Bioprocess Research Articles

Is it possible to shape the microalgal biomass composition with operational parameters for target compound accumulation?

Microalgae, as photosynthetic microorganisms, offer a sustainable source of proteins, lipids, carbohydrates, pigments, vitamins, and antioxidants. Leveraging their advantages, such as fast growth, CO2 fixation, cultivation without arable land, and wastewater utilisation, microalgae can produce a diverse range of compounds. The extracted products find applications in bioenergy, animal feed, pharmaceuticals, nutraceuticals, cosmetics, and food industries. The selection of microalgal species is crucial, and their biochemical composition varies during growth phases influenced by environmental factors like light, salinity, temperature, and nutrients. Manipulating growth conditions shapes biomass composition, optimising the production of target compounds. This review synthesises research from 2019 onwards, focusing on stress induction and two-stage cultivation in microalgal strategies. This review takes a broader approach, addressing the effects of various operating conditions on a range of biochemical compounds. It explores the impact of operational parameters (light, nutrient availability, salinity, temperature) on biomass composition, elucidating microalgal mechanisms. Challenges include species-specific responses, maintaining stable conditions, and scale-up complexities. A two-stage approach balances biomass productivity and compound yield. Overcoming challenges involves improving upstream and downstream processes, developing sophisticated monitoring systems, and conducting further modelling work. Future efforts should concentrate on strain engineering and refined monitoring, facilitating real-time adjustments for optimal compound accumulation. Moreover, conducting large-scale experiments is essential to evaluate the feasibility and sustainability of the process through techno-economic analysis and life cycle assessments.

Comprehensive insight into recent algal enzymes production and purification advances: Toward effective commercial applications: A review.

Algal enzymes are essential catalysts in numerous biological reactions and industrial processes owing to their adaptability and potency. The marketing of algal enzymes has recently risen due to various reasons, including the cost-efficient manner of their cultivation in photobioreactors, the eco-friendly production of high biomass contents, sources of novel enzymes that used in many sectors (biofuel and bioremediation applications), sustainability, and more renewability. Oxidoreductases and hydrolytic enzymes are among the important applied algal enzymes in industrial applications, with annually growing demand. These algal enzymes have opened up new avenues for significant health advantages in reducing and treating oxidative stress, cardiovascular illness, tumors, microbial infections, and viral outbreaks. Despite their promising uses, commercial applications of algal enzymes face many difficulties, such as stability, toxicity, and lower data availability on specific and adequate catalytic mechanisms. Therefore, this review focuses on the algal enzyme types, their uses and advantages over other microbial enzymes, downstream and upstream processing, their commercial and marketing, and their challenges. With the constant development of novel enzymes and their uses, enzyme technology provides exciting options for several industrial sectors.

Simultaneous nitrification and denitrification framework for decentralized systems: Long-term study utilizing rope-type biofilm media under field conditions

This research introduces a novel approach to achieve simultaneous nitrification-denitrification (SND) under dynamic load conditions using a cost-effective rope-type biofilm technology. The approach represents a significant advancement in wastewater treatment, particularly beneficial for remote and decentralized communities. The biofilm-based SND process was developed using a pilot-scale flow-through reactor by implementing upstream carbon management with constant-timer-based aeration control versus dynamic-sensor-based aeration control strategies. The findings indicate that adding an upstream anaerobic pretreatment process to handle excess carbon plays a substantial role in achieving a sustainable SND process under a dynamic load environment using simple aeration on-off control. The most optimal nitrification performance of 0.32 g NH3-N/m2/d (89 % removal) was achieved under a 1-hour ON/30-minute OFF aeration. The process sustained an average bulk liquid DO of 5.16 mg/L and 3.80 mg/L during the aeration ON and OFF periods, respectively, facilitating a 0.13 g N/m2/d (41 %) total inorganic nitrogen (TIN) removal, notably, implementing advanced aeration strategies driven by DO, NH3, and NO3 sensors enhanced TIN removal efficiency to 72 %. The nitrification performance remained comparable (89 % removal), resulting in 3 and 10 mg N/L effluent ammonia and TIN concentration, respectively. Additionally, utilizing two multivariate approaches accounting for 82 % and 64 % of the variance, this study discerned patterns in monitored variables and performance. Additionally, the analysis underscored the difference of bulk liquid DO levels in the biofilm versus suspended systems inhibiting the SND process. Distinct bacterial communities were established in biofilms under aerobic, anaerobic, and SND conditions, with the SND reactor showing a hierarchy of functional group and enzymes, enriched sequentially from heterotrophs to denitrifiers, nitrifiers, and anammox bacteria. These innovations underline the potential of tailored control strategies to enhance a passive biofilm-based SND process efficiency under dynamic conditions, providing scalable solutions for diverse target water quality demands in remote communities and decentralized systems.

