Towards bioprocess engineering of cable bacteria: Establishment of a synthetic sediment.

Cable bacteria, characterized by their multicellular filamentous growth, are prevalent in both freshwater and marine sediments. They possess the unique ability to transport electrons over distances of centimeters. Coupled with their capacity to fix CO2 and their record-breaking conductivity for biological materials, these bacteria present promising prospects for bioprocess engineering, including potential electrochemical applications. However, the cultivation of cable bacteria has been limited to their natural sediment, constraining their utility in production processes. To address this, our study designs synthetic sediment, drawing on ion exchange chromatography data from natural sediments and existing literature on the requirements of cable bacteria. We examined the effects of varying bentonite concentrations on water retention and the impacts of different sands. For the first time, we cultivated cable bacteria on synthetic sediment, specifically the freshwater strain Electronema aureum GS. This cultivation was conducted over 10 weeks in a specially developed sediment bioreactor, resulting in an increased density of cable bacteria in the sediment and growth up to a depth of 5 cm. The creation of this synthetic sediment paves the way for the reproducible cultivation of cable bacteria. It also opens up possibilities for future process scale-up using readily available components. This advancement holds significant implications for the broader field of bioprocess engineering.

Assessing the feasibility and sustainability of a surfactin production process: a techno-economic and environmental analysis.

Biosurfactants have been profiled as a sustainable replacement for chemical-based surfactants since these bio-based molecules have higher biodegradability. Few research papers have focused on assessing biosurfactant production to elucidate potential bottlenecks. This research aims to assess the techno-economic and environmental performance of surfactin production in a potential scale of 65m3, considering different product yields and involving the European energy crisis of 2021-2022. The conceptual design, simulation, techno-economic, and environmental assessments were done by applying process engineering concepts and software tools such as Aspen Plus v.9.0 and SimaPro v.8.3.3. The results demonstrated the high economic potential of surfactin production since the higher values in the market offset the low fermentation yields, low recovery efficiency, and high capital investment. The sensitivity analysis of the economic assessment elucidated a minimum surfactin selling price between 29 and 31 USD/kg of surfactin, while a minimum processing scale for economic feasibility between 4 and 5 kg/h is needed to reach an equilibrium point. The environmental performance must be improved since the carbon footprint was 43 kg CO2eq/kg of surfactin. The downstream processing and energy demand are the main bottlenecks since these aspects contribute to 63 and 25% of the total emissions. The fermentation process and downstream process are key factors for future optimization and research.

Modeling the behavior of monoclonal antibodies on hydrophobic interaction chromatography resins.

Monoclonal antibodies (mAbs) require a high level of purity for regulatory approval and safe administration. High-molecular weight (HMW) species are a common impurity associated with mAb therapies. Hydrophobic interaction chromatography (HIC) resins are often used to remove these HMW impurities. Determination of a suitable HIC resin can be a time and resource-intensive process. In this study, we modeled the chromatographic behavior of seven mAbs across 13 HIC resins using measurements of surface hydrophobicity, surface charge, and thermal stability for mAbs, and hydrophobicity and zeta-potential for HIC resins with high fit quality (adjusted R2 > 0.80). We identified zeta-potential as a novel key modeling parameter. When using these models to select a HIC resin for HMW clearance of a test mAb, we were able to achieve 60% HMW clearance and 89% recovery. These models can be used to expedite the downstream process development for mAbs in an industry setting.

Synthetic co-culture in an interconnected two-compartment bioreactor system: violacein production with recombinant E. coli strains.

The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such 'one pot' approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli-E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h)-1 at 25 °C which holds great promise for future applications.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis.

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

OptMSP: A toolbox for designing optimal multi-stage (bio)processes.

One central goal of bioprocess engineering is to maximize the production of specific chemicals using microbial cell factories. Many bioprocesses are one-stage (batch) processes (OSPs), in which growth and product synthesis are coupled. However, OSPs often exhibit low volumetric productivities due to the competition for substrate for biomass and product synthesis implying trade-offs between biomass and product yields. Two-stage or, more generally, multi-stage processes (MSPs) offer the potential to tackle this trade-off for improved efficiency of bioprocesses, for example, by separating growth and production. MSPs have recently gained much attention, also because of a rapidly growing toolbox for the dynamic control of metabolic fluxes. Despite these promising advancements, computational tools specifically tailored for the optimal design of MSPs in the field of biotechnology are still lacking. Here, we present OptMSP, a new Python-based toolbox for identifying optimal MSPs maximizing a user-defined process metrics (such as volumetric productivity, yield, and titer or combinations thereof) under given constraints. In contrast to other methods, our framework starts with a set of well-defined modules representing relevant stages or sub-processes. Experimentally determined parameters (such as growth rates, substrate uptake and product formation rates) are used to build suitable ODE models describing the dynamic behavior of each module. OptMSP finds then the optimal combination of those modules, which, together with the optimal switching time points, maximize a given objective function. We demonstrate the applicability and relevance of the approach with three different case studies, including the example of lactate production by E. coli in a batch setup, where an aerobic growth phase can be combined with anaerobic production phases with or without growth and with or without enhanced ATP turnover.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

Prediction of CHO cell line stability using expression of DNA repair genes.

