Optimization and scale‐up of oligonucleotide synthesis in packed bed reactors using computational fluid dynamics modeling

A computational fluid dynamics (CFD) model for the analysis of oligonucleotide synthesis in packed bed reactors was developed and used to optimize the scale up of the process. The model includes reaction kinetics data obtained under well defined conditions comparable to the situation in the packed bed. The model was validated in terms of flow conditions and reaction kinetics by comparison with experimental data. Experimental validation and the following model parameter studies by simulation were performed on the basis of a column with 0.3 g oligonucleotide capacity. The scale‐up studies based on CFD modelling were calculated on a 440 g scale (oligonucleotide capacity). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1048–1056, 2014     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

Efficient purification of antiproliferative polysaccharides from Hypsizigus marmoreus with radial flow chromatography

The increasing commercial significance of natural polysaccharides for use in medicinal products is stimulating the development of efficient and easy scale‐up techniques for polysaccharide purification. In this research, the crude polysaccharides from submerged cultivation broth of Hypsizigus marmoreus were purified using radial flow chromatography (RFC), and the antiproliferative activity of the purified fractions was evaluated in vitro. DEAE Sepharose CL‐6B was selected to be packed in the RFC column based on its good resolution, physical stability, and low cost. Compared with axial flow chromatography (AFC), an efficient chromatographic process with significantly less time and buffer consumption but yielding higher polysaccharide recovery and resolution was established in RFC, which could clearly purify the crude polysaccharides into different fractions. An acceptable linear scale‐up effect of RFC from 100 to 500 mL was successfully achieved without loss of resolution and enhancement of time consumption. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays in cell cultures indicated that the purified polysaccharide fractions possess moderate antiproliferative activities in three different human cancer cell lines, but have significantly lower cytotoxicity in normal human cell lines in vitro. Among the polysaccharide fractions, the main purified acidic fraction W‐I could be considered as a novel potential antitumor agent candidate for several tumors, especially for human alveolar epithelial tumors. This research confirmed for the first time that RFC would be a new fast and efficient tool for purification of polysaccharides into different fractions, both at laboratory and commercial scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:872–878, 2014     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014     

Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling

Preparative packed‐bed chromatography using polymer‐based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history‐dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid–particle interactions for the first time. A three‐dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in‐silico and in laboratory experiments. A pronounced axial compression–relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force‐chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure‐flow dependency. Furthermore, the particle Young's modulus and particle–wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363–371, 2016     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Effects of emulsion viscosity during surfactant‐enhanced soil flushing in porous media

Surfactants can potentially improve the efficiency of pump‐and‐treat technology for remediation of aquifers contaminated by nonaqueous phase liquids (NAPLs). However, the formation of emulsions during the removal process can Increase the viscosity in the system. This can result in pore clogging and reduction of flow, which inhibits the contaminant removal process. Formation of viscous emulsions has been identified in previous research as one of the probable causes for in situ field test failures using surfactant‐enhanced soil‐flushing technology. However, the effects of in situ emulsification and viscosity increases have not been quantified previously. The purpose of this article is to investigate effects of in situ emulsification on the remediation process. Laboratory column studies examined the mobilization of m‐xylene from porous media using a 1% alcohol ethoxylate surfactant solution (Witconol® SN90). Effects of in situ emulsification were determined. Glass columns (1.1 cm i.d. × 30 cm) were packed with 0.2‐mm glass beads to model soil media. Viscosities of emulsion solutions prepared with 1 % SN90 and various concentrations of m‐xylene were measured and compared with effluent collected during column‐flushing experiments. It was determined that as m‐xylene concentration in the emulsion solution Increases, viscosity increases. Viscosity increases caused a decrease in relative permeability within the soil column. As a result, the hydraulic gradient required to maintain a constant flowrate of 1.1 ml/min (using a syringe pump) through the soil column increased. Results show that a relatively small increase in viscosity could have a noticeable effect on the mobilization process. It is suggested that the surfactant/contaminant systems be screened to determine emulsion theology and the potential effects on the remediation process. The use of low‐concentration alcohol cosurfactants to reduce system viscosity was evaluated and was shown to be ineffective.     

