Reversible electroporation (EP) is a pivotal biophysical technology that leverages pulsed electric fields to enhance the permeability of cell membranes, thereby facilitating the introduction of foreign material into cells. In this review, we provide an overview of bulk electroporators and microfluidic-enabled EP systems, focusing on their controversial points of mechanisms, architectures, and parameter settings. Bulk electroporators have been extensively commercialized with settled form including pulse generator and accessories (i.e., EP cuvette and plates). Researchers have made efforts to increase the throughput and simplify the operation of bulk EP systems. Additionally, microfluidics has emerged as a promising technology for optimizing EP parameters and enhancing the performance. Given the significant structural differences between these two types of EP systems, their operating conditions such as temperature, voltage, and pulse parameters are discussed. Research tend to operate single cells under more concentrated electric field induced by low voltage, enabling a quantitative exogenous materials delivery and numerical simulation. However, due to cost constraints and properties of materials utilized in laboratories, the commercialization of laboratory prototypes has been impeded. Furthermore, the technological limitations, current commercialization status, and development trends have been examined.

Bayesian optimization is a stochastic, global black-box optimization algorithm. By combining Machine Learning with decision-making, the algorithm can optimally utilize information gained during experimentation to plan further experiments-while balancing exploration and exploitation. Although Design of Experiments has traditionally been the preferred method for optimizing bioprocesses, AI-driven tools have recently drawn increasing attention to Bayesian optimization within bioprocess engineering. This review presents the principles and methodologies of Bayesian optimization and focuses on its application to various stages of bioprocess engineering in upstream and downstream processing.

Protein developability is an important, yet often overlooked, aspect of protein discovery campaigns that is a key driver of utility. Recent advances have improved developability screening capacity, making it an increasingly viable option in early-stage discovery. Here, we engineered one component of developability, stability, of two affibody proteins-one that targets death receptor 5 and another that targets tumor necrosis factor receptor 1-previously evolved to bind receptor and non-competitively inhibit signaling via conformational modulation. Starting from an error-prone PCR library of each affibody, variants were screened via yeast surface display binder selections, including depletion of non-specific binders, followed by developability assessment using the on-yeast protease and yeast display level assays. Multiplex deep sequencing identified variants for further evaluation. Purified variants exhibited elevated stability-8°C to 14°C increase in T-with maintained 1-2 nM affinity for the TNFR1 affibody and 30-fold improvement in the DR5 affibody affinity to 0.8 nM.

Microalgal cultivation for biofuels and proteins holds significant promise but faces challenges in achieving economically viable biomass productivity under variable environmental conditions. This study introduces a forecast-informed pond operation (FIPO) system that uses numerical weather prediction (NWP) ensemble forecasts and the biomass assessment tool (BAT) to optimize daily dilution rates for enhanced biomass production. In contrast to the current practice, where fixed dilution rates are based on operator experience, the FIPO system determines the optimal dilution rate based on future weather forecasts and biomass growth conditions. Our experiments validate the effectiveness of FIPO in both short- and long-term growth scenarios. In short-term experiments, FIPO increased biomass production by 21.3% compared to batch growth and 7.4% over fixed dilution (60% every 3 days) operations. The NWP forecast-informed operations achieved biomass production nearly identical to that using perfect weather forecasts, highlighting the accuracy of current NWP forecasts for guiding pond operations. In long-term experiments, FIPO resulted in biomass production increases of 13.3% and 17.8% compared to two fixed dilution rates (60% every 3 days and 20% daily). These findings underscore the viability of using NWP forecasts to optimize microalgal cultivation systems. By adjusting daily dilution rates in response to forecasted weather, operators can achieve higher biomass yields and mitigate risks associated with environmental variability. This study provides a foundation for future research and practical applications in commercial-scale microalgal production.

Natural killer (NK) cells, integral to the innate immune system, are notable in cell therapies because of their applicability in allogeneic treatments, distinguishing them from T cells typically employed in conventional cell therapies. However, their limited half-life (proliferative capability) poses a challenge for therapy. The limited half-life creates difficulties in obtaining a sufficient number of cells for in vitro adoptive therapy. Gene modification is commonly employed to address this limitation. However, due to concerns such as genetic instability and unintended gene expression, its suitability for long-term cultivation is uncertain. Consequently, safer alternatives are needed. We aimed to promote NK cell proliferation through feeder cells rather than genetic modification. These cells are designed to interact with NK cells without adverse effects, aiming to promote NK cell proliferation more safely. In our study, during the tailoring of feeder cells, we excluded genetic modification and instead applied chemical-based extracellular stress. The extracellular stress applied consisted of hypoxia and cytochalasin D. By treating the feeder cells with these stressors, we were able to inhibit feeder cell proliferation, enabling them to function more efficiently as feeder cells. Furthermore, we observed that the feeder cells subjected to extracellular stress exhibited upregulated expression of 4-1BBL, which enhances the 4-1BB/4-1BBL interaction with NK cells. The upregulated 4-1BBL binds to 4-1BB on the surface of NK cells, promoting their proliferation. Additionally, following coculture with feeder cells exposed to extracellular stress, we observed an upregulation of CD56 expression on the surface of NK cells. These CD56 NK cells influence NK cell proliferation through enhanced cytokine release. We further validated this process under dynamic conditions where shear stress is applied, demonstrating that the feeder cell-mediated enhancement of NK cell proliferation is applicable under dynamic conditions such as those found in bioreactors.

Host cell proteins (HCPs) are process-related impurities of therapeutic protein production and may affect product quality or patient safety. In clinical trials, certain HCPs (e.g., PLBL2 or CCL2) that co-purify with the therapeutic protein have been associated with immune reactions in patients. In this study, we examined the adjuvant potential of six commonly detected HCPs from CHO cells (PRDX1, S100A4, PLBL2, CCL2, CLU, and YWHAE) using an in vitro dendritic cell (DC) maturation assay. Recombinant HCPs were expressed in CHO cells to mimic manufacturing conditions. PRDX1, S100A4, and PLBL2 caused a slight increase in the expression of maturation markers on DCs, while YWHAE, CLU, and CCL2 did not. Interestingly, CLU and CCL2 reduced the DC maturation induced by rituximab. In addition, we observed that process parameters such as elution conditions during chromatographic purification can influence HCP aggregation, which in turn can mask or enhance the intrinsic adjuvant potential of an HCP. These findings not only provide initial insights into the adjuvant potential of individual HCPs but also indicate that the quantity as well as the degree of aggregation of HCPs might influence adjuvanticity.

Vaccination represents a promising approach to combat resistant Klebsiella pneumoniae (KP). However, there is currently no licensed vaccine in the veterinary field. Outer membrane proteins have been proven to possess good immunogenicity, but Freund's adjuvant, which is commonly used to administer protein vaccines, has limitations such as a complicated formulation process as well as a tendency to cause pain and inflammation in animals. Here, we prepared a nano-vaccine based on zeolitic imidazolate framework-8 (ZIF-8)-encapsulated outer membrane protein PhoE and evaluated its efficiency in enhancing humoral and cellular immune responses in BALB/c mice. ZIF-8 nanoparticles rapidly delivered the protein antigen into dendritic cells and successfully activated them. In addition, significantly higher IgG antibody titers, cytokine levels, and splenocyte proliferation indices were founded in mice subcutaneously immunized with PhoE@ZIF-8 than in those receiving free PhoE alone. In a BALB/c mouse model, PhoE@ZIF-8 elicited a strong immune response with improved prophylactic efficacy against KP that was similar to the Freund's adjuvant-formulated vaccine. Based on the superiority of this nano-vaccine with good biocompatibility, inexpensive preparation and higher efficiency of delivering antigen into cells, ZIF-8 can serve as a promising replacement for Freund's adjuvant in research, with a prospective usage for vaccines against bacterial pathogens in the veterinary field.

Recombinant adeno-associated virus (rAAV) has emerged as an attractive gene delivery vector platform to treat both rare and pervasive diseases. With more and more rAAV-based therapies entering late-stage clinical trials and commercialization, there is an increasing pressure on the rAAV manufacturing process to accelerate drug development, account for larger trials, and commercially provide high doses. Still, many of the pre-clinical and clinical manufacturing processes are tied to outdated technologies, which results in substantial production expenses. Those processes face challenges including low productivity and difficult scalability, which limits its ability to provide for required dosages which in turn influences the accessibility of the drug. And as upstream efforts are expected to increase productivities, the downstream part needs to adapt with more scalable and efficient technologies. In this review, both traditional and novel rAAV downstream technologies are presented and discussed. Traditional rAAV downstream processes are based on density gradient ultracentrifugation and have been shown to effectively purify rAAVs with high yields and purities. However, those processes lack scalability and efficiency, which is why novel rAAV downstream processes based on column-chromatography have emerged as an attractive alternative and show potential for integration in continuous processes, following the principle of next-generation manufacturing.

Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.

There is now an urgent need to develop reliable, rapid, and cost-effective methods for bacterial detection, particularly for point-of-care applications. This study explores the unique properties of silicon nanowire (SiNW) arrays as a resistive sensing platform for detecting Listeria innocua. Vertically aligned SiNWs, fabricated via metal-assisted chemical etching, exhibited high sensitivity to bacterial adsorption. Conductance measurements revealed a more than 10-fold increase as bacterial concentrations rose from 10 to 10 CFU/mL, with clear saturation at higher levels. The study employed both direct current (DC) and alternating current (AC) methodologies, with AC conductance consistently outperforming DC due to reduced potential barrier effects. An equivalent circuit model was developed to describe the impedance behavior of the SiNW-bacteria system, offering valuable insights into charge transport mechanisms. These results demonstrate the potential of SiNW-based sensors as robust, scalable, and high-performance diagnostic tools. Beyond bacterial detection, the proposed platform offers promising applications in clinical diagnostics, environmental monitoring, and food safety.

The transcriptional factors control the expression of many genes and represent an important layer of complexity in cell factories. However, the effect of individual sigma factor deletions from a biomanufacturing perspective has not been addressed. In this contribution, growth, green fluorescence protein (GFP) expression, and oxygen consumption of Escherichia coli BW25113 strains with individual inactivation of each sigma factor were characterized under various conditions. Specific growth rate, specific GFP fluorescence, and fluorescence emission rates were compared in a mineral media and in lysogeny broth at two temperatures. rpoD has been reported to be lethal for E. coli; however, the evaluated rpoD mutant did not exhibit major growth defects in the mineral medium. This is attributed to the presence of a second copy of rpoD in this strain. GFP was expressed at three different induction levels in a mineral and LB media. The fliA mutant was the best producer in the mineral medium, whereas the rpoD mutant overperformed the other strains in LB medium. This suggests that a lower rpoD gene dosage is positive for the performance of the cell factory in a complex medium. In cultures at 20°C, the rpoS mutant exhibited the greatest recombinant expression. To our knowledge, this is the first systematic study evaluating the potential of sigma factor deletion for improving recombinant protein production.

Airborne pathogens, such as influenza and SARS-CoV-2, pose significant health risks. While traditional diagnostic methods have limitations, optical biosensors offer a promising solution due to their rapid, sensitive, and cost-effective nature. This review focuses on the application of optical biosensors, including colorimetry, surface plasmon resonance, surface-enhanced Raman spectroscopy, and fluorescence techniques, for the detection of influenza and SARS-CoV-2. We discuss the advantages of these techniques, such as their potential for point-of-care testing and early disease detection. By addressing the limitations of existing methods and exploring emerging technologies, optical biosensors can play a crucial role in combating the spread of airborne pathogens. This review provides a comprehensive overview of optical biosensor techniques for the detection of both SARS-CoV-2 and influenza, addressing a significant gap in the literature.

Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (HO) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade HO. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG, and katE, from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM HO was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing HO consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, SscaCYP_E284I, and trans-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.