Deciphering molecular drivers of lactate metabolic shift in mammalian cell cultures

Lactate metabolism plays a critical role in mammalian cell bioprocessing, influencing cellular performance and productivity. The transition from lactate production to consumption, known as lactate metabolic shift, is highly beneficial and has been shown to extend culture lifespan and enhance productivity, yet its molecular drivers remain poorly understood. Here, we have explored the mechanisms that underpin this metabolic shift through two case studies, illustrating environmental- and genetic-driven factors. We characterised these study cases at process, metabolic and transcriptomic levels. Our findings indicate that glutamine depletion coincided with the timing of the lactate metabolic shift, significantly affecting cell growth, productivity and overall metabolism. Transcriptome analysis revealed dynamic regulation the ATF4 pathway, involved in the amino acid (starvation) response, where glutamine depletion activates ATF4 gene and its targets. Manipulating ATF4 expression through overexpression and knockdown experiments showed significant changes in metabolism of glutamine and lactate, impacting cellular performance. Overexpression of ATF4 increased cell growth and glutamine consumption, promoting a lactate metabolic shift. In contrast, ATF4 downregulation decreased cell proliferation and glutamine uptake, leading to production of lactate without any signs of lactate shift. These findings underscore a critical role for ATF4 in regulation of glutamine and lactate metabolism, related to phasic patterns of growth during CHO cell culture. This study offers unique insight into metabolic reprogramming during the lactate metabolic shift and the molecular drivers that determine cell status during culture.

Spatial and temporal distribution of ribosomes in single cells reveals aging differences between old and new daughters of Escherichia coli

Lineages of rod-shaped bacteria such as Escherichia coli exhibit a temporal decline in elongation rate in a manner comparable to cellular or biological aging. The effect results from the production of asymmetrical daughters, one with a lower elongation rate, by the division of a mother cell. The slower daughter compared to the faster daughter, denoted respectively as the old and new daughters, has more aggregates of damaged proteins and fewer expressed gene products. We have examined further the degree of asymmetry by measuring the density of ribosomes between old and new daughters and between their poles. We found that ribosomes were denser in the new daughter and also in the new pole of the daughters. These ribosome patterns match the ones we previously found for expressed gene products. This outcome suggests that the asymmetry is not likely to result from properties unique to the gene expressed in our previous study, but rather from a more fundamental upstream process affecting the distribution of ribosomal abundance. Because damage aggregates and ribosomes are both more abundant at the poles of E. coli cells, we suggest that competition for space between the two could explain the reduced ribosomal density in old daughters. Using published values for aggregate sizes and the relationship between ribosomal number and elongation rates, we show that the aggregate volumes could in principle displace quantitatively the amount of ribosomes needed to reduce the elongation rate of the old daughters.

Spatial and temporal distribution of ribosomes in single cells reveals aging differences between old and new daughters of Escherichia coli.

