Bioprocess Research Articles

Clarification upsets and ion exchange resin symptoms due to design

Abstract: Ion exchange resin (IER) decolorization columns face challenges because of upstream process upsets and certain design choices. Some of these challenges have been caused by excessive amounts of calcium carbonate carried over from press filtration to the IER columns. As a result, the columns experience sudden impacts on performance, which causes a premature shutdown or retention of the calcium carbonate, degrading resin performance over time. In addition, the refinery suffers in terms of overall throughput due to pressure drop in the IER columns or reduced decolorization and higher amount wash syrup in the centrifuges. Fiber glass nozzles installed in the columns have been replaced with stainless steel nozzles. Pros and cons of the replacement are discussed, which resulted in a much longer service life of the nozzles.

Optimizing the switching operation in monoclonal antibody production: Economic MPC and reinforcement learning

Monoclonal antibodies (mAbs), crucial in medicine and biopharmaceuticals, require optimized large-scale production to meet high clinical dosage demands. Most of the processes for industrial mAb production rely on fed-batch operations, resulting in significant downtime. Transitioning to fully continuous and integrated processes holds the potential to boost product yield, enhance quality, and reduce storage costs for intermediate products. The integrated continuous mAb production process can be divided into the upstream and downstream processes. One crucial aspect that ensures the continuity of the integrated process is the switching of the capture columns, which are typically chromatography columns operated in a fed-batch manner downstream. Due to the discrete nature of the switching operation, advanced process control algorithms such as economic MPC (EMPC) are computationally difficult to implement. This is because an integer nonlinear program (INLP) needs to be solved online at each sampling time.This paper introduces two computationally-efficient approaches for EMPC implementation, namely, a sigmoid function approximation approach and a rectified linear unit (ReLU) approximation approach. It also explores the application of deep reinforcement learning (DRL). These three methods are compared to the traditional switching approach which is based on a 1 % product breakthrough rule and which involves no optimization.

Accelerated and intensified manufacturing of an adenovirus-vectored vaccine to enable rapid outbreak response.

The Coalition for Epidemic Preparedness Innovations' "100-day moonshot" aspires to launch a new vaccine within 100 days of pathogen identification, followed by large-scale vaccine availability within the "second hundred days." Here, we describe work to optimize adenoviral vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing output from the available manufacturing footprint. We describe a rapid virus seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by approximately fourfold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10,000 doses from each liter of bioreactor capacity. This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.

Effects of wet granulation process variables on the quantitative assay model of transmission Raman spectroscopy for pharmaceutical tablets

Transmission Raman spectroscopy (TRS) is a process analytical technology tool for nondestructive analysis of drug content in tablets. Although wet granulation is the most used tablet manufacturing method, most TRS studies have focused on tablets manufactured via direct compression. The effects of upstream process parameter variations, such as granulation, on the prediction performance of TRS quantitative models are unknown. We evaluated the effects of process parameter variations during granulation on the prediction performance of the TRS quantitative model. Tablets with a drug concentration of 1%w/w were used. We developed PLS calibration models for the drug concentration range of 70–130% label claims. Subsequently, we predicted the drug content of the tablets with different granulation parameters. The results of our study demonstrate that the variation in the predicted recovery due to the variation in granulation parameters was practically acceptable. The calibration model showed a good prediction performance for tablets manufactured at different granulation scales and thicknesses. Therefore, we conclude that TRS quantitative models are robust to variations in upstream processes, such as granulation and downstream variations in tableting parameters. These results suggest that TRS is a versatile non-destructive quantitative analysis method that can be applied in tablet manufacturing.

