Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.     

Modeling Metal Flow Systems

Substance flow analysis (SFA) is a frequently used industrial ecology technique for studying societal metal flows, but it is limited in its ability to inform us about future developments in metal flow patterns and how we can affect them. Equation‐based simulation modeling techniques, such as dynamic SFA and system dynamics, can usefully complement static SFA studies in this respect, but they are also restricted in several ways. The objective of this article is to demonstrate the ability of agent‐based modeling to overcome these limitations and its usefulness as a tool for studying societal metal flow systems. The body of the article summarizes the parallel implementation of two models—an agent‐based model and a system dynamics model—both addressing the following research question: What conditions foster the development of a closed‐loop flow network for metals in mobile phones? The results from in silico experimentation with these models highlight three important differences between agent‐based modeling (ABM) and equation‐based modeling (EBM) techniques. An analysis of how these differences affected the insights that could be extracted from the constructed models points to several key advantages of ABM in the study of metal flow systems. In particular, this analysis suggests that a key advantage of the ABM technique is its flexibility to enable the representation of societal metal flow systems in a more native manner. This added flexibility endows modelers with enhanced leverage to identify options for steering metal flows and opens new opportunities for using the metaphor of an ecosystem to understand metal flow systems more fully.     

Metal‐Organic Framework‐Derived Non‐Precious Metal Nanocatalysts for Oxygen Reduction Reaction

By virtue of diverse structures and tunable properties, metal‐organic frameworks (MOFs) have presented extensive applications including gas capture, energy storage, and catalysis. Recently, synthesis of MOFs and their derived nanomaterials provide an opportunity to obtain competent oxygen reduction reaction (ORR) electrocatalysts due to their large surface area, controllable composition and pore structure. This review starts with the introduction of MOFs and current challenges of ORR, followed by the discussion of MOF‐based non‐precious metal nanocatalysts (metal‐free and metal/metal oxide‐based carbonaceous materials) and their application in ORR electrocatalysis. Current issues in MOF‐derived ORR catalysts and some corresponding strategies in terms of composition and morphology to enhance their electrocatalytic performance are highlighted. In the last section, a perspective for future development of MOFs and their derivatives as catalysts for ORR is discussed.     

Zonal rate model for axial and radial flow membrane chromatography,part II: Model‐based scale‐up

Membrane chromatography (MC) systems are finding increasing use in downstream processing trains for therapeutic proteins due to the unique mass‐transfer characteristics they provide. As a result, there is increased need for model‐based methods to scale‐up MC units using data collected on a scaled‐down unit. Here, a strategy is presented for MC unit scale‐up using the zonal rate model (ZRM). The ZRM partitions an MC unit into virtual flow zones to account for deviations from ideal plug‐flow behavior. To permit scale‐up, it is first configured for the specific device geometry and flow profiles within the scaled‐down unit so as to achieve decoupling of flow and binding related non‐idealities. The ZRM is then configured for the preparative‐scale unit, which typically utilizes markedly different flow manifolds and membrane architecture. Breakthrough is first analyzed in both units under non‐binding conditions using an inexpensive tracer to independently determine unit geometry related parameters of the ZRM. Binding related parameters are then determined from breakthrough data on the scaled‐down MC capsule to minimize sample requirements. Model‐based scale‐up may then be performed to predict band broadening and breakthrough curves on the preparative‐scale unit. Here, the approach is shown to be valid when the Pall XT140 and XT5 capsules serve as the preparative and scaled‐down units, respectively. In this case, scale‐up is facilitated by our finding that the distribution of linear velocities through the membrane in the XT140 capsule is independent of the feed flow rate and the type of protein transmitted. Introduction of this finding into the ZRM permits quantitative predictions of breakthrough over a range of industrially relevant operating conditions. Biotechnol. Bioeng. 2014;111: 1587–1594. © 2014 Wiley Periodicals, Inc.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Actinomycetes scale‐up for the production of the antibacterial,nocathiacin

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale‐up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R_o of 68 vs. 92 min^?1) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm²) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40–50% decrease). High off‐gas CO₂ levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25‐fold scale‐up (600–15,000 L) was controlled to match off‐gas CO₂ profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L‐scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on‐line data (i.e., pH, oxygen uptake rate, and CO₂ evolution rate) and off‐line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Modeling of scale-down effects on the hydrodynamics of expanded bed adsorption columns

