Compared to batch operation, continuous bioprocessing can offer numerous advantages, including increased productivity, improved process control, reduced footprint, and increased flexibility. However, integration of traditional batch operations into a connected process can be challenging. In contrast to batch operations run at constant pressure or high flux, virus filtration in continuous processes may be operated at very low flux. This change in operating conditions may reduce the viral retention performance of the filter which has inhibited adoption of truly continuous virus filtration. To overcome this limitation, a novel approach is described that utilizes serial virus filtration, with a high area ratio between first to second stage filters, to achieve virus retention targets. In this study, virus filters were operated continuously (except for planned process interruptions) for 200 hours in a serial configuration at a first to second stage filter area ratio of 13:1 and at a first stage flux of 5 L/m 2/hr. While the minute virus of mice (MVM) retention performance of the first stage filter was about 4 log reduction value (LRV), there was no virus detected in the second stage filtrate, translating to an MVM LRV across the filtration train of ≥6.7. The second stage filter was the dominant flow resistance at the start of the run but, as it was protected from foulants by the first stage filter, it suffered minimal fouling and the life of the filter train was controlled by the first stage. A theoretical case study projected that continuous virus filtration using serial configuration at high area ratio would have about 30% longer filter changeout time, 14% higher productivity, and virus retention nearly 6 LRV greater than single stage operation. The findings of this research are expected to provide valuable insights into optimizing virus filtration in continuous bioprocessing.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes’ size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product’s activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (i) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants, and (ii) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up-to 80% and up-to 50-fold reduction of host cell proteins upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a 2-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of host cell proteins to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blotting, transmission electron microscopy, nanoparticle tracking analysis.

Fast diagnostic methods are crucial to reduce the burden on healthcare systems. Currently, detection of diabetes complications such as neuropathy requires time-consuming approaches to observe the correlated red blood cells (RBCs) morphological changes. To tackle this issue, an optical analysis of RBCs in air was conducted in the 250-2500 nm range. The distinct oscillations present in the scattered and direct transmittance spectra have been analyzed with both Mie theory and anomalous diffraction approximation. The results provide information about the swelling at the ends of RBCs and directly relate the optical data to RBCs morphology and deformability. Both models agree on a reduction in the size and deformability of RBCs in diabetic patients, thus opening the way to diabetes diagnosis and disease progression assessment.

It is of great importance to study the detachment/attachment behaviors of cells (cancer cell, immune cell and epithelial cell), as they are closely related with tumor metastasis, immunoreaction and tissue development at variety scales. To characterize the detachment/attachment during the interaction between cells and substrate, some researchers proposed using cell traction force (CTF) as the indicator. To date, various strategies have been developed to measure the CTF. However, these methods only realize the measurements of cell passive forces on flat cases. In order to quantify the active CTF on non-flat surfaces, which can better mimic the in vivo case, we employed elastic hydrogel microspheres as a force sensor. The microspheres were fabricated by microfluidic chips with controllable size and mechanical properties to mimic substrate. Cells were cultured on microsphere and the CTF led to the deformation of microsphere. By detecting the morphology information, the CTF exerted by attached cells can be calculated by the in-house numerical code. Using this microspheres, the CTF of various cells (including tumor cell, immunological cell, and epithelium cell) were successfully obtained on non-flat surfaces with different curvature radii. The proposed method provides a versatile platform to measure the CTF with high precision and to understand the detachment/attachment behaviors during physiology processes.

Biosensors are valuable tools in accelerating the test phase of the design-build-test-learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein-coupled receptor (GPCR)-based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR-based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment. Here, we explore the integration of production and sensing modalities into a single Saccharomyces cerevisiae strain and compartmentalization using three different methods: (1) cultivation in microtiter plates, (2) spatial separation on agar plates, and (3) encapsulation in water-in-oil-in-water double emulsion droplets, combined with analysis and sorting via a fluorescence-activated cell sorting (FACS) machine. Employing tryptamine and serotonin as proof-of-concept target molecules, we optimize biosensing conditions and demonstrate the ability of the autocrine screening method to enrich for high producers, showing the enrichment of a serotonin-producing strain over a non-producing strain. These findings illustrate a workflow that can be adapted to screening for a wide range of complex chemistry at high throughput using commercially available microfluidic systems.