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Uncertainty quantification for gene delivery methods: A roadmap for pDNA manufacturing from phase I clinical trials to commercialization.

The fast-growing interest in cell and gene therapy (C>) products has led to a growing demand for the production of plasmid DNA (pDNA) and viral vectors for clinical and commercial use. Manufacturers, regulators, and suppliers need to develop strategies for establishing robust and agile supply chains in the otherwise empirical field of C>. A model-based methodology that has great potential to support the wider adoption of C> is presented, by ensuring efficient timelines, scalability, and cost-effectiveness in the production of key raw materials. Specifically, key process and economic parameters are identified for (1) the production of pDNA for the forward-looking scenario of non-viral-based Chimeric Antigen Receptor (CAR) T-cell therapies from clinical (200 doses) to commercial (40,000 doses) scale and (2) the commercial (40,000 doses) production of pDNA and lentiviral vectors for the current state-of-the-art viral vector-based CAR T-cell therapies. By applying a systematic global sensitivity analysis, we quantify uncertainty in the manufacturing process and apportion it to key process and economic parameters, highlighting cost drivers and limitations that steer decision-making. The results underline the cost-efficiency and operational flexibility of non-viral-based therapies in the overall C> supply chain, as well as the importance of economies-of-scale in the production of pDNA.

Exploring nutrient supplementation and bioprocess optimization to improve the production of lentiviral vectors in serum-free medium suspension cultures.

The use of lentiviral vectors (LV) in gene therapy has been growing in recent years. To meet the increasing clinical demand, LV production platforms will benefit from improved productivity and scalability to enable cost-effective manufacture of LV-based therapies. Here we report the adaptation of 293T cells to serum-free suspension cultures and the improvement of LV yields through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters. Cells were sequentially adapted to different serum-free culture media, transfection parameters were optimized and the two best-performing conditions were selected to explore process intensification by increasing cell density at the time of transfection. LV production at higher cell densities increased volumetric titers up to 12-fold and lipid supplementation was the most efficient metabolic optimization strategy further enhancing LV productivity by 3-fold. Furthermore, cell concentration was identified and validated as an important source of transfection variability impairing cellular uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers down to 6-fold. This work contributes to improving LV-based gene therapy by establishing new scalable manufacturing platforms and providing key metabolic insights, unveiling important bioreaction parameters to improve vector yields.

Scaling strategy for cell and gene therapy bioreactors based on turbulent parameters.

So far, power input has been used as the main parameter for bioreactor scale-up/-down in upstream process development and manufacturing. The rationale is that maintaining a consistent power input per unit volume should result in comparable mixing times at different scales. However, shear generated from turbulent flow may compromise the integrity of non-robust cells such as those used during the production of cell and gene therapies, which may lead to low product quality and yield. Of particular interest is the Kolmogorov length parameter that characterizes the smallest turbulent eddies in a mixture. To understand its impact on scale-up/-down decisions, the distribution of Kolmogorov length along the trajectory flow of individual particles in bioreactors was estimated in silico with the help of computational fluid dynamics simulations. Specifically, in this study the scalability of iPSC-derived lymphocyte production and the impact of shear stress across various differentiation stages were investigated. The study used bioreactors of volumes from 0.1 to 10 L, which correspond to the scales most used for parameter optimization. Our findings, which align with in vitro runs, help determine optimal agitation speed and shear stress adjustments for process transfer between scales and bioreactor types, using vertically-oriented wheel and pitched-blade impellers. In addition, empirical models specific to the bioreactors used in this study were developed. The provided computational analysis in combination with experimental data supports selection of appropriate bioreactors and operating conditions for various cell and gene therapy process steps.

Robust method for chromatogram shape analysis to improve early detection of performance drifts and adverse changes in process parameters during purification column operations.

The biopharmaceutical industry is under increased pressure to maximize efficiency, enhance quality compliance, and reduce the cost of drug substance manufacturing. Ways to reduce costs associated with manufacturing of complex biological molecules include maximizing efficiency of chromatography purification steps. For example, process analytical technology (PAT) tools can be employed to improve column resin life, prevent column operating failures, and decrease the time it takes to solve investigations of process deviations. We developed a robust method to probe the shape of the chromatogram for indications of column failure or detrimental changes in the process. The approach herein utilizes raw data obtained from manufacturing followed by a pre-processing routine to align chromatograms and patch together the different chromatogram phases in preparation for multivariate analysis. A principal component analysis (PCA) was performed on the standardized chromatograms to compare different batches, and resulted in the identification specific process change that affected the profile. In addition, changes in the chromatogram peaks were used to create predictive models for impurity clearance. This approach has the potential for early detection of column processing issues, improving timely resolution in large-scale chromatographic operations.

