O-glycosylated mGMOP peptide to the C terminus of the β-subunit. rbFSH-LD showed increased sialylation, prolonged pharmacokinetic properties, and higher in vivo specific activity than unmodified rbFSH. The present study aimed to advance its biotechnological development through clonal selection and production at bioreactor scale, followed by comprehensive product characterization and a bovine proof-of-concept study. The previously established suspension CHO-K1 cell line producing rbFSH-LD was subjected to limiting-dilution cloning. Selected clones were evaluated for growth and specific productivity, and the best-performing clone was cultivated in a 1-L perfusion bioreactor using serum-free medium. rbFSH-LD was purified by immobilized metal affinity chromatography (IMAC) and characterized for purity, aggregation, dimer content, isoform distribution, sialic acid content, and structural properties. Biological activity was evaluated in vivo in rats and in a bovine superovulation model. Clone P8D3 showed the highest specific productivity and growth rate and was selected for bioreactor cultivation. rbFSH-LD was recovered with 65% yield and >99% purity, with a high proportion of dimer and low levels of high-molecular-weight aggregates (~1%). The purified variant exhibited a shift toward more acidic isoforms and approximately 4-fold higher Neu5Ac content than rbFSH, consistent with its increased O-glycosylation. Structural analyses showed similar overall secondary and tertiary structures between rbFSH and rbFSH-LD. The clone-derived rbFSH-LD displayed a specific biological activity of 9,930 IU/mg, approximately 2.4-fold higher than rbFSH. Importantly, in a proof-of-concept study in cattle, a single 180-µg dose of rbFSH-LD induced a superovulatory response comparable to that obtained with 180 µg of rbFSH administered in eight divided doses. Both recombinant hormones significantly increased the superovulatory response compared with untreated controls, while viable embryo yield was similar between treatments. rbFSH-LD was successfully produced at bioreactor scale while maintaining high purity, appropriate structural properties, and high biological activity. Most importantly, the bovine proof-of-concept study demonstrated that a single administration can achieve a superovulatory response comparable to multiple administrations of conventional rbFSH, supporting its potential as a long-acting recombinant alternative for bovine superovulation. These findings provide a promising basis for further optimization of the production process and dose regimen.

• Instituto de Biotecnología, UNAM
• Facultad de Farmacia-UAEM

* Presenting author

Advances in biotechnology enable biosimilar biopharmaceuticals to expand healthcare access upon patent expiration. Regulatory approval requires rigorous physicochemical characterization; however, biological potency assays are essential to confirm functional identity and preclinical efficacy comparability. This study developed and validated an analytical bioassay to determine the biological potency of a Filgrastim biosimilar relative to its reference product using M-NFS-60 cell proliferation. To optimize and establish the method, parameters evaluated included dose dilution schemes (scheme D), sodium resazurin development time (31 h post-assay), and cell incubation periods (24 and 48 h). Growth kinetics demonstrated cell doubling times of 20 h with Filgrastim versus 33 h without it. Dose-response curves optimally fit a four-parameter logistic (4PL) model. Validation following FEUM and USP criteria demonstrated linearity across a relative potency (RP) range of 70%–130% (R^2 = 0.9526$), precision with %GCV < 20%, accurate RP representation without bias, and high specificity with no cross-reactivity against bevacizumab.

• Instituto de Investigaciones en Salud, Grupo de Epidemiología del Cáncer, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica
• Laboratorio de Investigación en Bacteriología Anaerobia, Facultad de Microbiología, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica

* Presenting author

Low-dose Clostridioides difficile toxin B (TcdB) exposure could promote cancer stem cell-associated plasticity in colorectal cancer cells. Using SW620 and HT29 cells, we found increased spheroid formation and proliferation in both models, while SW620 cells also showed enhanced invasion and upregulation of stemness, epithelial-to-mesenchymal transition, and pluripotency-associated markers.

