WO2001031323A1 - Multistage electrophoresis apparatus and method of use for the separation and purification of cells, particles and solutes - Google Patents

Abstract

An electrophoresis device (30) is disclosed which separates cells, particles, proteins, and other separands by collecting samples of decreasing electrophoretic mobility in a train of inverted collection cavities while an electric field is applied between said inverted cavities and one or more sample cuvettes containing a mixture of cells, particles, proteins or other separands. One circular plate (40) is provided for the one or more sample cuvettes, and one circular plate (38) is provided for the train of collection cavities. The invention utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. The purification method includes a method for dealing with electrolysis products, a technique for controlling the electrical energy input, and an approach for keeping the process isothermal. The invention solves many separation applications problems on earth and also in reduced gravity in space flight.

Description

"MULTISTAGE ELECTROPHORESIS APPARATUS

AND METHOD OF USE FOR THE SEPARATION AND PURIFICATION

OF CELLS, PARTICLES AND SOLUTES"

This application is part of a government project, Contract No. NAS9-97088.

This application claims priority from copending United

States Provisional Application Serial No. 60/162,319 filed on

October 28, 1999, and United States Provisional Application

Serial No. 60/163,667 filed on November 5, 1999 both of which are hereby incorporated by reference herein.

Field of the Invention

The invention relates to the field of combining free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, proteins, and solutes in useful quantities at high concentrations.

Description of the Prior Art

Conventional particle separation techniques typically include centrifugation, which is limited due to its specificity, capacity, speed, energy consumption, biological impact, and microgravity environment disturbances.

Electrophoresis is a leading method for resolving mixtures of cells or charged macromolecules (proteins and nucleic acids) . The electrophoretic separation of proteins without gels has been a long-standing goal of separations research. The process of electrophoresis has so far been unable to "graduate" from an analytical tool to a viable unit operation. This is primarily because of various problems such as thermal convection, electro osmosis, particle sedimentation, droplet sedimentation, particle aggregation, and electro hydrodynamic zone distortion have been found to be major obstacles to scale-up. The traditional approach has been to devise density gradients or elaborate flowing devices to counteract these problems; however incorporation of such methods has been unable to address these problems effectively. Also, their addition has caused the process to become cumbersome, thus further reducing the appeal of electrophoresis.

Without the need to prepare density gradients and/or use elaborate flowing systems, free electrophoresis can enjoy much more widespread use because it is a high-resolution method that does not require adsorption to solid media and the subsequent solids handling. It can separate both particles (cells) and solutes (macromolecules) with equal ease. Some specific applications for electrophoresis include the separation of different cells of peripheral blood and bone marrow in hematological and immunological research. Other potential applications include clinical therapeutics and the separation of proteins from body fluids, tissue extracts and fermentation broths .

A mixing problem encountered during free electrophoresis is the mixing caused by the release of gases at the electrodes. However, the use of either non-gassing electrodes such as described in (Agarwala 1994) and incorporated herein or membrane- separated electrodes such as described in (Cole et. al . 1995) and incorporated herein will effectively solve this problem. Experiments performed using palladium electrodes have demonstrated our ability to solve this problem.

Free electrophoresis is a process in which a sample is introduced into a liquid buffer, static or flowing, in a zone and subjected to an electric field in which separands migrate according to their surface charge properties. Fluid instabilities result because longer migration paths are exposed for longer times to developing instabilities.

Tulp ( ) teaches that a short electrophoretic migration path in a non-moving buffer avoids exposure of migrating separands to unstable buffer flows. Tulp designed a reorienting, free electrophoresis device consisting of a flat disk-shaped container with thin sample bands and a short vertical migration distance. The bottom and the top electrode fluids served as the coolant, the total height of the separation column was 1-2 cm, and its diameter was greater than 15 cm. The distance between the unrelated separands was 1-2 mm, and this distance was increased during ractionation after electrophoresis by re-orienting the disk so that it became a narrow vertical column.

In a different field of separations, Albertsson et al . teaches that multistage extraction processes can proceed in a multistage separator consisting of two sets of cavities facing each other around the periphery of a pair of plates. Further, conventional electrophoresis devices rely on the use of gels, paper or flowing channels to stabilize the electrophoresis buffer in which separands migrate. The devices are limited in capacity and, in the case of flowing channels, difficult for the user to operate and maintain. Obviously, prior art is not meeting the needs of separations by free electrophoresis. Further, applications of free electrophoresis in low gravity require a gravity independent means of collecting electrophoretically separated fractions of the sample.

The present invention fulfills this requirement and is hence ideal for applications in space-flight electrophoresis experiments and applications. For instance, The overall efficacy of electrophoresis as a unit operation can be greatly improved if the migration distance is greatly reduced and the process is multistaged. SUMMARY OF THE INVENTION

A thin-layer countercurrent distribution apparatus is designed and constructed so that up to 20 fractions can be collected on the basis of electrophoretic mobility by application of an electric field. The multistage electrophoretic separation and purification of cells, particles, proteins, and solutes utilize an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating and/or purifying living cells, particles and proteins in useful quantities and at high concentrations. The mixture to be separated starts in a bottom cavity, and successive top cavities, collect fractions as separand particles or molecules are electrophoresis upward out of the bottom cavity. Mathematical models of this process have been developed, and experiments performed to verify the predictions of the models by collecting and counting particles in each cavity after fractionation. The process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space.