Comparative assessment of Chinese mitten crab aquaculture in China: Spatiotemporal changes and trade-offs

The increasing human demand for high-quality animal protein has provided impetus for the development of aquaculture. Chinese mitten crab (Eriocheir sinensis) is a catadromous species rapidly being cultured in China but scientific literature documenting its life cycle environmental and economic consequences remains scarce. This study aims to address this gap by examining the spatio-temporal evolution of crab aquaculture in China since the 2000s and evaluating the environmental and economic characteristics along its life-cycle stages: megalopa, juvenile crab, and adult crab cultivation. The geostatistical analysis shows a more dispersed pattern of crab aquaculture nationally as crab grows, with coastal provinces that have brackish water for megalopa cultivation but wider spatial coverage for juvenile and adult crab cultivation. Our findings reveal that the production of 1 ton of live-weight crab results in 7.65 ton of CO2 equivalent of greenhouse gas emissions, surpassing previous estimates for finfish fish production by approximately 50%. Most environmental pressures occur during the adult crab cultivation stage, with significant contributions from upstream processes such as electricity and feed production. By comparing between different production systems, our study shows that crab aquaculture in lake systems performs better than pond systems in terms of most global environmental impact categories and economic considerations. This work contributes to the existing literature by elucidating the spatio-temporal changes of crab aquaculture boom in China and constructing a representative life cycle data pool that broadens the benchmark knowledge on its environmental and economic characteristics. We highlight the trade-offs between environmental and economic performance as well as the balance between global and local environmental impacts to promote sustainable growth in the aquaculture industry.

Recent Advancements in Lignin Valorization, Bioengineering, Catalysis and Biorefining

Most of the research studies in the field of lignin valorization has been achieved on lignin from paper and pulp industries. The key merit of utilizing lignin from the facilities is that resources are already centralized, and the costs of transportation to further process are fundamentally less compared to possible sources. Biomass composed of lignin, hemicelluloses, and cellulose (also known as lignocellulosic biomass) is both plentiful and renewable. The three polymer groups may be isolated and chemically transformed to provide a wide variety of bio-generated value-added materials, chemical, and fuels. To maximize the value of the biomass feedstock as a whole, it is best to extract these products via a streamlined, integrated models of pathways concerned with catalytic reactions i.e., bio-refinery. In this article, upstream processing is analyzed in depth, including the phenylpropanoid route, bioengineered lignins, and lignin bioengineering. The last section addresses the difficulties inherent in lignin bioengineering from a technical and practical standpoint.

Downstream new product development and upstream process innovation

Downstream new product development and upstream process innovation

Recent advances in upstream process development for production of recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in-vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes currentavailable information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in-depth understanding of production kinetics and quality control.

Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells.

Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell's identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached.

Sensor Selection and State Estimation of Continuous mAb Production Processes

The production of monoclonal antibodies (mAbs) plays a pivotal role in therapeutic treatments, and optimizing their production is crucial for minimizing costs and improving their accessibility to patients. One way of improving the production process is to improve model accuracy through the correct estimation of its states and parameters. The contributions of this paper lie in the provision of guidelines for sensor selection in the upstream production process of mAbs to enhance the accuracy of state estimation. Furthermore, this paper applies an effective variable selection technique for simultaneous state and parameter estimations for enhanced estimation results in the biomanufacturing processes of mAbs. An estimation framework of MHE is designed for three different case studies to demonstrate the efficiency of the proposed approach. The estimation performance is compared and assessed using the Root Mean Squared Error (RMSE) as an evaluation criterion.

Sequential clinical scheduling with stochastic patient re-entrance: Case of Mohs Micrographic Surgery

We study the problem of appointment scheduling for outpatient clinics with stochastic patient re-entrance. It is motivated by the scheduling problem for Mohs Micrographic Surgery, which is a popular technique for the excision of skin cancers. Re-entrance occurs when a patient repeats upstream processes during the single-day outpatient appointment, usually after a medical test. In some surgical procedures, the number of re-entrances for each patient is unknown until medical test results become available, causing long patient waiting time and clinic overtime. To address these challenges, we develop a stochastic slot model, SMART, that captures the key characteristics of the appointment processes and stochastic re-entrance, utilizing a delay before possible re-entrance. We then design a sequential scheduling algorithm that balances patient waiting, clinic overtime and patient throughput while considering stochastic complications such as no-shows, processing time variability, and the number of re-entrances per patient. We establish several theoretical properties of the algorithm, including the optimality of the resulting schedules for the sequential scheduling setting. We apply SMART to appointment scheduling for Mohs clinics, where patients may have same-day re-entrance due to repetitive skin excisions. We compare SMART schedules to others that ignore re-entrance and to current scheduling practices via simulation studies under a more realistic setting. Numerical experiments show that SMART schedules dominate other schedules, and ignoring re-entrance can cost huge loss in efficiency. Furthermore, SMART schedules exhibit a special ‘stair-stepping’ pattern for the number of appointments, with most patients assigned to the early slots and many empty slots at the end.