• UNL, CONICET, FBCB, Centro Biotecnológico del Litoral, Santa Fe, Argentina
• UNL, CONICET, Centro de Medicina Comparada, Instituto de Ciencias Veterinarias del Litoral (ICiVet-Litoral), Esperanza, Santa Fe, Argentina
• UNL, FBCB, Centro Biotecnológico del Litoral, Santa Fe, Argentina
• UNL, FCV, CONICET, Centro de Medicina Comparada, Instituto de Ciencias Veterinarias del Litoral (ICiVet-Litoral), Esperanza, Santa Fe, Argentina
• UNL, FBCB, Centro Biotecnológico del Litoral, Santa Fe, Argentina.

* Presenting author

A CHO-K1 reporter cell line stably expressing bovine FSHR coupled to a cAMP-responsive luciferase system was developed. Functional assays with rbFSH and other glycoprotein hormones identified responsive clones, and DoE optimization refined assay conditions, supporting this platform as a standardized in vitro alternative for glycoprotein hormone bioactivity testing

• Departamento de Bionanotecnología, Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km 107 carretera, Tijuana-Ensenada, Baja California, 22860, México

* Presenting author

Sustained resonant acoustic mixing (RAM) exposure (≥360 h) yields robust CHO cell populations that achieve up to 50% greater biomass, maintain IgG production, and exhibit increased tolerance to acute hydrodynamic stress. These findings support RAM-driven adaptation as a strategy to develop more robust CHO cell platforms.

• UNL, CONICET, FBCB, Centro Biotecnológico del Litoral, Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina.
• UNL, FBCB, Centro Biotecnológico del Litoral, Ciudad Universitaria, Ruta Nacional Nº 168, km 472, CC 242 (CPA S3000ZAA), Santa Fe, Argentina.

* Presenting author

Endogenous CHO-K1 promoters were engineered through sequence shortening and combination with native enhancers. The resulting variants maintained or improved transcriptional activity and outperformed the viral CMV promoter in stable cell lines, showing potential as efficient alternatives for biopharmaceutical production.

• Universidad Nacional del Litoral (UNL), CONICET, Facultad de Bioquímica y Ciencias Biológicas (FBCB), Centro Biotecnológico del Litoral, Santa Fe, Argentina.
• Universidad Nacional del Litoral (UNL), CONICET, Facultad de Bioquímica y Ciencias Biológicas (FBCB), Centro Biotecnológico del Litoral, Santa Fe, Argentina. BioSynaptica SA, Santa Fe, Argentina.

* Presenting author

A cellular uptake assay using bEnd.3 cells was optimized as a preliminary approach to assess EPO analogs for their ability to cross the blood–brain barrier (BBB). The optimized conditions enabled reproducible detection of intracellular erythropoietin (EPO) while excluding extracellular and membrane-associated protein. The assay provides a useful preliminary screening tool for comparing EPO derivatives and their interaction with brain endothelial cells.

• Mammalian Cell Culture Lab, Department of Chemical, Biotechnology and Materials Engineering, University of Chile; Centre for Biotechnology and Bioengineering (CeBiB), University of Chile. Mostrar más líneas
• Centre for Biotechnology and Bioengineering (CeBiB), University of Chile; Process Modeling and Distributed Computing Lab, Department of Chemical, Biotechnology and Materials Engineering, University of Chile.
• Mammalian Cell Culture Lab, Department of Chemical, Biotechnology and Materials Engineering, University of Chile; Centre for Biotechnology and Bioengineering (CeBiB), University of Chile.
• Veterquímica S.A., Santiago, Chile.
• Process Modeling and Distributed Computing Lab, Department of Chemical, Biotechnology and Materials Engineering, University of Chile.

* Presenting author

A model-based fed-batch strategy was developed for Hi5 insect cells using metabolic characterization and culture optimization. The optimized process increased maximum cell concentration by 23% and enhanced σ1 antigen productivity by 180% compared with batch culture, demonstrating the potential of model-based feeding strategies for vaccine antigen production.

