WO2001037959A1

WO2001037959A1 - Method for separating cells and biomolecules using counter-current chromatography

Info

Publication number: WO2001037959A1
Application number: PCT/EP2000/011520
Authority: WO
Inventors: Renate BLÜTTERS-SAWATZKI; Peter Czermak
Original assignee: Scinu Tec Gmbh
Priority date: 1999-11-20
Filing date: 2000-11-20
Publication date: 2001-05-31
Also published as: AU2358101A; EP1231992A1

Abstract

The invention relates to a method for separating cells and biomolecules from mixtures containing said cells and biomolecules. To this end, counter-current chromatography is used with continuous sample delivery and sample drawing using solid, non-porous separation materials. The method is characterised in that only one separating fluid is used so that said separating fluid is not changed, that non-porous separation materials with a modified or non-modified surface are used and that weak affinity interactions are exploited in order to separate the desired cells and biomolecules, the cells to be separated and the biomolecules undergoing said weak affinity interactions with the modified or non-modified surface. The term weak affinity interaction is used to describe those interactions, in which the bonds have a dissociation constant greater than or equal to 10-5 M (Kd ≥ 10-5 M).

Description

Process for the separation of cells and biomolecules by means of countercurrent chromatography

DESCRIPTION

The invention relates to a method for the separation of cells and biomolecules from mixtures which contain these cells and biomolecules by means of continuous countercurrent chromatography with continuous sample application and removal using solid separation materials.

Numerous chromatographic processes have already been developed for the purification of biological substances or substance mixtures. These include, for example, HPLC, anion exchange chromatography, gel filtration, affinity chromatography and hydrophobic matrix chromatography.

These processes were primarily developed for analytical purposes and mostly also meet the requirements placed on them in an excellent manner, namely to ensure optimum separation and high resolution. However, due to the column material, high pressures or special matrices must be used in these processes, as well as drastic elution media. In particular, the frequently required salt concentrations for some of the elution media used lead to problems in practice since the salt often has to be removed from the products again using energy-intensive processes.

If the processes described are used in production and in particular for the production of bulk goods, then other aspects and in particular economic aspects play an important role. The most important factors for low production costs are moderate operating pressures, high flow rates, fast elutions and low salt loads.

For production on an industrial scale, separations with purity levels of less than 90% can also be accepted, if this significantly reduces production costs. However, purity levels greater than 90% are desirable for analytical purposes. Of particular interest on the industrial scale are therefore those processes which entail low process costs, even if this takes place at the expense of lower separation specificities.

Another disadvantage of the chromatographic methods described above is that they are mostly batch, i.e. H. discontinuously. This is disadvantageous in terms of high production output.

These methods also include those that, for. B. work with affinity chromatography. In these known methods, mild application media are used, from which the substance to be obtained is removed by relatively strong binding to the separation medium. The subsequent selective detachment from the separation medium then always requires the use of a relatively drastic so-called elution medium, with the aid of which the substance sought can then be obtained in a second separate step. Such elution media are usually very rich in salt, and therefore the substances sought have to be freed from the salt load in a very complex manner.

For example, DE 1 9938394 A1 and DE 3930947 A1 describe affinity chromatography methods which use absorption on surfaces. The molecules to be separated, such as antibodies, are covalently bound to the separation surfaces by means of strong bonds. Due to the strong connection to such surfaces, the selective detachment of the corresponding substances can only take place by using specific elution media, the substance previously bound to the support being detached by this elution medium in a second separate step. Thus, such methods cannot be used for continuous Chromatography methods, such as the countercurrent method described above, can be used.

Continuous chromatographic processes have therefore already been developed in which the fluid to be chromatographed or a multicomponent mixture is fed continuously and the separated fractions are also continuously removed. Some of these are so-called countercurrent processes. A representative of such a countercurrent process is, for example, the so-called simulated moving bed process (SMB). Such methods are described, for example, in US-A-2985589 and US-A-3231492. The so-called True Moving Bed (TMB) process also belongs to these continuous countercurrent processes. The time-dependent chromatographic separation of the column chromatography is converted into a spatially resolved separation. With regard to the theoretical foundations for SMB chromatography and their use in the production sector, in particular for carrying out separations of optical isomers on a large scale, reference is made to M. Schulte, J.N. Kinkel, R.M. Nicoud and F. Charton in Chem.Ing.Tech. 68 (1996), 670-683 "Simulated countercurrent chromatography - an efficient technique for the production of optically active compounds on an industrial scale" and in the references listed there.

This known countercurrent process mainly uses porous, particulate separation media. However, such processes have also been used for non-particulate sorbents, in which, however, porous materials have always been used, as described, for example, in WO 9803242. For example, it is known to use a porous separation matrix with defined pores for the so-called gel chromatography as the separation medium. The delaying effect that causes the separation of the biomolecules is a different flow of the molecules through the pores or past the pores, depending on the size of the molecule.

The phenomenon of so-called weak interactions, which play an important role in cell-cell recognition in particular, is now known from biological processes. Lots of protein-protein, protein-carbohydrate and carbohydrate-carbohydrate

Interactions are examples of such weak interactions. Weak interactions are understood to mean those bonds, which are typically typical of biomolecules, that are not covalent in nature and are relatively unstable, such as e.g. B. the already known hydrogen bonds, hydrophobic and electrostatic bonds and van der Waals forces. The description of such weak interactions, as are known for many interactions between biomolecules, can be thermodynamically with the aid of the "heat capacity difference" (ΔC _P ), as described in Cooper (A. Cooper, Thermodynamic analysis of biomolecular interactions, Current Opinion in Chemical Biology 1999, 3: 557-563) or as with Dunitz (JD Dunitz, Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions, Chemistry and Biology 1 995, 2: 709-712) about the change in free energy (ΔG _f ) can be described. Areas around 5 kcal per mole for the difference in free energy in weak interactions are discussed. Various methods are known for measuring such weak interactions between proteins (protein-protein), such as surface plasmon resonance, isothermal titration calorimetry, optical spectroscopy or mass spectrometry, as is the case with Lakey and Raggett (JH Lakey and EM Raggett, Measuring protein-protein interactions, Current Opinion in Structural Biology 1998, 8: 1 1 9-123). For the determination of the binding constants in carbohydrate-carbohydrate interactions z. B. the method of affinity electrophoresis (P. Tomme et al. Affinity electrophoresis for the identification and characterization of soluble sugar binding by carbohydrate-binding modules, Enzyme and Microbial Technology 2000, 27: 453-458) can be used.

Many modern detection methods for biomolecules are based on sensor techniques using weak interactions (S. Ohlson et al. Continuous weak-affinity immunosensing, TIBTECH 2000, 18: 49-52); In this cited work it is also defined that weak interactions are to be understood as those whose dissociation constant K _{d is} in the range from 0.10 to 0.01 mM. Weak interactions as a separation principle have so far only been used in so-called affinity chromatography processes (S. Ohlson et al. Weak affinity chromatography of small saccharides with immobilized wheat germ agglutinin and its application to monitoring of carbohydrate transferase activity, Bioseparation 1998, 7: 101 -105) , The principle has already been described in a manual (P. Matejtschuk et al., Affinity separations of proteins, in: P. Matejtschuk (Ed.) 'Αffinity separations ", IRL-Press, Oxford, 1997, p. 93-94) In all of this work, weak interactions are understood to mean bonds in which the dissociation constant K _{d is} greater than 10 ^{~ 5} M.

The object of the present invention is accordingly to provide a method for separating cells and biomolecules from mixtures, in particular from complex mixtures, by means of countercurrent chromatography with continuous sample application and removal using solid non-porous separation materials, with which in the mixtures contained cells and biomolecules can be separated or separated, especially for production purposes and on an industrial scale.