In nature, many organisms exhibit remarkable freezing tolerance and employ rich and intriguing physiological strategies to adapt to low-temperature environments. However, cryopreservation techniques face challenges when it comes to freezing and thawing biological tissues and cells, such as cryo-damage caused by crystallization and cell membrane rupture due to osmotic imbalances. In recent years, there has been growing interest in the molecular mechanisms underlying natural freezing tolerance and artificial cryopreservation processes. This review delves into the adaptive mechanisms observed in freeze-tolerant species and hibernating animals, elucidating the significance of specific genes, proteins, and metabolic pathways for survival. Furthermore, we discuss advancements in cryopreservation technologies, highlighting their impacts on cellular and tissue integrity, and analyzes the adaptive regulation of processes like glucose metabolism, miRNA expression, and cryoprotective protein modulation. Finally, future directions for cryopreservation are proposed.

The fifth modeling workshop (5MW) was held in June 2023 in Favrholm, Denmark and sponsored by Recovery of Biological Products Conference Series. The goal of the workshop was to assemble modeling practitioners to review and discuss the current state, progress since the last fourth mini modeling workshop (4MMW), gaps and opportunities for development, deployment and maintenance of models in bioprocess applications. Areas of focus were four categories: biophysics and molecular modeling, mechanistic modeling, computational fluid dynamics (CFD) and plant modeling. Highlights of the workshop included significant advancements in biophysical/molecular modeling to novel protein constructs, mechanistic models for filtration and initial forays into modeling of multi-phase systems using CFD for a bioreactor and mapped strategically to cell line selection/facility fit. A significant impediment to more fully quantitative and calibrated models for biophysics is the lack of large, anonymized data sets. A potential solution would be the use of specific descriptors (ex. patch sizes and types mapped to a homology model) in a database that would allow for detailed analyses without sharing proprietary information. Another gap identified was the lack of a consistent framework for use of models that are included or support a regulatory filing beyond the high-level guidance in ICHQ8-Q11. One perspective is that modeling can be viewed as a component or precursor of machine learning (ML) and Artificial Intelligence (AI). Recent requests for feedback from health authorities on use of ML and AI may provide an opportunity for more clarity regarding expectations for use and deployment. Two specific additional outcomes of the workshop were alignment on a key definition for “mechanistic modeling” and the acknowledgement/realization that modeling can have a significant impact on improving sustainability of bioprocessing through elimination of experiments both during development and manufacturing. Feedback from the participants was that there was progression in all of the fields of modeling within scope of the conference. Some areas (e.g. biophysics and molecular modeling) have opportunities for significant research investment to realize full impact. However, the need for ongoing research and development for all model types does not preclude the application to support process development, manufacturing and use in regulatory filings. Analogous to ML and AI, given the current state of the four modeling types, a prospective investment in educating inter-disciplinary subject matter experts (SMEs) (e.g. data science, chromatography) is essential to advancing and maintaining the modeling community.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. P cpcB, P psbA1, P 11770 promoters exhibited consistently higher fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for RBSs, using an operon construct comprising of fluorescent protein genes eyfp and mTurq under P cpcB (P cpcB: eyfp:RBS: mTurq:T rrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Microalgal biotechnology offers a promising platform for the sustainable production of diverse renewable bioactive compounds. The main difference from other microbial bioprocesses is the crucial role that light plays for cultures since it can be used as a source of environmental information to control metabolic processes. Therefore, we can use these criteria to design a bioprocess that aims to stimulate the accumulation of target molecules by controlling light exposure. We study the effect on biochemical and photobiological responses of Golenkinia brevispicula FAUBA-3 to the exposition of different spectral irradiances (high-fluence PAR of narrow yellow spectrum complemented with low intensity of monochromatic radiations of red, blue, and UV-A) under pre-stress and salinity stress conditions. High light coupled to salinity stress affected the photosynthetic activity and photoprotection mechanisms as shown by maximal quantum yield ( Fv/Fm) and non-photochemical quenching (NPQ max) reduction, respectively. High light treatments combined with the proper dose of UV-A radiation under salinity stress induced the highest carotenoid content (2.75 mg g DW -1) composed mainly of lutein and β-carotene, and the highest lipid accumulation (35.3 % DW) with the highest PUFA content (ALA (C18:3) and LA (C18:2)). Our study can guide the strategies for commercial indoor production of G. brevispicula for high-value metabolites.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kB) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Microfiltration is an essential step during biopharmaceutical manufacturing. However, unexpected flux decay can occur. Although the flux decay profile and initial flux are important factors determining microfiltration filterability, predicting them accurately is challenging since the root cause of unexpected flux decay remains elusive. In this study, the methodology for developing a prediction model of flux decay profiles was established. First, the filtration profiles of different monodisperse polystyrene latex and silica beads of various sizes were evaluated. These results revealed that the size and surface electrostatic properties of the beads affect the flux decay profile. Taking the size and surface electrostatic properties of protein aggregates into account, we constructed a predictive model using model bead filtration profiles. We showed that this methodology was applicable to two different microfiltration filters to predict the flux decay profile of therapeutic proteins. Since this prediction model is based on normalized flux, the initial flux must be predicted. We therefore successfully developed a method to predict initial flux based on the Hagen-Poiseuille equation using sample viscosity values for both filters. These prediction models can be used for effective microfiltration scale-up assessment and can be applied during early stage of process development.