• Instituto de Ciencias Físicas, Universidad Nacional Autónoma De México, Cuernavaca, Morelos, México
• Instituto de Nanociencia y Materiales De Aragón, INMA (CSIC-Universidad De Zaragoza), Zaragoza, Spain
• Instituto de Ciencia de Materiales de Madrid, ICMM/CSIC, C/Sor Juana Inés de la Cruz 3, Madrid, Spain
• Department of Continuum Mechanics and Structural Analysis, Universidad Carlos III De Madrid, Leganes, Madrid, Spain

* Presenting author

Here, we describe MagPiezo, a novel magnetogenetic toolkit for the remote and untethered manipulation of human endogenous Piezo1 channels in endothelial cells in vitro. We demonstrate, for the first time, that Piezo1 channels can be activated without additional cellular modifications, by using a custom-made magnetic applicator and MNPs smaller than 20 nm, functionalized with anti-Piezo1 antibodies for selective targeting. Through real-time calcium imaging, we show an increase in the cytosolic Ca2+ influx associated with magnetomechanical stimulation. Ca2+ entry elicits various endothelial responses including cytoskeleton remodeling and transcriptional activation. This novel toolkit opens the venue for the direct manipulation of native Piezo1 ion channels in different cell types, presenting significant opportunities for biological research.

• Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada (CICATA) Unidad Morelos, IPN
• Facultad de odontología, Universidad Autónoma de Tlaxcala

* Presenting author

Fungal laccases are oxidases that degrade estrogenic contaminants such as 17β-estradiol. The aim of this study was to evaluate the in vitro antineoplastic potential and cytotoxic selectivity of laccase extracts from Agrocybe aff. pediades on lung cancer cells (A549) and normal fibroblasts (LL29). Following propagation on solid Malt Extract Agar (MEA) and subsequent scale-up in liquid culture, enzymatic extracts were obtained and purified. Biological assays showed antiproliferative effects on A549 cells at high concentrations (>20 IU/mg), whereas low concentrations (<0.300 IU/mg) did not induce cytotoxicity in LL29 cells, suggesting a selective therapeutic profile.

• Instituto de Biotecnología, UNAM

* Presenting author

Novel applications of adeno-associated virus (AAV), including Virus-Like Particles (VLPs), have been explored, with preclinical vaccination results showing promising outcomes. Introducing a formylglycine-generating enzyme (FGE) recognition motif allows for the incorporation of formylglycine, which can serve as a specific binding site for large proteins. Recently, Chinese hamster ovary (CHO) cells have been investigated as an expression system for AAVs, but limited information is available to date. In this study, we evaluate the ability of CHO, HEK293, and Sf9 cells to produce AAV VLPs with their native sequence and VLPs modified with the FGE motif.

• Instituto de Biotecnología, UNAM

* Presenting author

Clonal selection of CHO cells producing the SARS-CoV-2 spike recetor RBD fused to an EGFP protein was performed from a stable pool using fluorescent signals of celular markers (EGFP and TMRM). Clones obtained were evaluated in fed-batch cultures, monitoring daily growth, protein production, metabolites, cell density, viability and fluorescence signals. Results showed significant production differences during the exponetial phase. Correlating EGFP signal with specific productivity showed an apparent secretion bottleneck. Overall, clone selected by a high level of signals of EGFP/TMRM resulted in a 48% increase in the specific productivity of RBD-EGFP.

• Instituto de Biotecnología, UNAM
• FMVZ UNAEM

* Presenting author

Herpesviruses are globally relevant pathogens with major implications for human and animal health. Their ability to establish life long latency and reactivate under stress conditions makes them persistent threats across species and underscores the importance of One Health strategies in controlling these viruses. Bovine herpesvirus type1(BHV-1) is a key agent in the bovine respiratory disease complex (shipping fever). Furthermore, BHV-1 reactivation is a major cause of abortion outbreaks, resulting in economic losses of millions of dollars worldwide. Effective control of herpesvirus infections requires integrated, multidisciplinary strategies to protect both human and animal health. In this work we present purification of BHV-1 and staining of whole purified BHV-1 in a flow citometry panel as an strategy to isolate antigen-specific B-cells.

