Performance prediction of industrial centrifuges using scale-down models

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.     

The scale-down of an industrial disc stack centrifuge

The means are described whereby a disc stack centrifuge may be scaled-down by up to 10-fold of its separation capacity. The centrifuge separation characteristics so measured are suitable for direct scale-up predictions of centrifuge performance where only small volumes of particle suspension are available for study. Such an ability to scale-down is especially important in the processing of biological particles where for example, in the early stage of process development, there is often insufficient fermentation broth for fullscale studies. Scale-down is achieved by the reduction of the number of discs available for separation purposes and by the careful positioning of these discs in the overall disc stack. A combination of dye tracer and particle separation studies are used to optimise the disc stack configuration. The resulting grade efficiency curve is an accurate reflection of the curve for the full-scale centrifuge especially in the critical design region specifying centrifuge throughput for near complete particle recovery.     

Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.     

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.     

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.     

Assessment of a disc stack centrifuge for use in mammalian cell separation

A prototype disc stack centrifuge was tested for the separation of mammalian cell cultures from 80- and 2000-L fermentations. The clarification capacity for mammalian cells was excellent, but some smaller particles remained in the supernatant and reduced its usefulness for downstream processing. In order to identify the source of such particle formation, several parameters were assessed and minimum particle size for separation was calculated. An analysis of particle distribution was performed. Temperature and pressure effects inside the centrifuge bowl were measured. Some modifications of mechanical engineering can be suggested for the improvement of the use of standard disc stack centrifuges for mammalian cells. (c) 1995 John Wiley & Sons, Inc.     

Prediction and verification of centrifugal dewatering of P. pastoris fermentation cultures using an ultra scale-down approach

Recent years have seen a dramatic rise in fermentation broth cell densities and a shift to extracellular product expression in microbial cells. As a result, dewatering characteristics during cell separation is of importance, as any liquor trapped in the sediment results in loss of product, and thus a decrease in product recovery. In this study, an ultra scale-down (USD) approach was developed to enable the rapid assessment of dewatering performance of pilot-scale centrifuges with intermittent solids discharge. The results were then verified at scale for two types of pilot-scale centrifuges: a tubular bowl equipment and a disk-stack centrifuge. Initial experiments showed that employing a laboratory-scale centrifugal mimic based on using a comparable feed concentration to that of the pilot-scale centrifuge, does not successfully predict the dewatering performance at scale (P-value <0.05). However, successful prediction of dewatering levels was achieved using the USD method (P-value ≥0.05), based on using a feed concentration at small-scale that mimicked the same height of solids as that in the pilot-scale centrifuge. Initial experiments used Baker's yeast feed suspensions followed by fresh Pichia pastoris fermentation cultures. This work presents a simple and novel USD approach to predict dewatering levels in two types of pilot-scale centrifuges using small quantities of feedstock (<50 mL). It is a useful tool to determine optimal conditions under which the pilot-scale centrifuge needs to be operated, reducing the need for repeated pilot-scale runs during early stages of process development.     

Dual salt precipitation for the recovery of a recombinant protein from Escherichia coli

When considering worldwide demand for biopharmaceuticals, it becomes necessary to consider alternative process strategies to improve the economics of manufacturing such molecules. To address this issue, the current study investigates precipitation to selectively isolate the product or remove contaminants and thus assist the initial purification of a intracellular protein. The hypothesis tested was that the combination of two or more precipitating agents will alter the solubility profile of the product through synergistic or antagonistic effects. This principle was investigated through several combinations of ammonium sulfate and sodium citrate at different ratios. A synergistic effect mediated by a known electrostatic interaction of citrate ions with Fab' in addition to the typical salting-out effects was observed. On the basis of the results of the solubility studies, a two step primary recovery route was investigated. In the first step termed conditioning, post-homogenization and before clarification, addition of 0.8 M ammonium sulfate extracted 30% additional product. Clarification performance measured using a scale-down disc stack centrifugation mimic determined a four-fold reduction in centrifuge size requirements. Dual salt precipitation in the second step resulted in >98% recovery of Fab' while removing 36% of the contaminant proteins simultaneously.     

Ultra scale-down approach for the prediction of full-scale recovery of ovine polycolonal immunoglobulins used in the manufacture of snake venom-specific Fab fragment

In this article, we describe a new approach that allows the prediction of the performance of a large-scale integrated process for the primary recovery of a therapeutic antibody from an analysis of the individual unit operations and their interactions in an ultra scale-down mimic of the process. The recovery process consisted of four distinct unit operations. Using the new approach we defined the important engineering parameters in each operation that impacted the overall recovery process and in each case verified its effect by a combination of modelling and experimentation. Immunoglobulins were precipitated from large volumes of dilute blood plasma and the precipitated flocs were recovered by centrifugal separation from the liquor containing contaminating proteins, including albumin. The fluid mechanical forces acting on the precipitate and the time of exposure to these forces were used to define a time-integrated fluid stress. This was used as a scaling factor to predict the properties of the precipitated flocs at large scale. In the case of centrifugation, the performance of a full-scale disc stack centrifuge was predicted. This was achieved from a computational fluid dynamics (CFD) analysis of the flow field in the centrifuge coupled with experimental data obtained from the precipitated immunoglobulin flocs using the scale-down precipitation tank, a rotating shear device, and a standard swing-out rotor centrifuge operating under defined conditions. In this way, the performance of the individual unit operations, and their linkage, was successfully analysed from a combination of modelling and experiments. These experiments required only millilitre quantities of the process material. The overall performance of the large-scale process was predicted by tracking the changes in physical and biological properties of the key components in the system, including the size distribution of the antibody precipitates and antibody activity through the individual unit operations in the ultra scale-down process flowsheet.     

Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system

Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge.     

Structural Bionic Design and Experimental Verification of a Machine Tool Column

Based on the configuration principles of biological skeletons and sandwich stems, a machine tool column with stiffening ribs inside was designed using structural bionic method. After the lightening effect was verified by finite element simulation, scale-down models of a conventional column and a bionic column were fabricated and tested. Results indicate that the bionic column can reduce the maximum static displacement by 45.9% with 6.13% mass reduction and its dynamic performances is also better with increases in the first two natural frequencies. The structural bionic design is effective in improving the static and dynamic structural performances of high speed machine tools.     

The biocontainment of a high speed disc bowl centrifuge

The containment of a high speed disc bowl centrifuge during normal operation has been examined using a mutant E. coli strain and a range of sampling devices to monitor the release of viable organisms. A discharge of a small amount of supernatant was used to provide a mimic of a release of a low number of organisms with a view to testing the sensitivity of the sampling systems used. Three sampling devices were used: settle plates, a slit sampler and an air filter sampler. These were all shown to be effective in the collection of viable organisms during the release under low pressure of 10 ml of supernatant (equivalent microbial count to 0.02 ml fermentation broth). Three runs carried out under normal operation of the centrifuge showed no release of viable organisms. The prevention of a second source of release during disassembly and cleaning of the bowl was demonstrated to be possible by the ability to clean and steam sterilize in place. The consequences of such operations are discussed in terms of the use of high speed disc bowl centrifuges for the processing of organisms under various levels of containment.     

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.     

Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.     

The effect of antiapoptosis genes on clarification performance

Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host‐cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host‐cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk‐stack centrifugation, a scale‐down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging‐bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20–50% reduction in HCP levels and 50–65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk‐stack centrifuge, the reduction in HCP was 10–15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 10⁶ W/m³ and minimal damage to the cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:100–107, 2014     

Trainable High Resolution Melt Curve Machine Learning Classifier for Large-Scale Reliable Genotyping of Sequence Variants

High resolution melt (HRM) is gaining considerable popularity as a simple and robust method for genotyping sequence variants. However, accurate genotyping of an unknown sample for which a large number of possible variants may exist will require an automated HRM curve identification method capable of comparing unknowns against a large cohort of known sequence variants. Herein, we describe a new method for automated HRM curve classification based on machine learning methods and learned tolerance for reaction condition deviations. We tested this method in silico through multiple cross-validations using curves generated from 9 different simulated experimental conditions to classify 92 known serotypes of Streptococcus pneumoniae and demonstrated over 99% accuracy with 8 training curves per serotype. In vitro verification of the algorithm was tested using sequence variants of a cancer-related gene and demonstrated 100% accuracy with 3 training curves per sequence variant. The machine learning algorithm enabled reliable, scalable, and automated HRM genotyping analysis with broad potential clinical and epidemiological applications.     

Adapted ultra scale-down approach for predicting the centrifugal separation behavior of high cell density cultures

The work presented here describes an ultra scale-down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5- to 10-fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous-flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to approximately 2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell-cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot-scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large-scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale-up, are discussed.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.     

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles

Background aimsThis article describes the development of a small-scale model for Ficoll-based cell separation as part of process development of an advanced therapy medicinal product and its qualification. Because of the complexity of biological products, their manufacturing process as well as characterization and control needs to be accurately understood. Likewise, scale-down models serve as an indispensable tool for process development, characterization, optimization and validation. This scale-down model represents a cell processor device widely used in advance therapies. This approach is inteded to optimise resources and to focus its use on process characterisation studies under the paradigm of the Quality by design. A scale-down model should reflect the large manufacturing scale. Consequently, this simplified system should offer a high degree of control over the process parameters to depict a robust model, even considering the process limitations. For this reason, a model should be developed and qualified for the intended purpose.MethodsProcess operating parameters were studied, and their resulting performance at full scale was used as a baseline to guide scale-down model development. Once the model was established, comparability runs were performed by establishing standard operating conditions with bone marrow samples. These analyses showed consistency between the bench and the large scale. Additionally, statistical analyses were employed to demonstrate equivalence.ResultsThe process performance indicators and assessed quality attributes were equivalent and fell into the acceptance ranges defined for the large-scale process.ConclusionsThis scale-down model is suitable for use in process characterization studies.     

10. Purification of Viruses by Cenlrifugation in Gradients of Inert Substances

ABSTRACT

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in “self formed” caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Centrifugation in gradients of inert substances is useful for the separation of components in a mixture¹. There are two principles involved in this type of separation. One, termed reorienting gradient centrifugation (reograd) relies on the differences in masses or, better still on the sedimentation coefficients of the different components in the mixture and the second, termed isopycnic centrifugation², on the densities or specific gravities of the different entities.