Search Results (16)

Synechocystis sp. PCC 6803 is a photosynthetic microbe with high potential for capturing excessive atmospheric carbon while generating valuable bioproducts, like glucose. Current cultivation technologies remain expensive, closed-source, and poorly suited for downstream processing. This study presents a low-cost, open-source bioreactor platform with integrated modules for Synechocystis cultivation and glucose extraction. The system incorporates a photobioreactor, a lysis module, and a pressure-driven filtration setup. Optical density was continuously monitored using a custom-built module, and glucose was quantified using high-performance liquid chromatography (HPLC). Under an incident light intensity of approximately 400 $\mathsf{\mu }\mathrm{m}\mathrm{o}\mathrm{l}$${\mathrm{m}}^{-2}$${\mathrm{s}}^{-1}$, cultures reached a biomass productivity of 90 mg ${\mathrm{L}}^{-1}{\mathrm{day}}^{-1}$, with a specific growth rate of $0.166{\mathrm{day}}^{-1}$ and glucose concentrations up to $5.08$$\mathrm{m}\mathrm{g}{\mathrm{L}}^{-1}$. A model was developed to predict the growth based on measured environmental parameters, achieving a strong predictive accuracy with a mean absolute error and variance of $0.0009\pm 0.0003$. The system demonstrates up to 65% reduction in cost compared to commercial alternatives. This modular platform provides an accessible solution for biomanufacturing research and serves as a template for sustainable cyanobacteria-derived glucose production. Full article

The biopharmaceutical industry is undergoing a fundamental transformation from traditional batch manufacturing to continuous manufacturing (CM) for recombinant drugs and biosimilars, driven by regulatory support through the International Council for Harmonization (ICH) Q13 guidance and compelling economic advantages. This comprehensive review examines the technical, economic, and regulatory aspects of implementing continuous manufacturing specifically for recombinant protein production and biosimilar development, synthesizing validated data from peer-reviewed research, regulatory sources, and global implementation case studies. The analysis demonstrates that continuous manufacturing offers substantial benefits, including a reduced equipment footprint of up to 70%, a 3- to 5-fold increase in volumetric productivity, enhanced product quality consistency, and facility cost reductions of 30–50% compared to traditional batch processes. Leading biomanufacturers across North America, Europe, and the Asia–Pacific region are successfully integrating perfusion upstream processes with connected downstream bioprocesses, enabling the fully end-to-end continuous manufacture of biopharmaceuticals with demonstrated commercial viability. The regulatory framework has been comprehensively established through ICH Q13 guidance and region-specific implementations across the FDA, EMA, PMDA, and emerging market authorities. This review provides a critical analysis of advanced technologies, including single-use perfusion bioreactors, continuous chromatography systems, real-time process analytical technology, and Industry 4.0 integration strategies. The economic modeling presents favorable return-on-investment profiles, accompanied by a detailed analysis of global market dynamics, regional implementation patterns, and supply chain integration opportunities. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

The delivery of biomolecules to target cells has been a longstanding challenge in biotechnology. DNA viruses naturally evolved the ability to deliver genetic material to cells and modulate cellular processes. As such, they inherently possess requisite characteristics that have led to their extensive study, engineering, and development as biotechnological tools. Here, we overview the application of DNA viruses to biotechnology, with specific implications in basic research, health, biomanufacturing, and agriculture. For each application, we review how an increasing understanding of virology and technological methods to genetically manipulate DNA viruses has enabled advances in these fields. Additionally, we highlight the remaining challenges to unlocking the full biotechnological potential of DNA viral technologies. Finally, we discuss the importance of balancing continued technological progress with ethical and biosafety considerations. Full article

There is a pressing need to produce novel food ingredients from sustainable sources to support a growing population. Filamentous fungi can be readily cultivated from low-cost agricultural byproducts to produce functional proteins for food biomanufacturing of structured products. However, there is a lack of scientific knowledge on the gelling characteristics of fungal proteins or their potential in additive biomanufacturing. Therefore, this study investigated the feasibility of utilizing fungal protein extracts and flours from Aspergillus awamori, Pleurotus ostreatus, Auricularia auricula-judae as sole gelling agents in 3D-printed products. Protein extracts were successfully prepared using the alkaline extraction–isoelectric precipitation method and successful physical gels were created after heating and cooling. Results indicated that shear-thinning gel materials could be formed with acceptable printability at mass inclusion rates between 15% and 25% with the best performance obtained with P. ostreatus protein extract at 25% inclusion. A. auricula-judae demonstrated promising rheological characteristics but further optimization is needed to create homogeneous products appropriate for extrusion-based 3D printing. This work provides valuable insights for continued development of 3D-printed foods with filamentous fungi. Full article