Moreover, an electrophoresis device is disclosed which separates cells, particles, proteins and other separands by collecting samples of decreasing electrophoretic mobility in a train of inverted cavities while an electric field is applied between said inverted cavities and one or more sample cuvettes containing a mixture of cells, particles, proteins or other separands . One circular plate is provided for the one or more sample cuvettes, and one circular plate is provided for the multiple collection cavities. The invention utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. The purification method includes a method for dealing with electrolysis products, a technique for controlling the electrical energy input, and an approach for keeping the process isothermal. The invention solves many separation applications problems on earth and also in reduced gravity in space flight.

The multistage electrophoretic purification of cells, particles, and proteins, utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles and proteins in useful quantities and at high concentrations. The isothermal process depends on the electrophoretic mobility of separands, and is gravitationally stabilized so that it functions in laboratories on earth and in space. The purification method includes a method for dealing with electrolysis, a technique for transporting and varying electrical energy, and an approach for keeping the process isothermal. The electrophoretic technology resolves many unique separation applications on earth as well as in reduced gravity environments in space flight.

The instant invention is extremely well suited to immunological research, pharmaceutical delivery, biomedical applications, cell biology, and cell separation problems associated with clinical, animal, and plant research. The separation process is well suited to space flight, specifically for on-orbit cell separation problems associated with biological research. Moreover, the electrophoretic technology and electrokinetic separation employ affinity partitioning and electrophoresis. The invention incorporates both features, a short migration distance, and a multistage operation technique in order to increase the throughput of the process and to make the process easier to operate.

The multistage electrophoresis separation and purification assembly utilizes oppositely charged electrodes at the ends of two cavities providing the electric driving force for the migration of particles. It provides a thin layer countercurrent distribution apparatus capable of collecting up to 22 fractions by applying an electric field. The hardware is a combination of free electrophoretic and multistage extraction and consists of 20 or more cavities of a multistage thin layer extraction system. Half cavities oppose each other in disks that are sealed together and one disk rotates with respect to each other. The mixture to be separated starts in a single cavity on a first plate, and successive cavities collect fractions as separand particles or molecules are electrophoresed upward out of the cavity of a second plate. The half cavities are disk shaped, the top cavities having flat tops and the bottom cavities having flat bottoms. Both consist of palladium metal electrodes that produce an electric field when the two cavities re in contact. Each half cavity is only a fee millimeters in height so that the fluid within it remains isothermal during the application of an electric field that transfers separand particles or molecules from the cavity of one plate to the cavity of the corresponding plate.

As each separand is transferred to a new cavity it is swept into the upper half by the electric field or left in the lower half, depending on its electrophoretic mobility. The first fractions collected into the top cavity consist of high mobility separands while later fractions consist of lower mobility solutes or particles. The resulting fractogram corresponds linearly to an electrophoretic mobility distribution. The unit can be operated in various modes such as skimming separands from the top of a single bottom cavity without mixing, or following a true counter-current separation with or without remixing at each stage. A mathematical model, from which distributions and resolution can be derived was formulated, and its predictions tested in multistage experiments.

It is an object of the invention to provide a device for the successful electrophoretic separation of cells, particles, proteins and other separands

It is an object of the present invention to provide a temperature control system capable of controlling the temperature from -37°C to 20°C and preferably at about 4°C.

It is an object of the present invention to provide a sample collection capability of one or more independent samples.

It is an object of the present invention to provide a means for holding the magnitude of the electric field is held constant at a selected field strength by a microprocessor-controlled electric circuit,

It is an object of the present invention to provide a means of collecting different types of cells or to collect only cells or particles, only media, or both.

It is an object of the present invention to provide a capability to separate cells from culture medium for sampling.

It is an object of the present invention to provide a medium which is replenishable by means of perfusion which is programmable or active on demand.

It is an object of the present invention to provide as an option a electromagnetic stirring system.

It is an object of the present invention to provide a means of providing the apparatus in a modular cassette in order to facilitate sequential experiments.

It is an object of the present invention to provide a means for the researcher to have experiment flexibility and select solutions, temperatures, and sampling times.

It is an object of the present invention to provide a means for collecting samples of cells, particles, or medium in bags, cuvettes, syringes, or other vessels. It is an object of the present invention to provide a purification process in which a low conductivity separating buffer is used and electrode metals are selected to prevent gas bubble release and minimize or eliminate the need for active cooling.

It is an object of the present invention to provide a high conductivity separating buffer and receiving electrode metals are selected to prevent gas bubble release, and the one feed cavity electrode is separated from the feed sample cavity by an ultrafiltration membrane and contains an electrolyte that is pumped therethrough.

It is an object of the present invention to provide a multistage electrophoretic separation and purification system designed to be utilized in a cassette integrated within an space flight processing facility.

These and other objects of the present invention will be more fully understood from the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be had upon reference to the following description in conjunction with the accompanying drawings in which like numerals refer to like parts throughout the several views and wherein:

Figure 1 is a flow diagram showing a single stage of the multistage electrophoretic process where μ is the electrophoretic mobility of a particle, E is the electric field strength, and t is time;

Figure 2 is a perspective view of a multistage electrophoresis device; Figure 3 is an exploded view illustrating the components of the device shown in Figure. 2 ;

Figure 4 is a perspective view of the rotating mechanisms of the device shown in Figure 2;

Figure 5 is a perspective view of the rotating sample- collection plate of the device shown in Figure 2 ;

Figure 6 is a perspective view of the stationary sample-feed plate of the device shown in Figure 2 ;

Figure 7 is an elevation view of a pair of cavities in which electrophoresis takes place and a stationary electrode chamber for flowing electrolyte;

Figure 8 is a diagram of the flow path for electrolyte fluid for the stationary electrodes in a high-current embodiment of the device shown in Figure 2 ;