Biofilm formation by agave epiphytic lactic acid bacteria fed with agave fructans.

The aim of this work was to find out if biofilms can be made by lactic acid bacteria (LAB) isolated from agave plants using agave fructans as sole carbohydrate substrates or if it was necessary to use fructose as a breakdown product of such polymers. This is part of a research project geared to develop industrial lactic acid production from agave fructans, an abundant raw material in Mexico's agave plantations. Present results showed that nine strains of LAB isolated from Agave salmiana and belonging to genus Lacticaseibacillus and Enterococcus produced exopolysaccharides directly from agave fructans to a greater extent than with fructose. The best polysaccharide productions in planktonic cultures were Lacticaseibacillus paracasei strains DG2, DG3, DG4 and DG8. Furthermore, all nine LAB strains produced biofilms on polystyrene microplates, much better with agave fructans than with fructose. In most strains, biofilm formation was favored at pH from 6.0 to 6.5, except for strains DG7 and DG9 where pH 5.5 was optimal. Biofilm formation required between 3 and 5 days of incubation in all Lacticaseibacillus paracasei strains, whereas Enterococcus faecium required a little less of 3 days. Present results support the straight use of agave fructans to develop LAB biofilms using agave epiphytic bacteria. This finding simplifies upstream processing of agave fructans to be used for future lactic acid fermentation in LAB biofilm reactors.

PO2/TransformON, an ontology for data integration on food, feed, bioproducts and biowaste engineering

We are witnessing an acceleration of the global drive to converge consumption and production patterns towards a more circular and sustainable approach to the food system. To address the challenge of reconnecting agriculture, environment, food and health, collections of large datasets must be exploited. However, building high-capacity data-sharing networks means unlocking the information silos that are caused by a multiplicity of local data dictionaries. To solve the data harmonization problem, we proposed an ontology on food, feed, bioproducts, and biowastes engineering for data integration in a circular bioeconomy and nexus-oriented approach. This ontology is based on a core model representing a generic process, the Process and Observation Ontology (PO2), which has been specialized to provide the vocabulary necessary to describe any biomass transformation process and to characterize the food, bioproducts, and wastes derived from these processes. Much of this vocabulary comes from transforming authoritative references such as the European food classification system (FoodEx2), the European Waste Catalogue, and other international nomenclatures into a semantic, world wide web consortium (W3C) format that provides system interoperability and software-driven intelligence. We showed the relevance of this new domain ontology PO2/TransformON through several concrete use cases in the fields of process engineering, bio-based composite making, food ecodesign, and relations with consumer’s perception and preferences. Further works will aim to align with other ontologies to create an ontology network for bridging the gap between upstream and downstream processes in the food system.

The effect of various harvesting techniques and drying methods on the yield and quality of Liberoid coffee

In the upstream process, the dominant factor determines the quality of green coffee beans. Harvest and post-harvest techniques can influence the physical, chemical, and taste qualities by up to 60%. The goal of this study was to see how different harvesting techniques and drying methods affected the yield and quality of Liberoid coffee green beans grown in a dry process. From June to August 2022, the research was carried out at the Agricultural Science Park, Indonesian Industrial and Beverage Crops Research Institute Sukabumi West Java. The study used a factorial randomized block design, with Liberica and Excelsa coffee as the first factor, various harvesting techniques (cherry and rainbow fruit harvest) as the second factor, and the drying method as the third. There were eight treatment combinations, each of which was repeated three times. According to the findings, all Liberica and Excelsa coffee varieties found the highest yield in the cherry fruit. Furthermore, when compared to direct drying, the decrease in water content and weight loss/day is faster and more evenly distributed when using Hybrid tools. A berry provided the best physical quality and chemical content.

AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

Tumor-associated macrophages (TAMs) are integral to the development of complex tumor microenvironments (TMEs) and can execute disparate cellular programs in response to extracellular cues. However, upstream signaling processes underpinning this phenotypic plasticity remain to be elucidated. Here, we report that concordant AXL-STAT3 signaling in TAMs is triggered by lung cancer cells or cancer-associated fibroblasts in the cytokine milieu. This paracrine action drives TAM differentiation toward a tumor-promoting "M2-like" phenotype with upregulation of CD163 and putative mesenchymal markers, contributing to TAM heterogeneity and diverse cellular functions. One of the upregulated markers, CD44, mediated by AXL-IL-11-pSTAT3 signaling cascade, enhances macrophage ability to interact with endothelial cells and facilitate formation of primitive vascular networks. We also found that AXL-STAT3 inhibition can impede the recruitment of TAMs in a xenograft mouse model, thereby suppressing tumor growth. These findings suggest the potential application of AXL-STAT3-related markers to quantitatively assess metastatic potential and inform therapeutic strategies in lung cancer.

Upstream and downstream processing of essential fatty acids from microbial biomass

Microbial biomass is a promising supply of essential fatty acids (EFAs) for utilization in human and animal nutrition such as fish. EFAs, including polyunsaturated fatty acids (PUFAs), are needed for cell regulation and disease prevention. Oleaginous microorganisms from species like fungi (Cryptococcus, Cunninghamella, and Mortierella), microalgae (Chlorella zofingiensis and Crypthecodinium cohnii), and bacteria (Moritella sp. and Vibrio sp.) can accumulate lipids exceeding 20% of their biomass. Optimizing factors such as nitrogen and carbon sources, cultivation methods, and environmental conditions may improve their lipid production. Efficient lipid extraction methods through mechanical, non-mechanical or chemical methods are essential to obtain EFAs from microbial biomass. Challenges include substrates (carbon and nitrogen sources) cost and downstream processing and overcoming these challenges can provide a sustainable source of EFAs for human and animal nutrition. By advancing metabolic engineering, cultivation techniques, and extraction methods, microbial lipid production holds the potential to offer cost-effective and environmentally friendly EFAs. The utilization of microbial biomass as a source of EFAs can contribute to a healthier future by addressing the limitations of traditional sources and providing a sustainable solution for meeting the increasing demand for EFAs in human and animal diets.

Towards the effective distillation sequences for the purification of acetone-butanol-ethanol from the condensate of gas stripping-vapor permeation system

Gas stripping-vapor permeation (GSVP) is an attractive method for in situ acetone-butanol-ethanol (ABE) separation from fermentation broth to obtain renewable biological n-butanol. However, the downstream distillation systems for effective purification of ABE from the upstream GSVP process has not been developed. In this study, focusing on the generation of high purify A-B-E solvents as the final products, the downstream distillation systems for GSVP condensate purification were investigated. According to literature survey, four candidate distillation sequences were selected and optimized by steady-state simulation, followed by cross-over comparisons of energy requirements, techno-economics and environmental influences. The results indicate that the Scenario 3 that containing three individual columns and one decanter exhibited the lowest sensitivity to the key factors, lowest total annual cost (6.483 × 106 $/yr) and CO2 emission (3707.23 kg/h) in a scheme with 30 kt/yr n-butanol production among the four candidate distillation sequences. This study provides a valuable reference for the development of downstream ABE separation and purification processes in future works.

