Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.

The efficient isolation of viable and intact circulating tumor cells (CTCs) from blood is critical for the genetic analysis of cancer cells, prediction of cancer progression, development of drugs, and evaluation of therapeutic treatments. While conventional cell separation devices utilize the size difference between CTCs and other blood cells, they fail to separate CTCs from white blood cells (WBCs) due to significant size overlap. To overcome this issue, we present a novel approach that combines curved contraction–expansion (CE) channels with dielectrophoresis (DEP) and inertial microfluidics to isolate CTCs from WBCs regardless of size overlap. This label-free and continuous separation method utilizes dielectric properties and size variation of cells for the separation of CTCs from WBCs. The results demonstrate that the proposed hybrid microfluidic channel can effectively isolate A549 CTCs from WBCs regardless of their size with a throughput of 300 μL/min, achieving a high separation distance of 233.4 μm at an applied voltage of 50 Vp–p. The proposed method allows for the modification of cell migration characteristics by controlling the number of CE sections of the channel, applied voltage, applied frequency, and flow rate. With its unique features of a single-stage separation, simple design, and tunability, the proposed method provides a promising alternative to the existing label-free cell separation techniques and may have a wide range of applications in biomedicine.

The development of gene therapies based on recombinant adeno-associated viruses (rAAVs) has grown exponentially, so the current rAAV manufacturing platform needs to be more efficient to satisfy rising demands. Viral production exerts great demand on cellular substrates, energy, and machinery; therefore, viral production relies heavily on the physiology of the host cell. Transcriptomics, as a mechanism-driven tool, was applied to identify significantly regulated pathways and to study cellular features of the host cell for supporting rAAV production. This study investigated the transcriptomic features of two cell lines cultured in their respective media by comparing viral-producing cultures with non-producing cultures over time in parental human embryonic kidney cells (HEK293). The results demonstrate that the innate immune response signaling pathways of host cells (e.g., RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytosolic DNA sensing pathway, JAK-STAT signaling pathway) were significantly enriched and upregulated. This was accompanied by the host cellular stress responses, including endoplasmic reticulum stress, autophagy, and apoptosis in viral production. In contrast, fatty acid metabolism and neutral amino acid transport were downregulated in the late phase of viral production. Our transcriptomics analysis reveals the cell-line independent signatures for rAAV production and serves as a significant reference for further studies targeting the productivity improvement in the future.

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.

When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.

Process analytical technology (PAT) tools such as Raman Spectroscopy have become established tools for real time measurement of CHO cell bioreactor process variables and are aligned with the QbD approach to manufacturing. These tools can have a significant impact on process development if adopted early, creating an end-to-end PAT/QbD focused process. This study assessed the impact of Raman based feedback control on early and late phase development bioreactors by using a Raman based PLS model and PAT management system to control glucose in two CHO cell line bioreactor processes. The impact was then compared to bioreactor processes which used manual bolus fed methods for glucose feed delivery. Process improvements were observed in terms of overall bioreactor health, product output and product quality. Raman controlled batches for Cell Line 1 showed a reduction in glycation of 43.4% and 57.9%, respectively. Cell Line 2 batches with Raman based feedback control showed an improved growth profile with higher VCD and viability and a resulting 25% increase in overall product titer with an improved glycation profile. The results presented here demonstrate that Raman spectroscopy can be used in both early and late-stage process development and design for consistent and controlled glucose feed delivery.

Silk fibroin (SF), extracted from Bombyx mori, has unique physicochemical properties to achieve an efficient wound dressing. In this study, reduced graphene oxide (RGO)/ZnO NPs/silk fibroin nanocomposite was made, and an innovative nanofiber of SF/polyvinyl alcohol (PVA)/RGO/ZnO NPs was ready with the electrospinning technique and successfully characterized. The results of MIC and OD analyses were used to investigate the synthesized materials' antibacterial effects and displayed that the synthesized materials could inhibit growth against Staphylococcus aureus and Escherichia coli bacteria. However, both in vitro cytotoxicity (MTT) and scratch wound studies have shown that RGO/ZnO NPs and SF/PVA/RGO/ZnO NPs are not only non-toxic to NIH 3T3 fibroblasts, but also can cause cell viability, cell proliferation, and cell migration. Furthermore, improving the synthesized nanofiber's structural properties in the presence of RGO and ZnO NPs has been confirmed by performing tensile strength, contact angle, and biodegradation analyses. Also, in a cell attachment analysis, fibroblast cells had migrated and expanded well in the nanofibrous structures. Moreover, in vivo assay, SF/PVA/RGO/ZnO NPs nanofiber treated rats and has been shown significant healing activity and tissue regeneration compared with other treated groups. Therefore, this study suggests that SF/PVA/RGO/ZnO NPs nanofiber is a hopeful wound dressing for preventing bacteria growth and improving superficial wound repair.

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3′ end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS.

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using 13C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water–water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.

Mixed-mode chromatography combines features of ion-exchange chromatography and hydrophobic interaction chromatography and is increasingly used in antibody purification. As a replacement for flow-through operations on traditional unmixed resins or as a pH-controlled bind-and-elute step, the use of both interaction modes promises a better removal of product-specific impurities. However, the combination of the functionalities makes industrial process development significantly more complex, in particular the identification of the often small elution window that delivers the desired selectivity. Mechanistic modeling has proven that even difficult separation problems can be solved in a computer-optimized manner once the process dynamics have been modeled. The adsorption models described in the literature are also very complex, which makes model calibration difficult. In this work, we approach this problem with a newly constructed model that describes the adsorber saturation with the help of the surface coverage function of the colloidal particle adsorption model for ion-exchange chromatography. In a case study, a model for a pH-controlled antibody polishing step was created from six experiments. The behavior of fragments, aggregates, and host cell proteins was described with the help of offline analysis. After in silico optimization, a validation experiment confirmed an improved process performance in comparison to the historical process set point. In addition to these good results, the work also shows that the high dynamics of mixed-mode chromatography can produce unexpected results if process parameters deviate too far from tried and tested conditions.

Integrated continuous bioprocessing has been identified as the next important phase of evolution in biopharmaceutical manufacturing. Multiple platform technologies to enable continuous processing are being developed. Multi-column counter-current chromatography is a step in this direction to provide increased productivity and capacity utilization to capture biomolecules like monoclonal antibodies (mAbs) present in the reactor harvest and remove impurities. Model-based optimization of two prevalent multi-column designs, 3-column and 4-column periodic counter-current chromatography (PCC) was carried out for different concentrations of mAbs in the feed, durations of cleaning-in-place and equilibration protocols. The multi-objective optimization problem comprising three performance measures, namely, product yield, productivity, and capacity utilization was solved using the Radial basis function optimization technique. The superficial velocities during load, wash, and elute operations, along with durations of distinct stages present in the multi-column operations were considered as decision variables. Optimization results without the constraint on number of wash volumes showed that 3-Column PCC performs better than 4-Column PCC. For example, at a feed concentration of 1.2 mg/mL, productivity, yield and capacity utilization, respectively, were 0.024 mg/mL.s, 0.94, and 0.94 for 3-Column PCC and 0.017 mg/mL.s, 0.87, and 0.83 for 4-column PCC. Similar trends were observed at higher feed concentrations also. However, when the constraint on number of wash volumes is included, 4-Column PCC was found to result in consistent productivity and product yield under different operating conditions but at the expense of reduced capacity utilization.

Wound healing is a process getting affected by internal and external factors and might be interrupted by infections. To overcome infections during wound healing, novel antibacterial agents such as antimicrobial peptides have gained popularity because of the rising antibiotic resistance. Therefore, in this study, a three-dimensional polymeric scaffold was designed for the controlled release of HF-18 peptide, with the contribution of hyaluronic acid, chondroitin sulfate, and chitosan polymers with the crosslinker genipin. The obtained scaffold structure (OPT) was found to have interconnected pores, was pH-responsive and swelled more in acidic conditions (5446.5% at pH: 5.0). It was observed that HF-18-loaded OPT (P-OPT) was able to release HF-18 peptide both in acidic and neutral conditions in a controlled release manner. This study also demonstrated that both OPT and P-OPT were biocompatible and promoted L929 cell attachment and migration. Antimicrobial activity assessments demonstrated that P-OPT was effectively bactericidal on Staphylococcus aureus and methicillin-resistant S. aureus. Moreover, OPT produced a synergistic effect on the antimicrobial activity of HF-18 peptide, as P-OPT showed activity below the reported MIC value. As a result, OPT is considered a promising scaffold as a carrier for HF-18 for wound healing.

