Economic analysis of pilot‐scale production of B‐phycoerythrin

β‐Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B‐phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two‐phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two‐phase systems in a scaled‐up process. This study analyzed the production of B‐Phycoerythrin using real data obtained during the scale‐up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two‐phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10‐year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B‐phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472–1479, 2016     

Economic analysis of royalactin production under uncertainty: Evaluating the effect of parameter optimization

Royalactin is a protein with several different potential uses in humans. Research, in insects and in mammalian cells, has shown that it can accelerate cell division and prevent apoptosis. The method of action is through the use of the epidermal growth factor receptor, which is present in humans. Potential use in humans could be to lower cholesterolemic levels in blood, and to elicit similar effects to those seen in bees, e.g., increased lifespan. Mass production of Royalactin has not been accomplished, though a recent article presented a Pichia pastoris fermentation and recovery by aqueous two‐phase systems at laboratory scale as a possible basis for production. Economic modelling is a useful tool with which compare possible outcomes for the production of such a molecule and in particular, to locate areas where additional research is needed and optimization may be required. This study uses the BioSolve software to perform an economic analysis on the scale‐up of the putative process for Royalactin. The key parameters affecting the cost of production were located via a sensitivity analysis and then evaluated by Monte Carlo analysis. Results show that if titer is not optimized the strategy to maintain a low cost of goods is process oriented. After optimization of this parameter the strategy changes to a product‐oriented and the target output becomes the critical parameter determining the cost of goods. This study serves to provide a framework for the evaluation of strategies for future production of Royalactin, by analyzing the factors that influence its cost of manufacture. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:744–749, 2015     

Economic simulation of batch and continuous aqueous two-phase purification for viral products

Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid–liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry.     

Integration of biocatalyst and process engineering for sustainable and efficient n‐butanol production

Recent environmental economic developments generate a need for sustainable and cost‐effective (microbial) processes for the production of high‐volume, low‐priced bulk chemicals. As an example, n‐butanol has, as a second‐generation biofuel, beneficial characteristics compared to ethanol in liquid transportation fuel applications. The industrial revival of the classic n‐butanol (ABE) fermentation requires process and strain engineering solutions for overcoming the main process limitations: product toxicity and low space–time yield. Reaction intensification on the biocatalyst, fermentation, and bioprocess level can be based on economic and ecologic evaluations using quantifiable constraints. This review describes the means of process intensification for biotechnological processes. A quantitative approach is then used for the comparison of the massive literature on n‐butanol fermentation. A comprehensive literature study—including key fermentation performance parameters—is presented and the results are visualized using the window of operation methodology. The comparison allowed the identification of the key constraints, high cell densities, high strain stability, high specific production rate, cheap in situ product removal, high n‐butanol tolerance, to operate in situ product removal efficiently, and cheap carbon source. It can thus be used as a guideline for the bioengineer during the combined biocatalyst, fermentation, and bioprocess development and intensification.     

Early‐stage sustainability assessment of biotechnological processes: A case study of citric acid production

Sustainability assessment using a life‐cycle approach is indispensable to contemporary bioprocess development. This assessment is particularly important for early‐stage bioprocess development. As early‐stage investigations of bioprocesses involve the evaluation of their ecological and socioeconomic effects, they can be adjusted more effectively and improved towards sustainability, thereby reducing environmental risk and production costs. Early‐stage sustainability assessment is an important precautionary practice and, despite limited data, a unique opportunity to determine the primary impacts of bioprocess development. To this end, a simple and robust method was applied based on the standardized life‐cycle sustainability assessment methodology and commercially available datasets. In our study, we elaborated on the yeast‐based citric acid production process with Yarrowia lipolytica assessing 11 different substrates in different process modes. The focus of our analysis comprised both cultivation and down‐stream processing. According to our results, the repeated batch raw glycerol based bioprocess alternative showed the best environmental performance. The second‐ and third‐best options were also glycerol‐based. The least sustainable processes were those using molasses, chemically produced ethanol, and soy bean oil. The aggregated results of environmental, economic, and social impacts display waste frying oil as the best‐ranked alternative. The bioprocess with sunflower oil in the batch mode ranked second. The least favorable alternatives were the chemically produced ethanol‐, soy oil‐, refined glycerol‐, and molasses‐based citric acid production processes. The scenario analysis demonstrated that the environmental impact of nutrients and wastewater treatment is negligible, but energy demand of cultivation and down‐stream processing dominated the production process. However, without energy demand the omission of neutralizers almost halves the total impact, and neglecting pasteurization also considerably decreases the environmental impact.     

