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.

Sub-models representing transformation processes by microorganisms and hydrolases, a one-dimensional (1-D) biofilm, and a bioreactor were integrated to simulate organic-matter fermentation and methane (CH 4) production in an anaerobic moving bed biofilm reactor (AnMBBR). The integrated models correctly represented all experimental observations and identified mechanisms underlying how and why AnMBBR performance changed when the volumetric loading rate (VLR) of total chemical oxygen demand (TCOD) increased from 3.9 to 19.5 kg COD T/m 3-d. The fractional removal of TCOD and CH 4 production decreased as the VLR of TCOD increased due in part to an increasing biofilm thickness that filled the interior channels of plastic carriers and led to a decrease in biofilm surface area and an increase in the mass-transfer boundary layer. Also, the ~25-day duration for each VLR of TCOD was too short to allow the biofilm to establish a new quasi steady state with respect to biofilm thickness. The mechanistic understanding of how biofilm characteristics and process performance respond to increased VLR of TCOD can be applied in engineering practice to improve AnMBBR process design and operation.

Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC 50 value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.

The bioaugmentation performance is severely reduced in the treatment of high saline pesticide wastewater because the growth and degradation activity of pesticide degraders are significantly inhibited by high salt concentrations. In this study, an artificial halotolerant degrader J9U-MP capable of mineralizing p-nitrophenol (PNP) substituted organophosphorus pesticides (OPs) [e.g., methyl parathion (MP)] was created by integrating a MP-mineralizing pathway into the genome of a salt-tolerant chassis Halomonas cupida J9. MP degradation coupled with stable isotope analysis indicated that J9U-MP was able to metabolize MP as a sole carbon source to finally produce CO 2 and H 2O in high-salt media (up to 120 g/L NaCl). J9U-MP was genetically stable during passage culture and exogenous gene integration did not negatively influence growth and metabolism of J9U-MP. A real-time monitoring system was established with enhanced green fluorescent protein (EGFP) to track the movement and activity of J9U-MP in environmental remediation. A low-oxygen tolerant system was developed by enhancing oxygen utilization, which makes J9U-MP maintain the MP-mineralizing activity under oxygen-limited conditions. More importantly, efficient mineralization of MP by J9U-MP in high saline wastewater was demonstrated. This study highlights that synthetic biology has opened up new avenues for creating stress-resistant pollutants-mineralizing microbes. Competitive advantages of J9U-MP in high-salinity and low-oxygen environments make this degrader suitable for bioaugmentation of pesticide wastewater.

The devastating global toll precipitated by the SARS CoV-2 outbreak and the profound impact of vaccines in stemming that outbreak has established the need for molecular platforms capable of rapidly delivering effective, safe and accessible medical interventions in pandemic preparedness. We describe a simple, efficient and adaptable process to purify SARS CoV-2 virus-like particles (VLPs) that can be readily scaled for manufacturing. A rapid but gentle method of tangential flow filtration using a 100 kDa semi-permeable membrane concentrates and buffer exchanges 0.5 L of SARS CoV-2 VLP containing supernatant into low salt and optimal pH for anion exchange chromatography. VLPs are washed, eluted under high salt, dialysed into physiological buffer, sterile filtered and aliquoted for storage at – 80 0C. Purification is completed in less than two days. A simple quality control process includes Western blot for coincident detection of Spike, Membrane and Envelope protein as a proxy for intact VLP, ELISA to detect conformationally sensitive Spike using readily available anti-Spike and/or anti-RBD antibodies, and negative stain and immunogold electron microscopy to validate particulate, Spike crowned VLPs. This process to purify SARS CoV-2 VLPs for preclinical studies serves as a roadmap for preparation of more distantly related VLPs for pandemic preparedness.

Detergent-mediated viral inactivation is an important process step for ensuring viral safety of parenteral biotherapeutics, including plasma proteins and monoclonal antibodies (mAb). The conventional Triton™ X-100 detergent has ecological toxicity concerns and REACH classification that mandate replacement in the biopharmaceutical industry. Criteria for a replacement detergent include viral inactivation efficacy, acceptable safety and biodegradation profile, process removal and quality suitable for parenteral drug product manufacturing. A non-ionic, C11-15 secondary alcohol ethoxylate, Deviron ® 13-S9 detergent, has been demonstrated to meet the necessary requirements for detergent performance. Benchmarking studies with Triton™ X-100 demonstrate comparable performance with a panel of enveloped viruses in multiple matrices, including human IgG, clarified cell culture harvest, and fractionated plasma. Deviron ® 13-S9 detergent demonstrated viral inactivation efficiency comparable to or better than, Triton™ X-100 detergent, achieving > 5 log reduction values (LRV). Critical micelle concentration (CMC) was determined across different temperatures and media. Deviron ® 13-S9 detergent was demonstrated to be readily biodegradable according to OECD 301B guidelines. Effective removal with typical chromatography processes used in downstream purification was confirmed. These findings support Deviron ® 13-S9 detergent as a viable alternative to Triton™ X-100 detergent, ensuring robust viral inactivation, environmental compatibility, and alignment with regulatory requirements.

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.

Scale-up of microbial bioprocesses to production scale is accompanied by significant changes in growth conditions. One aspect are rapidly changing substrate concentrations due to prolonged mixing times. The impact of substrate gradients on the physiology of Saccharomyces cerevisiae has been studied for glucose but no other industrially relevant carbohydrate. With different transport mechanism, non-glucose sugars will lead to a different intracellular response. Here we studied the response of S. cerevisiae to gradients for four different sugars: glucose, fructose, sucrose and maltose. To study the impact of the carbon sources as well as the large-scale gradients, steady-state and dynamic feast/famine cultivation conditions were applied. The physiology, intracellular metabolome as well as the proteome were compared. Especially, gradients of maltose lead to a significant decrease in biomass yield. Under dynamic glucose, fructose and sucrose conditions S. cerevisiae was able to maintain the biomass yield of steady-state conditions. Although the physiology was very comparable for these sugars, the intracellular metabolome and proteome changed. The concentration of upper glycolytic enzymes decreased for glucose and maltose (up to -60% and -40% respectively), while an increase was observed for sucrose and fructose when exposed to gradients. At the same time, enzymes of lower glycolysis were increased. Interestingly, common stress-related proteins were decreased during dynamic conditions. The observed adaptations to repeating gradients highlight the importance to study physiology and metabolism under dynamic conditions to obtain results that are relevant for the envisioned large-scale process.

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.