Expanded-bed adsorption (EBA) is a technique for primary recovery of proteins starting from unclarified broths. This process combines centrifugation, concentration, filtration, and initial capturing of the proteins in a single step. An expanded bed (EB) is comparable to a packed bed in terms of separation performance but its hydrodynamics are that of a fluidized bed. Downstream process development involving EBA is normally carried out in small columns to minimize time and costs. Our purpose here is to characterize the hydrodynamics of expanded beds of different diameters, to develop scaling parameters that can be reliably used to predict separation efficiency of larger EBA columns. A hydrodynamic model has been developed which takes into account the radial liquid velocity profile in the column. The scale-down effect can be characterized in terms of apparent axial dispersion, D(axl,app), and plate number, N(EB), adapted for expanded bed. The model is in good agreement with experimental results obtained from 1- and 5-cm column diameters with buffer solutions of different viscosities. The model and the experiments show an increase of apparent axial dispersion with an increase in column diameter. Furthermore, the apparent axial dispersion is affected by an increase in liquid velocity and viscosity. Supported by visual observations and predictions from the model, it was concluded that operating conditions (liquid viscosity and superficial velocity) resulting in a bed-void fraction between 0.7 and 0.75 would provide the optimal separation efficiency in terms of N(EB).     

Unravelling Degradation Pathways of Oxide‐Supported Pt Fuel Cell Nanocatalysts under In Situ Operating Conditions

Knowledge of degradation pathways of catalyst/support ensembles aids the development of rational strategies to improve their stability. Here, this is exemplified using indium tin oxide (ITO)‐supported Platinum nanoparticles as electrocatalysts at fuel cell (FC) cathodes under degradation protocols to mimic operating conditions in two potential regimes. The evolution of crystal structure, composition, crystallite and particle size is tracked by in situ X‐ray techniques (small and wide angle scattering), metal dissolution by in situ scanning flow cell coupled with mass spectrometry (SFC ICP‐MS) and Pt surface morphology by advanced electron microscopy. In a regular FC operation regime, Pt poisoning rather than Pt particle growth, agglomeration, dissolution or detachment was found to be the likely origin of the observed degradation and ORR activity losses. In the start‐up regime degradation is actually suppressed and only minor losses in catalytic activity are observed. The presented data thus highlight the excellent nanoparticle stabilization and corrosion resistance of the ITO support, yet point to a degradation pathway involving Pt surface modifications by deposition of sub‐monolayers of support metal ions. The identified degradation pathway of the Pt/oxide catalyst/support couple contributes to our understanding of cathode electrocatalysts for polymer electrolyte fuel cells (PEFC).     

FeS2 Nanoparticles Embedded in Reduced Graphene Oxide toward Robust,High‐Performance Electrocatalysts

Developing low‐cost, highly efficient, and robust earth‐abundant electrocatalysts for hydrogen evolution reaction (HER) is critical for the scalable production of clean and sustainable hydrogen fuel through electrochemical water splitting. This study presents a facile approach for the synthesis of nanostructured pyrite‐phase transition metal dichalcogenides as highly active, earth‐abundant catalysts in electrochemical hydrogen production. Iron disulfide (FeS₂) nanoparticles are in situ loaded and stabilized on reduced graphene oxide (RGO) through a current‐induced high‐temperature rapid thermal shock (≈12 ms) of crushed iron pyrite powder. FeS₂ nanoparticles embedded in between RGO exhibit remarkably improved electrocatalytic performance for HER, achieving 10 mA cm^?2 current at an overpotential as low as 139 mV versus a reversible hydrogen electrode with outstanding long‐term stability under acidic conditions. The presented strategy for the design and synthesis of highly active earth‐abundant nanomaterial catalysts paves the way for low‐cost and large‐scale electrochemical energy applications.     

Scale‐up of citric acid fermentation by redox potential control

To obtain high citric acid productivity in Aspergillus niger fermentation on beet molasses substrate, a certain redox potential profile with two maxima (260 and 280 mV) and two minima (180 and 80 mV) must be maintained. The most effective regulation of redox potential is by regulation of aeration and agitation. It has been shown that control of redox potential by aeration and agitation is a most successful method for scale‐up from 10‐L laboratory scale to the 100‐ and 1000‐L pilot‐plant scale, even in geometrically dissimilar stirred‐tank reactors. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 552–557, 1999.     

Robust scale-up of dead end filtration: impact of filter fouling mechanisms and flow distribution

Robust design of a dead end filtration step and the resulting performance at manufacturing scale relies on laboratory data collected with small filter units. During process development it is important to characterize and understand the filter fouling mechanisms of the process streams so that an accurate assessment can be made of the filter area required at manufacturing scale. Successful scale-up also requires integration of the lab-scale filtration data with an understanding of flow characteristics in the full-scale filtration equipment. A case study is presented on the development and scale-up of a depth filtration step used in a 2nd generation polysaccharide vaccine manufacturing process. The effect of operating parameters on filter performance was experimentally characterized for a diverse set of process streams. Filter capacity was significantly reduced when operating at low fluxes, caused by both low filtration pressure and high stream viscosity. The effect of flux on filter capacity could be explained for a variety of diverse streams by a single mechanistic model of filter fouling. To complement the laboratory filtration data, the fluid flow and distribution characteristics in manufacturing-scale filtration equipment were carefully evaluated. This analysis identified the need for additional scale-up factors to account for non-uniform filter area usage in large-scale filter housings. This understanding proved critical to the final equipment design and depth filtration step definition, resulting in robust process performance at manufacturing scale.     