Practical application of aqueous two-phase partition to process development for the recovery of biological products

The practical application of aqueous two-phase systems (ATPS) to process development has been exploited for several years for the recovery of biological products. Unfortunately, this has not resulted in an extensive presence of the technique in commercial processes. Some of the main identified reasons for such situation involve the full understanding of the mechanism governing phase formation and the behaviour of solute partitioning in ATPS processes, the cost of phase forming polymers and the necessary extended time to understand the technique for process development. In this review paper, some of the practical disadvantages attributed to ATPS are addressed. The practical approach exploited to design ATPS processes, the application to achieve process integration, the increasing use for the recovery of high-value products and the recent development of alternative low cost ATPS, are discussed. It is proposed that the potential trend in the application of ATPS processes for the recovery of biological products will involve the recovery of high-value bio-particulate products with medical applications. This proposed trend in the application of ATPS will address the urgent need to rapidly and economically bring new biopharmaceutical products to market using scaleable and efficient bioprocess technology.     

Economic evaluation of the development of a phage therapy product for the control of Salmonella in poultry

Poultry products are one of the major transmission media of Salmonella enteritidis to humans. A promising alternative to reduce the load of Salmonella in poultry are bacteriophages. Elsewhere, a mixture of six bacteriophages has been used successfully, but large-scale production would be necessary to supply potential poultry market and costs analyses have not been calculated yet. For this, a powerful tool to predict production costs is bioprocess modeling coupled with economic analyses. This work aims to model the scaled-up production of a six bacteriophages mixture based on a laboratory/pilot-scale production using Biosolve Process. For the model construction, a combination of experimental and reported data was applied, in which different production alternatives and the range of 1–100% of the Colombian poultry market (at broiler's farm and slaughterhouse) were analyzed. Results indicate that the best cost-effective process configuration/scale is to use one bioreactor (156 L) for the six bacteriophages, then a 0.45 μm filtration for removal of biomass, and a 0.22 μm filtration for sterility; this to supply the 35% of the market size for broiler farms (equivalent to 210 million chickens). This configuration gives a production cost per chicken of US$ 0.02. Additionally, a sensitivity analysis and a theoretical contrast for understanding the impact that titer and recovery have on production scale determined that titer affects the most the cost and requires optimization. The present works serves as a first, and required, approach for the development of phage therapy products that are alternatives to present-day pathogens control strategies.     

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1343–1353, 2013     

Application of an aqueous two‐phase systems high‐throughput screening method to evaluate mAb HCP separation

Aqueous two‐phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud‐point method for binodal determination was developed. PEG4000–PO₄ binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000–PO₄ systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task. Biotechnol. Bioeng. 2011; 108:69–81. © 2010 Wiley Periodicals, Inc.     

Extractive disruption process integration using ultrasonication and an aqueous two‐phase system for protein recovery from Chlorella sorokiniana

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time‐consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100‐fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.     