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 ( SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6>C conversion in the SMA-iPSCs. The SMN2 T6>C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and anti-apoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

The harvesting of microalgae is the main bottleneck of its large-scale biomass production, and seeking an efficient, green, and low-cost microalgae harvesting technology is one of the urgent problems to be solved. Microbubble air flotation has been proved to be an effective measure, but the generation of the right size of microbubbles and the mechanism of microbubbles-algal cells attachment are still unclear. In this study, microbubble air flotation was used as a harvesting method for Microcystis cultured in agricultural wastewater. The process mechanism of microbubble air flotation harvesting microalgae in wastewater was fully revealed from three aspects (the design of bubble formation, the adhesion law, and the recovery rate of microalgae under different working conditions). The results show that the length of the release pipe is the main factor affecting the proportion of microbubbles with particle size less than 50 μm. In the process of adhesion, when the particle size of microbubbles is 0.6~1.7 times the size of Microcystis, the adhesion efficiency of microbubbles to Microcystis is the highest. Under the conditions of pressure 0.45 MPa, gas-liquid ratio 5% and release pipe length 100 cm, the harvesting performance of Microcystis was the best. Microbubble air flotation has better harvesting performance of Microcystis with higher density. By understanding the mechanism of microbubble flotation, the technical parameters of microbubble flotation for harvesting energy microalgae are optimized to provide support for the development of efficient and low-cost devices and equipment for collecting microalgae.

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its non-pathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and non-dividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of pre-formed empty capsids and viral DNA copies per cell are not limiting to the capsid filling reaction. However, optimal expression of Rep proteins (< 240 ± 13 ag per cell) enables enrichment of the filled capsid population (> 12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multi-billion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγR), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labelling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5 oC and 25 oC. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviours, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve (AUC) of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (less than 5 minutes per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

Sonification, or the practice of generating sound from data, is a promising alternative or complement to data visualization for exploring research questions in the life sciences. Expressing or communicating data in the form of sound rather than graphs, tables, or renderings can provide a secondary information source for multitasking or remote monitoring purposes or make data accessible when visualizations cannot be used. While popular in astronomy, neuroscience, and geophysics as a technique for data exploration and communication, its potential in the biological and biotechnological sciences has not been fully explored. In this review, we introduce sonification as a concept, some examples of how sonification has been used to address areas of interest in biology, and the history of the technique. We then highlight a selection of biology-related publications that involve sonifications of DNA datasets and protein datasets, sonifications for data collection and interpretation, and sonifications aimed to improve science communication and accessibility. Through this review, we aim to show how sonification has been used both as a discovery tool and a communication tool and to inspire more life-science researchers to incorporate sonification into their own studies.

A high rate upflow anaerobic polyfoam-based bioreactor (UAPB) was developed for lab-scale in-situ biogas upgrading. The use of a porous diffuser, alongside high gas recirculation, led to a higher H 2 liquid mass transfer, and subsequently to a better uptake for high CH 4 content (up to 90%). Although our attempts to optimize both operational parameters (H 2 flow rate and gas recirculation ratio) were not successful, at a very high recirculation ratio (32) and flow rate (54 mL/h), a significant improvement of the hydrogen consumption was achieved. These operational conditions have in turn driven the methanogenic community towards the dominance of Methanosaetaceae, which out-competed Methanosarcinaceae. Nevertheless, a highly stable methane production rate was observed despite the methanogenic turnover. During the different applied operational conditions, the bacterial community was especially impacted, resulting in substantial shifts of taxonomic groups. Notably, Aeromonadaceae was the only bacterial group positively correlated with increasing hydrogen consumption rates. The capacity of Aeromonadaceae to extracellularly donate electrons suggests that direct interspecies electron transfer (DIET) enhanced biogas upgrading. Overall, the proposed innovative biological in-situ biogas upgrading technology using the UAPB configuration shows promising results for stable, simple and effective biological biogas upgrading.