Figure 9 is a circuit diagram of a field-regulation circuit for maintaining constant field across the pair of cavities shown in Figure 7 ;

Figure 10 is an exploded view, in perspective, of an embodiment of the device of Figure 2 adapted for functioning in space flight showing the electrophoretic device nested within a cassette assembly which is inserted within a containment enclosure;

Figure 11 is a graph showing an experimental result of the number of particles extracted at constant potential as a function of the collecting cavity number with the temperature rise in degrees C and versus the time of field application in seconds depicting heating curves for a single cavity due to application of field strength to a cavity containing 0.01 M phosphate buffer, Squares, 5 V/cm calculated from Eq. (12) triangles, 10 V/cm calculated from Eq. (12) , shown in diamonds, measurement with a thermistor in the cavity during application of a 5 V/cm field;

Figure 12 is a graph showing the pseudoequilibrium concentration of a separand vs. its concentration in the feed fluid, with the path to purity at pseudoequilibrium stepped off at each stage, beginning in the upper right, wherein the results of a preliminary single particle migration experiment in a test of the constant-potential model for comparison with Figure 20, and the top graph, 5 V/cm, the lower graph, 10 V/cm in 0.01 M phosphate buffer, Ph8.0 , ordinate units, 10⁴ particles/mL;

Figure 13 is a graph showing the rise of temperature during application of a current to a pair of cavities based on adiabatic theory and experiment .

Figure 14 shows a sectional side view of the top plate 38 and bottom plate 40 and cavities,-

Figure 15 shows a sectional view of the top cavity 52 and bottom cavity 54 of the plate of Figure 14;

Figure 16 is a sectional view of a portion of the top plate and the bottom plate showing the details of the cavity and fill ports;

Figure 17 is a top view of the device showing the pumps, feed reservoir, and circulation system for circulating coolant electrolyte to one or more selected cavities in the top stationary plate removing gas and heat, helping to maintain an isothermal process;

Figure 18 is a side view showing the slip ring assembly for transferring electric current to the plates;

Figure 19 is a top view of a slip ring wiper for communicating with a rotating copper contact; Figure 20 shows a schematic representation of the migration of particles of a single type from the original single stationary cavity to a series of rotary cavities during the application of a fixed electric field for a fixed period of time per stage (constant Ef) , representing the constant-field operating model;

Figure 21 is illustrates equilibrium lines for the separation of two separands having the indicated mobility ratios using the pseudoequilibrium model, Y, particle fraction in the extract, X, particle fraction in the feed, muB/muA, mobility ratio,- and

Figure 22 is a graph of the Number of Stages v. the Mobility Ratio representing the purity resulting from application of the pseudoequilibrium model to the separation of two particle types with the given mobility ratios.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The multistage electrophoresis apparatus and method of use for the separation and purification of cells, particles, and solutes is generally designated 30 in the drawings. The individual components of the device 30 will first be identified and the operating parameters and methods of use will be described thereafter.

The multistage electrophoresis device is generally designated 30 in FIGS 2, 3 and 10 of the drawings. The individual components of the device 30 will first be identified and the operation of, and fluid flow through device 30 will then be described. Multistage electrophoresis device 30 includes a base 19 with a support framework 17 extending upwardly therefrom. Top plate 21 is attached to support framework 17 opposite base 19. A top stationary sample plate 38 is attached to top plate 21. Said stationary top plate is manufactured of a nonconducting polymer and has four cavities 52 for the containment of feed samples. An electrolyte compartment 46 is fastened above each of four cavities 52 for the containment of conducting solution 62, which conducting solution is also known as electrolyte. Each electrolyte compartment 46 is separated from its respective sample cuvettes 52 by a hydrophilic polymeric membrane 60, said membrane having a molecular-weight cutoff of 1,000. Each electrolyte compartment 46 contains a circular noble- metal electrode and is perforated on two sides by tubing connectors that carry flowing electrolyte 62 from electrolyte reservoir 76 via polymeric tubing 8 to all four electrolyte compartments 52 connected in series to electrolyte reservoir 76, said electrolyte reservoir containing hydrophilic polymeric membrane 74 which cleans the circulating electrolyte 62. Electrolyte also passes through a vapor release having hydrophilic membrane filter 71 through which gas bubbles are released via opening 72 which is open to the ambient environment. Upper sample plate 38 is held in downward compression by Belleville spring action applied by pressure ring 27 via the tension on machine screws 14 which hold down cover plate 21 such that fluid leakage between upper sample plate 38 and lower sample plate 40 is prevented. Upper sample plate 38 is prevented from rotating by four pins 22, said pins penetrating cover plate 38, pressure ring 27 and upper sample plate 38. Lower sample plate 40 is attached by four machine screws 16 to rotating gear wheel 34 and caused to rotate with said gear wheel by four pins 22, said gear wheel being caused to rotate by worm-drive motor 23 when commanded to do so by an incoming computer-generated signal . Lower sample plate 40 contains 22 more-or-less sample collection cavities 52, each said sample cavity having a fill port 56 sealed by a fill-port plug 42, a noble-metal electrode 50, and a conducting wire which penetrates hole 58 to carry current to said noble-metal electrode. Support framework 17 separates the above- described upper plate assembly from the above-describe lower plate assembly. The current between noble-metal electrodes 50 in the upper and lower cavities 52 and 54 through electrophoresis buffer 52 is controlled by a control circuit shown in FIG 9 of the drawings such that the electric field between said electrodes, in volts per meter, is held constant at a value chosen by the operator. Said electric field is held between positive 96 and negative 98 amplifiers, which receive an analogue signal between 0 and 5 volts from microcomputer 92 (in this embodiment, Siemens C505 digital micro controller) via digital- to-analogue converter 94. The current passing through load resistor 91 governs the signal to microcomputer 92 via analogue- to-digital converter 90.

