Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.     

Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0 g/L 2-PE+2-PEA vs. 1.4 g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess.     

In situ product recovery (ISPR) by crystallization: basic principles, design, and potential applications in whole-cell biocatalysis

The removal of inhibiting or degrading product from a bioreactor as soon as the product is formed is an important issue in industrial bioprocess development. In this review, the potential of crystallization as an in situ product removal (ISPR) technique for the biocatalytic production of crystalline compounds is discussed. The emphasis of this review is on the current status of crystalline product formation by metabolically active cells for application in fine-chemicals production. Examples of relevant biocatalytic conversions are summarized, and some basic process options are discussed. Furthermore, a case study is presented in which two conceptual process designs are compared. In one process, product formation and crystallization are integrated by applying ISPR, whereas a second, nonintegrated process is based on a known conventional process equivalent for the production of 6R-dihydro-oxoisophorone. The comparison indicates that employing ISPR leads to significant advantages over the nonintegrated case in terms of increased productivity and yield with a corresponding decrease in the number of downstream processing steps, as well as in the quantity of waste streams. This leads to an economically more interesting process alternative. Finally, a general outlook on the various research aspects of ISPR by crystallization is given.     

The integrated simulation and assessment of the impacts of process change in biotherapeutic antibody production

Growing commercial pressures in the pharmaceutical industry are establishing a need for robust computer simulations of whole bioprocesses to allow rapid prediction of the effects of changes made to manufacturing operations. This paper presents an integrated process simulation that models the cGMP manufacture of the FDA-approved biotherapeutic CroFab, an IgG fragment used to treat rattlesnake envenomation (Protherics U.K. Limited, Blaenwaun, Ffostrasol, Llandysul, Wales, U.K.). Initially, the product is isolated from ovine serum by precipitation and centrifugation, before enzymatic digestion of the IgG to produce FAB and FC fragments. These are purified by ion exchange and affinity chromatography to remove the FC and non-specific FAB fragments from the final venom-specific FAB product. The model was constructed in a discrete event simulation environment and used to determine the potential impact of a series of changes to the process, such as increasing the step efficiencies or volumes of chromatographic matrices, upon product yields and process times. The study indicated that the overall FAB yield was particularly sensitive to changes in the digestive and affinity chromatographic step efficiencies, which have a predicted 30% greater impact on process FAB yield than do the precipitation or centrifugation stages. The study showed that increasing the volume of affinity matrix has a negligible impact upon total process time. Although results such as these would require experimental verification within the physical constraints of the process and the facility, the model predictions are still useful in allowing rapid "what-if" scenario analysis of the likely impacts of process changes within such an integrated production process.     

Critical limitations in biological production of chemicals: process or genetic solutions?

Abstract: Production of bulk chemicals by biological processes is presently limited by failure of contemporary biological and bioreactor technology to deliver high product concentrations in high space-time yields in fluids of sufficiently low water content for subsequent down-stream processing operations. Limitations in the bioreactor portion of the process can arise due to failure to process sufficient substrate, substrate inhibition, inadequate rates or yields, and product inhibition. Various process approaches for addressing many of these limitations have been demonstrated or conceptualized. Less developed but potentially effective are genetic strategies addressing these process limitations. Ideally, the most effective combination of genetic and process approaches should be integrated in a synergistic fashion to maximize the economic potential of biological production of chemicals.     

Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.     

In situ product removal using a crystallization loop in asymmetric reduction of 4-oxoisophorone by Saccharomyces cerevisiae

In situ product crystallization was investigated for solid product crystals that were obtained during fermentation. The model reaction was the asymmetric reduction of 4-oxoisophorone (OIP) using baker's yeast (S. cerevisiae) as a biocatalyst. The target product was 6R-dihydro-oxoisophorone (DOIP), also known as levodione, a key intermediate in carotenoid synthesis. DOIP was degraded by baker's yeast mainly to (4S,6R)-actinol, an unwanted byproduct in the process. Actinol formation reached up to 12.5% of the initial amount of OIP in the reactor during a batch process. However, better results were obtained when the dissolved DOIP concentration was controlled using an integrated fermentation-crystallization process because: (a) actinol formation was reduced to 4%; and (b) DOIP crystal formation in the reactor was avoided. DOIP productivity was improved by 50% and its selectivity was raised from 87% to 96% relative to the batch process. In the integrated process, most of the product was recovered as pure crystals; this may already minimize, if not eliminate, the need for organic solvents in the final purification steps. An almost sixfold reduction in biocatalyst consumption per kilogram product was achieved, which also can contribute to the minimization of waste streams.     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

A new large‐scale manufacturing platform for complex biopharmaceuticals

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion‐dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non‐ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions. In order to maximize yield, process efficiency, facility and equipment utilization, we have developed, scaled‐up and successfully implemented a new integrated manufacturing platform in commercial scale. This platform consists of a (semi‐)continuous cell separation process based on a disposable flow path and integrated with the upstream perfusion operation, followed by membrane chromatography on large‐scale adsorber capsules in rapid cycling mode. Implementation of the platform at commercial scale for a new product candidate led to a yield improvement of 40% compared to the conventional process technology, while product quality has been shown to be more consistently high. Over 1,000,000 L of cell culture harvest have been processed with 100% success rate to date, demonstrating the robustness of the new platform process in GMP manufacturing. While membrane chromatography is well established for polishing in flow‐through mode, this is its first commercial‐scale application for bind/elute chromatography in the biopharmaceutical industry and demonstrates its potential in particular for manufacturing of potent, low‐dose biopharmaceuticals. Biotechnol. Bioeng. 2012; 109: 3049–3058. © 2012 Wiley Periodicals, Inc.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

