Microbial synthesis using renewable C1-carbon sources like electrochemically produced formate (e-formate) represents a promising approach for climate-neutral chemical production. This study investigates formate utilization for ectoine biosynthesis by the halophilic methylotroph Methyloligella halotolerans. Preliminary growth assays confirmed formate utilization using 15-20 mM formate as the sole energy source substrate, when supplemented with yeast extract or vitamin solutions in a mineral salt medium. In a systematic study for ectoine production, formate utilization reached 0.305 ± 0.020 mmol d-1 at 20 mM. With different C1-substrates at 20 mM (3 mmol), ectoine production reached 10.3 ± 3.2 µmol (from methanol), 6.5 ± 0.8 µmol (from equimolar methanol/formate mix), 4.4 ± 0.1 µmol (from formate), and 1.2 ± 0.1 µmol (from e-formate). Medium buffering, pH stability and toxicity limited performance when formate and e-formate were supplied. Although ectoine yields were suboptimal, the feasibility of e-formate-based ectoine biosynthesis under high-salinity conditions with 9% NaCl, as shown in this study, discloses the great potential for integrating highly efficient electrochemical CO2 reduction in saline media with microbial synthesis of organic chemicals.
Combining the volumetric fabrication of hydrogel constructs using extrusion bioprinting with highly precise drop-on-demand (DoD) bioprinting offers exciting opportunities in biofabrication. This technical report presents a technique in which a solenoid micro-pipette is operated as an additional tool in an extrusion (bio)printing system to deposit small volumes of bioinks into extrusion-printed hydrogel constructs. Using three exemplary approaches, we show that this enables the patterned placement of cells or growth factors within 3D constructs and thus influences developmental processes. Human cells within low-viscosity bioinks, deposited into extrusion-printed hydrogel constructs by filling inter-strand cavities or by injection into the hydrogel strands, maintained their viability and functionality up to 28 days. As demonstrated for salivary gland cells, the properties of the hydrogel matrix can influence the fate of the injected cells: In a stiff alginate (Alg)-based hydrogel, they formed aggregates, which is beneficial for organoid formation, and in softer hydrogels, they migrated to neighboring cell clusters. Locally injected signaling factors such as vascular endothelial growth factor (VEGF) attracted endothelial cells and fibroblasts, which migrated into previously cell-free hydrogel areas. The combination of extrusion and DoD bioprinting opens new approaches to integrate different cell types and functionalizations in one construct, facilitating the creation of more complex and dynamic models.
Sequencing depth is a crucial parameter for variant calling accuracy and sensitivity. The trade-off between sequencing breadth and depth is a well-known limitation in capture-based targeted next-generation sequencing (NGS). Herein, we propose a differential depth sequencing method, SPRE-Seq, to acquire different sequencing depths for different targeted regions in an NGS panel. The SPRE-Seq performance was evaluated using a panel of reference standards and clinical samples based on our custom-designed homologous recombination deficiency (HRD) assay. By applying SPRE-Seq, the effective sequencing depths of the homologous recombination repair (HRR) and HRD regions of all seven HRD reference standards met the required thresholds with only half the sequencing data volume (reduced from 12 to 6 GB). The results for the HRR genes and HRD showed 100% consistency with the expected results. In clinical samples, the effective sequencing depth of the HRR regions was significantly higher, with a sequencing data volume of 6 GB using the SPRE-Seq approach compared with 6 GB using a regular capture approach. However, there was no significant difference between a data volume of 6 GB using SPRE-Seq and 12 GB using a regular capture method. The SPRE-Seq approach was feasible and reliable for determining the HRD status and HRR somatic variants in reference standards and clinical samples at a low sequencing volume. SPRE-Seq is a reliable, feasible, and cost-effective method that can acquire an adequate sequencing depth of an NGS panel at a low sequencing data volume.