PROCESS AND METHOD OF USE

The multistage electrophoresis device 30 must be capable of physically separating the sample at the conclusion of an experiment so that the sample may be analyzed when it is returned to the experimenters. This is accomplished by using the pair of plates or disks, defining a top stationary sample plate 38 and a lower rotating sample plate 40) , formed from polycarbonate or other similar nonconducting 'material, that have spaced apart aligned cavities or voids formed therein for containing fluid samples cut into them.. The plates 38, 40 could be formed or other materials preferably polymers. As shown, the cavities 52 form compartments, chambers or cuvettes in the top stationary sample plate 38. Movable cavities 54 form compartments in the rotary bottom plate 40 which contain the sample to be separated and are alignable with the cavities 52 in the stationary top plate 38. Figures 14 shows a side view of the top plate 38 and bottom plate 40, Figure 15 shows a sectional view of the top cavity 52 and bottom cavity 54 of the plate of Figure 14, Figure 16 shows the details of the cavity and fill ports.

Although the cavities shown in the preferred embodiment are cylindrical in shape it is contemplated that they could be shaped having an elliptical, rectangular or pie shape as well depending upon the particular application. The plates 38 and 40 are cooperatively engaged and are able to both maintain a seal during rotation.. A sealant such as a silicon grease of other nonvolatile and inert grease may be utilized therebetween at selected points, but is not required..

The process by which the cavities 52 align is illustrated in Figurel which shows a single stage of the multistage electrophoretic process. In the embodiment shown, initially, movable cavity 54 contains the sample mixture to be separated. It is then brought into contact with buffer- containing stationary cavity 52, and an electric field is applied between electrodes 50. The magnitude of the electric field is between 1 and 10 volts per cm, and the time of its application is between 1 and 100 seconds.

Thus, at the beginning of the experiment, a single cavity

52 of the stationary plate 38 holds the sample and multiple cavities 54 of plate 40 for containing the separated fractions are aligned sequentially as follows. As shown only the cells with μ>x/Et are desired. In the separation step, the top sample cavity 52 is aligned with the bottom cavity 54, (A) , the electric field pull all cells with μ>x/Et toward the positive (+) pole.

In (B) , the transfer step is shown, whereby 100% of the cells with μ>x/Et, . 50% of all other cells are pulled into the upper compartment or cavity 52. The bottom plate 40 rotates sealing thereinbetween and aligning the next bottom plate cavity 54 in alignment with the top plate 38. The new bottom plate cavity or chamber 40 contains no cells. Separation step (C) is repeated whereby all cells with μ>x/Et remain in the top chamber

52 with some nonconforming cells. All of the separated cells in the bottom cavity 54 are rotated in the second transfer (D) whereby another bottom cavity 54 is aligned with the top plate cavity 52. When the separation step is completed, the bottom plate rotates and separates the sample into two parts for later analysis . This procedure can be repeated in stages to enhance and to establish a history of the electrophoretic process. After approximately 10 transfers about 99% or more of the μ>x/Et cells are isolated in the top sample cell 52, and approximately 0.1% or less of the nonconforming cells remain in the last bottom cell 54 of the bottom plate 40. Of course, the degree of separation and/or purification is dependent upon several variables including concentration of the cells and number of transfers; however, the number of cavities can be modified to obtain the desired results. For this prototype design, the capability to utilize up to 22 stages or more if required.

The rotating plate 40 will have at least 1 and preferably a plurality of chambers 54. In a preferred embodiment the rotating plate 40 has 22 chambers, and the stationary plate 38 which can also have an undetermined number of chambers 52 will has only 1 chamber, but typically may have four or more. Since the plate 38 with 1 chamber is exposed to the electric field during every stage, gases will accumulate at the electrode and the temperature of the fluid will rise significantly if not controlled. The plate configuration is shown in Figure 7 with a simplied view of the cross section of the plates when the chambers are aligned. An optical ring can be used with the preferred embodiment to accomplish alignment wherein a reflective ring used in combination with an optical sensor provides means for aligning the cavities 52, 54 (cuvettes) at particular selected sites so the bottom rotary plate 40 port(s) 54 corresponds to one or more selected cavities 52 in the top stationary plate 38.

The plate arrangement is set forth in Figure 7 shows a top and bottom plate, 38 and 40, respectively, with the electrolyte block and the electrical connections . The preferred electrolyte solution is a salt solution such as sodium chloride or preferably potassium chloride because they are good conductors. The electrolyte draws bubbles through the a membrane and carries them away where they are removed and float in an earth environment or are removed by a scrubbing apparatus in a space environment. The removal system may also contain filtering elements. The palladium disc or wire within each cavity also has the ability to removes hydrogen gas and prevents the collection of gases which form bubbles which interferes with the fluid flow, mass flow rate, temperature control, and quantitative measurements.

By experimentation, it was determined that when gases form at the electrodes 50, the electric field may be disrupted. A temperature change also affects the results of the separation process . To remove the gases and maintain an isothermal condition, an electrolyte coolant 100 contained within an electrode block 102 disposed in alignment with and over the cavity 53 of top stationary plate 38 providing coolant to be circulated around the electrode 50. Coolant is only needed on the electrode 50 of the top chamber (s) 52, and not the chambers 54 within the plate 49. The electrodes 50 are made of metal. Moreover, the preferred metal of composition for the electrodes 50 is Palladium which is a non-corrosive metal that has a unique property of absorbing hydrogen up to 400 times its own volume which also aids in gas removal.