A modular approach to situation identification of the dynamics of bacterial populations synthesizing poly-β-hydroxybutyrate

Biotechnological processes involving bacteria are strongly nonlinear. Therefore, both their productivity and the final product quality may be considerably improved by applying appropriate control strategies to modulate behavior of the bacteria during transitional states. This requires advance identification of indicative signals by off-line investigation (i.e. experimental analysis) and on-line monitoring, (i.e. real time evaluation). A modular scheme is presented for doing this, which incorporates an Extended Kalman Filter and a prediction filter. If this is based on a suitable process-feature vector, which must be chosen in advance, the system can provide sufficient information to trigger appropriate feedback signals. Thus, it can provide a key element in modular situation control, allowing continuously periodic process management. In this publication the individual modules involved, and their assembly into an integrated system are described. Potential problems concerning selection of the feature vector, and experimental results are also discussed.     

An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter

In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.     

A modified Escherichia coli protein production strain expressing staphylococcal nuclease,capable of auto-hydrolysing host nucleic acid

The large-scale production of recombinant biotherapeutics, particularly recombinant proteins, provides significant process and regulatory challenges to the biotechnology industry in order to meet the regulatory agencies stringent requirements in a cost-effective manner. Host cell derived nucleic acid causes problems from both a process and a regulatory perspective, as high molecular weight chromosomal DNA is responsible both for the viscosity of cell lysates, and it is a source of heterologous DNA sequences whose inclusion in the final product must be prevented. We have constructed a modified Escherichia coli JM107 expression host (JMN), containing a staphylococcal nuclease expression cassette, integrated into the host chromosome at the dif locus. The nuclease is expressed as a fusion to the ompA signal peptide, and is translocated to the periplasm of the cell, protecting the cytoplasmic nucleic acid from any toxic activity. The nuclease is released during cell lysis, where it subsequently acts to hydrolyse host nucleic acid present in the lysate. Results with this strain show that sufficient levels of nuclease activity are produced to completely auto-hydrolyse the host's chromosomal DNA to a size non-visible on 1% agarose gel, generating a markedly lower lysate viscosity. This provides a suitable methodology to remove heterologous DNA sequences early in the product stream and decrease lysate viscosity, improving the efficiency of downstream processing and product yield, whilst avoiding the addition of exogenous nuclease and its prohibitive costs at large-scale.     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Design and operation of a continuous integrated monoclonal antibody production process

The realization of an end‐to‐end integrated continuous lab‐scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter‐based cell‐retention, a continuous two column capture process, a virus inactivation step, a semi‐continuous polishing step (twin‐column MCSGP), and a batch‐wise flow‐through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity‐yield trade‐off of classical batch‐wise bind‐elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight‐through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end‐to‐end integration. The steady‐state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303–1313, 2017     

Process analytical technology implementation for protein refolding: GCSF as a case study

Process analytical technology is gaining interest in the biopharmaceutical industry as a means to enable consistency in processing and thereby in product quality via process control. Protein refolding is known to be significantly impacted by critical process parameters and feed material attributes including composition and pH of the solubilisation and refolding buffers. Hence, to achieve robust process control and product quality, these attributes and parameters need to be monitored. This paper presents an approach towards statistical process control and monitoring of protein refolding, from buffer preparation to refold quenching, during manufacturing of therapeutic proteins from Escherichia coli based systems. The proposed approach utilises measurements of online redox potential, temperature, and pH for development of a statistical model. The model has then been integrated with LabView to permit real-time monitoring of the refolding process. The proposed system has been demonstrated to successfully identify process deviations and thereby enable process control for manufacturing product of consistent quality.     

Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic"> coli</Emphasis> by fully integrated reactive extraction

A fully integrated process for the microbial production and recovery of the aromatic amino acid L-phenylalanine is presented. Using a recombinant L-tyrosine (L-Tyr) auxotrophic Escherichia coli production strain, a fed-batch fermentation process was developed in a 20-l-scale bioreactor. Concentrations of glucose and L-Tyr were closed-loop-controlled in a fed-batch process. After achieving final L-phenylalanine (L-Phe) titres >30 g/l the process strategy was scaled up to 300-l pilot scale. In technical scale fermentation L-phenylalanine was continuously recovered via a fully integrated reactive extraction system achieving a maximum extraction rate of 110 g/h (final purity >99%). It was thus possible to increase L-Phe/glucose selectivity from 15 mol% without to 20.3 mol% with integrated product separation.     

Impact considerations of post-production processes on cell and gene drug products

Cell and gene therapies are demonstrating clinical efficacy, but prohibitive product costs and operational complexity bottlenecks may limit expanded patient access to these innovative and transformative products. An initial survey and subsequent article published through the International Society for Cell & Gene Therapy in 2017 presented a roadmap on how specific steps, from tissue procurement and material acquisition to facility operation and production, contribute to the high cost of cell and gene therapies. Herein the authors expanded the investigation to provide considerations to better understand how post-production procedures can impact a product's accessibility to patients. The administration of a drug product to and follow-up in a patient involve key decisions in several post-production process areas, such as product storage, distribution and handling logistics and compliance, across the value chain through integrated data management solutions. Understanding as well as carefully evaluating these specific components is not widely considered during early process development but is critical in developing a viable product life cycle.