Umbelopsis ramanniana was investigated to increase carotenoid production. Nine different carbon sources and six different nitrogen sources were evaluated for the maximum carotenoid production. The most effective nitrogen and carbon sources were KNO3 and lactose, respectively. Then, the optimization of medium components for enhancement of carotenoid production by Umbelopsis ramanniana was achieved using Plackett–Burman design. Box–Behnken response surface methodology was applied to further optimize carotenoid and biomass production. Carbon to nitrogen ratio, lactose concentration, and shaking speed were studied as variables in Box–Behnken design. The optimum conditions for carotenoid and biomass production were determined as 32.42 g/L of lactose concentration, 20:1 of carbon to nitrogen ratio, and shaking speed of 130 rpm. The maximum carotenoid and biomass production under optimized conditions were 1141 μg/L (β-carotene-Eq) and 13.14 g/L, respectively. When compared to the control fermentation, carotenoid, and biomass production were increased by about 2 and 1.3 folds, respectively.

This article developed a novel electrochemical immunosensor for the specific detection of aflatoxin B1 (AFB1). Amino-functionalized iron oxide nanoparticles (Fe3O4-NH2) were synthesized. Fe3O4-NH2 were chemically bound on self-assembly monolayers (SAMs) of mercaptobenzoic acid (MBA). Finally, polyclonal antibodies (pAb) were immobilized on Fe3O4-NH2-MBA. The sensor system was evaluated through atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A reduction in the anodic and cathodic peak currents was observed after the assembly of the sensor platform. The charge transfer resistance (Rct) was increased due to the electrically insulating bioconjugates. Then, the specific interaction between the sensor platform and AFB1 blocks the electron transfer of the [Fe(CN)6]3−/4− redox pair. The nanoimmunosensor showed a linear response range estimated from 0.5 to 30 μg/mL with a limit of detection (LOD) of 9.47 μg/mL and a limit of quantification (LOQ) of 28.72 μg/mL for AFB1 identification in a purified sample. In addition, a LOD of 3.79 μg/mL, a LOQ of 11.48 μg/mL, and a regression coefficient of 0.9891 were estimated for biodetection tests on peanut samples. The proposed immunosensor represents a simple alternative, successfully applied in detecting AFB1 in peanuts, and therefore, represents a valuable tool for ensuring food safety.

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (qp) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA) cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD+/NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD+/NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Mesenchymal Stem Cells (MSCs) are non-hematopoietic and multipotent stem cells, which have been considered in regenerative medicine. These cells are easily separated from different sources, such as bone marrow (BM), umbilical cord (UC), adipose tissue (AT), and etc. MSCs have the differentiation capability into chondrocytes, osteocytes, and adipocytes; This differentiation potential along with the paracrine properties have made them a key choice for tissue repair. MSCs also have various advantages over other stem cells, which is why they have been extensively studied in recent years. The effectiveness of MSCs-based therapies depend on several factors, including differentiation status at the time of use, concentration per injection, delivery method, the used vehicle, and the nature and extent of the damage. Although, MSCs have emerged promising sources for regenerative medicine, there are potential risks regarding their safety in their clinical use, including tumorigenesis, lack of availability, aging, and sensitivity to toxic environments. In this study, we aimed to discuss how MSCs may be useful in treating defects and diseases. To this aim, we will review recent advances of MSCs action mechanisms in regenerative medicine, as well as the most recent clinical trials. We will also have a brief overview of MSCs resources, differences between their sources, culture conditions, extraction methods, and clinical application of MSCs in various fields of regenerative medicine.

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2–3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.