The selected coolant is preferably an electrolyte solution to avoid interrupting the electric field. The maximum conductivity of the electrolyte used for this purpose is approximately 10 mS/cm. The electrolyte used could be the same as the buffer solution in which the particles are contained. A lower conductivity buffer and electrolyte could be used. In a preferred embodiment, the upper separation chamber 52 is served by a chemical electrode 50 in which the electrolyte 100 also serves as a coolant. The electrolyte flow rate is sufficient to carry away the gases and heat. This flow rate can be as low as 10 ml/hr. or even discontinued when necessary. The method of cooling provides a method of controlling the flow rate while regulating fluid pressure. Figure 17 illustrates the pump, feed reservoir, and circulation system for circulating coolant electrolyte to one or more selected cavities in the top stationary plate.

In one embodiment, each chamber is 0.275" tall and 0.375" in diameter. When the two chambers align, the sample is 0.550" tall and 0.375" diameter, having a total volume of 0.061 in³ (1- ml) . The electrodes 50 in the plate 40 with 22 chambers 54 are in direct contact with the sample solution. The single electrode 50 sits in a cavity that is 0.275" away from the sample solution. It makes contact with the circulating electrolyte. The electrolyte 50 is separated from the sample solution by a thin membrane 60, preferably a hydrophilic polymeric membrane. This membrane 60 should have negligible effects on the electric field, but will allow gases to escape from the chamber and pass into the electrolyte stream. The membrane 60 is rated up to a 3000 molecular weight cut-off.

An uniform electric field test was conducted to determine whether the electric field is affected by the presence f the membrane by observing the conductivity changes and voltage drops across the field as a function of time and bubble formation. The results indicated that there is no statistical difference in the voltages with or without the membrane in place for the first 10 minutes of the experiment and very little difference thereafter,- and that there is very little variation in the electric field from the center to the edge of the chamber.

The expected range of conductivity in the sample solution is 200 mS/cm to 10 mS/cm. The highest necessary electric field at the greatest conductivity is 5 V/cm.. Conductivity changes slightly due to gas build-up in the electrolyte, temperature, and particle separation are unavoidable, but increasing the voltage accordingly at the electrodes can minimize their impact. A voltage regulator circuit is shown in Figure 9 to compensate for the changes in conductivity. The power input requirement is 12VDC regulated at 1 amp. The maximum electrode voltage potential is limited to approximately 24VDC due to restrictions on space shuttle and space station to conserve power. For earth laboratory use the voltage can be determined depending upon the particular power sources available. Based upon the conductivity values of the solutions, the voltages are regulated so that the desired electric field is maintained in the buffer, despite any overall conductivity changes due to heating, gassing, or ion migration .

An experiment was also conducted by filling the test sample chamber with a 50-50 mixture of pure phosphate buffer and a prepared suspension of 9.7 micron particles in phosphate buffer. After connecting the bottom electrode to the positive terminal and the top electrode to the negative terminal, power was applied until a clear front was observed at the region where the two blocks that form the sample cavity are joined together. It was determined that the particle movement was uniform indicating the electric field is uniform . It was also determined that the accumulation of bubbles in the cavities could interfere with the electric field which would effect the flow pattern.

The circuit that controls the plate voltage has a potentiometer available for the user to manually control the current. A test point is accessible which gives the user the ability to calibrate the current accurately. In an automated embodiment, the processor-driven control circuit 104 accomplishes this calibration. A switch operated manually or by software, can be utilized to reverse the electrode polarity. There are two general types of embodiments, a high current and a low current unit. The high current unit is based on the flowing electrolyte concept as shown in Figure 8, while the low current unit has only metal electrodes.

A sealed interface between the two plates is provided having at least some face angle and preferably a face angle of between .1 and 10 degrees, and preferably about 1 degree on each plate to compensate for flexing and maintenance of the seal. As an alternate design or combination design one plate may be made thicker to lessen the effects of flexing. The face angle on that plate can be 0 degrees and a smoother surface can be obtained during machining; however, the face angle may be utilized at about 1 degree. A sealed slip ring is installed within the rotating plate 40 to allow for 360+ degrees of movement, thus eliminating the need to flex wires during operation . Figure 18 shows the slip ring assembly for transferring electric current to the plates and Figure 19 illustrates a slip ring wiper for communicating with a rotating copper contact.

A linear actuator on a syringe for supplies the flow of electrolyte. The pressure fluctuations are suppressed by the flexible hosing (tubing) . The pressure in the electrolyte chamber 54 will be slightly lower than the sample chamber 52. This should help the gases pass through the electrode membrane 60. A suction pump can maintain the pressure gradient in the electrolyte. Depending on the direction of flow, one syringe creates suction while the other supplies fluid so that it is possible to create a higher pressure or approximately equal pressure (electrolyte vs. sample pressure) . A peristaltic pump or piston is used for electrolyte circulation. The electrolyte pumping rate can be varied by adjusting a potentiometer. Pumping can be controlled automatically or pumping may be automated.

A DC gear drive motor pumps the fluids and a worm gear drive motor assembly rotates the bottom plates 40.

The unit 30 also can be adapted to be held by a mounting bracket which will allow the user to position the plates in any orientation (on side, face up, or face down) . Since gases rise in a liquid when subjected to gravity, the membrane 60 will be positioned on the top of the chamber for nearly all ground-based tests. The plate mounting bracket pivots to allow for this. This feature is not available (or needed) on the flight prototype.

As shown in Figure 10, an embodiment of the device of Figure 2 adapted for functioning in space flight showing the electrophoretic device 30 nested within a cassette 110 which is inserted within a containment enclosure 80. Within the cassette 110 is the base 19, a stepper motor, electrolyte reservoir 76, indexing tray, stationary sample plate 38, rotating plate 40, circuit board 83, collection plate, and electromagnet holding magnet .

Operating Models

As in equilibrium-stages separations, two operating models have been selected on the basis of the properties of the specific separands to be separated, a constant-potential model and a pseudoequilibrium model.

The constant-potential model and the pseudo-equilibrium model enables the user to calculate the optimum duration for applying the electric field in order to obtain maximum purification. Additionally, it enables the use to calculate the number of successive purification operations (stages) to which the original mixture would have to be subjected in order to obtain the desired level of purity.

In the constant-pote itial mcdel two different types of particles are tracked fr nm one cavity to the corresponding cavity on the other plate ^"he transport of particles by electrical fields is a ate prc :ess (not an equilibπun process) and the partioes ha ing higher mobility ar j separated faster when compa 'ed to those having re atively lowe- mobility In . cour er current distribution (CCD^'1 apparatus the se _'aration of particles having afferent electrophoretic moDihty is z thieved by contacting the buffer so'ut'O" of the ' Dp ch mbers with th e bottom cham bers containing the particles in buffer bv applying elec- tπca nelα at regular predetermineα

A CCD appε'Etu≤ has n extraction stages Let LS consider a situation where the bottom chambers of each of these stages have (say) two types of particles with electrophoretic mobilities x and μ₂

The number of particles initially (at . = Oi present in the bottcm chambers is denoted by N, and tne change in the number of particles in a chamber n after step r will be equa to the number of particles that migrate to the top chamber during step r The general equation can thus be written for this situation as a material balance

- [x- Λ/)_n , + [x, Λ/]_{n r}-.ι = Number of particles migrating from stage n during step r to the top chamber (1 ) where N is the total number of particles present in any of the n bottom chambers Λ/, the number of particles with mobility u , , Λ/₂ the number of bioparticles with mobility μ₂, N = N, + Λ/₂ and x, = /^/(Λ/, + Λ/₂) = NfN

let us consider one chamber, with total depth D and radius R having particles suspended in buffer solution When an electric field is applied the particles move due to their correεponαmg electrophore'ic mc bi ties Their This is a general equation ,-.hicn enables«us to estimate velocity will be proportional to the tie α appl ed thr fraction of particles having a mobility of μ, at any stage provided their concentration is known in the previ¬

X ous stage Similar equations ca . be wntten for particles

— ≡ μ E r α. ha ing otner mobilities No'e hcΛever that the particles are assumed to be in the upper lalf of tne bottom chamand tl e proportionality constant m is elect ophoretic mober Equa ion (3) needs to be modified to make the equa- bility < f the tioparticle and thus the chara (eristic of tne tioi more general Assuming th it the (articles are uni- biopai cle l*s magnitude is decioeα b. the surface for ily dis πbuted in the chamber then, i i general, chargt of th~ biooarticles Integratirg betv een the limits v = 0 o D a^rd I = 0 to / where / is t' e tiπif of applicat on (A, V)„_r= -[(r)(u,E.)'(D)](Λ, .. (Λ/₀) (4) of ele:trιc tie d results in iv I ere π^c the step number anc / ₀ is the initial concentra¬

D= u Ξt te i of the particles having mob ity u, An example of a pr .dictec migration pattern and 3 dis r )utιon of particles

In otl εr wo'ds ;c^r the oarticle cu m< oi t to move a:s actic is based on Eq (-' s s own r Fig ^. _ tanc D a i elec"'C πelc ot iniensi ^v E mi ≤t be appliec or a time . It is obvcus thai ι' E is in^creased 1 will decrease or v/i e ver-a uncer ot^e .'se SIΠVIT CO'¹ ntions 31.2 Tne pseudoequi brum model

I' the Lseudoequilio lum - see ive a sume that the opti

Initially the panic es are 'anc'orniy jistπbuted o_^e' the ^" turn Doientiai is applied ε ?^r . stage for separation of a ume of the lowe- chaπ-^e^r However their prcbabilu ct pair of separanαs In other wo'ds if the mixture that we migrating/movinc io tr.e too chamDer increases as fen want to separate oy e!ect^rcoho^resιs has two types of pardistance from trs bottom c: the chamber mceases al ticles with different mcbih; es all part.cies (of both types i any given set c' experi^mental conditions of E anc ' ⁼or could be drawn into one s'aoe i the current were applied example the numoer c panicles that migrate to the 'op iong enough The pnmarv coiec'ive was to determine the chamber will be 'our times higner if they are a' D,~ i::a- time tor wn'ch the dπvinc cectπr notentia¹ was to be tion (from the tco surtace in the chamber when c:"¹- applied in order Ό achieve a maximum resolution and the pared to those ? locat ^"■ D That is the ratio o' ^*r ^c actual value o 'ms maximjm ^reso'utιon that can be ob heights gives the relati.e njmber of the biopaπicies "at tamed We have called this ooiimum resolution the psej- migrate to the too cha ce- at any given set of E and . ^τc αoequilibπum for the pan\cu.ar stage of the process Tf.e arrive at the absolute rjrt er this ratio has 'o be m. ti- next goal was to πgure ou' now to further enrich the mixplied bv the conce ιtr=> on ot the particles (/ e n< mbe^r of ture obtained fr^_m 'he previous process, and keep doing particles per un,- olu ei since the ratio of the r-eigntε is this in a stepw.e manner In terms o' a classical chemical nothing but the ra eo o' '^e volumes of the chamjer cc^re engineering s paration process this is equivalent to cal- sponding to the c ;at o~ considered Therefore 'ne n_.ni ber of particles ~ ra¹ -g curing step r is for exa no'e

(μ£τπfl-)

if we wish to ca; ture 12 ot the particles with π obilit. . in the first transfer Nov. Eq (1) becomes

-(x,Λ)_n, + (x₁Λ =,2uEl(N,)_{r l}]/D where (Λ/),)_r , is the number of particles of mcoilitv i oer unit volume in st ige n at step (r-1 )

Hence,

(x,Λ/)_n , = [(μEtj^/, D/2)](Λ,)_n , , + (x,Λ/)_n , (3) culatmg the operating line A corollary to calculating the cally all the particles suspended in the fe"ed may be trans operating line is the development of Drotocols which outferred to the extract In the mathematical analysis which line the handling of the various fluids and mixtures This follows we show that for a given particle fraction of A m was done for the most general case - when w e ι 'ished to the feed, there exists an optimum duration for applying isolate some types of particles and discard other types the driving potential gradient The degree of separation from a suspension with many different types of particles achieved actually decreases if the potential is applied for a longer duration Thus, the "pseudoequilibrium' fo^r the

Since the process of electrophoresis has not been ope - separation is said to have been reached when this maxiated in a multistage manner before, there is no estabmum possible degree jf separation is achieveα As a conlished convention of def mng and mathematicali ' represequence, each "stage in this process is completed when senting the various ph* sical parameters that are e > pseudoequilibrium is achieved between the residue and countereσ in a mu'tistage electrophoresis Henc^c befo e the extract we describe the mathem atical model, we preser a shcrt overview explainir g the rationale behind what physical We specify the toliow ng physical parameters X³ _A5 rspre seπts the partic e frac on in the feed or the re∑ idue rep parameters we have ch csen to monitor during iβ prc c- resents the pai icle fr .ction in the solvent or tl e extr ict A ess, what we have called them and by what s /i ibols ve letter in the suf script shows the panicle type The number have representec them Here we present the concepts in the superscript tells us the chamber nurrner whereas and definitions as applicable to the simplest c cases - wherein the mixture that is to be resolved contains o nly the number n the si bscπpt gives the stags or ite ation two types of dissolved particles (say of types and B) number of the elect' ophoretic process Thus X _s repre Typically at the beginning of the electrophoi e c separasents the par icle fraction of type A T the 3^rd chambe^r tion in each of the chambers thus formed one of the prior to the 5th stage of separatio - Y _Λ5 represents the halves contains a mixture of particles whereas the other particle fraction of type A in the 3 ^" chamber prior to the one contains clear fluid We call the former the feed' and 5th stage of separation (The astensk implies that pseu the latter the "solvent" If we allow the separation to take doequilibπum has been achieved) _e represents the mo place fo^r a certain amount of time, the feed would be bility of a particle of type A, and møj represents the num depleted of particles of a particular type (say type A) We ber of particles of type A actually transferred during the now call this the "residue Tne solvent now would be 4th stage in chamber 2 from the feed to the extract enriched with these particles of type A and is now called Let the volume of each chamber te equal to 2 V and let the "extract" The extract from the final stage which the crude be such that a total of F carticles is suspended meets ou^r required degree of purification, is called the in fluid of volume V This crude seri es as the feed to the product We call the original mixture of particles, the first stage in chamber 1 If X_A is the particle fraction of "crude" The crude serves as the feed for the first stage particles in the feed, which has a total number of particles equal to P₀, then P_A0 is the number of particles of type A

Our desired objective is to obtain a suspension containing in the feed given by only (or almost only) the particles of our desired type As long as there are particles of only that type present, we PAO - (XAO)PO (5 ι would not be too concerned about its actual concentration (i e , in terms of the number of particles per unit volume of Similarly, P_Bo = (^BO)PO (6) suspension) It is not particularly useful if we are able to obtain a high number of our desired particles in a sample Now, if an electric field of strength E is applied for a dura if it is accompanied by large numbers of other particles tion of time ., the number of particles of each type that is We therefore chose to monitor the particle fraction of a transferred into the extract is given by given type of particle, just as in the case of the constant- potential model described above In the two-particle-type m = {X' P, ^~ π case we define the particle fraction of particles of type A to be the ratio of the number of particles of type A in a given volume of liquid, to the total number of particles m- = (Xi)P , μ_B H

We now define a stage and the concept of pseudoequiliAs a consequence of this transfer the particle ratio of parbrium In a classical equilibrium-driven process, a theoretticles of type A is given by ical stage is usually defined to be completed when the extract is in equilibrium with the residue However, since m^<

Y = - electrophoresis is a kmetically driven process, theoreti- mi + m_B' _l which on simolification, reduces to ^t this sta e note that (i) this duration of t me is independent of the feed concentration, (n) if we are interested in solating p ure B (without regard for yield) we can draw off

A ure B at this ooint as the residue has only particles ot λA pure B suspenαed in it (in) the pseudoe luilibπum particle ^•-actions m the extract deperd on onl ' two things the w. ch says t lat this is the partid fraction o be found in -atio of tl e electrophoretic m abilities 0' the two types of tn- extract respective of the dilation of - 3 application particles and tne particle fraction ot pa tides of type A in of the elect ιc field However thι _ equatio ' assumes that ^■πe teed (prior 'c the applicati on of the electric field T that th ere are particles of all types 1 1 the feeα -esiαue How □articular stage er u, - 11 B there will come a time wr e- all particles c type A v ould have been transierred 'c -e e^ ract and - sejdo --quilib' _m plots whi ch show he variation c " ' e ; ere . ould be only particles c >ype E re~ ^~r -c

the ..anicle tracti: the extrac 1 with tne particle "rac c^~ residue It would be logical to stop the ao: ca^' n of the e teed for thr article with the higner mobility ma . mus eiectr.c neld at this point This duration of ' —= _? -ouno bv „e constructec "or various values of the mocnty -atio settmc ra'io of the eiεcrophoretic mobility of the slower pa role c that of the taε'er particle) Figure V shows such plots m'_A, = (9) ^•or mobility ratios varying from 0 1 through 0.9 Once the

Dseuαo)equιlιo jm lines have been obtainec the neΛt hicr on simplification using Eq (7) , yields step is to αeveiop an operating line The operating nne .voulα be a grεomcal representation of the equa'ion Λ r.ic 'ells us what ^e particle fractions of the feeα are as a

1 1 01 ^•unc'ion ot the oarti e fractions of ihe extract irom the previous stage Several possible modes of operation can oe considered tor this system Initially we chose the moαe

At this time, pseudoequilibrium is reachec .n stage j At 01 using the extract from one stage as the feed for the pseuooequilibπum, the particle fraction of panicles of type next In this case the operating line corresponds to the A in the extract is given by Eq (8) which or -urther simpli45^c line in the oseudoequilibπum plot and the numoer of fication yields stages is founc by stepping off the stages in a way similar ;o classical metnods such as the McCabe Thiele method

| 1 I 16 Figure ^Z-snows the variation in Ihe number of stages

1 / μ 'equ.red to ta*e the particle fraction of the more mobile particle from 5 to 95°o as a function of the mobility ratio

3.2 Thermal mode l

When low-conductivity buffers are useα reaction of heal,, lo ambient conditions is ddequale however, when a cur' rent of several mA musi be applied active heat rejection is required and this can be achieved b , using a recircu latmg electrode buffer system ( 10 121 Initially we wishec o determine the rale of sensible heat generation due to passage of current through a pair of cavπies with bare Pd electrodes The rale of te nperaiure rise in an adiabalic cav.ty depends on the buffer conductivity and the desireo eiectnc field strength and C3n be datermin ed from

α ^"7d. = IE/f C_p ( 12) w e-e / is tne current αens ιy, the fielα s' 'ength (= //k) p Me i olutior density C_c ihe speci ic heat c water at 25°C l a .εjmeσ onstant) and / the ι c lαuctiv ' Two condi ! c ι^c were i onsiderec πel stren i ns o' 5 ana 1 0 V/cm n C 1 ^{■ •} pi .c sohate bi eι The fo r ier ca. e to- e> ample tc [ q M 2 resu'ts ιr

c ^" d = 0 0 2 oeg C s

a ih'S is piolieα as s ouares in -ιg H A simila r plot for 1 V/cm shc . s tne ss 'Sifvity c^; heatmg jpor tne f ield anc inO'Cates 'ha' usmc. nigher fieiαs or mgner co.nαuctivi ties wil' reouire ihe m lementation of a cooling systeπ. The generation of sens ole heat was monuored in a single Cc i' v using a sma' the - istor probe the results are plot ted as diamonds in Fig i\ The corresponding relationship is only d l'd . = 0 0092 oeg C/s indicating thai about 1 /2 o' the neat was rejectee ic the ADSEP po'vcarbona'e plate These calculations app v only to cavities with Dare Pd electrodes ano heat transfer relationshios developed for elecirodes w ith circjianon and cooling will oe publislied later

3 3 Particle migration experi ments

When a suspension of particles w as placed in a single bottom carvity of the unil and a field was applied (5 or 10 V/cm) for 1 mm per cavity Ihe resulting fractograms compared favorably with model preoiciions The con slant potential model was lesied in a series of expeπ

ments using 3.4 μm sulfated polystyrene latex particles (Interfacial Dynamics, Portland, OR) as test particles. The initial concentration was 5.5 x 10⁶ particles/mL, and field strengths of 5 or 10 V/cm were applied for 60 s per transfer for eight transfers. The results of two such experiments are shown in the bar graphs of Fig \X when the field strength was doubled, the cell extraction was com- pleteα in about half the number of transfers. Specifically, 450 x 10" particles were extracted in six transfers at 5 V/ cm, while the same number was extracted in three transfers at 10 V/cm, in keeping with Eq (4) and as described in Fig χo . The foregoing detailed description is given primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom, for modification will become obvious to those skilled in the art upon reading this disclosure and may be made upon departing from the spirit of the invention and scope of the appended claims. Accordingly, this invention is not intended to be limited by the specific exemplifications presented hereinabove. Rather, what is intended to be covered is within the spirit and scope of the appended claims.

Claims

CLAIMSWe claim:

1. A claim according to claim 1 in which the magnitude of the electric field is held constant at a selected field strength by a microprocessor-controlled electric circuit,

2. A multistage electrophoretic purification process for separating and purifying cells, particles, and proteins, comprising opposing circular plates with coin-shaped cavities arranged to form a multi-stage thin-layer extraction system, said half cavities oppose each other in plates that are sealed together and rotate with respect to each other, each of said half cavity contains a metal electrode that produces an electric field when the two cavities are in contact, as the electric field is applied in a cavity separands migrate into the upper or lower half of said cavity depending on their electrophoretic mobility, upon complete separation the electric field is de-energized and the plates are rotated countercurrently until the upper cavity aligns with a lower cavity with fresh solution that is thoroughly mixed with the separated cells or molecules, and the process is repeated as many times as necessary to effect the desired separation.