Lineages of rod-shaped bacteria such as Escherichia coli exhibit a temporal decline in elongation rate in a manner comparable to cellular or biological aging. The effect results from the production of asymmetrical daughters, one with a lower elongation rate, by the division of a mother cell. The slower daughter compared to the faster daughter, denoted respectively as the old and new daughters, has more aggregates of damaged proteins and fewer expressed gene products. We have examined further the degree of asymmetry by measuring the density of ribosomes between old and new daughters and between their poles. We found that ribosomes were denser in the new daughter and also in the new pole of the daughters. These ribosome patterns match the ones we previously found for expressed gene products. This outcome suggests that the asymmetry is not likely to result from properties unique to the gene expressed in our previous study, but rather from a more fundamental upstream process affecting the distribution of ribosomal abundance. Because damage aggregates and ribosomes are both more abundant at the poles of E. coli cells, we suggest that competition for space between the two could explain the reduced ribosomal density in old daughters. Using published values for aggregate sizes and the relationship between ribosomal number and elongation rates, we show that the aggregate volumes could in principle displace quantitatively the amount of ribosomes needed to reduce the elongation rate of the old daughters.

Mixed matrix PVDF microfiltration membranes with in-situ synthesized polyethyleneimine particles as a platform for flow through, high capacity, weak base and salt tolerant anion exchange membrane adsorbers for downstream bioprocessing

The rapid growth of the monoclonal antibody (mAb) therapeutic market has led to a need for improved downstream bioprocessing, including mAb capture and release from bioreactor harvests followed by product purification and polishing. Membrane chromatography (MC) is poised to displace resin-based column chromatography in mAb product polishing. To remove negatively charged product impurities (e.g., host cell proteins, DNA, viruses, and endotoxins), anion exchange (AEX) membrane adsorbers offer higher process rates and eliminate the risks of cross contamination as they can be deployed as single use separation devices, which are increasingly being utilized in downstream bioprocessing to increase manufacturing speed and decrease production cost. Here, we describe a one-pot and single step phase inversion casting process for the preparation of a family of mixed matrix polyvinylidene fluoride (PVDF) microfiltration (MF) membranes with in-situ synthesized polyethyleneimine (PEI) microparticles that can serve as flow through, high capacity, and salt tolerant weak base AEX membrane adsorbers. These new membranes have a high content of weak base (WB) groups (primary and secondary amines) by virtue of the utilization of bis(2-chloroethyl)amine hydrochloride (BCAH) as crosslinker in the in-situ synthesis of PEI microparticles in the dope dispersions prior to membrane casting. By varying the concentration of PEI and BCAH in the casting dispersions, we successfully utilized nonsolvent induced phase separation (NIPS) with isopropyl alcohol as the nonsolvent in the membrane coagulation bath to prepare a mixed matrix PVDF-PEI MF membrane with (i) a relatively uniform and open microstructure in which the PEI particles completely cover the PVDF spherulites present at the membrane surface and cross section, (ii) high water flux (>1000 liters/m2/hr. at 2 bar) and (iii) a high loading of BCAH crosslinked PEI microparticles (51.0 wt%) containing weak base primary, secondary, and tertiary amine groups. The protein binding measurements show that this new mixed matrix PVDF-PEI MF membrane can serve as a flow through, high capacity, and salt tolerant WB AEX membrane adsorber with a bovine serum albumin (BSA) dynamic binding capacity of ∼60 mg BSA per mL of membrane in a 50 mM TRIS buffer containing 100 mM of NaCl (15 mS/cm) at 10 % breakthrough and flow rate of 10 MV/min. The overall results of this study indicate that our mixed matrix PVDF-PEI MF membranes with in-situ synthesized and BCAH crosslinked PEI microparticles have a promising potential to serve as a platform for the design, preparation, and scale up of a new generation of flow through, high capacity, salt tolerant, WB AEX membrane adsorber materials for downstream bioprocessing.