Tangential flow microfiltration is easily adapted for batch and continuous bioreactor clarification. The permeate can be introduced directly to the subsequent capture step. However, the commercial use of tangential flow filtration (TFF) is limited by membrane fouling, leading to a compromised performance. Here, we explored the possibility of reducing membrane fouling by integrating a hydrocyclone as the primary clarification operation. The overflow from the hydrocyclone was introduced directly as the feed to the microfiltration module. Chinese hamster ovary cells were used as the feed stream to investigate the feasibility of this integrated process. A range of cell viabilities from 0% (cell lysate) to 96% were investigated. The cell densities ranged from 0.9 to 10 million cells per mL. Two commercially available hollow fiber microfiltration membranes were used, an essentially symmetric membrane and a reverse asymmetric membrane where the more open support structure faced the feed stream. The reverse asymmetric membrane was more resistant to fouling in the absence of an integrated hydrocyclone. Integrating a hydrocyclone led to a reduction in the flux decline for the symmetric membrane, but did not affect the performance of the reverse asymmetric membrane. The careful choice of membrane morphology and pore size is important when designing an integrated process. Full article

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs. Full article

Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing. Full article

A continuing limitation and major challenge in the development and utilization of predictable stem cell therapies (SCTs) is the determination of the optimal dosages of stem cells. Herein, we report the quantification of stem cell fractions (SCF) of human mesenchymal stem cell (MSC) preparations derived from oral tissues. A novel computational methodology, kinetic stem cell (KSC) counting, was used to quantify the SCF and specific cell culture kinetics of stem cells in oral alveolar bone-derived MSC (aBMSCs) from eight patients. These analyses established, for the first time, that the SCF within these heterogeneous, mixed-cell populations differs significantly among donors, ranging from 7% to 77% (ANOVA p < 0.0001). Both the initial SCF of aBMSC preparations and changes in the level of the SCF with serial culture over time showed a high degree of inter-donor variation. Hence, it was revealed that the stability of the SCF of human aBMSC preparations during serial cell culture shows inter-donor variation, with some patient preparations exhibiting sufficient stability to support the long-term net expansion of stem cells. These findings provide important insights for the clinical-scale expansion and biomanufacturing of MSCs, which can facilitate establishing more effective and predictable outcomes in clinical trials and treatments employing SCT. Full article

The development and optimization of lipid nanoparticle (LNP) formulations through hydrodynamic mixing is critical for ensuring the efficient and cost-effective supply of vaccines. Continuous LNP formation through microfluidic mixing can overcome manufacturing bottlenecks and enable the production of nucleic acid vaccines and therapeutics. Predictive process models developed within a QbD Biopharma 4.0 approach can ensure the quality and consistency of the manufacturing process. This study highlights the importance of continuous LNP formation through microfluidic mixing in ensuring high-quality, in-specification production. Both empty and nucleic acid-loaded LNPs are characterized, followed by a TFF/buffer exchange to obtain process parameters for the envisioned continuous SPTFF. It is shown that LNP generation by pipetting leads to a less preferable product when compared to continuous mixing due to the heterogeneity and large particle size of the resulting LNPs (86–104 nm). Particle size by continuous formation (71 nm) and the achieved encapsulation efficiency (EE) of 88% is close to the targeted parameters for Pfizer’s mRNA vaccine (66–93 nm, 88%EE). With the continuous encapsulation of nucleic acids in LNPs and the continuous production of mRNA in in vitro transcription, the basis for the holistic continuous production of mRNA is now established. We already showed that a fully autonomous process requires the incorporation of digital twins and a control strategy, with predictive process models and state-of-the-art PAT enabling real-time-release testing. This autonomous control can considerably improve productivity by about 15–20% and personnel as well as chemical reduction of about 30%. The results of this work complement this, laying the basis for fully continuous, bottleneck-free production of mRNA and other cell- and gene-therapeutic drug/vaccine candidates in a GMP- and QbD-compliant Biopharma 4.0 facilities on a flexible scale. Full article