• Instituto Butantan

* Presenting author

West Nile Virus is an arthropod-borne arbovirus, the infection can be asymptomatic or cause clinical symptoms ranging from mild illness to severe neurological complications. Virus-like particles have emerged as promising vaccine candidates because they mimic viral morphology and induce immune responses without the risk of replication. This study aimed to produce recombinant baculovirus stocks to expression WNV-VLPs in Sf9 insect cells. The L1 stock was generated using two different bacmid amounts, 5µg and 10µg. Although the 5µg condition produced a more pronounced cytopathic effect in cell culture, both conditions yielded similar titers, 1.24 x 10⁴ PFU/mL and 1.45 x 10⁴ PFU/mL, respectively. The L3 stock reached a titer of 3.55 x 10⁶ PFU/mL and it was used for VLP production. Envelope protein expression was confirmed by the transmission electron microscopy revelead particles measuring 45-55nm. The system successfully expressed WNV envelope proteins and produced correctly sized VLPs, demonstrating its potential as a vaccine platform.

• Escola de Artes, Ciências e Humanidades (EACH), Universidade de São Paulo
• Laboratório de Biotecnologia Viral, Instituto Butantan

* Presenting author

Rabies causes >59,000 annual deaths, driving the need for scalable vaccine platforms like insect cell-derived virus-like particles (VLPs). Applying a Quality by Design (QbD) framework, a full three-level factorial design evaluated host cell line (Sf9 vs. High Five™), Time of Infection (TOI: 24–72 h), and Harvest Time (HT: 48–96 h p.i.) effects on rabies glycoprotein (RVGP) and matrix protein (RVM) co-expression. Protein expression was analyzed by Dot Blot and Western Blot assays. Distinct expression profiles were observed between the cell lines. Sf9 cells achieved the highest RVGP expression (401.6 ng/mL), whereas High FiveTM cells showed superior RVM production (1294.5 ng/mL). . Statistical modeling identified Sf9 cells infected at TOI 72 h and HT 96 h post-infection as the optimal condition for simultaneous expression of both structural proteins. Transmission electron microscopy confirmed the assembly and formation of rabies VLPs. These findings demonstrate the usefulness of the QbD framework for defining critical process parameters and establishing a design space for rabies VLP production using the baculovirus/insect cell platform.

• Instituto Butantan

* Presenting author

Viral zoonoses and emerging infectious diseases such as Zika, avian influenza (H5), and rabies represent critical public health burdens in Brazil, driving an urgent need for versatile, rapid-response vaccine technologies. Lipid nanoparticles (LNPs) encapsulating non-amplifying messenger RNA (na-mRNA) or self-amplifying RNA (sa-mRNA) offer a powerful platform to combat these endemic and epidemic threats. This study evaluated a standardized, single-composition LNP formulation -comprising an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid-delivering na-mRNA or sa-mRNA encoding antigens for Zika, H5, and rabies, assessing encapsulation efficiency, particle size, polydispersity index (PDI), and in vitro protein expression via indirect immunofluorescence (IFI) in BHK-21 and HEK-293T cells. Our promising results demonstrate that a single, adaptable LNP platform can successfully formulate and express distinct viral antigens, supporting its versatility for rapid vaccine development; nevertheless, further formulation optimization and in vivo evaluations remain essential to fully harness this technology against diverse viral threats.

• Laboratory of Viral Biotechnology (LABV), Butantan Institute (IBu)
• Institute for infection research and vaccine development (IIRVD), University Medical Center Hamburg Eppendorf (UKE)