Biosurfactant production: emerging trends and promising strategies

Biosurfactants are economically most sought after biotechnological compounds of the 21st century. However, inefficient bioprocessing has mitigated the economical commercial production of these compounds. Although much work is being done on the use of low-cost substrates for their production, a paucity of literature exists on the upcoming bioprocess optimization strategies and their successes and potential for economical biosurfactant production. This review discusses some of the latest developments and most promising strategies to enhance and economize the biosurfactant production process. Recent market analysis, developments in the field of optimally formulated cost credit substrates for enhanced product formation and subsequent process economization are few of the critical aspects highlighted here. Use of nanoparticles and coproduction of biosurfactant along with other commercially important compounds like enzymes, are other upcoming bioprocess intensification strategies. The recent developments discussed here would not only give an overview of pertinent parameters for economic biosurfactant production but would also bring to fore multiple strategies that would open up new avenues of research on biosurfactant production. This would go a long way in making biosurfactants a commercially successful compound of the current century.     

Partition behavior of CD133+ stem cells from human umbilical cord blood in aqueous two‐phase systems: In route to establish novel stem cell primary recovery strategies

Aqueous two‐phase systems (ATPS) represent a promising strategy for the recovery of CD133⁺ stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer–polymer ATPS were exploited to study the partitioning behavior of CD133⁺ stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133⁺ sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133⁺ stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133⁺ in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133⁺ stem cells and contaminants from complex samples was as follows: 59% of CD133⁺ stem cells fractionated to the top phase when employing ficoll 400,000–DEX 70,000 or 100% to the bottom phase with Ucon‐DEX 75,000 and PEG 8,000‐DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000‐DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon‐DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000‐DEX 500,000. Cell viability was at least 98% after ATPS recovery. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:700–707, 2014     

Examining the feasibility of bulk commodity production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is currently used by many research institutions and companies around the world as a platform organism for the development of bio-based production processes for bulk biochemicals. A given bulk biochemical bioprocess must be economically competitive with current production routes. Ideally the viability of each bioprocess should be evaluated prior to commencing research, both by metabolic network analysis (to determine the maximum theoretical yield of a given biocatalyst) and by techno-economic analysis (TEA; to determine the conditions required to make the bioprocess cost-competitive). However, these steps are rarely performed. Here we examine theoretical yields and review available TEA for bulk biochemical production in E. coli. In addition, we examine fermentation feedstocks and review recent strain engineering approaches to achieve industrially-relevant production, using examples for which TEA has been performed: ethanol, poly-3-hydroxybutyrate, and 1,3-propanediol.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K₂HPO₄ ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K₂HPO₄ and 16% (w/w) KCl with pH of 8.0, volume ratio (V _R ) of 0.25, temperature of 25 °C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 €) were additionally features that confirmed the suitability of proposed technique.     

A Material Flow‐based Approach to Enhance Resource Efficiency in Production and Recycling Networks

Resource and energy efficiency are key strategies for production and recycling networks. They can contribute to more sustainable industrial production and can help cope with challenges such as competition, rising resource and energy prices, greenhouse gas emissions reduction, and scarce and expensive landfill space. In pursuit of these objectives, further enhancements of single processes are often technologically sophisticated and expensive due to past achievements that have brought the processes closer to technical optima. Nevertheless, the potential for network‐wide advancements may exist. Methods are required to identify and assess the potential for promising resource and energy efficiency measures from technical, economic, and ecological perspectives. This article presents an approach for a material flow‐based techno‐economic as well as ecological analysis and assessment of resource efficiency measures in production and recycling networks. Based on thermodynamic process models of different production and recycling processes, a material and energy flow model of interlinked production and recycling processes on the level of chemical compounds is developed. The model can be used to improve network‐wide resource efficiency by analyzing and assessing measures in scenario and sensitivity analyses. A necessary condition for overcoming technical and economic barriers for implementing such measures can be fulfilled by identifying strategies that appear technologically feasible and economically and ecologically favorable. An exemplary application to a production and recycling network of the German steel and zinc industry is presented. From a methodological point of view, the approach shows one way of introducing thermodynamics and further technological aspects into industrial planning and assessment.     