Roles of interstitial fraction and load conditions on the dynamic binding capacity of proteins on capillary‐channeled polymer fiber columns

Capillary‐channeled polymer (C‐CP) fibers are used as a stationary phase for ion‐exchange chromatography of proteins. Collinear packing of the fibers permits operation at high linear velocities (U_o > 100 mm s^?1) and low backpressure (<2,000 psi) on analytical‐scale columns. Rapid solvent transport is matched with very efficient solute mass transfer as fibers are virtually non‐porous with respect to the size of the target protein molecules. Lack of porosity of course limits the equilibrium binding capacity of stationary phases. Breakthrough curves and frontal analysis are used to better understand trade‐offs between the kinetic and thermodynamic properties as C‐CP fibers are applied in preparative situations. Fiber columns packed to different interstitial fraction values affect both the total fiber surface area (e.g., equilibrium binding capacity [EBC]) and the permittivity to flow and mass transport characteristics (e.g., dynamic binding capacity [DBC]). The EBC of the nylon 6 C‐CP fibers was found to be 1.30 mg g^?1, with isotherms that were best matched by a Moreau model, showing linearity up to solute concentrations of ～0.4 mg mL^?1. Isotherms generated under flow conditions were equally well approximated using Langmuir, Freundlich, and Moreau isotherm models. Fairly linear responses were seen up to the maximum load concentration of 1.2 mg mL^?1. Counterintuitively, dynamic studies revealed that conditions of high column porosity yielded a DBC that is ～70% higher than the EBC. These findings point to potential advantages in terms downstream processing applications, where protein throughput and yield are critical metrics. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:97–109, 2015     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination

A 1.5 μL ion exchange chromatography column to accommodate resins used for biopharmaceutical processing has been designed to produce breakthrough curves and to quantify dynamic and maximum protein binding capacities. Channels within a glass chip were fabricated using photolithography and isotropic etching. The design includes a 1 cm long microfluidic column in which compressible, polydispersed porous agarose beads (70 μm mean diameter) were packed using a keystone method where particles aggregate in a narrow channel. The depth of the column is such that two bead layers exist. The fabrication technique used forms Cartesian geometries as opposed to circular cross sections found in standard columns. The voidage was therefore higher than standard values when measured by 3D confocal microscopy. In conjunction with microscopic techniques, the column allows visualization of events within the bed such as adsorption profiles that would otherwise be difficult to observe. In this work, the binding of fluorescently labeled protein during isocratic loading was used to generate breakthrough from the microcolumn. Useful breakthrough curves were achieved using mobile phase velocities from 60 to 270 cm h^?1. Calculated dynamic binding capacities were compared well with previously published data on conventional scale columns. The microfluidic chromatography column described here thus allows study of process scale chromatography behavior at scales 20,000 times smaller than in current practice. The work described in this article is representative of the proof of principle of a potentially powerful tool for the generation of microfluidic process bed data for the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Continuous bind‐and‐elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938–948, 2016     

Fine‐scale spatial distribution of murundus structures in the semi‐arid region of Brazil

Murundus are earth mounds widespread in most landscapes in the semi‐arid region of Brazil. Evidence obtained from predictive modelling has suggested a termite origin for these structures, opening up new opportunities for further research. Distribution of densely packed murundus at larger spatial scales is most related to climatic regime and soil nutrient availability. However, factors and processes underlying their distribution and density at smaller spatial scales are not yet fully understood. In this study, we adopted an approach based on mapping point data using high‐resolution satellite imagery, multi‐scale second‐order analysis and general linear models to examine the fine‐scale spatial distribution and density of murundus. Our results suggest that the distribution of those structures within densely packed areas is regulated by more than one process acting or interacting across multiple spatial scales. All densely packed murundus showed a significant regular distribution at the distance scale of up to 50 m radially and a completely random distribution across all other upper distance scales. We interpret the regular pattern as a result of competition for foraging territories between different termite colonies during the process formation of densely packed murundus. The random pattern at larger distance scales (above 50 m radially) can be attributed to habitat selection preferences by termite species builders of murundus mediated by local environmental resources and conditions (i.e. availability of food resources and nesting and open habitat), which would be randomly distributed in space. Thus, at finer spatial scales murundus distributions are associated with biotic interactions acting on an abiotic template. On the basis of significant linear correlations, we suggest that the density of murundus is strongly related to local temperature regime with soil‐type influencing its effect on the murundus densities. Our findings provide novel evidences that mound‐building termites are involved in the formation of murundus in the semi‐arid region of Brazil.     

