Felix Trampler

Ultrasonic separation of suspended particles

The forces on suspended particles in acoustic fields are reviewed briefly and the theoretical modelling of ultrasonic separators based on piezoelectrically excited layered resonators is described. Two flow-through resonator chamber concepts for ultrasonic particle (bio-cell) separation are investigated: (a) the coagulation or sedimentation approach, (b) the so-called h-shaped ultrasonic separator. The h-shaped ultrasonic separator is analysed by combining for the first time the mathematical modelling of the laminar flow with the acoustic force based velocity field of the particles relative to the suspension medium. This allows a complete modelling of the resonators particle separation performance. Examples for separation chamber designs optimized by use of the mathematical model are presented and the calculated particle traces in the h-resonator are shown and compared with experimental results. For direct comparison of different ultrasonic flow through separator concepts a separation performance figure is introduced and its value is given for the two investigated separator concepts for the sample suspensions of polystyrene spheres, yeast and spirulina cells in (salt) water. The presented results are of importance for the state of the art design of acoustic cell filters for perfusion type bioreactors, as recently launched at the biotechnology market, as well as for the ultrasonic separation of plant (algae) cells under low gravity conditions, where the sedimentation concept fails.

Viscosity sensor utilizing a piezoelectric thickness shear sandwich resonator

This paper describes a novel quartz crystal sensor for measurement of the density-viscosity product of Newtonian liquids. The sensor element consists of two piano-convex AT-cut quartz crystals vibrating in a thickness-shear mode with the liquid sample in between. This special set-up allows suppression of disturbing resonances in the liquid layer. Such resonances are generated in the common single-plate arrangements due to compressional waves caused by spurious out-of-plane displacements of the shear vibrating finite plate. The primary measurands of the sensor are the fundamental resonance frequency and the associated resonance Q-value, which are influenced by the viscously entrained liquid contacting the quartz surface. The sensor allows the measurement of samples with viscosities from almost zero (air!) up to 200 cP with a sample volume of 130 /spl mu/l.

Viscosity sensor based upon an angular momentum compensated piezoelectric thickness shear sandwich resonator

The paper describes a novel quartz crystal sensor for measurement of the density-viscosity product of Newtonian liquids. The sensor element consists of two piano-convex AT-cut quartz crystals vibrating in a thickness-shear mode with the liquid sample in between. This special set-up allows suppression of disturbing resonances in the liquid layer. Such resonances are generated in the common single-plate arrangements due to compressional waves caused by spurious out-of-plane displacements of the shear vibrating finite plate. The primary measurands of the sensor are the fundamental resonance frequency and the associated resonance Q-value, which are influenced by the viscously entrained liquid contacting the quartz surface. The sensor allows the measurement of samples with viscosities from almost zero (air!) up to 200 cP with a sample volume of 130 /spl mu/l.

Comparison of Fluidized Bed and Ultrasonic Cell-Retention Systems for High Cell Density Mammalian Cell Culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch‐Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well‐suited for stable, long‐term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.

Particle trajectories in a drifting resonance field separation device

Drifting resonance field (DRF), a new method for the separation of particle/fluid suspensions is introduced. Unlike particle filters using ultrasound‐induced coagulation, the DRF method only utilizes the primary acoustic radiation force to achieve the separation effect. This is done by cyclically switching the driving frequency of the DRF resonator between consecutive resonances, causing a directed motion of the particles and thus concentrating the particles in a certain region of the separation volume. Trajectories of polystyrene spheres (approximately 12 microns in diameter) suspended in distilled water have been measured in a prototype DRF resonator, varying the number of the utilized resonance frequencies and the acoustic power input. The measured particle trajectories are compared to computer simulations based on the same process parameters as used in the experimental setup, showing a high correspondence between experimental and simulated data. Both experiments and simulations prove the high potentia...