Single cell oils produced in microorganisms constitute appealing alternatives to plant oils. Oleaginous fungi accumulate triacylglycerols in lipid droplets (LD). Their biosynthesis is typically induced under nitrogen limitation. We exploit the fungal model Ustilago maydis for oil production. The stain 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) can be used to track LD formation during cultivation but this expensive compound is only affordable at small-scale. Therefore, mutant screening for optimization of oil production and composition would benefit from an inexpensive online monitoring system. Accordingly, we aimed at developing an intrinsic reporter that is suitable to track oil formation even in larger cultures. From three tested candidates, the potential delta(24)-sterol C-methyltransferase Erg6 turned out to be the best reporter. Fluorescence microscopy confirmed its localization at the LD membrane. After optimization, Erg6 fused to mKate2, expressed from a promoter derived from glycolipid biosynthesis, showed a good correlation of fluorescence with oil accumulation. Time course experiments in micro-cultivators demonstrated that the fluorescence read-out can be used to track oil formation starting at the onset of nitrogen limitation to approximate the LD amount. In essence, our study introduces a biosensor for oil monitoring that can easily be transferred to other oleaginous yeasts. Summary:Microbial oils are promising, environmentally friendly alternatives to plant oils and have the potential of a huge market share once competitive production and isolation processes are accomplished.Online monitoring is key to efficient engineering of single cell oil producing microorganisms and bioprocess optimization in order to achieve competitive products.Here, we present an inexpensive, fluorescence-based reporter that can be used to track the approximate oil accumulation of microbial cultures in vivo. This omits the use of expensive dyes or offline methodology with a high workload.While we established the biosensor in the yeast form of the fungal microorganism U. maydis, the evolutionary conservation of the underlying protein Erg6 will allow for a straightforward transfer of the methodology to other oleaginous yeasts.
Foam formation in stirred tank fermentation processes is a well-studied phenomenon. However, foaming in shake flask cultivations is rarely considered. Non-baffled shake flasks, in particular, are generally considered to prevent foaming problems. However, under certain process conditions, foaming in non-baffled shake flasks can occur. In this study, phenomena of foam formation in shake flasks, their impact on the maximum oxygen transfer capacity (OTRmax), and experimental reproducibility are investigated. It is shown that foaming events in shake flasks can increase the OTRmax by up to threefold. This enhanced OTRmax alters process conditions and, thereby, affects the reproducibility of experiments. Foaming in shake flasks can be induced by elements such as conventional baffles or sensor spots that are used for online measurement. Moreover, a connection between the out-of-phase phenomenon and foam formation was discovered in non-baffled shake flasks. This is especially important when cultivating microorganisms at elevated viscosities. Hence, foaming in shake flasks should be considered as significantly altering process conditions, compared to non-foaming cultures. Ensuring in-phase cultivation conditions and unhindered liquid flow in shake flasks may help to avoid foaming. Practical application: This work provides insights into foam formation in non-baffled shake flasks and its resulting implications. Foaming can increase the maximum oxygen transfer capacity and, thus, affect process conditions. The reproducibility can be severely reduced, and a comparison between foaming and non-foaming cultivations is only possible to a limited extent. Foaming can be induced by baffles or internals, such as small sensor spots, used for online monitoring. Additionally, foaming can be caused by the out-of-phase phenomenon. This is of particular importance when cultivating microorganisms at elevated viscosities. This paper is intended to raise awareness of the topic of foam formation in the shake flask and help to correctly interpret this phenomenon.