* Presenting author

Tonsils are accessible secondary lymphoid tissues that provide a valuable model for studying vaccine-induced immune responses. Tonsils contain diverse immune cell populations, including B and T cells and antigen-presenting cells, allowing the assessment of multiple aspects of adaptive immunity. Lymphoid tissue is obtained from routine tonsillectomies, tonsillotomies and adenotomies. For organoid culture, tissue is dissected mechanically and processed through a strainer and by density gradient centrifugation. Dissociated mononuclear cells are seeded in a transwell for organoid formation. H5N8 HA mRNA-LNPs (pSFV-H5 and pVAX-H5) were synthesized via microfluidics. Encapsulation efficiency (EE%) was determined by fluorometric quantification, particle size and polydispersity by dynamic light scattering (DLS). Vaccine-induced immune responses are evaluated using human tonsil-derived immune organoids and subsequent flow cytometry analysis of B- and Tfh- cell responses. mRNA was successfully synthesized and encapsulated in nanoparticles (<200 nm; PDI 0.23–0.29). Both mRNA vaccines induced germinal center and plasmablast development in human tonsil organoids, suggesting that the vaccines can elicit a B-cell response. VAX-LNP also increased Tfh total and Tfh2 frequencies, suggests enhanced T-cell help associated with B-cell differentiation. Antigen-specific B- and T-cell responses are currently being evaluated by ELISpot.

• Centro de Nanociencias y Nanotecnología UNAM
• Centro de Nanociencias y Nanotecnología
• Instituto de Química UNAM
• Departamento de Química UAM

* Presenting author

This work evaluated CHO cells adapted and non-adapted to hydrodynamic stress comparing their growth kinetics and exosome production. Exosomes were isolated from culture supernatants and characterized by dynamic light scattering (DLS). AD432 cells reached a 32% higher maximum viable cell density than CRL12445, while both cultures exhibited a specific growth rate of 0.032 h⁻¹. DLS revealed particles of ~60 nm in average for adapted and non-adapted cell lines, consistent with the reported exosome size range, supporting the effectiveness of the isolation protocol.

• Laboratorio 7 de Biotecnología Farmacéutica, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, México
• Coordinación de Investigación, Centro Médico Nacional “20 de Noviembre” ISSSTE, Mexico City, México
• Hematopoietic Stem Cell Transplantation and Cell Therapy Program, Instituto Nacional de Pediatría, Mexico City, Mexico

* Presenting author

This work evaluates a preclinical platform based on Jurkat/CAR models targeting CD19 and CD33, with an emphasis on assessing cellular identity, functionality, and quality attributes throughout their development. In the Jurkat/CAR-CD19 model, an in-silico design of CAR was made, cell expansion strategies were explored as well as the determination of different quality parameters were studied, aiming to strengthen experimental reproducibility and establish reliable tools applicable to research and development of CAR-T cell therapies.

• Universidad Autonoma del Estado de Morelos

* Presenting author

This work presents an integrated adenovirus serotype 5 (Ad5) platform encompassing vector construction, HEK293-based production, analytical characterization, and efficacy assessment in 2D and 3D in vitro models. Applications include cancer gene therapy, immunomodulation, and diabetic foot complications, alongside bioprocess optimization to support scale-up and future in vivo evaluation.

• Laboratorio de Anticuerpos Monoclonales, Departamento de Biología Molecular y Validación de Técnicas, Dirección de Gestión Integral y Soporte Técnico, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”
• Departamento de Biología Molecular y Validación de Técnicas, Dirección de Gestión Integral y Soporte Técnico, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”

* Presenting author

Members of the family Mycoplasmataceae (formerly classified within the genus Mycoplasma) are frequent cell culture contaminants that alter metabolism, viability, and genetic stability, compromising experimental results. Common contaminants include Mesomycoplasma hyorhinis, Mycoplasmopsis arginini, Mycoplasmopsis fermentans, Metamycoplasma orale, Metamycoplasma salivarium, and Metamycoplasma hominis. This study aimed to develop an in-house duplex real-time PCR assay for routine cell culture quality assurance without commercial detection kits. Developed for murine hybridoma cultures, the assay targets a conserved region of the rrs gene from Mesomycoplasma hyorhinis, shared by common Mycoplasmataceae contaminants, and murine GAPDH as an endogenous internal control. Both targets are amplified in one reaction. Specific primers and hydrolysis probes were designed, and recombinant plasmids constructed as positive controls. The assay generated two independent amplification curves. The internal control helps distinguish valid negative results from potentially false-negative results when Mycoplasma is not detected. This in-house system may reduce reagent costs and processing time and could be adapted to other cell lines.