The production of messenger ribonucleic acid (mRNA) and other biologics is performed primarily in batch mode. This results in larger equipment, cleaning/sterilization volumes, and dead times compared to any continuous approach. Consequently, production throughput is lower and capital costs are relatively high. Switching to continuous production thus reduces the production footprint and also lowers the cost of goods (COG). During process development, from the provision of clinical trial samples to the production plant, different plant sizes are usually required, operating at different operating parameters. To speed up this step, it would be optimal if only one plant with the same equipment and piping could be used for all sizes. In this study, an efficient solution to this old challenge in biologics manufacturing is demonstrated, namely the qualification and validation of a plant setup for clinical trial doses of about 1000 doses and a production scale-up of about 10 million doses. Using the current example of the Comirnaty BNT162b2 mRNA vaccine, the cost-intensive in vitro transcription was first optimized in batch so that a yield of 12 g/L mRNA was achieved, and then successfully transferred to continuous production in the segmented plug flow reactor with subsequent purification using ultra- and diafiltration, which enables the recycling of costly reactants. To realize automated process control as well as real-time product release, the use of appropriate process analytical technology is essential. This will also be used to efficiently capture the product slug so that no product loss occurs and contamination from the fill-up phase is <1%. Further work will focus on real-time release testing during a continuous operating campaign under autonomous operational control. Such efforts will enable direct industrialization in collaboration with appropriate industry partners, their regulatory affairs, and quality assurance. A production scale-operation could be directly supported and managed by data-driven decisions. Full article

Three-dimensional (3D) bioprinting is an emerging technology based on 3D digital imaging technology and multi-level continuous printing. The precise positioning of biological materials, seed cells, and biological factors, known as “additive biomanufacturing”, can provide personalized therapy strategies in regenerative medicine. Over the last two decades, 3D bioprinting hydrogels have significantly advanced the field of cartilage and bone tissue engineering. This article reviews the development of 3D bioprinting and its application in cartilage tissue engineering, followed by a discussion of the current challenges and prospects for 3D bioprinting. This review presents foundational information on the future optimization of the design and manufacturing process of 3D additive biomanufacturing. Full article

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batch cultivations were run under the desired conditions, generating sufficient information to define the fastest-growing strain in an environment with oscillating glucose concentrations similar to industrial-scale bioreactors. Full article

Diabetes mellitus (DM) is a chronic metabolic disease with increasing prevalence worldwide. Diabetic foot ulcers (DFUs) are a serious complication of DM. It is estimated that 15–25% of DM patients develop DFU at least once in their lifetime. The lack of effective wound dressings and targeted therapy for DFUs often results in prolonged hospitalization and amputations. As the incidence of DM is projected to rise, the demand for specialized DFU wound management will continue to increase. Hence, it is of great interest to improve and develop effective DFU-specific wound dressings and therapies. In the last decade, 3D bioprinting technology has made a great contribution to the healthcare sector, with the development of personalized prosthetics, implants, and bioengineered tissues. In this review, we discuss the challenges faced in DFU wound management and how 3D bioprinting technology can be applied to advance current treatment methods, such as biomanufacturing of composite 3D human skin substitutes for skin grafting and the development of DFU-appropriate wound dressings. Future co-development of 3D bioprinting technologies with novel treatment approaches to mitigate DFU-specific pathophysiological challenges will be key to limiting the healthcare burden associated with the increasing prevalence of DM. Full article

An experimental feasibility study on continuous bioprocessing in pilot-scale of 1 L/day cell supernatant, that is, about 150 g/year product (monoclonal antibody) based on CHO (Chinese hamster ovary) cells for model validation is performed for about six weeks including preparation, start-up, batch, and continuous steady-state operation for at least two weeks stable operation as well as final analysis of purity and yield. A mean product concentration of around 0.4 g/L at cell densities of 25 × 10⁶ cells/mL was achieved. After perfusion cultivation with alternating tangential flow filtration (ATF), an aqueous two-phase extraction (ATPE) followed by ultra-/diafiltration (UF/DF) towards a final integrated counter-current chromatography (iCCC) purification with an ion exchange (IEX) and a hydrophobic interaction (HIC) column prior to lyophilization were successfully operated. In accordance to prior studies, continuous operation is stable and feasible. Efforts of broadly-qualified operation personal as well as the need for an appropriate measurement and process control strategy is shown evidently. Full article