Three layer thickness extensional mode piezoelectric resonator for determining density and sound velocity of liquids

The principle of calculating two unknown physical parameters of a layered resonator out of two quasiharmonic series resonance frequencies is applied to the determination of density and sound velocity of liquids. The resonance frequencies are measured using a three layer sensor vibrating in thickness extensional resonance modes. The three layers are formed by two piezoelectric plates and the liquid sample in between. Knowing the behaviour of the two piezoelectric single resonators from measurements which have to be done first, we have calculated the sound velocity and the mass density of the liquid between these two plates out of two series resonance frequencies of the composite resonator.

Piezoelectric resonant sensor for sound velocity of liquids

The sensor consists of two circular piezoelectric plates with the liquid in between. Each piezoelectric plate is a bulk resonator with two electrodes driven in thickness extensional modes. The two resonator plates are electrically connected in parallel and the total admittance is measured in the vicinity of two quasiharmonic series resonance frequencies by a PC‐controlled electric admittance measurement system. The extensional thickness mode purity was checked directly by corresponding mode pattern measurements using a laser speckle vibration amplitude measurement system. The sensor element is mounted in a thermostated aluminum housing to avoid temperature effects and to obtain the sound velocity of the liquid at a defined temperature. Two quasiharmonic resonance frequencies are used to allow the elimination of the unknown density of the liquid. The sound velocity is calculated out of a rigorous one‐dimensional theoretical model of the three‐layer sensor arrangement. In addition to the determination of the sound velocity of the liquid, the mass density of the liquid can be obtained from the same primary measurements. However, at present the accuracy of the density value is one order of magnitude lower than that of the velocity (typically 0.1% for water, acetone, glycerin).

Aggregation and Separation of Hybridoma Cells by Ultrasonic Resonance Fields - Eifect on Viability, Growth Rate and Productivity.

An acoustic resonator was used to generate an ultrasonic standing wave field within a suspension of hybridoma cells. Acoustically induced aggregation and increased cell sedimentation rates were observed. Exposure of cells to ultrasonic resonance fields did not influence viability, growth rate, glucose consumption and antibody productivity. This paper demonstrates the potential of utilizing acoustic resonance separation methods in bioprocessing applications. Parameters investigated include time of ultrasound exposure, power level and cell concentration.

Frequency Dependence of the Acoustic Loss Distribution in Piezoceramic/Liquid Resonators

During the past decade, several separation methodes based on the response of fine particles to ultrasonic standing wave fields have been developed. The forces acting on the particles depend beside other quantities on the energy density and therefore on the stored energy in the liquid. Since separation cells are mainly constructed as layered piezoelectric resonators, a model is presented, which can describe the frequency dependence of the quality factor of a composite piezoelectric resonator. In addition, using this model, the stored (elastic) energy in each of the layers related to the electric input power can be calculated.

Continuous Perfusion versus Discontinuous Fed-Batch

The expression of recombinant glycoproteins that bear a cytotoxic potential is a major challenge using animal cells. Product yields in a range of 0.1 g/l to 1.0g/l as commonly reached for proteins that do not adversely affect the cultured cells are out of reach. The permanent expression of a cytotoxic protein at low specific productivities requires a bioprocess that allows the accumulation of product at concentrations adequate for purification. We compared a continuous perfusion process based on an ultrasonic cell retention device with a discontinuous fed-batch using a balanced nutrient concentrate. Theproduct yield in batch mode was set to a relative concentration of 1.0. In fed-batch the product yield was increased by 40% to a relative value of 1.4 accompanied by a significant loss of culture viability indicating the detrimental effect. However, under perfused conditions not only the volumetric productivity was increased as expected, but also the relative product concentration reached a valueof 6.0 at viabilities of more than 90%. Furthermore the product was harvested almost cell free at perfusion rates of 2d−1. Combining the perfusion mode (cell accumulation) and the fed-batch mode (product accumulation) the product yield was significandy increased toa relative value of 10.0 at final viabilities of still 65%. We thus developed a process capable of accumulating a cytotoxic protein in concentrations sufficient for downstream processing requirements.