The CHO VRC01 cell line produces an anti-HIV IgG1 monoclonal antibody containing N-linked glycans on both the Fab (variable) and Fc (constant) regions. Site-specific glycan analysis was used to measure the complex effects of cell culture process conditions on Fab and Fc glycosylation. Experimental data revealed major differences in glycan fractions across the two sites. Bioreactor pH was found to influence fucosylation, galactosylation, and sialylation in the Fab region and galactosylation in the Fc region. To understand the complex effects of process conditions on site-specific N-linked glycosylation, a kinetic model of site-specific N-linked glycosylation was developed. The model parameters provided mechanistic insights into the differences in glycan fractions observed in the Fc and Fab regions. Enzyme activities calculated from the model provided insights into the effect of bioreactor pH on site-specific N-linked glycosylation. Model predictions were experimentally tested by measuring glycosyltransferase-enzyme mRNA-levels and intracellular nucleotide sugar concentrations. The model was used to demonstrate the effect of increasing galactosyltransferase activity on site-specific N-linked glycan fractions. Experiments involving galactose and MnCl 2 supplementation were used to test model predictions. The model is capable of providing insights into experimentally measured data and also of making predictions that can be used to design media supplementation strategies.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pH ER). We then investigated how changes in lactate metabolism impacted pH ER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pH ER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in stationary phase in all perfusion and fed-batch conditions. pH ER decline in stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Protein aggregate levels correlated positively with pH ER, indicating that aggregation is not solely driven by oxidative stress and cellular protein load. Improved characterization of intracellular changes in bioprocesses can help guide media optimizations, improve bioprocess control strategies or provide new targets for cell engineering.

Pterostilbene (PST), a 3,5- O-methylated derivative of resveratrol (RSV), is a natural potent antioxidant produced by some plants in trace amounts as defense compounds. It exhibits various health-promoting activities, such as anticancer, antiviral, and antimicrobial effects. Large-scale biosynthesis of PST is crucial due to the challenges associated with extracting it from plants. This study aims to develop an efficient method for PST production using an engineered E. coli strain by feeding RSV as an intermediate compound. We introduced a two-step substrate addition strategy combined with immobilized RSV (IMRSV) on macroporous adsorption resin (MAR) to enhance PST production. Five MARs were selected for RSV immobilization, and the substrate additions strategy and fermentation parameters for PST synthesis were optimized. A maximum PST concentration of 402.78 ± 9.20 mg/L was achieved, representing a 239% increase over the control. The PST titer reached 395.20 ± 23.89 mg/L in a 3-L bioreactor. In conclusion, the combination of a two-step substrate adding system and IMRSV is a promising approach for the economical and industrial-scale production of PST.

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.

The concept of sustainable production necessitates the utilization of waste and by-products as raw materials, the implementation of biotechnological processes, and the introduction of automated real-time monitoring for efficient use of resources. One example is the biocatalyzed conversion of the reusable by-product glycerin by acetic acid bacteria to dihydroxyacetone (DHA), which is of great importance to the cosmetic industry. The application of compact spectrometers enables the rapid measurement of samples while simultaneously reducing the consumption of resources and energy. Yet, this approach requires comprehensive data preprocessing and, on occasion, multivariate data analysis. For the process monitoring of the production of DHA, a low-field 1H nuclear magnetic resonance (NMR) spectrometer was implemented in on-line mode. Small-volume samples were taken from a bypass and transferred to the spectrometer by an autosampler. Complete analysis within minutes allowed real-time process control. To this purpose, reliable automated spectral preprocessing preceded the creation of a univariate model. The model enabled the acquisition of process knowledge from chemical kinetics and facilitated the tracking of both substrate and product concentrations, requiring independent calibration. As a second multivariate approach, principal component analysis was utilized to monitor the process in a semi-quantitative manner without the necessity for calibration. The results of this study are beneficial for real-time monitoring applications with the objective of exerting control over the process in question, while minimizing expenditure.

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.

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals manufactured by well-established processes using Chinese Hamster Ovary (CHO) cells. Next-generation biomanufacturing using alternative hosts like Komagataella phaffii could improve the accessibility of these medicines, address broad societal goals for sustainability, and offer financial advantages for accelerated development of new products. Antibodies produced by K. phaffii, however, may manifest unique molecular quality attributes, like host-dependent, product-related variants, that could raise potential concerns for clinical use. We demonstrate here conservative modifications to the amino acid sequence of aglycosylated antibodies based on the human IgG1 isotype that minimize product-related variations when secreted by K. phaffii. A combination of 2-3 changes of amino acids reduced variations across six different aglycosylated versions of commercial mAbs. Expression of a modified sequence of NIST mAb in both K. phaffii and CHO cells showed comparable biophysical properties and molecular variations. These results suggest a path towards production of high-quality mAbs that could be expressed interchangeably by either yeast or mammalian cells. Improving molecular designs of proteins to enable a range of manufacturing strategies for well-characterized biopharmaceuticals could accelerate global accessibility and innovations.

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.