• UNAM, INCAN
• Houston Methodist Research Institute
• INCAN

* Presenting author

CAR T cells have shown promising results in hematological malignancies; however, the landscape in solid tumors remains challenging. We have generated CAR T cells against two antigens upregulated in gastric cancer, showing partial response and relapses. Here, we analyzed the population of T cells and tumor cells after treatment to understand the possible mechanisms of failure. In vitro assays demonstrated CAR functionality and cytotoxic activity. Then, mice engrafted with SNU638 that received CAR T cells showed significantly delayed tumor growth compared to control groups but relapsed 5 weeks after treatment. Ex vivo analysis showed antigen persistence, CAR downregulation, and a high percentage of PD-1 T cells, suggesting that relapse might be caused by T cell disfunctionality.

• Laboratorio de Anticuerpos Monoclonales, Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”. Dirección General de Epidemiología, Secretaría de Salud
• Laboratorio de Inmunología Experimental. Instituto Nacional de Pediatría, Secretaría de Salud.
• Laboratorio de Enfermedades Osteoarticulares e inmunológicas, Sección de estudios de posgrado e Investigación, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional.
• Centro de Investigación en Ciencias de la Salud Facultad de Ciencias de la Salud Universidad Anáhuac Norte.

* Presenting author

Toxoplasmosis is caused by Toxoplasma gondii (T.gondii). In Mexico, a 40% prevalence in humans is reported. T. gondii can be transmitted through ingestion of oocysts or infected meat and by congenital transmission. Surface antigen 1 (SAG1), abundant on tachyzoites and conserved among strains, is an immunodiagnostic target. This study aimed to generate and characterize monoclonal antibodies against recombinant SAG1 of T. gondii for potential diagnostic applications. Female BALB/c mice were immunized with recombinant SAG1. Hybridomas were generated by splenocyte–murine myeloma fusion, selected in HAT medium, and screened by indirect ELISA. Cross-reactivity against antigens from six parasites (Trypanosoma cruzi, Leishmania mexicana, Leishmania major, Babesia bovis, Babesia bigemina, and Trichinella spiralis) and recognition of three T. gondii strains by indirect immunofluorescence were examined. Six hybridomas producing IgG1 monoclonal antibodies were obtained, without detectable cross-reactivity against the evaluated panel. The antibodies recognized all three strains, with varying fluorescence intensities. These antibodies have immunodiagnostic potential for human toxoplasmosis.

• Centro de Nanociencias y Nanotecnología UNAM
• Facultad de Ciencias UNAM
• Instituto de Química UNAM
• Departamento de Química UAM

* Presenting author

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. During scale-up, agitation and sparging expose cells to hydrodynamic stress, which can influence growth and secretion. Responses to sustained, sublethal stress and the effects of adaptation remain incompletely understood. Adapted AD432 cells reached a 32% higher maximum viable cell density than CRL12445 cells, while both cultures exhibited a specific growth rate of 0.032 h⁻¹. We developed a protocol to isolate extracellular particles from their culture supernatants. DLS showed an average hydrodynamic diameter of approximately 110 nm in both cell lines, within the size range reported for exosomes. These findings indicate that CHO cultures release exosome-sized particles and that our protocol successfully recovers them.

• Laboratorio de Biotecnología Farmacéutica, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos (UAEM), Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, México.

* Presenting author

Biopharmaceutical production requires efficient, reproducible expression systems to optimize process performance and ensure product quality. This study evaluates mammalian cell expression strategies using human interferon gamma (IFN-γ) as a model molecule. CHO-S and HEK293 cells were used to establish transient viral expression with Ad5/IFN-γ, non-viral expression using pCDNA3.1/IFN-γ and UCOE/IFN-γ vectors, and inducible expression using pPB/BOOSTER-Tet-On-IFN-γ. Six expression systems were generated, representing complementary approaches to genetic delivery and transgene regulation. Characterization includes assessing genetic material presence and integration, transgene expression, and operating modes, including microgravity. This work establishes a foundation for comparing expression platforms and guiding investigations into host selection, gene regulation, and operating conditions to support bioprocess optimization.