Allogeneic cell therapy bioprocess economics and optimization: Single‐use cell expansion technologies

For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot‐sizes capable of meeting commercial demands of up to 10⁹ cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost‐effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier‐based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost‐effective and where microcarrier‐based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier‐based systems. These data are presented using a technology S‐curve as well as windows of operation to identify the combination of cell productivities and scale of single‐use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision‐making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Estimation of Waste Battery Generation and Analysis of the Waste Battery Recycling System in China

China produces and consumes a large amount of batteries annually, which leads to many waste batteries needing to be recycled. The collection and recycling system of primary, alkaline secondary, and lithium‐ion secondary batteries in China is particularly poor, and waste battery recycling enterprises generally sustain economic losses if they solely use waste batteries as raw materials. Increasing the profits of waste battery recycling systems is a key problem that needs to be considered. This article quantitatively analyzes waste battery generation in China by using annual sales data and probable lifetime distribution of various batteries. The results show that the rapid growth of battery usage has led to an increased generation of waste batteries and the percentage of different types of waste batteries is changing over time. In 2013, the total quantity of all waste batteries in the medium lifetime scenario reached 570 kilotons, of which primary, alkaline secondary, and lithium‐ion secondary waste batteries accounted for approximately 36%, 28%, and 35%, respectively. Based on a real‐world case study of a typical domestic waste battery recycling enterprise in China, material flow analysis and cost‐benefit analysis were conducted to study the development of the recycling process of comingled waste batteries. Through scenario analysis, we conclude that increasing the use of waste batteries as raw materials and the recycling of other materials that are less valuable reduces the profits of the waste battery recycling enterprise. Higher profits can be achieved by adding the production of high value‐added downstream products and government support. At the same time, the essential role of the government in developing a waste battery recycling system was identified. Finally, relevant suggestions are made for improvements in both the government and enterprise sectors.     

A Spatial Analysis of Loop Closing Among Recycling,Remanufacturing, and Waste Treatment Firms in Texas

Industrial ecology has emerged as a key strategy for improving environmental conditions. A central element of industrial ecology is the concept of closing the loop in material use (cycling) by directing used material and products (wastes) back to production processes. This article examines the issue of geographic scale and loop closing for heterogeneous wastes through an analysis of the location and materials flows of a set of recycling, remanufacturing, recycling manufacturing, and waste treatment (RRWT) firms in Texas. The results suggest that there is no preferable scale at which loop closing should be organized. RRWT firms are ubiquitous and operate successfully throughout the settlement hierarchy. The cycling boundaries of RRWT firms are dependent primarily upon how and where their products are redirected to production processes rather than the firm's location in the settlement hierarchy. In other words, loop closing is dominated by the spatial economic logic of the transactions of the firm involved. These results suggest that we cannot assign loop closing to any particular spatial scale a priori nor can we conceive of closing the loop via RRWT firms in terms of monolithic networks bounded in space or place with internal material flows.     

Advanced near‐infrared monitor for stable real‐time measurement and control of Pichia pastoris bioprocesses

Near‐infrared spectroscopy is considered to be one of the most promising spectroscopic techniques for upstream bioprocess monitoring and control. Traditionally the nature of near‐infrared spectroscopy has demanded multivariate calibration models to relate spectral variance to analyte concentrations. The resulting analytical measurements have proven unreliable for the measurement of metabolic substrates for bioprocess batches performed outside the calibration process. This paper presents results of an innovative near‐infrared spectroscopic monitor designed to follow the concentrations of glycerol and methanol, as well as biomass, in real time and continuously during the production of a monoclonal antibody by a Pichia pastoris high cell density process. A solid state instrumental design overcomes the ruggedness limitations of conventional interferometer‐based spectrometers. Accurate monitoring of glycerol, methanol, and biomass is demonstrated over 274 days postcalibration. In addition, the first example of feedback control to maintain constant methanol concentrations, as low as 1 g/L, is presented. Postcalibration measurements over a 9‐month period illustrate a level of reliability and robustness that promises its adoption for online bioprocess monitoring throughout product development, from early laboratory research and development to pilot and manufacturing scale operation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:749–759, 2014