Optimization of curdlan production and ultrasound assisted extraction processes from Priestia megaterium

In this study, the upstream and downstream production processes of curdlan from Priestia megaterium were optimized to enhance its yield. Additionally, a novel extraction method was developed for curdlan recovery. Optimization studies were conducted using Central composite design (CCD). Curdlan yield improved from 0.15 g/L (unoptimized) to 0.46 g/L (3-fold increase) when fermentation was carried out in CCD-optimized media of (w/v) sucrose 20%, urea 0.1%, KH2PO4 0.02%, agitation speed 250 rpm. To further enhance curdlan yield during extraction, ultrasonication was incorporated as a novel step into the conventional method of acid/alkali-assisted curdlan recovery. A two-step optimization was chosen for extraction, namely, one-factor-at-a-time (OFAT) and CCD, wherein the optimized extraction parameters were determined to be 25 s sonication, 1 N NaOH, and 2 h solubilization time. The curdlan yield improved by 1.5-fold (0.70 g/L) post optimization, in comparison with unoptimized conventional extraction step. Finally, the biopolymer was validated through characterization by nuclear magnetic resonance (NMR) which showed characteristic curdlan spectra in the13C and1H NMR studies. To the best of our knowledge, this study represents the first documented report on curdlan extraction using this novel method of ultrasonication.

Recovery of bio-based volatile fatty acids from anaerobically treated winery wastewater using a closed-loop liquid-liquid hydrophobic membrane contactor system

This research evaluated a closed-loop hydrophobic liquid–liquid membrane contactor (MC) for the recovery of volatile fatty acids (VFAs) from the effluent of an up-flow anaerobic sludge blanket (UASB) bioreactor treating winery wastewater. Sodium bicarbonate was used as a stripping solution for its operational flexibility and compatibility with downstream bioprocesses. An electrochemical acidification cell (EAC) was tested to continuously adjust of pH of the feed solution to avoid the use of inorganic acids. VFA recovery was optimized under different flow rates and feed pH conditions using a synthetic VFA solution and validated with actual UASB effluent. Lower feed pH resulted in higher VFA recovery due to increased protonation of the acids. This effect was more pronounced when using synthetic VFA solutions compared to real UASB effluent. Specifically, the MC system recovered 16 ± 5 % of acetic acid at 0.3 ± 0.1 gCOD/m2h flux and 34 ± 4 % of butyric acid at 0.5 ± 0.1 gCOD/m2h flux at an unadjusted UASB effluent feed pH of 4.9, compared to 22 ± 3 % at 0.5 ± 0.1 gCOD/m2h flux and 46 ± 5 % at 0.7 ± 0.1 gCOD/m2h flux, respectively, at pH 2. The EAC effectively lowered the feed pH of UASB effluent to 3.9, achieving acetic acid recovery of 22 ± 3 % at a mass flux of 0.2 ± 0.1 gCOD/m2h, and butyric acid recovery of 45 ± 1 % at a mass flux of 0.6 ± 0.1 gCOD/m2h. These moderate recovery efficiencies are likely influenced by the lower pH gradient resulting from the use of sodium bicarbonate. However, the compatibility of sodium bicarbonate with bioprocesses offers the advantage that the proposed UASB-MC system can be seamlessly integrated with downstream bioprocesses to produce valuable end products for other markets, such as PHA for plastics and microbial proteins for animal feed.

Real-Time Adaptive Inline Acidification Enhances Continuous pH Control for Viral Inactivation.

Existing low pH viral inactivation methods for continuous downstream processing of biologics typically rely on predictive models to estimate the necessary pH adjustments. However, these methods are of limited use during the process development stage due to the dynamic nature of capture chromatography, where batch variations can alter the eluted protein titer. This study introduces an inline viral inactivation system (IVIS) that utilizes real-time adaptive control and inline sensor readings to precisely regulate the pH manipulation for inline acidification and continuous viral inactivation. The IVIS, which includes a coiled flow inversion reactor (CFIR), is integrated with a multicolumn capture chromatography system to demonstrate a fully continuous process from protein capture chromatography to inline pH manipulation. The system achieved precise inline pH manipulation within ±0.15 and a narrow residence time distribution of 13.5min with a relative width of 0.7. The introduction of real-time inline pH manipulation with the IVIS signifies a notable advancement in managing critical process parameters (CPPs) and ensuring consistent product quality across varied production environments for continuous downstream bioprocessing.

Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles

Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures of membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs of these vesicles can be challenging due to the strong signal contributed from the lipid bilayer. This challenge often requires iterative and laborious particle selection workflows to generate a dataset of high-quality particle images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on the Segment Anything model. Vesicle Picker enables automatic identification of vesicles in cryo-EM micrographs with high recall and precision. It then exhaustively selects all potential particle locations, either at the perimeter or uniformly over the surface of the projection of the vesicle. The program is designed to interface with cryoSPARC, which performs both upstream micrograph processing and downstream single particle image analysis. We demonstrate Vesicle Picker’s utility by determining a high-resolution map of the vacuolar-type ATPase from micrographs of native synaptic vesicles (SVs) and identifying an additional protein or protein complex in the SV membrane.

Beyond viability: Advancing CHO cell culture process strategies to modulate host cell protein levels

Chinese Hamster Ovary (CHO) cells are widely utilized in bioprocessing industry for monoclonal antibody (mAb) production. In many instances, challenges persist in achieving sufficient clearance of Host Cell Proteins (HCPs) in the final drug substance. While purification strategies usually offer substantial HCP clearance, certain "problematic" HCPs, particularly lipases, continue to pose significant challenges. This study investigates the accumulation of various "problematic" HCPs in CHO cell culture using transcriptomics, revealing correations between cell culture parameters and HCP level. Contrary to conventional assumptions, viability alone does not reliably predict HCP levels, with factors such as clone selection, host cell line choice, media and feed compositions significantly influencing HCP accumulation. Leveraging transcriptomics-based approaches, we demonstrate the potential of upstream process control strategies to mitigate HCP presence and improve biologic product quality. Our findings underscore the importance of considering diverse cell culture parameters in bioprocess optimization to ensure product stability and quality. While promising, further research is needed to elucidate the mechanisms underlying HCP release and propagation through downstream processing stages, emphasizing the necessity of a comprehensive integrated approach to HCP control in biologics production.

Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells.

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 (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Qualitative drivers of postoperative prophylactic antibiotics use and resistance in Ethiopia.

The World Health Organization (WHO) cautions against unnecessary prolongation of postoperative antibiotics to prevent surgical site infections (SSI), however this practice is still common in many countries. This study aims to describe drivers of prolonged postoperative antibiotic prescribing and clinicians' perspectives on antibiotics resistance and stewardship in Ethiopia. We conducted semi-structured interviews of 16 surgeons and nine surgical ward nurses at three academic referral hospitals in Addis Ababa. Audio recordings were transcribed verbatim and coded. Codes were inductively and iteratively derived between two researchers, tested for inter-rater reliability (IRR), and the codebook was consistently applied to all transcripts. Thematic analysis was performed to understand drivers of prolonged prophylactic antibiotic use in surgical patients. Interviews revealed factors contributing to postoperative prophylactic antibiotics overprescribing, including inadequate infection prevention and control (IPC) practices, wide variability in local prescribing practices, and distrust in the applicability of WHO guidelines. Antimicrobial resistance was also identified as a major concern by staff. Barriers to improving stewardship included a lack of multidisciplinary teamwork to inform prescribing decisions, while solutions included constructing appropriate context-specific guidelines and improving evidence-based practices through input from local stakeholders, including surgeons, clinical pharmacists, and nurses. Study participants perceived that existing evidence and guidelines did not apply in their settings due to high rates of surgical site infections and gaps in perioperative IPC practices (e.g., availability of water for handwashing, sterility breaches). These gaps were a key contributor to prophylactic antibiotic overprescribing, reinforcing the need to strengthen upstream and perioperative surgical antisepsis processes. The findings of this study underscore the importance of engaging multidisciplinary teams in strengthening antimicrobial stewardship efforts, aligning processes to achieve compliance with best practices, and the need for rigorous, contextually appropriate studies from these settings to inform policy.

Design, synthesis, and pharmacological characterization of sulfonylurea-based NLRP3 inhibitors: Towards an effective therapeutic strategy for Alzheimer's disease

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder that severely diminishes the quality of life for millions. The NLRP3 inflammasome, a critical mediator of inflammation, has emerged as a promising therapeutic target for AD. In this study, we report the development and optimization of a novel series of sulfonylurea-based NLRP3 inhibitors, with a focus on compound MC1 for the treatment of AD. Utilizing the co-crystal structure of MCC950 in complex with NLRP3 as a guide, we employed a hybrid approach of computer-aided drug design and traditional medicinal chemistry to perform two iterative optimization cycles. This strategy led to the synthesis and evaluation of 40 sulfonylurea derivatives, culminating in the identification of MC1 as the lead candidate. MC1 exhibited enhanced NLRP3 inhibitory activity and demonstrated high binding affinity to NLRP3, effectively blocking NLRP3 activation induced by diverse stimuli such as ATP and Nigericin, without perturbing upstream processes like reactive oxygen species (ROS) generation. In vivo experiments in AD mouse models revealed that MC1 significantly ameliorated cognitive deficits, surpassing the performance of MCC950. Importantly, MC1 showed no signs of hepatotoxicity or adverse effects on the central nervous system. These findings suggest that MC1 holds strong potential as a lead compound for further development in AD therapy, providing a new scaffold for NLRP3 inhibition with improved safety and efficacy profiles.

In-line monitoring of bioreactor by Raman spectroscopy: Direct use of a standard-based model through cell-scattering correction

Raman spectroscopy and machine learning have become popular in in-line monitoring of bioreactors. However, traditional modeling processes typically entail extensive fermentation batches to collect learning datasets, which are significantly time–consuming and laborious. In addition, these models are limited to configurations with the same conditions as the training batches. The present work proposes a reproducible and adaptable modeling approach by combining standard spectra as a training dataset, with a simple means of correcting for cell scattering. Alcoholic fermentation by Saccharomyces cerevisiae is used as a benchmark. Initially, a partial least squares (PLS) regression model was developed based on the spectra of pure solutions of glucose and ethanol. Then, a mathematical expression was defined to estimate yeast concentration, allowing the correction of Raman intensity attenuated by cell scattering. The corrected spectra demonstrate close alignment with reference spectra in both shape and intensity. Validation of the methodology was conducted across numerous batches and one fed–batch bioreactor. As a result, the developed method enables the simultaneous monitoring of glucose, ethanol, and yeast concentrations, effectively addressing the challenge of implementing an independent standards based PLS model to manage the intricate compositional dynamics in bio–processes. The conclusion underscores the effectiveness of the proposed method and offers new prospects in biotechnological industries.

Exploring bias risks in artificial intelligence and targeted medicines manufacturing

BackgroundThough artificial intelligence holds great value for healthcare, it may also amplify health inequalities through risks of bias. In this paper, we explore bias risks in targeted medicines manufacturing. Targeted medicines manufacturing refers to the act of making medicines targeted to individual patients or to subpopulations of patients within a general group, which can be achieved, for example, by means of cell and gene therapies. These manufacturing processes are increasingly reliant on digitalised systems which can be controlled by artificial intelligence algorithms. Whether and how bias might turn up in the process, however, is uncertain due to the novelty of the development.MethodsExamining stakeholder views across bioethics, precision medicine, and artificial intelligence, we document a range of opinions from eleven semi-structured interviews about the possibility of bias in AI-driven targeted therapies manufacturing.ResultFindings show that bias can emerge in upstream (research and development) and downstream (medicine production) processes when manufacturing targeted medicines. However, interviewees emphasized that downstream processes, particularly those not relying on patient or population data, may have lower bias risks. The study also identified a spectrum of bias meanings ranging from negative and ambivalent to positive and productive. Notably, some participants highlighted the potential for certain biases to have productive moral value in correcting health inequalities. This idea of “corrective bias” problematizes the conventional understanding of bias as primarily a negative concept defined by systematic error or unfair outcomes and suggests potential value in capitalizing on biases to help address health inequalities. Our analysis also indicates, however, that the concept of “corrective bias” requires further critical reflection before they can be used to this end.