Fixed-bed bioreactors for anchorage-dependent cells are an obvious choice for development because of their large-scale capabilities, allowing manufacturing with reduced cost and footprint. In this study, a serum-free production process for Japanese encephalitis virus (JEV) in a single-use fixed-bed bioreactor was developed and compared to conventional roller bottle production as a productivity benchmark. After optimization of serum-free cell culture conditions, an initial media screening in roller bottles showed a strong impact of growth and production media on virus yields. Selected optimized medium combinations were assessed in roller bottles and the fixed-bed bioreactor. Both systems proved to be excellent production systems for JEV, but media choice was key to achieve the highest titers. In particular, DMEM with its enriched glucose content beneficially affected viral yields, enabling potential large-scale manufacturing using the fixed-bed reactor with serum-containing or serum-free media. Practical application: Data presented in this work show feasible ways of serum-free virus production with Vero cells, a common cell substrate in vaccine development. The fixed-bed bioreactor process described here could facilitate manufacturing activities to reduce cost and footprint while simultaneously achieving higher process control compared to conventional manufacturing systems like roller bottles. With a much better upscale potential (up to 500 m2) the fixed-bed bioreactor showed comparable or better yields to roller bottles depending on media used, even with serum-free media. This research article further emphasizes the need to optimize cell culture media or media combinations for each virus individually to achieve the highest titers. As shown, performing a simple media screening experiment to optimize yields early in process development could lead to better productivity, with a high business impact in later development stages.
In vitro measurement of protein diffusion within matrices that simulate the subcutaneous (SQ) environment is of interest, given that protein-based therapeutics formulated for SQ injection comprise the largest class of biologics. To mimic the in vivo transport of a biologic from the SQ injection site through the extracellular matrix (ECM), in vitro diffusion assays typically utilize hyaluronic acid (HA) matrices, as it is the principal component of ECM. However, broad utility has been hampered by inherent lot-to-lot variability in commercially sourced HA, wherein key properties that impact protein diffusion (for example, molecular weight distribution and viscosity) differ across lots, even when nominal molecular weights are identical, making it challenging to compare results across matrices prepared from different HA lots. To address this gap, we report a facile approach wherein binary HA blends generated from individual HA matrices derived from distinct HA lots are functionally equivalent with respect to protein diffusion, that is, the diffusion of a representative set of proteins matches that in a previously reported single HA lot-derived matrix that served as a representative reference. Taken altogether, our protocols enable preparing blended HA matrices with consistent diffusion properties, enabling the use of in vitro assays that leverage this capability. Practical application: The measurement of in vitro diffusion of IgG-type proteins enables calculation of diffusion coefficients that could help to guide the formulation of protein-based therapeutics, administered by subcutaneous (SQ) injection, and used for treating a range of diseases, including cancer. The side-by-side comparison of these proteins over a period of time provides confirmation of consistency of properties when in vitro hyaluronic acid matrices, within which injected protein diffusion is measured, are also consistent. However, their broad utility has been hindered by the inherent variability of commercial sources of HA used to make-up matrices that simulate the SQ environment in a predictable manner. Our research addresses this gap by defining an approach (validated with rheological and diffusion measurements) that facilitates the preparation of blended matrices from different lots of HA. The resulting matrix properties enable reliable measurement of protein diffusion from one lot to the next.
Silica-alginate capsule (G-0) has recently been used in fermentation processes to encapsulate microbial cells for several benefits, including facilitating continuous flow processes and simplifying cell recovery and reuse. However, these conventional silica-coated alginate capsules suffer from poor diffusion channels, which are critical for efficiently transporting substrates and products. This study aimed to develop a novel method for producing silica-coated alginate capsules with improved diffusion channels (G-3). The Ca-alginate capsule was fabricated via a simple dripping method, where a solution of calcium chloride (CaCl2) and carboxymethylcellulose (CMC) was dripped into an alginate solution. For the traditional silica coating (G-0), the alginate capsule was mixed with a silica source (hydrolyzed 3-aminopropyl triethoxysilane) under specific conditions. In the modified method, glucose was introduced as a pore-forming agent (PFA), with varying amounts (0.75, 1.5, and 3 g) resulting in capsules labeled G-0.75, G-1.5, and G-3, respectively. The diffusion coefficient for G-3 was found to be the highest, for example, at 313.15 K, it was calculated as ( 7.77 ± 0.57 ) × 10 - 3 m m 2 / min compared to ( 3.04 ± 0.09 ) × 10 - 3 m m 2 / min for G-0. This finding highlights the effectiveness of PFA in enhancing membrane porosity and diffusivity, which is promising for microbial cell immobilization where mass transfer is a significant concern.