This problem is solved by a method according to the teaching of the claims. A core element of the method according to the invention is that the weak interactions described in the context of the present documents are used. The above-discussed dissociation constant K _d , which according to the invention is greater than or equal to 10 ^{~ 5} M, is used to define these weak interactions. This definition is therefore used in the context of the invention to characterize weak interactions.

However, it must be expressly stated that the previously described techniques of using weak interaction for separation purposes have so far only been described for analytical processes and that this technique has so far not been used for preparative processes or even on an industrial scale.

In other words, in the process according to the invention, reversible adsorption on modified and unmodified surfaces on solid non-porous separating materials takes place using the weak interactions.

Such weak interactions are also described for cells; so z. B. in the delayed rolling of blood cells on blood vessel walls. So-called selectins are responsible for this as mediating molecules. A more detailed description of this process can be found in Lawrence (MB Lawrence, Selectin-carbohydrate interactions in shear flow, Current Opinion in Chemical Biology 1999, 3: 659-664). Exactly the same interactions should form the basis for the separation principle described here: the cells are held on their surface by specific molecules as they flow through the separation units, but are not firmly bound and can therefore continue to flow with a delay. This selective (biospecific) delay of certain cells in the flow due to short-term weak connection to specific molecules of the separation surfaces and the detachment represent the separation principle described. Weak interactions are understood to mean, in particular, those which interact with the cell-cell recognition, the protein-protein interaction, the protein-carbohydrate

Interaction and the carbohydrate-carbohydrate interaction are based and the dissociation constant K _{d is} greater than or equal to 1 0 ^"5 M.

In contrast to the known chromatography processes described at the outset, which are carried out batchwise and therefore not continuously, the process according to the invention is a continuous process or a continuous process which works according to the countercurrent process, with only a single liquid process Separation medium is used.

By using the weak interactions used according to the invention, it is thus possible for the first time to use continuous processes with countercurrent processes, since only a single separation medium is used here, in the simplest case even the raw material in its natural form (e.g. fermentation supernatant) directly and can be fed to the chromatography process without the addition of salts. Since there is no strong binding of substances to the separation medium, such a process does not require any special elution media and can therefore be carried out as a continuous process.

Only the use of the weak interactions and thus the elimination of a separate elution medium enable a continuous process since the separation medium does not have to be changed.

Another essential element of the process according to the invention is that, in contrast to the prior art, no porous separating materials are used. In other words, it will be thus separating materials with an impermeable and non-porous surface are used. Non-porous separating materials (non-porous supports) are understood to mean those materials that are already partially used in materials such as nylon, polystyrene, silicate glass (AL Plant et al., Immobilization of binding proteins on nonporous supports. Comparison of protein loading, activity , and stability, Appl Biochem Biotechnol 1991, 30: 83-98), 1, 5 micrometer silica gel (PD Angus et al., Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis, Electrophoresis 1999, 20 : 2349-59), modified silica gels (FB Anspach et al. High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas, Anal Biochem 1989, 179: 171-81), alkylated styrene-divinylbenzene copolymers (CG. Huber et al., High-resolution liquid chromatography of oligonucleotides on nonpo rous alkylated styrene-divinylbenzene copolymers, Anal Biochem 1993, 212: 351 -8), or agarose (JP Li, Hjerten, S. High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormones as ligand and a cobination of ethylene glycol and salt for desorption of the antibodies, J Biochem Biophys Methods 1991, 22: 311-22). For non-particulate, non-porous separating materials, there are only a few examples, such as. B. Quartz fibers (P. Wikstrom. Larsson PO, Affinity fiber - a new support for rapid enzyme purification by high-performance liquid affinity chromatography, J Chromatogr 1987, 388: 123-34). However, all of these materials, even in their non-porous form, have so far been used neither for continuous chromatography processes nor for industrial processes. The applications described also did not relate to separations in which so-called weak interactions were used as the separation principle. In the method according to the invention, therefore, the already known retarding effect due to diffusion processes, which can be observed with porous materials, is used, but a completely different principle. In addition, a delaying effect is necessary in the method according to the invention for separating the molecules. However, this is achieved by the weak interactions described in more detail above, which the molecules to be separated enter into with the surfaces of the non-porous separation materials.