• Laboratorio de Anticuerpos Monoclonales, Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”. Dirección General de Epidemiología, Secretaría de Salud
• Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”. Dirección General de Epidemiología, Secretaría de Salud
• Laboratorio de Inmunología Experimental. Instituto Nacional de Pediatría, Secretaría de Salud.
• Laboratorio de Enfermedades Osteoarticulares e inmunológicas, Sección de estudios de posgrado e Investigación, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional.
• Centro de Investigación en Ciencias de la Salud Facultad de Ciencias de la Salud Universidad Anáhuac Norte.

* Presenting author

Toxoplasmosis is caused by Toxoplasma gondii (T.gondii). In Mexico, a 40% prevalence in humans is reported. T. gondii can be transmitted through ingestion of oocysts or infected meat and by congenital transmission. Surface antigen 1 (SAG1), abundant on tachyzoites and conserved among strains, is an immunodiagnostic target. This study aimed to generate and characterize monoclonal antibodies against recombinant SAG1 of T. gondii for potential diagnostic applications. Female BALB/c mice were immunized with recombinant SAG1. Hybridomas were generated by splenocyte–murine myeloma fusion, selected in HAT medium, and screened by indirect ELISA. Cross-reactivity against antigens from six parasites (Trypanosoma cruzi, Leishmania mexicana, Leishmania major, Babesia bovis, Babesia bigemina, and Trichinella spiralis) and recognition of three T. gondii strains by indirect immunofluorescence were examined. Six hybridomas producing IgG1 monoclonal antibodies were obtained, without detectable cross-reactivity against the evaluated panel. The antibodies recognized all three strains, with varying fluorescence intensities. These antibodies have immunodiagnostic potential for human toxoplasmosis.

• Laboratorio de Anticuerpos Monoclonales, Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”, Dirección General de Epidemiología, Secretaría de Salud
• Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos “Dr. Manuel Martínez Báez”, Dirección General de Epidemiología, Secretaría de Salud

* Presenting author

Members of the family Mycoplasmataceae (formerly classified within the genus Mycoplasma) are frequent cell culture contaminants that alter metabolism, viability, and genetic stability, compromising experimental results. Common contaminants include Mesomycoplasma hyorhinis, Mycoplasmopsis arginini, Mycoplasmopsis fermentans, Metamycoplasma orale, Metamycoplasma salivarium, and Metamycoplasma hominis. This study aimed to develop an in-house duplex real-time PCR assay for routine cell culture quality assurance without relying on commercial detection kits. Developed for murine hybridoma cultures, the assay targets a conserved region of the rrs gene from Mesomycoplasma hyorhinis, shared by common Mycoplasmataceae contaminants, and murine GAPDH as an endogenous internal control. Both targets are amplified in a single reaction. Specific primers and hydrolysis probes were designed, and recombinant plasmids were constructed as positive controls. The assay generated two independent amplification curves. The internal control helps distinguish valid negative results from potentially false-negative results when Mycoplasma is not detected. This in-house duplex real-time PCR assay may reduce reagent costs and processing time and could be adapted to other cell lines.

• Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México
• eversyn GmH, Magdeburg, Germany
• Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, 39106 Magdeburg, Germany
• Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, 39106 Magdeburg, Germany, eversyn GmH, Magdeburg, Germany

* Presenting author

Monoclonal antibody (mAbs) therapy is an effective treatment option for infectious diseases. In addition to binding to antigens to neutralize the infection, mAbs induce immune responses mediated by their Fc region, which are determined by the N-glycosylation pattern of that domain. Because recombinant mAbs produced in mammalian cells exhibit heterogeneous N-glycosylation profiles in the constant region, the development of glycoengineering strategies to obtain specific glycoforms is important for optimizing the efficacy, safety, and mechanism of action of therapeutic antibodies; it will also provide a deeper understanding of the immune regulation mediated by glycosylation. In this work, we describe a straightforward, cell-free, sequential in vitro enzymatic protocol for the generation of galactosylated and sialylated glycovariants of a prototype IgG-1 mAb. Our protocol provides an opportunity to further advance mAb-based therapies to optimize the immune response to pathogens and for other applications.