Production of indole diterpenes by Aspergillus alliaceus

Production of two related indole diterpenes (differing by a dimethyl leucine side chain) by Aspergillus alliaceus was improved through several pilot scale fermentations. Media were optimized through focus primarily on initial increases, as well as mid-cycle additions, of carbon and nitrogen sources. Fermentation conditions were improved by varying ventilation conditions using various combinations of air flowrate and back-pressure set points. Production improvements were quantified based on total indole diterpene concentration as well as the ratio of the major-to-minor by-product components. Those changes with a positive substantial impact primarily on total indole diterpene concentration included early cycle glycerol shots and enhanced ventilation conditions (high air flowrate, low back-pressure). Those changes with a significant impact primarily on ratio included higher initial cerelose, soybean oil, monosodium glutamate, tryptophan, or ammonium sulfate concentrations, higher broth pH, and enhanced ventilation conditions. A few changes (higher initial glycerol and monosodium glutamate concentrations) resulted in less notable and desirable titer or ratio changes when implemented individually, but they were adopted to more fully realize the impact of other improvements or to simplify processing. Overall, total indole diterpene titers were improved at the 600 L pilot scale from 125-175 mg/L with a ratio of about 2.1 to 200-260 mg/L with a ratio of about 3.3-4.5. Thus, the ability to optimize total indole diterpene titer and/or ratio readily exists for secondary metabolite production using Aspergillus cultures.     

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.     

Discrete particle simulations predicting mixing behavior of solid substrate particles in a rotating drum fermenter.

A soft-sphere discrete particle model was used to simulate mixing behavior of solid substrate particles in a slow rotating drum for solid-state fermentation. In this approach, forces acting on and subsequent motion of individual particles can be predicted. The (2D) simulations were qualitatively and quantitatively validated by mixing experiments using video and image analysis techniques. It was found that the simulations successfully predicted the mixing progress as a function of the degree of filling and size of the drum. It is shown that only relatively large, straight baffles perpendicular to the drum wall (67% of the drum radius) increase the mixing performance of the rotating drum. Considering the different aspects of mixing dealt with in this work, it is concluded that the soft sphere discrete particle model can serve as a valuable tool for investigating mixing of solid substrate particles. Finally, it is expected that this model may evolve into a potential tool for design and scale-up of mixed solid-state fermenters.     

Microscale Lithium Metal Stored inside Cellular Graphene Scaffold toward Advanced Metallic Lithium Anodes

The volume expansion and dendrite growth of metallic Li anode during charge/discharge processes hinder its practical application in energy storage. Seeking an appropriate host for distributing bulk Li in a 3D manner is an effective way to solve these problems. Here, a novel porous graphene scaffold with cellular chambers for incorporating Li metal is presented. Using such a unique host, ultrathin Li layers of 3 µm in thickness are anchored on graphene to form porous microstructures, which provides much more reaction sites for Li ions compared with that of bulk Li, significantly promoting the reversibility of Li stripping and plating. Also the high current density can be effectively dissipated by the graphene scaffold to remarkably improve the rate capability of Li anode. The symmetrical Li cell using such a Li anode can run stably for 200 cycles at 5 mA cm^?2 and even 70 cycles at 10 mA cm^?2 in an unmodified carbonate‐based electrolyte, which has rarely been achieved in such aggressive working conditions. Lithium‐ion capacitor cells using this anode also show outstanding rate capability and cycling stability, which can work at an ultrahigh current density of 30 A g^?1 and keep steady for over 4000 cycles at 3.75 A g^?1.     

Modeling the Residence Time Distribution of Integrated Continuous Bioprocesses

In response to the biopharmaceutical industry advancing from traditional batch operation to continuous operation, the Food and Drug Administration (FDA) has published a draft for continuous integrated biomanufacturing. This draft outlines the most important rules for establishing continuous integration. One of these rules is a thorough understanding of mass flows in the process. A computer simulation framework is developed for modeling the residence time distribution (RTD) of integrated continuous downstream processes based on a unit‐by‐unit modeling approach in which unit operations are simulated one‐by‐one across the entire processing time, and then combined into an integrated RTD model. The framework allows for easy addition or replacement of new unit operations, as well as quick adjustment of process parameters during evaluation of the RTD model. With this RTD model, the start‐up phase to reach steady state can be accelerated, the effects of process disturbances at any stage of the process can be calculated, and virtual tracking of a section of the inlet material throughout the process is possible. A hypothetical biomanufacturing process for an antibody was chosen for showcasing the RTD modeling approach.