Energy balance and life cycle assessments in producing microalgae biodiesel via a continuous microalgal-bacterial photobioreactor loaded with wastewater

Life cycle assessments of microalgal cultivation systems are often conducted to evaluate the sustainability and feasibility factors of the entire production chain. Unlike widely reported conventional microalgal cultivation systems, the present work adopted a microalgal-bacterial cultivation approach which was upscaled into a pilot-scale continuous photobioreactor for microalgal biomass production into biodiesel from wastewater resources. A multiple cradle-to-cradle system ranging from microalgal biomass-to-lipid-to-biodiesel was evaluated to provide insights into the energy demand of each processes making up the microalgae-to-biodiesel value chain system. Energy feasibility studies revealed positive NER values (4.95–8.38) for producing microalgal biomass but deficit values for microalgal-to-biodiesel (0.14–0.23), stemming from the high energy input requirements in the downstream processes for converting biomass into lipid and biodiesel accounting to 88–90% of the cumulative energy demand. Although the energy balance for microalgae-to-biodiesel is in the deficits, it is comparable with other reported biodiesel production case studies (0.12–0.40). Nevertheless, the approach to using microalgal-bacterial cultivation system has improved the overall energy efficiency especially in the upstream processes compared to conventional microalgal cultivation systems. Energy life cycle assessments with other microalgal based biofuel systems also proposed effective measures in increasing the energy feasibility either by utilizing the residual biomass and less energy demanding downstream extraction processes from microalgal biomass. The microalgal-bacterial cultivation system is anticipated to offer both environmental and economic prospects for upscaling by effectively exploiting the low-cost nutrients from wastewaters via bioconversion into valuable microalgal biomass and biodiesel.

Investigation and evaluation of a 3D-printed optical modified cultivation vessel for improved scattered light measurement of biotechnologically relevant organisms.

In the field of bioprocess development miniaturization, parallelization and flexibility play a key role reducing costs and time. To precisely meet these requirements, additive manufacturing (3D-printing) is an ideal technology. 3D-printing enables rapid prototyping and cost-effective fabrication of individually designed devices with complex geometries on demand. For successful bioprocess development, monitoring of process-relevant parameters, such as pH, dissolved oxygen (DO), and biomass, is crucial. Online monitoring is preferred as offline sampling is time-consuming and leads to loss of information. In this study, 3D-printed cultivation vessels with optical prisms are evaluated for the use in upstream processes of different industrially relevant microorganisms and cell lines. It was shown, that the 3D-printed optically modified well (OMW) is of benefit for a wide range of biotechnologically relevant microorganisms and even for mammalian suspension cells. Evaluation tests with Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells were performed, providing highly reproducible results. Growth behavior of OMW cultures was comparable to behavior of shake flask (SF) cultivations and the signal to noise ratio in online biomass measurement was shown to be reduced up to 95.8% by using the OMW. Especially the cultivation phases with low turbidity respective optical densities below 1.0rel.AU could be monitored accurately for the first time. Furthermore, it was demonstrated that the 3D-printed optics are transferable to different well geometries and sizes, enabling efficient biomass monitoring for individual requirements with tailor-made 3D-printed cultivation vessels in small scale.

A Short Review on Current Status and Obstacles in the Sustainable Production of Biohydrogen from Microalgal Species.

Biohydrogen is an economical fuel which has enormous promise as an alternative energy source. The synthesis of biohydrogen can be done more affordably and sustainably using microalgae. For the generation of biohydrogen and the treatment of wastewater, microalgae derived from effluent have been showing very impressive outcomes. In comparison to traditional fuel sources, microalgae have benefits. Microalgae are capable of fixing ambient Carbon dioxide and converting it to carbohydrates, which are subsequently processed biochemically to provide fuel. When compared to terrestrial crops, they require less water and minerals for production. But besides these benefits, there are certain technological restrictions on the scale-up implementations of microalgae bioenergy. In this work, we explored the production of biohydrogen from several types of microalgae. The process of producing biohydrogen is affected by a number of variables, including pH, substrate concentration, the kinds of microalgal species, and others. The most recent studies and difficulties related to each stage of the biohydrogen manufacturing process are outlined. The synthesis of microalgal biohydrogen is improved using promising approaches that are discussed. Also, the specific future direction are covered. The possibility for microalgae-based production of biohydrogen to serve as an environmentally friendly and carbon-free biofuel solution that might handle the impending fuel scarcity was demonstrated. However, additional study is required on both the upstream and downstream processes of the synthesis of biohydrogen.