• Laboratorio Nacional para la Producción y Análisis de Moléculas y Medicamentos Biotecnológicos, IBT, UNAM
• Instituto de Biotecnología, UNAM

* Presenting author

Sialylation is a critical quality attribute of therapeutic glycoproteins. This study developed and implemented a chromatographic method for sialic acid profiling across two high-resolution liquid chromatography platforms. Sialic acids were released by acid hydrolysis, derivatized with DMB, and analyzed by reversed-phase chromatography with fluorescence detection. The method successfully resolved six reference sialic acid derivatives with reproducible retention times. Optimized sample amounts were 100 µg for bevacizumab and omalizumab and 1.5 µg for recombinant human erythropoietin. Monoclonal antibodies showed predominantly Neu5Ac, whereas erythropoietin exhibited a more heterogeneous profile. The method met system-verification criteria, supporting its applicability to glycosylation characterization and quality assessment of structurally diverse therapeutic glycoproteins across chromatographic platforms.

• Instituto de Biotecnología. Universidad Nacional Autónoma de México.

* Presenting author

Redox homeostasis contributes to protein maturation in the Golgi apparatus, where redox changes may affect glycosylation enzymes and recombinant therapeutic protein quality. Many therapeutic proteins are produced in Chinese hamster ovary (CHO) cells, and their N-glycosylation profiles can influence pharmacokinetic properties. We investigated the effect of sodium butyrate (NaBu), an additive used to enhance cell-specific productivity, on Golgi redox potential in CHO cells. The fluorescent redox sensor roGFP1-iL was targeted to the Golgi by fusion to the transmembrane domain of Cricetulus griseus α-2,3-sialyltransferase IV (ST3Gal-IV). Treatment with 1 mM NaBu shifted the sensor-derived Golgi redox potential from −213 ± 1 mV to −220 ± 2 mV, indicating a more reducing local environment. Further studies are needed to determine whether this shift affects N-glycan processing and therapeutic protein quality.

• LAMMB
• Instituto de Biotecnología
• UUSMB

* Presenting author

To evaluate commercial avian influenza (AIV) vaccines, an IFN-g ELISPOT assay was developed to assess cellular immunity. Spleen perfusion and density gradient separation yielded 97% cell viability, minimizing background. Optimal conditions (0.15 x10^6 cells/well and 5–10 µ g/mL PHA) enabled quantifiable responses without saturation, while short MHC Class II peptides provided antigen-specific stimulation. This standardized method is currently used to compare cell-mediated memory responses across commercial vaccines

• Pontificia Universidad Católica de Valparaíso
• Universidad de los Andes

* Presenting author

A metabolically reprogrammed UC-MSC platform (glycolytic phenotype induced by oligomycin) was transitioned from planar (2D) to a 3D stirred-tank bioreactor (STBR) system using Cytodex-1 microcarriers to produce extracellular vesicles (EVs) at expanded scale. Cell expansion was monitored throughout the 7-day culture, and recovered cells retained high identity marker expression and tri-lineage differentiation capacity. Downstream TFF improved particle recovery. Resulting EVs were characterized by NTA (size, concentration) and tetraspanin expression. The 3D STBR platform thus supported consistent cell growth and yielded a well-characterized EV product.

• Instituto de Biotecnología, UNAM

* Presenting author

Chinese hamster ovary (CHO) cells are widely used for recombinant protein production, particularly therapeutic glycoproteins, but their use for manufacturing complex particles, such as enveloped virus-like particles (eVLPs), remains limited. One challenge is the release of endogenous retrovirus-like particles (ERVLPs), which raises regulatory concerns. Here, we present evidence that molecular engineering approaches, including CRISPR-Cas9 and short hairpin RNAs (shRNAs), can reduce or eliminate ERVLPs in a CHO-DXB11-derived model. Engineered clones have been used to produce purified SARS-CoV-2 spike-bearing eVLPs, with evidence supporting their vaccine potential. These findings highlight the potential of engineered CHO cells as a platform for complex particle manufacturing and the development of eVLP-based carriers for RNA therapeutics.