Chinese hamster ovary (CHO) cells represent the most widely utilized host system for industrial production of high-quality recombinant protein therapeutics. Novel CHO cell line development is achieved through genetic and cellular engineering approaches, effectively addressing limitations such as clonal variation and productivity loss during culture. Previous studies have established that MAT2A inhibition in tumor cells promotes expression of the cyclin-dependent kinase inhibitor p21, inducing antitumor activity. Notably, p21 induction has been shown to enhance recombinant protein expression in CHO cells by triggering cell cycle arrest. In this study, we identified MAT2A as a potential regulatory target, showing significant differential expression in transfected CHO cells with elevated versus diminished recombinant protein production. To investigate this phenomenon, we generated CHO cells with MAT2A knockdown (shMAT2A) and evaluated their recombinant protein output. Results demonstrated that MAT2A silencing enhanced recombiant protein/antibody production by 1.73-/1.70-fold through suppression of CyclinD1, thereby activating p21 and inducing G1 phase arrest. Furthermore, pharmacological inhibition of MAT2A using small molecules increased cell volume, boosted metabolic activity, and improved specific antibody productivity of recombiant protein/antibody production by 1.88-/2.16-fold in transfected CHO cells. These findings advance our understanding of MAT2A-mediated regulatory mechanisms and provide a strategic framework for developing high-efficiency CHO cell expression systems.

The structural motif of hetero-di-C-glycosyl compound is prominent in plant polyphenol natural products and involves two different glycosyl residues (e.g., β-d-glucosyl, β-d-xylosyl) attached to carbons of the same phenolic ring. Polyphenol hetero-di-C-glycosides attract attention as specialized ingredients of herbal medicines and their tailored synthesis by enzymatic C-glycosylation is promising to overcome limitations of low natural availability and to expand molecular diversity to new-to-nature glycoside structures. However, installing these di-C-glycoside structures with synthetic precision and efficiency is challenging. Here we have characterized the syntheses of C-β-galactosyl-C-β-glucosyl and C-β-glucosyl-C-β-xylosyl structures on the phloroglucinol ring of the natural polyphenol phloretin, using kumquat (Fortunella crassifolia) C-glycosyltransferase (FcCGT). The FcCGT uses uridine 5'-diphosphate (UDP)-galactose (5 mU/mg) and UDP-xylose (0.3 U/mg) at lower activity than UDP-glucose (3 U/mg). The 3'-C-β-glucoside (nothofagin) is ~10-fold less reactive than non-glycosylated phloretin with all UDP-sugars, suggesting the practical order of hetero-di-C-glycosylation as C-galactosylation or C-xylosylation of phloretin followed by C-glucosylation of the resulting mono-C-glycoside. Each C-glycosylation performed in the presence of twofold excess of UDP-sugar proceeds to completion and appears to be effectively irreversible, as evidenced by the absence of glycosyl residue exchange at extended reaction times. Synthesis of C-β-glucosyl-C-β-xylosyl phloretin is shown at 10 mM concentration in quantitative conversion using cascade reaction of FcCGT and UDP-xylose synthase, allowing for in situ formation of UDP-xylose from the more expedient donor substrate UDP-glucuronic acid. The desired di-C-glycoside with Xyl or Gal was obtained as a single product of the synthesis and its structure was confirmed by NMR.

Detergents are routinely included in protein purification processes to inactivate enveloped viruses that may arise from adventitious or endogenous contamination. The detergent Triton X-100 (TX-100) has been widely used as part of the production process for therapeutic proteins. However, recent ecological studies indicate that TX-100 and its metabolites detrimentally impact aquatic organisms, thus alternative detergents for viral inactivation are required. The overall aim of this study was to identify one or more detergents that are a suitable replacement for TX-100 in the viral inactivation step. In stage one, 16 potential alternatives were identified and screened against TX-100 using multiple criteria such as solubility, feasibility of virus inactivation, critical micelle concentration, and storage conditions. The multi-criteria decision analysis (MCDA) methodology was used to identify four candidates for the second stage assessment. In stage two, a detailed evaluation was undertaken and two candidates C16-AO, and C11/15-sEO9, were found to be practical alternatives to TX-100 for use in protein therapeutic production processes for inactivating enveloped viruses. In addition, C13-EO8 demonstrated good viral inactivation capability and warrants further investigation in detergent clearance and impact on product quality.