Instrumentation for advanced microseparations in pharmaceutical analysis and proteomics

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.     

Improved selective ion monitoring mass-spectrometric assay for the determination of n,n-dimethyltryptamine in human blood utilizing capillary column gas chromatography

A gas—liquid chromatographic—mass spectrometric procedure is described for the assay of dimethyltryptamine (DMT) in whole blood. The use of a glass capillary column in combination with selective ion monitoring results in an assay with a high degree of specificity and sensitivity. 5-Methoxy-DMT is used as an internal standard and carrier in the isolation procedure. The superior resolving characteristics of the capillary column (as compared to previously employed packed columns) allows monitoring of the intense m/e 58 ion arising from the DMT side-chain. A sensitivity limit of 10 pg/ml blood is realized with a 10-ml blood sample.     

Drag of suction cup tags on swimming animals: Modeling and measurement

Bio‐logging tags are widely used to study the behavior and movements of marine mammals with the tacit assumption of little impact to the animal. However, tags on fast‐swimming animals generate substantial hydrodynamic forces potentially affecting behavior and energetics adversely, or promoting early removal of the tag. In this work, hydrodynamic loading of three novel tag housing designs are compared over a range of swimming speeds using computational fluid dynamics (CFD). Results from CFD simulation were verified using tag models in a water flume with close agreement. Drag forces were reduced by minimizing geometric disruptions to the flow around the housing, while lift forces were reduced by minimizing the frontal cross‐sectional area of the housing and holding the tag close to the attachment surface. Hydrodynamic tag design resulted in an experimentally measured 60% drag force reduction in 5.6 m/s flow. For all housing designs, off‐axis flow increased the magnitude of the force on the tag. Experimental work with a common dolphin (Delphinus delphis) cadaver indicates that the suction cups used to attach the types of tags described here provide sufficient attachment force to resist failure to predicted forces at swimming speeds of up to 10 m/s.     

Sorption and desorption of arsenic from sandy soils: Column studies

Rate‐limited sorption/desorption can have a profound effect upon the transport of sorbing contaminants. Numerical and analytical models used to predict chemical movement through the subsurface rarely incorporate the effects of nonlinear sorption and desorption kinetics, resulting in potentially large overestimates of mass extractability. Mass transfer characteristics of arsenic‐contaminated soils at the site of a former arsenical herbicide manufacturer in Houston, Texas, were examined in the laboratory using soil columns. Unaffected soils comprised of silty sands to coarse sands were collected from the uppermost aquifer. Two soil columns were loaded with a known mass of mixed organic and inorganic forms of arsenic resident in site ground water. A third control column was prepared with dry 20 × 30 mesh ASTM silica sand. Leachate samples were collected from each void volume until arsenic breakthrough was achieved. The dynamic test applied a continuing head of water, operating in an upflow mode through 4‐in. diameter by 12‐in. long soil columns repacked to in situ density. A flow‐through velocity of one void volume per day was chosen for arsenic loading to the columns and 0.08 void volume per day during the desorption phase of the test. Uncontaminated ground water was then passed through the columns, and the tests were restarted in the desorption mode. Analysis of the leachate and resulting arsenic concentrations in the test columns allowed for the calculation of distribution coefficients that describe arsenic behavior. Measured distribution coefficients during desorption ranged from 0.26 after one void volume to 3.3 after six void volumes had been passed through the column.     

EBA columns with a distribution system based on local stirring

A new type of liquid distribution system for expanded bed columns has been developed. The construction differs from traditional distribution designs by not having any small pores (like filters or distribution plates) in the flow path of the crude feedstock. A stirrer at the bottom of the column distributes the incoming feedstock. Due to the stirring, jet streams are prevented and a stable expanded bed is formed above a mixed zone. This article describes the new column design, and investigates the performance of the stirred distribution concept experimentally by measuring theoretical plate number and breakthrough profile. Furthermore, the possibilities of scaling up the concept will be discussed based on theoretical plate measurements.     

Effect of different operating modes and biomass concentrations on the recovery of recombinant hepatitis B core antigen from thermal-treated unclarified Escherichia coli feedstock

Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper.