EP1468278A1

EP1468278A1 - Device, use of said device, and separation agent for separating particles by means of free flow electrophoresis

Info

Publication number: EP1468278A1
Application number: EP20030729399
Authority: EP
Inventors: Christoph Eckerskorn; Hans-Jörg Grill; Gerhard Weber; Peter Weber; David Yost
Original assignee: Tecan Trading AG
Current assignee: Tecan Trading AG
Priority date: 2002-01-21
Filing date: 2003-01-20
Publication date: 2004-10-20
Also published as: WO2003060503A1; AU2003201252A1

Abstract

The invention relates to a free flow electrophoresis device, an associated method and separation agents for separating particles by means of one such device. Said FFE device comprises at least one separation chamber (14) through which a separation agent (8) can flow, said separation chamber being defined by a bottom, a cover, and a spacer for maintaining a distance between the same. The inventive FFE device comprises a dosing pump for transporting the separation agent (8) which is supplied to the separation chamber by means of admissions (15, 15') and leaves the same via outlets (16); electrodes (9, 10) for creating an electrical field in the separation agent (8); sample loading sites (17) for adding a mixture of particles (1, 1', 1'') to be separated; and fractioning sites (18) for removing the particles which are separated by means of FFE in the separation agent (8). Said FFE device comprises selectable charge carriers (4, 5) for combining with the particles to be separated (1', 1'') and for producing charge-modified particles (7', 7'') which - due to their selectively variable net surface charge - exhibit a different migration behaviour in the FFE device, to particles (7) which are not charge-modified. The inventive device is characterised in that it comprises guiding or focussing cushions (12, 13) arranged between the separation agent (8) and at least one electrode (9, 10). Said focussing cushions (12, 13) consist of a medium (20) which exhibits highly increased electrical conductivity in relation to the separation agent (8). Furthermore, said device comprises separate channels (15') for supplying said medium (20) to the focussing cushions (12, 13).

Description

Device, use of the device and separating means for separating particles in free-flow electrophoresis

The invention relates to a device, the use of the device and separating means for separating particles by means of the free-flow electrophoresis technique, according to the preamble of independent claims 1, 9, 26 and 27.

An important problem in diagnostics and in research concerns the precisely defined sample preparation. However, the technologies currently in use, such as centrifugation, filtration, magnetic separation and FACS (fluorescence activated cell sorting), have various disadvantages, such as restricted specificity, low throughput rate, low cleaning ability or an expensive device for isolating and cleaning cells, organelles or biomolecules (e.g. protein complexes). The most important applications for the cleaning of cells, organelles and biomolecules (eg protein complexes) can be found in the areas of biopharmaceuticals, biotechnology, environmental analysis, food technology and in medicine. The techniques used are either preparative (biopharmaceutical and biotechnology) or analytical (environmental analysis, food technology and in medicine).

Free-flow electrophoresis (FFE) is one of the most promising technologies for the separation of all possible particles [cf. Krivanova L. & Bocek P. (1998) "Continuous Free-Flow Electrophoresis" Electrophoresis 19: 1064-1074]. In the field of proteomics, the FFE is the technology of choice for the pre-division of complex protein samples in relation to their different pl-value (degree of ionization). The FFE separates cells based on the electrophoretic mobility of the cells. The corresponding principles have already been thoroughly characterized [cf. e.g. Bondy B., Bauer J., Seuffert I. and Weber G. (1995) "Sodium chloride in Separation medium enhances cell compatibility of free-flow electrophoresis", Electrophoresis 16: 92-97], however the FFE has so far been little accepted , because most Zeil types differ only slightly in their surface charge, so it is difficult to separate these cell types. The first improvement was the introduction of the Immuno-FFE, in which specific antibodies are bound to defined surface epitopes of the cells to be separated and thus a modified electrophoretic mobility of these cells in the FFE could be achieved by changing the net charge of the cell surface [cf. e.g. Hansen E. and Hannig K. (1982) "Antigen-specific electriophoretic cell separation (ASECS): isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies", J. Immunol. Methods 11, 51: 197-208].

A generic FFE device is described in the international patent application PCT / EP01 / 14408. It is an electrophoresis device with a separation space through which a separation medium flows, which is separated by a base and a cover and by these two at a distance spacers held to each other is limited. In addition, this FFE device comprises a pump for feeding the separation medium, which enters the separation space via media feeds and leaves it again via outlets. The FFE device also includes electrodes for applying an electrical field in the separation medium and sample application points for adding a mixture of particles or analytes to be separated and fractionation points for removing particles separated by the FFE in the separation medium. These separated particles can be sent for analysis or for further processing. To influence the flow profile of the separation medium, it can be provided that two or more separate delivery channels of a metering pump are connected to the separation space in the region of the fractionation outlets near the electrodes in order to add medium.

U.S. Patent 5,948,231 discloses compounds, methods and devices for performing ultra-fast binding assays in capillary electrophoresis or in other electro-separation techniques such as e.g. in free flow electrophoresis.

The object of the present invention is to further improve the preparative or analytical isolation of particles such as cells, organelles and biomolecules (e.g. protein complexes) and the like, or of bioparticles or biopolymers in the FFE.

According to the invention, this object is achieved - according to a first aspect - by proposing a free-flow electrophoresis device with the features of independent claim 1.

According to the invention, this object is achieved - according to a second aspect - by using a method for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof, using a free-flow electrophoresis device the features of independent claim 9 is proposed. According to the invention, this object is achieved - according to a third aspect - by separating means for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof in a free-fiow electrophoresis device, according to the features of independent claims 26 and 27, respectively.

Further preferred features result from the dependent claims.

In the context of the present invention, particles are considered to be e.g. all particulate mass units, preferably of biological origin, such as cells, viruses, cell organelles, vesicles, cell nuclei and membranes, and parts or aggregates thereof; also biomolecules, such as lipids, proteins, DNA, RNA and sugar, and complexes or aggregates of biomolecules, such as lipoproteins, glycoproteins, lipopolysaccharides, etc. These particles also include organic and inorganic molecules, such as pharmaceuticals, polymers and the like.

A device according to the invention for separating such particles based on the selectively modified surface charge - in particular for separating cells or cell components, organelles or biomolecules, or parts or complexes thereof, using the free-flow electrophoresis technique comprises the following advantages:

• Magnetic beads are not suitable for the preparative separation of particles; in addition, only a single type of particle can be isolated with magnetic beads. Magnetic bead applications can therefore be replaced by the FFE according to the invention, if positive and negative charged carriers are used at the same time. The FFE according to the invention allows simultaneous isolation of at least two particle types with maximum efficiency, because the individual electrophoretic mobility of the ponds is increased significantly and selectively. • Many FACS applications can be replaced by FFE applications and can be carried out with much cheaper and faster devices, if required also automated.

• Specifically selected, living cells can be isolated qualitatively and quantitatively or on a preparative or analytical scale thanks to the specificity of the antibodies used and the separating power of the FFE.

• For the first time, it is possible to carry out a gentle preparation to obtain a highly pure population of organelles.

• On a preparative or analytical scale, particle mixtures in a relatively large amount can be separated precisely within a short time. An example is the purification of circulating tumor cells from whole blood (tumor cells occur in the ratio of 1:10 ⁶ to 1: 10 ⁸ in whole blood): The white blood cells (2 ml MNC fraction from 10 ml Whole blood) including the tumor cells were separated from the red blood cells using a density gradient and, after appropriate labeling of the tumor cells (incubation with epitope-specific antibodies with "charge labels"), were applied to the FFE for separation. The addition took place at 4 to 10 ml / hour. The purified tumor cells could be obtained in a final volume of 2-8 ml.

• The proposed Free Flow Electrophoresis (FFE) device enables different separation techniques to be carried out with different and selective separation parameters.

• The proposed FFE method enables not only the isolation but also the selective depletion of particles from a particle mixture, such as a depletion of abundant proteins (for example separation of serum albumin from body fluids, for example blood plasma). • The setting of particles separated on the electrodes due to their special biological relevance and the resulting substantial losses of these particles can be successfully prevented with a focusing pad made of an electrically highly conductive pad medium.

Preferred and exemplary embodiments of the device according to the invention for separating particles on the basis of their selectively changed net surface charge - in particular for separating cells or cell components, organelles or biomolecules, or parts or complexes thereof, using the free-flow electrophoresis technique in the following with the aid of schematic drawings - which merely illustrate the invention but do not restrict its scope - explained in more detail. Show:

1 cells to be separated, which:

1A without certain epitopes,

1B with two epitopes of a first type,

Fig. IC are equipped with three epitopes of a second type;

2 shows an epitope-specific antibody and charge-carrying molecules;

3 cells to be separated with corresponding epitopes, in which:

FIG. 3A the two epitopes of the first type are negatively charged, which makes the net surface charge of the cells strongly negative, and in FIG. 3B the three epitopes of the second type are positively marked, which makes the net surface charge of the cells strongly positive;

4 shows a basic diagram of a device according to the invention for the simultaneous separation of two charge-labeled cell types; Fig. 5 is a schematic representation of usable according to the invention

Separation means in a possible arrangement and interaction with a particle to be separated;

6 shows schematic top views of devices according to the invention, wherein: FIGS. 6A and 6D show a CHIEF device with a linear pH gradient and a homogeneous field; 6B and 6E show a PZE device with a homogeneous pH value and a homogeneous field; and

6C and 6F show a CITP device with a pH gradient and a field gradient;

7 shows a schematic quantity distribution of charge-carrying cells of the same type:

7A without use, and

7B using a focusing cushion according to the invention.

Figure 1 shows cells to be separated as an example of particles 1, 1 ', 1 "to be separated. These cells can be equipped without specific epitopes (see FIG. 1A). Such cells have and are a relatively low negative net surface charge very difficult to separate in the FFE. However, if the cells have epitopes of a first type 2 '(FIG. IB) or a second type 2 "(FIG. IC), these cells can be epitope-specific with charge-carrying molecules or charge carriers be marked. In general, the net surface charge of particles can thus be selectively changed by binding charged binding molecules to these particles. This binding can be based on an antigen-antibody interaction. Further interactions that can be used for the modification of the net surface charge of particles according to the invention for coupling binding molecules to the particles to be separated also include receptor-ligand, enzyme-substrate and protein-protein interactions. Chelation or a bond based on general molecular interactions, be it ionic or based on Van der Waals forces or on hydrogen bonds, but also a covalent bond can be used for this purpose.

With the FFE device according to the invention or with the FFE method according to the invention, relevant cells can be extracted from body fluids (eg disseminated tumor cells from blood, sputum, ascites, urine, lavage etc.) or from tissue homogenates (eg solid tumors in the kidney, thymus) etc.) or organelles from cell homogenates (eg caiciosomes, coated vesicles, endosomes, endoplasmic reticulum, Golgi cisterns, lysosomes, peroxisomes and mitochondria) and proteins from proteomes or expression systems.

Figure 2 shows an epitope-specific antibody and two monomeric or polymeric charge carriers or "charge labe!" (cf. "CL" in FIG. 5) in the form of cations 4 or anions 5. A charge carrier 4.5 is a molecule ionized at a given pH. It can be anionic or cationic, monomeric or polymeric. With the help of such charge carriers 4, 5, the net surface charge of particles can be changed in a targeted manner. Charged particles, e.g. Beads can also be used as charge carriers.

Examples of anionic charge carriers 5 are: polyglutamic acid (PGA); anionic or derivatized proteins such as albumin; anionic polysaccharides such as heparin or alginic acid, polyaspartic acid, polyacrylic acid and polyamic acids with a net negative charge at a usable pH (for example in the range from pH 4 to 10). Anionic polymers with a molecular weight of 500 to about 500,000 Daltons are preferred. Several monomeric or polymeric charge carriers can also be bound to a specific binding molecule in order to further develop the net surface charge of the particles to be separated. Examples of preferred cationic charge carriers 4 include homopolymeric or copolymeric proteins with a preferred molecular weight of 500-5000000 daltons; quaternary ammonium compounds bonds with between 1% and 10% nitrogen (without counter ion). Commercially available quaternary ammonium polymer compounds include, for example, the products MERQUAT.RTM. (Calgon, Pittsburgh, PA, USA), CELQUAT.RTM. (National Starch and Chemical Corp., Bridgewater, NJ, USA), GAFQUAT.RTM. (GAF Corp., Wayne, NJ, USA) and MAGNIFLOC.RTM (CYTEC Ind., Indianapolis, IN, USA) as well as hexadimetrin bromide (POLYBRENE.RTM) and diethylaminoethyl dextran (both from Sigma Chemical Company, St. Louis, MO, UNITED STATES). These and other anionic and cationic charge carriers are described in US Pat. No. 5,670,381, to which reference is expressly made here.

Such an antibody (cf. FIG. 2) is referred to below as binding molecule 3 or “binding molecule”. The bond between a binding molecule 3 and a particle 1 ′, 1 ″ and the bond between a charge carrier 4, 5 and the binding molecule 3 can be covalent or non-covalent (eg adsorptive, on Van der Waals forces or hydrogen bonds, on one Biotin-streptavidin interaction, etc.) The binding molecule 3 can be, for example, an antibody (cf. FIG. 2), streptavidin, biotin, receptor, ligand, chelating agent (eg nickel-nitrilo-tetraacetic acid = Ni-NTA), a low-affinity binder (protein-protein interaction) or a chemically activated molecule (eg activated ester, affinity label). We call the complex consisting of binding molecule 3 and charge carrier 4 or 5 a charged binding molecule 6 or "charge label binding" molecule _"..

FIG. 3 shows cells to be separated with corresponding epitopes 2 ', 2 ". The two epitopes of the first type 2' are negative in FIG. 3A and the three epitopes of the second type 2" are positively marked in FIG. 3B. The net surface charge of the marked cells is therefore greatly changed, so that these cells behave differently with their specifically changed net surface charge in the FFE. We call the complex of particle 3 and charged binding molecule 6 charge-modified particle 7 ′, 7 ″ or “charge modified particle.” The net surface charge of the cells is preferably changed such that a cell type changes in a separation medium 8 of an inventive one Device moved against the cathode 9 and the other cell type against the anode 10, as shown in the schematic diagram of Figure 4. The two groups of charge-modified particles 7 ′, 7 ″ or the two charge-modified cell types move away essentially simultaneously from the unlabelled or non-charge-modified background cells 7, which are essentially in the middle (or at least a great distance from the electrodes) 9, 10 in the separation medium 8 of the device according to the invention, the arrow indicating the main flow direction of the separation medium 8. The various cell types which are separated from one another are collected in a collection area 11 and sent for further use The resolution of the FFE is essentially determined by the migration behavior of the particles 7, 7 'and the size and number of the discharge openings. The separation space 14 comprises the space for the separation medium 8 and the two lateral regions 19 near the electrodes 19 with the cushion medium 20 formed focusing pillow 12, 13 (cf. also Fig. 7).

The mass of the charge-modified particles 7 ', 7 "is fed through the FFE in approximately a normal distribution to the discharge openings, apart from an (unwanted) adhesion of charge-modified particles 7', 7" on the surfaces of the electrodes 9, 10. 7A shows a normal distribution in a schematic cross-sectional view. However, because this migration behavior of the charge-modified particles 7 ', 7 "can lead to poor resolution of the FFE, a focusing pad 12, 13 made of a likewise flowable, electrically highly conductive separating material is preferably provided in the immediate vicinity of the electrodes 9, 10 Due to its high electrical conductivity, this focusing pad 12, 13 prevents the charge-modified particles 7 ', 7 "from being able to diffuse all the way to the respective electrode. At the boundary between the separation medium 8 and the cushion medium 20, there is a concentration of the charge-modified particles 7 ', 7 ", as shown in FIG. 7B. Preferably, the separation medium 8 and the cushion medium 20 flow in the same direction. FIG. 5 shows a schematic illustration of separating agents which can be used according to the invention in a possible arrangement and interaction with a particle to be separated. The charge-modified particle 7 ', 7 "here consists of a protein P to which a chain of histidine molecules H (a so-called" His tag ") has been attached in a manner known per se. A charged binding molecule 6 is bound to this His tag. This charged binding molecule 6 consists of nickel-nitrilo-tetraacetic acid (Ni-NTA), which binds to the histidine molecules, here the anionic charge carrier 5 is covalently bound to the nickel-nitrilo-tetraacetic acid, the binding molecule 3.

FIG. 6 shows schematic top views of devices according to the invention. This free-flow electrophoresis device comprises at least one separation space 14 through which a separation medium 8 can flow. This separation space is delimited by a base and a cover and by these two spacers which are spaced apart from one another. This FFE device also includes a metering pump (not shown) for conveying the separating medium 8, which enters the separating space 14 via media feeds 15, 15 'and leaves it again via outlets 16. Furthermore, the FFE device comprises electrodes 9, 10 for applying an electric field in the separation medium 8 as well as sample application points 17 for adding a mixture of particles 7, 7 ', 7 "to be separated and fractionation points 18 for removing particles separated by the FFE in the separation medium 8 Two separate delivery channels 15 'of the metering pump are connected to the separating space 14 in the area of the fractionation outlets 16' near the electrodes in order to add medium Particles 7 ', 7 "in the regions 19 of the separating medium 8 near the electrodes and are connected to a separate medium container (not shown) for providing an electrically highly conductive cushion medium. The FFE separation technology of continuous isoelectric focusing

(CHIEF) is based on the different pI value of the particles to be separated. This pI value corresponds to the pH of the surrounding, inhomogeneous medium, against which the particles appear neutral. The FFE of particles due to their different isoelectric point enables the isolation of analytes or particles with the smallest differences in their pI values. The total charge or net surface charge is at the isoelectric point p 1 (ie at the location of the separation medium 8 which has just the pH at which the number of negative and positive charges is the same for a given particle (for example a protein molecule)) of this particle is zero. The focusing effect inherent in the separation medium 8 of a CHIEF device has the effect that a particle l ', l "which diffuses away from the pl automatically receives a (positive or negative) net surface charge again and is moved back to the pl by the electric field.

Due to the interaction of analytes or particles with a single binding molecule or "binding molecule" with a high charge density, a significant change in the pI value and thus a rapid and complete separation of the complex of particles and binding molecule from the other species of the sample can be expected , The method of continuous isolectrical focusing is particularly suitable for the micro-preparative to preparative isolation of biopolymers in general and of bioparticles whose biological function or their integrity is ensured in the range of the selected range of the pH gradient in the separation medium. This applies in the case of many representatives of viruses, bacteria, cell organelles and membrane domains, since the addition of "osmotic expanders" can ensure the maintenance of an ideal osmotic pressure: by adding uncharged substances (e.g. sugar, as non-ionic, osmotic expander) and / or of salts (eg NaCI, as ionic, osmotic expander) an optimal osmotic pressure for a cell type, ie isomolar conditions (eg for mammalian cells 250-310 mosmol) can be generated. FIG. 6 shows an inventive device for separating charge-modified particles 7 ', 7 "from non-charge-modified particles 7 in a separation medium 8 which flows in a separation space 14 between two electrodes 9, 10 (large arrow, 8). The separation space 14 comprises the space for the separation medium 8 and two lateral regions 19 near the electrodes for the guide or focusing cushions formed with the cushion medium 20. The electrodes 9, 10 are preferably designed as electrode spaces, from an electrode buffer contacted by an electrical lead 23, 23 ' 24 flow through and have a preferably semipermeable membrane 25 in relation to the separation space 14. The electrode buffer 24 is introduced into the electrode spaces via separate feed lines 26, 26 '(only partially shown in FIG. 6) and also leaves them via separate outlets 27, 27'. An additional one is preferably used for circulating and possibly also for cooling the electrode buffer Pump device (not shown) used.

FIG. 6A shows such a CHIEF device with a linear pH gradient. A separation medium 8 flows in a laminar movement (preferably from the bottom upwards in an inclined separation space) between the two electrodes (large arrow), is braked in the area of the outlet openings by a counterflow of separation medium (small arrow) and leaves the separation space 14 in fractions via the outlet openings. A sample with three groups of particles to be separated is added to the separation medium via the sample inlet and moved with the laminar flow of the separation medium. The three particle groups are continuously separated, focused and collected in separate fractions in the pH gradient, which is generated by the electric field generated between the electrodes in the separation medium. As can be seen from FIG. 6D, the electrical conductivity of the separation medium 8 between the two preferably provided focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is relatively low and high. The pH gradient is built up in the separation medium 8 between the two focusing pads 12, 13. The FFE separation technique of continuous FFE zone electrophoresis (PZE) is based on the difference in the value of the electrophoretic mobility of the particles to be separated compared to the separation medium used. FFE zone electrophoresis thus enables the isolation of analytes or particles on the basis of their different sizes and / or shapes and / or net surface charge.

Due to the interaction of particles with a single binding molecule with a high charge density, a significant change in the value of the electrophoretic mobility and thus a rapid and complete separation of the complex of particles and binding molecule 3 from the other species of the sample can be expected. The process of continuous FFE-PZE is particularly suitable in the case of the separation of "sensitive" bioparticles and complexes, the separation of which has to make special demands on the separation medium. This is particularly the case if the biological function and integrity of these particles should also be guaranteed after the separation. In these cases, special requirements apply: narrowly limited pH range of the separation media, good buffer capacity of the separation media, physiological tolerance of the buffer substances used, minimum contents of various "essential" cations and anions etc. Although the usual cell culture media are not very compatible with all techniques of Apply electrophoresis, the successful separation of cells with FFE-PZE is possible.

FIG. 6B shows such a PZE device, in which the samples are separated on the basis of their charge and to a lesser extent on the basis of their shape and size. As can be seen from FIG. 6E, the electrical conductivity of the separation medium 8 between the two focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is relatively low and homogeneous. The pH value is the same in the entire separation medium. The FFE separation technique of continuous isotachophoresis (CITP) is also based on the difference in the value of the electrophoretic mobility of the particles to be separated. In contrast to the FFE-PZE, the separation takes place in inhomogeneous separation media and offers a better resolution due to an inherent "focusing effect": If individual ponds diffuse away from a separated band of particles (eg proteins) during CITP, these particles enter into one Medium with different electric field strength, which accelerates or slows the particles. The inherent focusing effect means that the slower or faster migrating particles find their way back into the main fraction.

This FFE separation technique is principally suitable for bioparticles and cells, their separation in media without special "essential cations" (eg Mg ⁺⁺ or Ca ⁺⁺ ) or "essential anions" (eg Cl ^" ), which are essential for stability and vitality of cells are important, is not possible.

FIG. 6C shows such a CITP device in which discrete spacers are used. Such discrete spacers are ions with well-known, defined mobility, which push themselves between particles to be separated in the CITP and contribute to the separation taking place efficiently. Ampholytes can also be used as such discrete spacers. As can be seen from FIG. 6F, the electrical conductivity of the separating medium 8 between the two focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is at least partially gradient-like and inhomogeneous. In addition, an at least partial pH gradient is built up in the separation medium 8 between the two focusing pads 12, 13, the areas of the two gradients essentially coinciding and both the pH values and the electrical conductivity in the area of the focusing pads 12, 13 each being different are high.

Various combinations of these separation techniques (CHIEF and FFE-PZE or CHIEF and CITP) are possible. For example, different separation media can be used simultaneously in the separation area of the FFE apparatus and thus different separation parameters can be used at the same time. The device proposed according to the invention and the method proposed according to the invention allow continuous operating modes to be carried out, in which the separation of the particles 7, 7, 7, 7 "is carried out with a continuous flow of separation medium, permanent application of the electric field during the entire separation and continuous collection. A distinction is made between continuously preparative (sample is added continuously with the same separation conditions) and semi-preparative / analytical (discontinuous sample addition with the same separation conditions).

In a discontinuous operating mode, which is also possible, the separation medium 8 flows continuously: the sample application takes place when the high voltage is switched off; After stopping the sample application, the medium transport is switched off or greatly reduced or reduced. The high voltage is then switched on until the sample has been separated. After a predetermined time, the voltage is switched off and the separated sample is eluted from the separation chamber with an increased medium flow and collected. It can also be provided that by means of a further metering pump or separate delivery channels of the medium pump - preferably at the end of the FFE device opposite the media feeds 15, 15 '- an auxiliary medium 21 is fed into the separation chamber 14 via auxiliary medium feeds 22 and together with the Separation medium 8 is removed via the fractionation points 18 (see FIGS. 6A to 6C).

FIG. 7 shows the effect of the focusing pads 12, 13, the border of the focusing pad with the electrically highly conductive pad medium 20, which runs essentially parallel to the electrode 9, 10, being indicated by dashed lines. If no focusing pad is produced (FIG. 7A), a normal distribution (hatched) of the particles separated from a rest of a sample by means of the selectively changed net surface charge is established. It can also happen that particles adhere to the electrode 9, 10 (not shown), so that in some cases substantial losses occur on these particles, for example because of their particular biological relevance. This can be done with a A focusing pillow 12, 13 made of an electrically highly conductive pillow medium 20 can be successfully prevented (cf. FIG. 7B): thanks to the high electrical conductivity, the focusing pillow 12, 13 behaves like an electrode 9, 10, whereby - thanks to the high electrical conductivity of the cushion medium 20 is prevented that particles can reach the actual electrode 9, 10. As shown in FIG. 7B, a concentration of the isolated particles (hatched) forms at the cushion boundary, which far exceeds the measure of a normal distribution (shown in broken lines). A much larger amount of these particles in higher concentration can thus be removed from the FFE device and sent for further use via a fractionation point 18 which is positioned precisely in this area. In particular for the isolation of cells, organelles, membrane parts or complexes of biomolecules, which are to be separated from the rest of a sample by means of the FFE on the basis of their selectively changed net surface charge, the use of such a focusing pad 12, 13 represents a significant improvement the use of a focusing pad in the fine separation of identical subunits biomolecules or their complexes appears to be less advantageous. The guide or focusing pads 12, 13 are thus formed by a buffer 20, which has a greatly increased electrical conductivity compared to the separation medium 8. Since the electrophoretic separation performance is dependent on the field strength and is inversely proportional to the conductivity, the electrophoretic separation performance in the focusing pads 12, 13 decreases so much that the charge-modified particles 7 ', 7 "at the interface between the separation medium 8 and the Focusing pillow 12, 13 can be focused.

Three exemplary embodiments of immune FFE are presented below:

Example A: Purification of Circulating Tumor Cells from Whole Blood

A summary of the value of disseminated tumor cells can be found in: Bockmann B, Grill HJ, Giesing M. Molecular characterization of minimal residual cancer cells in patients with solid tumors. Biomol Eng 2001; 17: 95-111. The membrane protein "epithelial glycoprotein" (EGP), which is specific for epithelial cells, is used as the cell surface marker. This EPG is also expressed by tumor cells of epithelial tumors (Moldenhauer G, Momburg F, Möller P, Schwartz R, Hämmerling GJ. Epithelium-specific surface glycoprotein of Mr 34,000 is widely distributed carcinoma marker. BrJ Cancer 1987; 56: 714-21). Against a cell surface epitope of EGP there is a monoclonal antibody with the name Ber-EP4 (Latza U, Niedobitek G, Schwarting R, Nekarda H, Stein H. Ber-EP4: new monoclonal antibody which distinguishes epithelia from Mesothelia, J Clin Pathol 1990; 43: 213-9).

This monoclonal antibody is modified in two ways for the immune FFE: A) A charge carrier 4, 5 is directly and covalently coupled to the antibody (the binding molecule 3). B) Streptavidin is covalently coupled to the antiserum. Freely selectable, biotinylated charge carriers 4,5 are then bound to the streptavidin.

To isolate circulating tumor cells, citrated or heparin whole blood is obtained from patients with e.g. Breast, ovarian or colon carcinoma either used directly or prepared as follows:

1. Isolation of the white blood cells (mononuclear cells, MNC) via a density gradient, using methods known per se.

2. Add the correspondingly modified antibodies with charge carriers 4,5.

3. Incubation using methods known per se. 4. Separation of the cells in the immune FFE:

In a first experiment, separation conditions and separation medium were chosen, as described in Bondy B., Bauer J., Seuffert I. and Weber G. (1995) "Sodium Chloride in the Separation medium enhances cell compatibility of Free Flow Electrophoresis", Electrophoresis 16: 92-97 are known. This publication is expressly referred to here.

In a further experiment, a separation medium was used, as described in the international application PCT / EP01 / 10036. This application is expressly referred to here. Example B: Isolation of Peroxysomes

Peroxysomes are cell organelles that carry out oxidative reactions with molecular oxygen. They generate oxygen peroxide for oxidative purposes.

Highly enriched peroxysomes can e.g. from rat liver using an expensive density gradient centrifugation according to the method of Lüers et al. Isolation (Lüers GH, Hartig R, Mohr H, Hausmann M, Fahimi HD, Cremer C, Völkl A. Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 1998; 19: 1205-210):

1. Homogenization of rat liver using methods known per se.

2. Centrifugation of the homogenate at 1,900 x g to remove cell nuclei, cell debris, mitochondria and cells that are still intact. 3. Centrifuge the supernatant at 25,000 x g.

4. The resulting pellet then contains the so-called "light microsomal fraction" (including peroxysomes, microsomes and some mitochondria with lysosomes) and thus represents a mixture which also includes the peroxysomes.

There is already a method for enriching peroxysomes with a modified immune FFE (Voelkl A, Mohr H, Weber G, Fahimi HD. Isolation of peroxisome subpopulations from rat liver by means of immune free-flow electrophoresis. Electrophoresis 1998; 19 : 1140-1144). However, this known method for enriching peroxysomes with immune FFE is modified as follows by introducing charge carriers 4, 5:

1. Homogenization of the rat liver as known.

2. Centrifugation at 400 xg to remove cell nuclei, cell debris and cells that are still intact. 3. The supernatant is treated with an antibody modified with charge carriers 4.5 against a membrane protein of the peroxysomes (eg PMP70; 70 kDa cytoplasmic membrane protein of peroxysomes, antibodies) against the C-terminal, eleven amino acids long, cytoplasmic end). 4. The separation with immune FFE was carried out as described by A. Völkl, H. Mohr, G. Weber and HD Fahimi (Electrophoresis 1998, 19, 1140-1144), at a separation medium temperature of 4 ° C. and at a pH of 8.0 in an electric field of 100,000 V and 100 mA and a separation medium flow rate of approx. 5 ml / fraction per hour. The samples were introduced into the separation chamber at about 2 ml / hour and the separated fractions were collected using a 96-channel peristaltic pump. Each individual separation attempt took about 60 to 90 minutes.

Example C: Isolation of recombinant proteins

For the expression of recombinant proteins, there are vectors which can attach a cassette of 5-10 histidines to the protein to be expressed at the 5 'or 3' end of a construct (His tags, see FIG. 5). These additional histidines are used for later purification of the expressed proteins. Other vectors include e.g. Glutathione-S-transferase as an additional element. These vectors are suitable for the expression of recombinant proteins e.g. in E. coli, baculovirus-infected bacteria and mammalian cells. Appropriate methods for producing recombinant proteins are known to the person skilled in the art.

The current possibilities for the purification of recombinantly expressed proteins are mainly based on affinity chromatography methods. These methods are relatively easy to use and - depending on the amount of proteins to be purified - scalable. However, the affinity chromatographic purification of recombinant proteins has several significant disadvantages: 1. Bleeding out of the affinity matrix 2. Clogging of the columns due to the sample matrix (cell lysates)

3. Unspecific binding of proteins

4. Enzymatic Lysis of Proteins (Proteolysis) The person skilled in the art is familiar with the purification of recombinant proteins using poly-His tags using immobilized Ni-NTA (nickel nitrilotriacetic acid) chelates. The solid phase can be packed in preparative purifications in chromatography columns (eg Ni-NTA agarose), for analytical applications (eg Ni-NTA) magnetic particles are available.

The immune FFE according to the invention shown in FIG. 5, which comprises the following steps, is particularly suitable for the purification of recombinant proteins:

1. Lysis of the cells (e.g. yeast, bacteria, mammalian cells) according to a known method.

2. Binding of charge carriers 4,5 to the His-tagged proteins: Variant 1: Binding of the charge carriers 4,5 to a binding molecule 3 in

Form of Ni-NTA and binding of this charged binding molecule 6 to the His tags; or variant 2: binding of the charge carriers 4,5 to a binding molecule 3 in

Form of an anti-poly-His antibody and binding of this charged binding molecule 6 to the His tags.

3. Incubation according to a known method.

4. Separation by means of FFE (qualitatively but also quantitatively possible): In a first experiment, separation conditions and separation medium were selected as described in Bondy B., Bauer J., Seuffert I and Weber G. (1995) "Sodium Chloride in the Separation medium enhances cell compatibility of Free Flow Electrophoresis ", Electrophoresis 16: 92-97 are known. This publication is expressly referred to here. In a further experiment, a separation medium was used, as described in the international application PCT / EP01 / 10036. This application is expressly referred to here.

5. Collect the isolated His-tagged proteins

The His-tagged proteins can either now be used directly or the His-Tags are cleaved off enzymatically before further use.

There are several ways to separate the His tags from the proteins: For variant 1: a) detachment of the chelate by known incubation with imidazole and, if necessary, renewed purification of the protein for FFE; b) Known enzymatic cleavage of the His tag with thrombin or exoprotease and, if necessary, renewed purification of the isolated protein for FFE.

For variant 2: c) detachment of the antibodies using methods known per se, e.g. by changing the salt concentration or pH. d) Enzymatic cleavage of the His tags using methods known per se. e) Clean again with FFE.

The charge label or the charge carriers or the agent carrying the charge carrier (for example biomolecule) can also be additionally marked (for example with a dye). This enables detection during or after the FFE. Charge carriers could also be all types of particles (eg beads of all types, such as latex, agarose particles, colloids, etc.). Charge carriers can also be virtually any combination of beads and / or antibodies and / or charge-carrying molecules and / or fluorescent markers and / or dyes. Beads can, for example, be colored with fluorescent or other dyes. Beads can simultaneously carry a fluorescent label (inside or outside), the charge label and the binding molecule (eg antibody or Ni-NTA). If beads are used which have an enzymatically cleavable bridge, then after cleaning (eg cleaning of recombinant proteins with Ni-NTA) the beads can be cleaved from the purified molecule. Beads that are fluorescent and that are provided on the surface with charge carriers and binding molecules are particularly preferred. Possible measures for maintaining the biological activity or vitality of bioparticles in the various FFE separation techniques are shown in Table 1 below:

Explanations: PZE: continuous free flow zone electrophoresis CITP: continuous free flow isotachophoresis CHIEF: continuous isoelectric focusing +++ = unlimited use

+ + = only slightly restricted use _. + = only applicable in special cases

= Application not possible

The separation of particles according to the invention enables concentrated collection or enrichment of biologically relevant particles, the analysis or use of which in diagnostic or therapeutic processes has not previously been possible because of the background of too many related but irrelevant particles present at the same time. The FFE methods and devices presented thus enable isolation and consequently the use of particles naturally present in extremely low concentrations or the provision of their biologically relevant information for use in diagnostic or therapeutic processes. In addition, the isolated particles can be used to find and characterize targets for the development of medicines, targets for use serve in diagnostics, therapy selection and therapy monitoring for humans as well as for animals and plants.

Applications of the results obtained include, for example, tumor cells isolated from whole blood for target identification in the development of drugs against migrating _. Tumor cells.

The same reference numerals apply to the same features in all figures, even if they are not expressly cited.

The disclosed methods according to the invention are - if appropriate after appropriate adaptation - also suitable for use in a wide variety of areas of chromatography, for example in the affinity chromatography of organic or inorganic molecules and polymers.

Claims

claims

1. Free-flow electrophoresis device for separating particles (1, 1 ', 1 ") with at least one separation space (14) through which a separation medium (8) can flow and which is separated by a base and a cover and by these two spacers holding one another is limited, this FFE device also having a metering pump for conveying the separating medium (8), which is supplied via media feeds (15, 15 ') to the

Separation space (14) arrives and leaves it again via outlets (16), electrodes (9, 10) for applying an electric field in the separation medium (8) and sample application points (17) for adding a mixture of particles (1, 1 ', 1 ") and fractionation points (18) for removing particles separated by means of the FFE in the separation medium (8), the device selectively selectable charge carriers (4, 5) for combining with particles to be separated (1 ', 1") and for Generation of charge-modified particles (7 ', 7 ") which - because of their selectively changed net surface charge - have a different migration behavior in the FFE than non-charge-modified particles (7), characterized in that the Device or guide or focusing pad (12, 13) arranged between the separating medium (8) and at least one electrode (9, 10), said focusing pad (12, 13) consisting of a pad medium (20) which is opposite the separating medium (8) has a greatly increased electrical conductivity and this device comprises separate channels (15 ') for feeding this cushion medium (20) to the focusing pads (12, 13).

2. FFE device according to claim 1, characterized in that in the cushion medium (20) with the areas (19) in the vicinity of the anode (10) and cathode (9) a faster first flow movement than in between the areas (19) Separation medium (8) can be generated, the two flow movements being rectified.

3. FFE device according to claim 1 or 2, characterized in that the strength of the electric field generated with this device during the operation of the FFE device to a selectively changed net surface charge of the particles to be separated (1,1 ', 1 ") is customizable.

4. FFE device according to claim 1, 2 or 3, characterized in that it comprises separating means with which the net surface charge of the particles to be separated (1,1 ', 1 ") prior to the operation of the FFE device to the starch of the electric field that can be generated with this device is adaptable.

5. FFE device according to one of the preceding claims, characterized in that it comprises separating means with which the net surface charge of the particles (1,1 ', 1 ") for essentially simultaneously, selectively moving a first fraction of these particles in the direction towards the cathode (9) and a second fraction towards the anode (10) is changeable.

6. FFE device according to claim 4 or 5, characterized in that these separating means comprise charge carriers (4, 5) bound to binding molecules (3) which have a strongly positive or negative charge.

7. FFE device according to claim 6, characterized in that the

Charge carriers (4,5) are covalently bound to the binding molecules (3).

8. FFE device according to one of the preceding claims, characterized in that the metering pump additional delivery channels, or that the FFE device another metering pump for conveying a

Auxiliary medium (21), which enters the separation space (14) via auxiliary medium feeds (22) and leaves it again via fractionation points (18) and outlets (16). Process for separating particles (1,1 ', 1 ") - in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof - in particular using a free-flow electrophoresis device according to one or more of claims 1 to 8, which has a separation space (14) which is delimited by a base and a cover and by these two spacers which are spaced apart from one another, a metering pump for conveying a separation medium (8) which is supplied via media feeds (15, 15 ') enters the separation space (14) and leaves it again via outlets (16), electrodes (9, 10) for applying an electrical field in the separation medium (8) and sample application points (17) for adding a mixture of particles and fractionation points (18) to be separated. for removing particles (1,1 ', 1 ") separated by means of the FFE in the separation medium (8), which comprises the following steps:

• Providing a separation medium (8)

• conveying the separation medium (8) by means of the metering pump via the media feeds (15, 15 ') into the separation space (14);

• Flow through the separation space ( _. 14) with the separation medium (8); • fractionation of the separation medium (8) via discrete outlets (16);

• application of an electric field in the separation medium (8);

• Combining a particle to be separated (1 ', 1 ") with selectively selectable charge carriers (4, 5) to produce charge-modified particles (7', 7") which - because of their selectively changed net surface charge - are different in the FFE Have migration behavior, as non-charge-modified particles (7);

• adding a mixture of particles (1,1 ', 1 ") to be separated to the separation medium (8) in the separation space (14) via the sample application points (17); • removing particles (1,1', 1") separated by the FFE in the separation medium (8) via fractionation points (18);

Collecting the fractions with the separated particles; characterized in that guiding or focusing cushions (12, 13) with a cushion medium (20) are produced between the separation medium (8) and at least one electrode (9, 10), this cushion medium (20) being opposite the separation medium (8) has greatly increased electrical conductivity and is supplied to these focusing pads (12, 13) via separate channels (15 ').

10. The method according to claim 9, characterized in that during the operation of the FFE device, the net surface charge of the particles (1,1 ', 1 ") is adapted to the strength of the electric field or that the strength of the electric field to the changed surface charge of the particles (1,1 ', 1 ") is adjusted.

11. The method according to claim 9, characterized in that prior to the operation of the FFE device, the net surface charge of the particles

(1,1 ', 1 ") is adapted to the strength of the electric field or that the strength of the electric field is adjusted to the changed surface charge of the particles (1,1', 1") beforehand to operate the FFE device.

12. The method according to any one of claims 9 to 11, characterized in that this selective change in the net surface charge is generated with charge carriers (4, 5) bound to binding molecules (3).

13. The method according to claim 12, characterized in that an epitope-specific antibody or a Ni-NTA is used as a charged binding molecule (6), which are immobilized on corresponding epitopes (2 ', 2 ") on the particle surfaces.

14. The method according to any one of claims 9 to 13, characterized in that the charge carrier (4,5) has a strongly positive or negative charge, is formed as a bead, latex or agarose particles, molecule and the like.

15. The method according to any one of claims 9 to 13, characterized in that one or more charge carriers (4,5) or polymers thereof are selected from a group comprising: lysine, arginine, histidine,

Aspartate and glutamate.

16. The method according to any one of claims 9 to 15, characterized in that the charge carrier (4,5) is covalently bound to the epitope-specific antibody or to a Ni-NTA molecule.

17. The method according to any one of claims 9 to 16, characterized in that it comprises the use of one or more binding molecules (3) which are selected from a group comprising: neutravidin, streptavidin / biotin and secondary antibody systems and chelating ner.

18. The method according to any one of claims 9 to 17, characterized in that a first group of particles having a negative net surface charge in the electrical field of the separation medium (8) in the direction towards the anode (10) and - substantially synchronously to this - a second particle group having a positive net surface charge is moved in the electrical field of the separation medium (8) in the direction towards the cathode (9).

19. The method according to any one of claims 9 to 18, characterized in that during operation of the FFE device in the cushion medium (20) with the areas (19) in the vicinity of the anode (10) and cathode (9) a faster first flow movement than is generated in the separating medium (8) lying between the regions (19), the two flow movements being in the same direction.

20. The method according to one or more of claims 9 to 19, characterized in that by setting a certain pH in the separation medium (8) or in parts thereof with a buffer system on the charge carriers (4) or polymers thereof, which are selected from a group comprising: lysine, arginine and histidine, a positive

Charge is pronounced.

21. The method according to one or more of claims 9 to 20, characterized in that by setting a certain pH in the separation medium (8) or in parts thereof with a buffer system on the charge carriers (5) or polymers thereof, which are selected from a group comprising: aspartate and glutamate, a negative charge is pronounced.

22. The method according to one or more of claims 12 to 21, characterized in that for covalent binding to the binding molecules (3) biomolecules are used, which are selected from the group comprising primary, secondary antibodies, avidins, receptors and enzymes.

23. The method according to one or more of claims 12 to 21, characterized in that for the covalent binding to the binding molecules (3) ligands are used, which are selected from the group comprising biotin, steroids and enzyme substrates.

24. The method according to one or more of claims 9 to 23, characterized in that with this method particles (1,1 ', 1 ") are isolated which are used to characterize or represent targets for the development of medicaments or for the Use in diagnostics and / or therapy selection and / or therapy monitoring for

Serve people, animals or plants.

25. The method according to one or more of claims 9 to 24, characterized in that a selective depletion or depletion of particles from a particle mixture is generated with this method.

26. Separating agent for separating particles (1,1 ', 1 ") by means of the free-flow electrophoresis technique in an FFE device, in particular in an FFE device according to one of claims 1 to 8, with at least one with Separation medium (8) flowable separation space (14), which is delimited by a bottom and a cover and by these two spacers which are spaced apart from one another, this FFE device also having a metering pump for conveying the separation medium (8), which is supplied via media feeds (15, 15 ') enters the separating space (14) and leaves it again via outlets (16), electrodes (9, 10) for applying an electrical field in the separating medium (8) and sample application points (17) for adding one to be separated Mixture of particles (1,1 ', 1 ") and fractionation points (18) for removing particles separated by means of the FFE in the separation medium (8), the device selectively selectable charge carriers (4,5) for combining with those to be separated Particles (1 ', 1 ") and for producing charge-modified particles (7', 7") which - because of their selectively changed net surface charge - have a different migration behavior in the FFE than non-charge-modified particles (7) , characterized in that it is an electrically highly conductive cushion medium (20) which has a greatly increased electrical conductivity compared to the separation medium (8) and which via separate conveying channels (15 ') for forming at least one electrically highly conductive guide or focusing pad (12, 13) for the charge-modified particles (7 ', 7 ") between the electrodes (9, 10) and the separation medium (8) can be introduced into the regions (19) of the separation medium (8) near the electrodes.

7. Separating agent for separating particles (1,1 ', 1 ") by means of the free-flow electrophoresis technique in an FFE device, in particular in an FFE device according to one of claims 1 to 8, with at least one with Separation medium (8) flowable separation space (14), which is delimited by a bottom and a cover and by these two spacers which are spaced apart from one another, this FFE device also having a metering pump for conveying the separation medium (8), which is supplied via media feeds (15, 15 ') enters the separating space (14) and leaves it again via outlets (16), electrodes (9, 10) for applying an electrical field in the separating medium (8) as well as sample application points (17) for adding one to be separated Mixture of particles (1,1 ', 1 ") and fractionation points (18) for removing particles separated by means of the FFE in the separation medium (8), wherein the device selectively selectable charge carriers (4,5) for combining with those to be separated Particles (1 ', 1 ") and for producing charge-modified particles (7', 7") which - because of their selectively changed net surface charge - have a different migration behavior in the FFE than non-charge-modified particles (7) , and an electrically highly conductive cushion medium (20) which has a greatly increased electrical conductivity compared to the separation medium (8), this cushion medium (20) via separate delivery channels (15 ') for forming at least one electrically highly conductive guide or Focusing cushion (12, 13) for the charge-modified particles (7 ', 7 ") can be introduced between the electrodes (9, 10) and the separation medium (8) in the regions (19) of the separation medium (8) near the electrodes, characterized in that it is a charged binding molecule (6) with which the net surface charge of the particles (1,1 ', 1 ") for essentially simultaneously selectively moving a first fraction of these particles towards the cathode e (9) or a second fraction in the direction of the anode (10), can be changed.

28. Release agent according to claim 27, characterized in that the charged binding molecule (6) comprises a binding molecule (3) and a charge carrier (4, 5) with a strongly positive or negative charge.

29. Release agent according to claim 28, characterized in that the charge carrier (4, 5) is designed as a bead, latex or agarose particle, molecule and the like and is bound to the binding molecule (3).

30. Release agent according to claim 28 or 29, characterized in that the binding molecule (3) is an epitope-specific antibody or a Ni

Is an NTA molecule which can be immobilized on the particle surfaces.

31. Release agent according to one of claims 27 to 30, characterized in that the charged binding molecule (6) or the binding molecule (3) has a detectable marking, in particular a dye or phosphor, for detecting the current position of the charge-modified particles (7 ' , 7 ") in the separation medium (8).

32. Release agent according to one of claims 27 to 31, characterized in that the charged binding molecule (6) comprises one or more charge carriers (4, 5) or polymers thereof, which are selected from a group comprising: lysine, arginine, histidine , Aspartate and glutamate.

33. Release agent according to one of claims 28 to 32, characterized in that the charge carrier (4,5) is covalently bound to the binding molecule (3).

4. Release agent according to one or more of claims 28 to 33, characterized in that the binding molecule (3) is selected from a group comprising: neutravidin, streptavidin / biotin and secondary antibody systems and chelating agents.