Circulating tumor cells (CTCs) are cancer cells present in the bloodstream that originate from primary or metastatic sites. Sensitive and selective capture of these rare cells is essential for early diagnosis, metastasis prevention, and prognosis prediction. In this study, we demonstrated the effectiveness of a surface functionalized with epithelial cell adhesion molecule (EpCAM) Fab' (fragment-antigen-binding) fragments for the specific capture of EpCAM-positive human breast cancer cells. EpCAM antibody Fab' fragments were produced through pepsin digestion and characterized by SDS-PAGE analysis. Glass surfaces were silanized before being coated with a thin layer of gold via sputtering to ensure stability. The Fab' fragments were immobilized on the gold-coated glass surfaces through strong gold-thiol bonds. The modified surfaces were then characterized using Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and atomic force microscopy (AFM) analyses. Cell capture performance was assessed using fluorescence microscopy with both EpCAM-positive and EpCAM-negative cell lines. The results show that the Fab'-modified surface offers a promising platform for the selective immunocapture of EpCAM-positive cells.Practical application: This study presents a preliminary design of a Fab' fragment-immobilized surface for the selective capture of EpCAM-positive breast cancer cells. The surface modification relies on spontaneous Au-S bonding, offering a simple and effective chemical method. The modified surface demonstrates strong potential for integration into future biosensor platforms for detecting circulating tumor cells. Such a system is promising for advanced diagnostics, monitoring, disease progression, and personalized treatment uses.
Optical spectroscopic techniques have been successfully employed in bioprocessing as process analytical technology for real-time process monitoring in numerous applications. The implementation of spectroscopy-based PAT techniques commonly necessitates the generation of representative process data used for calibration and validation of multivariate statistical models for analyzing the sample composition in real-time. To automate the generation of such data, we present a novel assembly of a commercially available chromatography system in combination with a Raman spectrometer for fast and accurate acquisition of Raman spectra. Using the ultra-/diafiltration (UF/DF) process as a case study, our methodology involved the preparation of representative calibration and validation mixtures of phosphate and citrate buffer and lysozyme as a model protein. Chemometric PLS models were calibrated and validated using these datasets, and applied to in-line recorded Raman spectra during a UF/DF experiment. The primary results demonstrated that the novel assembly provides robust and precise offline measurement of Raman spectra, which directly compare with in-line record data. The chemometric PLS models showed good alignment in calibration and validation datasets (R2 and Q2), and could be used to simultaneously monitor the buffer and protein concentrations in real-time during UF/DF. This study provides a simple, commercially available setup for automated acquisition of Raman spectra and demonstrates its straightforward application to bioprocess monitoring.
Menthol, a natural organic compound and the primary component of mint, exhibits diverse biological activities, including analgesic, anti-inflammatory, antibacterial, neuroprotective, and anticancer effects. The chemical modification of menthol, through processes such as esterification and amination, further enhances these activities, expanding its potential applications in drug development, agriculture, and food preservation. This review explores the structure-activity relationships (SAR) of menthol and its derivatives, emphasizing the significance of molecular modifications in enhancing their pharmacological effects. Research indicates that menthol and its derivatives can improve drug permeation, reduce inflammation, enhance memory, and even target cancer cells through various mechanisms. In addition, we examine the safety and pharmacokinetics of menthol and its derivatives to better understand their clinical potential. Although significant progress has been made in preclinical models, further research is necessary to fully elucidate their mechanisms of action and optimize their therapeutic efficacy in clinical settings. Continued innovation in drug delivery technologies and the development of novel menthol derivatives present promising prospects for future therapeutic applications.