Feasibility and performance of cross-clone Raman calibration models in CHO cultivation.

Raman spectroscopy is widely used in monitoring and controlling cell cultivations for biopharmaceutical drug manufacturing. However, its implementation for culture monitoring in the cell line development stage has received little attention. Therefore, the impact of clonal differences, such as productivity and growth, on the prediction accuracy and transferability of Raman calibration models is not yet well described. Raman OPLS models were developed for predicting titer, glucose and lactate using eleven CHO clones from a single cell line. These clones exhibited diverse productivity and growth rates. The calibration models were evaluated for clone-related biases using clone-wise linear regression analysis on cross validated predictions. The results revealed that clonal differences did not affect the prediction of glucose and lactate, but titer models showed a significant clone-related bias, which remained even after applying variable selection methods. The bias was associated with clonal productivity and lead to increased prediction errors when titer models were transferred to cultivations with productivity levels outside the range of their training data. The findings demonstrate the feasibility of Raman-based monitoring of glucose and lactate in cell line development with high accuracy. However, accurate titer prediction requires careful consideration of clonal characteristics during model development.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering.

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Israel and the global synthetic biology ecosystem.

The field of synthetic biology emerged a few decades ago, following some key works of researchers in the USA, Europe, and the Far East. It reached Israel through academia and a few years later it finally got the attention of industry, venture capitals, and government authorities, especially the Israeli Innovation Authority, hoping to encourage entrepreneurs to establish startups in this field. Here we provide an overview of the activity of the field of synthetic biology in Israel, including historical notes, current strategy, prospects and developments, and further insight that are relevant to any stakeholders in the synthetic biology field.

Designing of an extract production protocol for industrial application of cell-free protein synthesis technology: Building from a current best practice to a quality by design approach.

Cell-Free Protein Synthesis (CFPS) has, over the past decade, seen a substantial increase in interest from both academia and industry. Applications range from fundamental research, through high-throughput screening to niche manufacture of therapeutic products. This review/perspective focuses on Quality Control in CFPS. The importance and difficulty of measuring the Raw Material Attributes (RMAs) of whole cell extract, such as constituent protein and metabolite concentrations, and of understanding and controlling these complicated enzymatic reactions is explored, for both centralised and distributed industrial production of biotherapeutics. It is suggested that a robust cell-free extract production process should produce cell extract of consistent quality; however, demonstrating this is challenging without a full understanding of the RMAs and their interaction with reaction conditions and product. Lack of technology transfer and knowledge sharing is identified as a key limiting factor in the development of CFPS. The article draws upon the experiences of industrial process specialists, discussions within the Future Targeted Healthcare Manufacturing Hub Specialist Working Groups and evidence drawn from various sources to identify sources of process variation and to propose an initial guide towards systematisation of CFPS process development and reporting. These proposals include the development of small scale screening tools, consistent reporting of selected process parameters and analytics and application of industrial thinking and manufacturability to protocol development.

Recent advances in methanol production from methanotrophs.

Methanol, the simplest aliphatic molecule of the alcohol family, finds diverse range of applications as an industrial solvent, a precursor for producing other chemicals (e.g., dimethyl ether, acetic acid and formaldehyde), and a potential fuel. There are conventional chemical routes for methanol production such as, steam reforming of natural gas to form syngas, followed by catalytic conversion into methanol; direct catalytic oxidation of methane, or hydrogenation of carbon dioxide. However, these chemical routes are limited by the requirement for expensive catalysts and extreme process conditions, and plausible environmental implications. Alternatively, methanotrophic microorganisms are being explored as biological alternative for methanol production, under milder process conditions, bypassing the requirement for chemical catalysts, and without imposing any adverse environmental impact. Methanotrophs possess inherent metabolic pathways for methanol production via biological methane oxidation or carbon dioxide reduction, thus offering a surplus advantage pertaining to the sequestration of two major greenhouse gases. This review sheds light on the recent advances in methanotrophic methanol production including metabolic pathways, feedstocks, metabolic engineering, and bioprocess engineering approaches. Furthermore, various reactor configurations are discussed in view of the challenges associated with solubility and mass transfer limitations in methanotrophic gas fermentation systems.

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production.

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

Characterization of tyrosine ammonia lyases from Flavobacterium johnsonian and Herpetosiphon aurantiacus.

p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.