Biotechnology Journal最新文献

Monoclonal antibody (MoAb) therapy is a cornerstone in treating cancer, inflammatory diseases, and infections. However, the development of new monoclonal antibodies is labor-intensive, costly, and species-specific, limiting their accessibility in veterinary medicine and slowing innovation in human therapies. In this work, we introduce adaptabodies, bispecific nanobody constructs that repurpose existing MoAbs, of irrelevant specificity, by bridging their idiotype to a new antigen. As a proof of concept, we tested this strategy using a model murine MoAb combined with a neutralizing adaptabody that redirected its binding to tetanus toxin and resulted in mice surviving at least for 8 days when challenged with 1 lethal dose 100 (LD₁₀₀). Mice challenged with 20 LD₁₀₀ of tetanus toxin and treated with two neutralizing adaptabodies survived 8 days and increasing five-fold the amount of the MoAb increased survival to at least 20 days. This approach reduces the need to develop new monoclonal antibodies for each disease, requiring only the retargeting domain to be generated. Moreover, a single MoAb could be developed for species lacking therapeutic MoAbs for adaptabody-mediated treatment. The increasing approval of monoclonal antibodies for treatment of both humans and companion animals underscores the relevance of this flexible and scalable strategy.

Human Embryonic Kidney 293 (HEK293) cells are currently one of the preferred host cell lines for the production of biologics, specifically, AAV-based viral vectors. These fast-growing cells consume significant amounts of nutrients and often convert them into byproducts such as lactate and ammonia. In fed-batch cultures, accumulation of lactate and ammonia to high levels can inhibit cell proliferation. In this study, we demonstrate that lactate and ammonia accumulation alone doesn't fully explain the growth inhibition observed in HEK293 fed-batch cultures. Growth inhibition was noted even when the residual levels of these byproducts were well controlled. Instead, we show that several previously unknown compounds accumulate in HEK293 cell fed-batch cultures, some of which can inhibit HEK293 cell growth either individually or synergistically. Many of these newly identified compounds are intermediates or byproducts of amino acid catabolism. When residual levels of the source amino acids for these novel byproducts were controlled in the low concentration range (∼1 mM) in HEK293 fed-batch cultures, lactate accumulated to higher levels, causing growth inhibition. This prompted the use of High-end pH Delivery of Glucose (HIPDOG), a control strategy that limits lactate production by keeping low residual concentrations of glucose. In HIPDOG cultures, controlling the source amino acids at low concentrations resulted in lower accumulations of the corresponding growth-inhibitory byproducts when compared to the control HIPDOG conditions with typical levels of amino acids. This led to higher viable cell densities (VCD) and viabilities in low amino acid conditions. Strategies that reduce byproduct accumulation, whether classical or novel byproducts, in HEK293 fed-batch processes can result in enhanced VCDs, potentially leading to higher volumetric productivities.

Extracorporeal membrane oxygenation (ECMO) is a fundamental treatment for cardiovascular and severe pulmonary diseases, with small animal models providing critical insights into organ protection during cardiopulmonary bypass and ECMO therapy. Conventional roller pumps, however, induce hemolysis and organ injury through repetitive compression-shear exposure, severely limiting their utility. While centrifugal pumps reduce shear stress and are effective in large animal ECMO, their flow range and priming volume are unsuitable for small animals. Here, we present a miniaturized, fully integrated ECMO system with an optimized centrifugal pump tailored for small animal models. The system reduces shear stress, minimizes blood damage, and enhances organ protection. Integrated multi-parameter sensors enable real-time monitoring of blood flow, pressure, and temperature, thereby streamlining setup and improving perioperative support. In a 6-h experiment, the system demonstrated significant hemolysis suppression, improved renal function (with reduced levels of Neutrophil Gelatinase-Associated Lipocalin, or NGAL), and stable hemodynamics. This innovation offers safer extracorporeal support for small animal studies on cardiovascular diseases, organ recovery, and ECMO mechanisms, furthering research into therapeutic interventions. By addressing key limitations of existing pumps, the system provides a reliable platform for exploring hemodynamic and pathophysiological processes in ECMO treatment, establishing a foundation for future preclinical investigations.

The development of robust, high-yielding cell lines represents a critical factor in establishing efficient and cost-effective manufacturing processes within the competitive biopharmaceutical industry. While extensive research has focused on optimizing plasmid design, host cell characteristics, and selection strategies to enhance cell line development, this study specifically investigates strategic expression plasmid design to improve both productivity and product quality of therapeutic proteins in Chinese Hamster Ovary (CHO) cells using transposon technology. Our findings demonstrate that increasing light chain and heavy chain copy numbers while maintaining balanced expression significantly enhances productivity and mitigates purity risks. Furthermore, we reveal that consolidating all chains of asymmetric molecules into a single plasmid, rather than distributing them across multiple plasmids, substantially improves both productivity and purity. For molecules exhibiting poor purity due to imbalanced chain expression, we demonstrate that incorporating an additional copy of the under-expressed chain can yield significant purity improvement. Based on these findings, we propose a customized plasmid design and pool development workflow to ensure high success rates for cell lines producing structurally complex molecules.

The human kidney proximal tubule is responsible for glucose reabsorption and serves as a primary target for exogenous toxins. While conventional in vitro cell-based models offer cost-effective alternatives to animal testing, they often fail to replicate the structural and functional complexity of the native proximal tubule. Here, we developed a paper-based human kidney proximal tubule-on-a-chip that mimicked key physiological functions, bridging between traditional cell cultures and animal models. Utilizing porous paper, the chip recreated an in vivo–like three-dimensional microenvironment that supported proximal tubule-specific functions and reproduced essential physiological processes including dynamic glycogen metabolism, glucose reabsorption, and drug transport. The model enabled precise pharmacodynamics evaluation of sodium-glucose co-transporter 2 (SGLT2) inhibitors, yielding median effect concentrations of 0.954 ng/mL for dapagliflozin and 2.685 ng/mL for canagliflozin. The platform maintained consistently high glucose reabsorption inhibition rates (94.59%–95.03%) under different conditions following SGLT2 inhibitors treatment. Furthermore, the methotrexate (MTX)–induced nephrotoxicity evaluation was performed by MTT assay, LDH assay, and glucose reabsorption measurements. The chip accurately reproduced MTX transport dynamics, demonstrating its potential for pharmacokinetic studies. Thus, the paper-based model serves as a reliable platform for pharmacokinetic and nephrotoxicity assessments, offering a valuable tool to replace animal testing and support Reduce, Refine, and Replace experimentation.

Acetoin is a key platform chemical with diverse industrial applications. In this study, the marine bacterium Vibrio diabolicus, characterized by its rapid growth and strong ability to utilize starch, was systematically engineered for efficient conversion of starch into acetoin. A suicide plasmid-mediated homologous recombination system was first developed to investigate the roles of four endogenous amylase genes. Based on transcriptomic analysis, two strong constitutively active endogenous promoters were identified and functionally validated to enhance gene expression. To increase acetoin production, the 2,3-butanediol dehydrogenase gene and polyhydroxyalkanoate synthase gene were deleted, thereby eliminating carbon flux into competing pathways for 2,3-butanediol and poly-3-hydroxybutyrate biosynthesis. Subsequently, multiple copies of the budAB operon were integrated into the chromosome to strengthen the acetoin biosynthetic route. The final engineered strain produced 13.21 g/L of acetoin within 12 h of shake flask cultivation, reflecting a significant enhancement in production efficiency. This study presents the first successful case of metabolic engineering in V. diabolicus for direct and efficient production of acetoin from starch, highlighting its significant potential for industrial-scale bioproduction.