Microbial bioprocessing is a key technology for the production of a wide range of biomolecules, including proteins, enzymes, antibiotics, and other bioactive compounds. In recent years, there has been an increasing interest in using microfluidic platforms for bioprocessing, due to the ability to precisely control and manipulate fluids at the microscale. Microfluidics offers a transformative platform for the manufacturing of biomolecules intended for clinical applications by addressing key technical challenges in scalability, precision, reproducibility, and the ability to study complex biological systems. In this review, various methods used to fabricate microfluidic platforms and the current state-of-the-art in the synthesis/production of biopharmaceuticals, polymers, bioactive compounds, and real-time monitoring in microscale bioprocesses are discussed. Additionally, the future trends and directions are highlighted. Overall, we envisage the utilization of microfluidic platforms to advance the field of microbial bioprocessing and applications in the biomedical field.
Celosia argentea is an undervalued crop that shows potential for production enhancement due to elevated leaf nutrient accumulative ability. By investigating propagation using various in vitro culture systems, thidiazuron (TDZ)-supplemented nutrient media enhanced yield from 10 plants per explant in semi-solid medium, to 27 under continuous immersion in liquid media in recipient for automated temporary immersion (RITA) bioreactors, to 63 under temporary immersion in liquid media in a balloon-type bubble bioreactor (BTBB). TDZ in the BTBB system also increased shoot biomass and subsequent nutrient content relative to TDZ-free media in ex vitro plants. Ex vitro plants originating from both continuous and temporary media immersion in BTBBs outperformed those in all other culture systems in accumulating leaf Mg, Fe, Ca and Zn to meet the recommended dietary allowance for males and females. The genotypic variance and genetic advance of the mean at 5% selection intensity varied for each nutrient per culture system, with and without TDZ. Selective breeding at 5% selection intensity would improve leaf nutrient content but is specific to the culture system and the presence of TDZ. This is the first study to use liquid-based bioreactor systems for C. argentea propagation thereby providing new opportunities to upscale plant production for high nutrient-accumulating genotypes. Practical application: This study establishes a commercially viable protocol for the large-scale clonal propagation of Celosia argentea, a nutrient-rich, fast growing leafy vegetable with untapped agronomic value. Using temporary immersion bioreactors and thidiazuron-supplemented media, the system delivers up to 63 plants per explant, more than 6-fold the yield of conventional methods, while significantly boosting leaf biomass and nutrient content (Mg, Ca, Fe, Zn). These results position C. argentea as a functional crop for health-focused markets and ready-to-cook vegetable lines. The low-input cultivation needs and rapid production cycle (8 weeks in vitro, 8 weeks ex vitro) make it ideal for high-turnover commercial nurseries, contract growers, and vertical farming operations. The systems reproducibility and high heritability of nutritional traits further support selective breeding programs for premium-value cultivars. This propagation platform offers agribusinesses a scalable entry point into the expanding market for nutrient-dense indigenous vegetables with health and wellness appeal.
Polydimethylsiloxane (PDMS) is extensively utilized for the recovery of bio-alcohols, but it encounters significant obstacles in volatile organic compounds (VOCs) removal, because of the narrow size for molecules diffusion. In this work, we designed a high-efficiency diffusion channel by introducing phenyl as a spacer into PDMS chains. The monomer divinylbenzene and vinyl-terminated PDMS (vinyl-PDMS) can be chemically crosslinked with thiol-grafted PDMS (thiol-PDMS) based on thiol-ene click reaction. The result shows that the free volume radius (r3 , r4 ) has a significant increase after the introduction of divinylbenzene as a spacer, which is beneficial to the transport of phenol diffusion. After a series of optimizations involving the divinylbenzene content, pervaporation (PV) operating temperature, photoinitiator content, and viscosity of vinyl-PDMS, the prepared phenyl-PDMS showed an excellent PV performance for phenol recovery containing 10.9 of separation factor and 3959.66 g m-2 h-1 of flux as separating 0.1 wt% of phenol/water solution at 70°C. This separation performance is significantly higher than the unmodified PDMS membrane, that is, 2.05 times higher in separation factor and 3.54 times higher in flux. This study provides an effective structure design for the removal of aromatic compounds by enlarging diffusion channels and will make a great contribution to biological medicine and bioengineering.
Circulating tumor cells (CTCs) hold significant promise for cancer diagnosis, prognosis, and treatment monitoring. We previously developed a technique for a single-cell filtering device known as the microcavity array (MCA), specifically designed for the efficient recovery of CTCs from whole blood samples. Efficient enrichment and release of cells from the MCA remains challenging because of cell adhesion that occurs on the MCA surface during the enrichment phase. This study investigated the effects of surface modification with 2-methacryloyloxyethyl phosphorylcholine (MPC) on the recovery efficiency of cancer cell lines from MCA. Scanning electron microscope (SEM) demonstrated reduced cell-substrate interactions, leading to improved recovery efficiency. Comparative analyses showed that the MCA method provided superior recovery efficiency and reduced processing time compared to traditional methods such as density gradient centrifugation (DGC), while maintaining cell viability and proliferative capacity. CTCs were successfully detected in patients with gastric cancer, and short-term cultures were achieved even when fewer than 20 CTCs per milliliter of blood were isolated. These findings emphasize the importance of surface modification for enhancing CTC isolation and the need for optimized culture conditions. The optimized MCA method offers a promising approach for rapid CTC recovery and potential integration with automated systems. Practical application: The Microcavity array (MCA) is a device specifically designed for efficient recovery of CTCs from whole blood. However cell adhesion on the MCA surface can limit release efficiency. This study demonstrated that surface modification with MPC signigicantly reduces cell-substrate adhesion, improving recovery efficiency while maintaining cell viability and proliferative capacity. Compared to traditional density gradient centrifugation, the MPC-modified MCA offers shorter processing time and better performance. CTCs were successfully detected in gastric cancer, and short-term cultures were achieved even when fewer than 20 CTCs per mL of blood were isolated. The method supports downstearm applications such as cancer cell characterization and treatment monitoring. With potential for integration into automated system, the optimized MCA provides a practical, scalable solution for clinical liquid biopsy and personalized oncology.
Marburg marburgvirus (MARV) is a highly virulent human pathogen with limited therapeutic options. Recombinant MARV glycoprotein (GP) produced in Drosophila Schneider 2 (S2) cells has been extensively investigated as potential vaccine antigen with promising efficacy demonstrated in nonhuman primate models. However, the existing production process for MARV-GP involving static batch cell cultures with limited scalability and process control show lower than desirable yields. Here, we assessed various process intensification strategies in single-use orbital shaken bioreactors (OSBs) or rocking bioreactors (WAVE) and report maximum viable cell concentrations (VCCs) of 31.6 × 106 cells/mL in batch, 69.5 × 106 cells/mL in fed-batch (FB), and up to 210.0 × 106 cells/mL in perfusion mode. By changing from a glucose-only feed to a CellBoost5 feed, MARV-GP yields were increased by over two-fold. Implementation of perfusion cultures achieved a peak MARV-GP concentration of 57.4 mg/L and a 540% higher space-time yield compared to the FB process in the 50 L WAVE system. However, maximum cell-specific productivities were achieved at a VCC of 85 × 106 cells/mL and decreased with increasing cell concentrations. Glycoanalysis revealed a uniform paucimannosidic N-glycan profile, predominantly α-1,6-core-fucosylated Man3F (F(6)M3) structures, across all production modes. Notably, transitioning pH control from CO2 to phosphoric acid shifted glycan profiles toward higher mannose forms, highlighting the influence of culture conditions on glycosylation.
ABSTRACT Sonochemistry has become increasingly important in bioengineering research, and many in vitro and in vivo bioapplications have been developed. Cytotoxicity is always a concern in its implementation. For in vivo treatments and studies, mechanical index (MI) is known to ensure biocompatibility, and even in vitro MI has been used. Because cell characteristics and acoustic phenomena differ in vitro and in vivo, we questioned using MI in vitro. The in vitro cytotoxicity of ultrasound exposure should be investigated to support the development of cutting‐edge sonochemistry. In this study, a system for irradiating cultured cells with 1–2 MHz‐range ultrasound was developed to demonstrate the invalidity of employing MI alone in vitro. The results showed that cell damage is defined by the MI, ultrasound frequency, and exposure time, which are new indices for quantifying cell damage. Furthermore, cavitation and acoustic streaming are shown to be the main scientific factors that injure cells.
Posttreatment of the effluents from wastewater treatment plants is becoming increasingly important, as the conventional treatment cannot completely remove organic trace contaminants. Promising techniques like chemical oxidation methods, including ozonation, face the challenge of potentially generating more toxic transformation products than their parent substances due to incomplete oxidation. In this work, the laccase from Trametes versicolor was immobilized on a polyester textile to create a biocatalytic textile filter for the posttreatment of organic trace contaminants and their ozonation by-products. Different filter designs for reactive filtration with biocatalytic textiles were implemented on the laboratory scale and tested for their effectiveness in degrading the dye Remazol Brilliant Blue, the pharmaceutical diclofenac, and its ozonation products. The plate module, inspired by lamellar clarifiers and featuring the textile with covalently immobilized enzyme on the lamella surfaces, exhibited the best performance characteristics. Employing this module, a continuous process of diclofenac ozonation and subsequent posttreatment with the biocatalytic filter was conducted. This not only demonstrated the feasibility of continuous biocatalytic wastewater filtration but also highlighted improved degradation efficiencies of ozonation products compared to the batch process using laccase in solution.
Establishing manufacturing processes for cell culture-based pharmaceutical products involves managing multiple parameters that can affect yield and efficiency, as well as process robustness and product quality. Implementing Quality by Design (QbD) principles can support process optimization, while streamlining the chemistry, manufacturing, and control aspects for regulatory approval. In this study, we mimic a QbD approach based on an influenza A virus production process using two clonal suspension Madin-Darby canine kidney (MDCK) cell lines with distinct characteristics. We performed a quantitative risk assessment including biological and technical parameters to identify the Critical Process Parameters (CPPs). To comprehensively study the effects and interactions of four CPPs, we used an Ambr 15 scale-down system following a Design of Experiments (DoE) approach. After data analysis and modeling, we obtained design spaces characterized by high robustness with a less than 1% risk of failure and even some indications for virus titer and yield improvement, while keeping process-related impurities such as DNA and total protein concentration low. These findings were subsequently verified at a more than 100-fold higher working volume. Taken together, our approach may stimulate ideas for the implementation of streamlined process development and regulatory approval in the field of viral vaccine production.
To increase their throughput, reduce laboratory work and improve reproducibility, automation of bioprocesses is gaining in importance nowadays. This applies in particular to microbioreactors (MBRs), which can be easily integrated in highly parallelized and automated platforms and, therefore, be applied for screenings, cell-based assays, and bioprocess development. One promising pharmaceutical application for MBRs is the performance of phage sensitivity tests called phagograms in phage therapy. However, there is no automated and parallelized platform available so far that fulfills the requirements of phagograms. Therefore, a novel highly parallelizable capillary-wave microbioreactor (cwMBR) with a volume of 7 µL, which has already been successfully applied for phagograms, was extended by an in-house built platform for automated fluid addition in the single-digit nanoliter range. The cwMBR has a phage-repellent hydrophilic glass surface. Furthermore, a custom-made highly parallelizable device for biomass measurement in the lower microliter scale was developed and validated in the cwMBR. To prove the applicability of the platform for the generation of phagograms, a phagogram using Escherichia coli and automated phage addition was performed. The results indicate a clear lysis of the bacteria by the phages and thus confirm the applicability of performing automated phagograms in the highly parallelizable cwMBR platform.