During the manufacturing of therapeutic antibodies, disposable depth filters are used after affinity chromatography to remove haze and process-related impurities such as host cell proteins (HCP) and DNA known as critical quality attributes. The present study reports on the regeneration of depth filters allowing their reuse for at least 10 times while retaining sufficiently high clarification capacity. Three filter types were evaluated including standard cellulose-based and fully synthetic matrix materials using acidic or alkaline solutions in alternating cycles of loading and regeneration. Both alkaline and acidic solutions were effective, however, overall acidic regeneration of the filter material appeared superior for multiple use. This was especially evident for the silica-containing XOSP filter, where HCP and DNA were almost completely removed and remained low over 10 applications. Simultaneously preserved product quality indicated a high resistance of the filter matrix toward regeneration. These unexpected findings offer improved flexibility for available filter capacity in downstream processing along with ecologic advantages over the single use applications. Regarding the carbon footprint of the filtration process, calculated potential savings by a factor of four can be achieved, mainly accounting for reduced plastic waste. Therefore, depth filter reuse supports sustainability and carbon dioxide reduction during production processes.

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 detergent 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. Critical micelle concentration was determined across different temperatures and media. Deviron 13-S9 detergent was demonstrated to be readily biodegradable according to OECD 301B guidelines. The absence of detergent binding to typical chromatography resins used in downstream purification was confirmed. The process removal of Deviron 13-S9 detergent from a protein-containing matrix was demonstrated using a protein A resin. 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.

Xylitol, a polyalcohol with anticariogenic properties, finds broad applications in different sectors. Research into new production methods has highlighted the biochemical route using immobilized microorganisms. However, challenges remain in optimizing operating conditions and understanding mass transport and biochemical reactions. Different macroscopic models have been proposed to address these challenges. Nevertheless, those models do not consider porous particles' microstructure and biofilms' formation within them, which can determine the macroscopic performance of the process due to its hierarchical nature. In this work, we derive two macroscopic models for the mass transport and reaction of the xylitol production process with immobilized microorganisms in porous particles. Such models are derived from microscopic ones using the volume averaging method, resulting in both two-equation and one-equation models, written in terms of effective medium coefficients. These latter are predicted by solving ancillary problems in representative 2D unit cells of the immobilization particles, incorporating their microstructural information. Besides, kinetic parameters are estimated through kinetic fitting using experimental data from the literature. Models' accuracy is assessed by comparing them with pore-scale simulations and experimental observations of xylitol production from sugarcane bagasse at the laboratory scale, finding good agreement. Finally, our results are compared with a macroscopic model reported in the literature, and similar predictions are found. However, unlike the reported model, the one derived here improves the modeling of the process since the effective coefficients do not need to be calculated using empirical correlations or estimators.

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 produce 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, dialyzed into physiological buffer, sterile filtered and aliquoted for storage at -80°C. Purification is completed in less than 2 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 produce SARS CoV-2 VLPs for preclinical studies serves as a roadmap for preparation of more distantly related VLPs for pandemic preparedness.

The aromatic compound β-phenylethanol (2-PE) is inherently toxic and can inhibit cell activity in Saccharomyces cerevisiae, making it highly challenging to enhance strain tolerance through rational design due to the lack of reliable connections between tolerance phenotype and genetic loci. This study employed adaptive laboratory evolution strategy to investigate the tolerance characteristics of S. cerevisiae S288C under inhibitory concentrations of 2-PE. The tolerant mutant SEC4.0 was characterized through comprehensive analysis of whole genome sequence, transcriptome, and phosphoproteome. The findings revealed that the high resistance of SEC4.0 was not primarily due to large-scale transcriptional upregulation of stress response genes, but rather through alterations in the phosphorylation levels of lipid-related pathways. PKC1 mutations that affect stress signal transduction and SPT3 mutations that affect arginine biosynthesis have been shown to significantly enhance 2-PE resistance. This study also investigated the effects of exogenous amino acid addition and synergistic effects with two key mutanted genes on 2-PE resistance. This study provides a foundation for enhancing yeast tolerance to this aromatic compound through rational design strategies.