Single-Batch Expression of an Experimental Recombinant Snakebite Antivenom Based on an Oligoclonal Mixture of Human Monoclonal Antibodies.

Oligoclonal antibodies, which are carefully defined mixtures of monoclonal antibodies, are valuable for the treatment of complex diseases, such as infectionss and cancer. In addition to these areas of medicine, they could be utilized for the treatment of snakebite envenoming, where recombinantly produced monoclonal human antibodies could overcome many of the drawbacks accompanying traditional antivenoms. However, producing multiple individual batches of monoclonal antibodies in an industrial setting is associated with significant costs. Therefore, it is attractive to produce oligoclonal antibodies by mixing multiple antibody-producing cell lines in a single batch to have only one upstream and downstream process. In this study, we selected four antibodies that target different toxins found in the venoms of various elapid snake species, such as mambas and cobras, and generated stable antibody-producing cell lines. Upon co-cultivation, we found the cell line ratios to be stable over 7 days. The purified oligoclonal antibody cocktail contained the anticipated antibody concentrations and bound to the target toxins as expected. These results thus provide a proof of concept for the strategy of mixing multiple cell lines in a single batch to manufacture tailored antivenoms recombinantly, which could be utilized for the treatment of snakebite envenoming and in other fields where oligoclonal antibody mixtures could find utility.

Application of a Novel Disposable Flow Cell for Spectroscopic Bioprocess Monitoring

The evaluation of the analytical capabilities of a novel disposable flow cell for spectroscopic bioprocess monitoring is presented. The flow cell is presterilized and can be connected to any kind of bioreactor by weldable tube connections. It is clamped into a reusable holder, which is equipped with SMA-terminated optical fibers or an integrated light source and detection unit. This modular construction enables spectroscopic techniques like UV-Vis spectroscopy or turbidity measurements by scattered light for modern disposable bioreactors. A NIR scattering module was used for biomass monitoring in different cultivations. A high-cell-density fed-batch cultivation with Komagataella phaffii and a continuous perfusion cultivation with a CHO DG44 cell line were conducted. A high correlation between the sensor signal and biomass or viable cell count was observed. Furthermore, the sensor shows high sensitivity during low turbidity states, as well as a high dynamic range to monitor high turbidity values without saturation effects. In addition to upstream processing, the sensor system was used to monitor the purification process of a monoclonal antibody. The absorption module enables simple and cost-efficient monitoring of downstream processing and quality control measurements. Recorded absorption spectra can be used for antibody aggregate detection, due to an increase in overall optical density.

Targeting the Main Sources of Reactive Oxygen Species Production: Possible Therapeutic Implications in Chronic Pain.

Humans have long been combating chronic pain. In clinical practice, opioids are firstchoice analgesics, but long-term use of these drugs can lead to serious adverse reactions. Finding new, safe and effective pain relievers that are useful treatments for chronic pain is an urgent medical need. Based on accumulating evidence from numerous studies, excess reactive oxygen species (ROS) contribute to the development and maintenance of chronic pain. Some antioxidants are potentially beneficial analgesics in the clinic, but ROS-dependent pathways are completely inhibited only by scavenging ROS directly targeting cellular or subcellular sites. Unfortunately, current antioxidant treatments do not achieve this effect. Furthermore, some antioxidants interfere with physiological redox signaling pathways and fail to reverse oxidative damage. Therefore, the key upstream processes and mechanisms of ROS production that lead to chronic pain in vivo must be identified to discover potential therapeutic targets related to the pathways that control ROS production in vivo. In this review, we summarize the sites and pathways involved in analgesia based on the three main mechanisms by which ROS are generated in vivo, discuss the preclinical evidence for the therapeutic potential of targeting these pathways in chronic pain, note the shortcomings of current research and highlight possible future research directions to provide new targets and evidence for the development of clinical analgesics.