According to the invention, the surfaces can either be modified or not modified. The appropriate selection of the surfaces or their modification ensures that the corresponding molecules to be separated are retained by molecular adhesion effects on the corresponding surfaces, as a result of which the molecules are separated. The degree of separation can be affected by the density of the corresponding molecular attachment sites.

This separation effect may not always be sufficient for customary, batchwise chromatography. However, it must be taken into account here that the process according to the invention is a countercurrent chromatography process which is capable of achieving the desired separation.

The process according to the invention, which exploits the weak interactions described, has the advantage that the biomolecules can be separated without the use of drastic measures, for example high salt concentrations. High pressure differences can thus be avoided in the method according to the invention, so that a separation of cells from biological matrices can also be achieved. The separation takes place - as described - basically on surfaces that are formed on non-porous matrices or porous membranes.

The method according to the invention can be used in particular for the industrial separation of biomolecules and cells. For example, proteins, carbohydrates, nucleic acids, glycoproteins, glycolipids, vitamins, enzymes, lipids and bioactive substances can be used as biomolecules. In particular, hormones and phytoestrogens are suitable as bioactive substances. The cells used are preferably those from plant, animal or unicellular cell sources or from peripheral blood. It may be necessary to use different separation matrices for certain molecules of these substance classes. The separation materials are selected in such a way that the weak interactions are sufficient to separate the substances from one another without them permanently adhering to the surfaces. For this purpose, the density of the release-active surface components or adhesion sites is preferably set such that no additions of salts are required for the separation.

With regard to the separation of cells, the method according to the invention is primarily intended for the separation of peripheral stem cells from the blood of human donors. The blood is preferably introduced directly into a device for carrying out the method according to the invention, which is described in more detail below, while the cells in question are retarded on the corresponding surfaces in such a way that the stem cells can be removed continuously from the device, while the other blood components in the can be removed substantially unchanged at other points in the device.

Furthermore, cells which can be obtained from fermentations are preferably used as cells. It can be act plant, animal or unicellular organisms. According to the invention, primarily biomolecules and thus those molecules that come from biological sources are taken into account for the industrial separation processes. These biomolecules represent important starting products for the pharmaceutical industry and for the food industry. According to the invention, animal milk and dairy products, fermentation supernatants, molasses, plant extracts, residues from plant processing, blood products, wash water are therefore preferably used as the liquid to be chromatographed or as a multi-component mixture

Industrial productions and industrial residues used. All these sources have in common that they contain a variety of biomolecules, some of which, particularly when they are in a purified form, are extremely interesting for further economic use. The separation processes currently used for such molecules are so expensive that they are particularly uneconomical for mass production, for example in the food sector.

As already explained above, the separating materials used can have unmodified and also modified surfaces. The modification of surfaces is known per se and is used for the separation using porous, particulate separation media or using porous membranes. In contrast to

According to the invention, however, prior art is not carried out on the surface of porous separating materials, but on the surface of non-porous materials. Biological polymers are preferably used as separation materials with an unmodified surface. These preferably include celluloses, chitosans, dextrans,

Dextrins, cyclodextrins, starches, agaroses, poly-L-leucine and poly-DL-methionine. It should again be expressly pointed out that there are no pores in the surfaces used through which the liquid flow with the molecules present can flow.

Modified biological polymers are also preferably used as separating materials. These preferably include modified celluloses such as cellulose acetates, carboxymethyicellulose,

Cellulose acetate butyrate, ethyl cellulose, cellulose and other cellulose esters and mixed esters. Modified and unmodified plastics are also used as separating materials. These are preferably the following: polyethylene, polysulfones, polyether sulfones, polyamides, nylons,

PVC (polyvinyl chloride), polyacrylonitrile, polyaramid, polystyrenes including styrene / divinylbenzene polymers, polyester, polypropylene, teflone, PTFE, FEP, PFA, polyvinyl alcohols, polyvinylidene fluoride, polyvinyl difluoride, polyolefins, methacrylic polymers, polybenzimidazole, polyfuranethylene, polyphenols

Polybutadienes, polybenzimidazolone, polyether / amide, polyether / urea, polyether viscose, polydimethylsiloxane, silicone, siloxanes, polycarbonate, polyalkylsulfone, polyisoprene, polyethylene terephthalate and mixtures of two or more of these plastics.

Furthermore, modified and unmodified inorganic matrices are preferably used, these being, in particular, biocompatible materials such as carbon / carbon fiber composites, ceramics (TiO ₂ , Al ₂ O ₃ , ZrSiO ₄ , ZrO ₂ ), silicates, glasses and other matrices (e.g. Fabrics made of metal wire or glass fiber with or without surface modification), which are typical for chemical apparatus construction. It must also be taken into account here that the biomolecules and cells to be separated do not penetrate through or into the surfaces in order to avoid the possibility of uncontrolled absorption and unwanted pressure drops. The modification of the separation materials (more precisely the surfaces thereof) can be carried out using methods known per se. Such modifications are described, inter alia, in DE 1980 54 31 A1, wherein also and porous and also non-particulate separating materials are used. However, the method described therein clearly has the aim of producing affinity ligands for the purpose of affinity chromatography, which are those ligands which bind nucleic acids firmly and remove them from the medium by binding to this support. However, ligands which only have weak interactions with nucleic acids and thus only cause a delay in the eluent, as is the subject of the principle of weak interactions, are not used in DE 1 980 54 31 A1.

The possible methods of modification preferably include chemical linking (cross-linking). Groups present on the polymers, for example hydroxyl, carboxy, carboxyl, amino, amide, aryl, sulfhydryl, alkyl, methoxy and ethoxy radicals or groups, are activated by methods known per se as described, for example, in: E. Müller, E. Klein "Membranes Modified for Biochromatography" in: Bioseparation and Bioprocessing. Vol 1 (Editor: G. Subramanian), Wiley-VCH, Weinheim 1 997. After activation, then The required ligands are covalently bound to the surfaces, and the activation methods which can be used according to the invention include, for example, methods in which the following activation reagents are used: cyanogen bromide, tosyl chloride, N-hydroxy succinimide, carbonyl-di-imidazole, carbodiimides, 1, 4 -Butanediol-diglycidol ether, glutaraldehyde and 2-fluoro-1-methylpyridine If desired, so-called spacers can be introduced as attachment sites or binding sites for the weak interaction Can then attach to the activated surfaces preferably the following biomolecules are bound: polymeric carbohydrates (e.g. chitosans and dextrans), glycoproteins, glycopeptides, glycans, lectins, polymerized amino acids (e.g. poly-L-leucine), peptides, proteins, nucleotides and biomolecule-binding dyes.

The geometry of the surfaces of the separating materials on which the separation takes place can be designed in a very varied manner. The separating materials are washed or flushed by the liquid to be chromatographed. So z. B. smooth surfaces in the smallest possible distances in the form of narrow channels or tubes. Flexible hoses with small diameters can also be used. In order to be able to keep the distances at a defined dimension, so-called spacers in the form of threads, nets or precision fabrics are preferably inserted between the surfaces. These spacers can be made of the same material as the surfaces of the separating materials. Furthermore, tubes, hoses or winding units made of an inert material can also be used, in the lumens of which there are threads, nets or wick-like fabrics, the surface of which is as previously described. Irregular filter shapes, such as molten metal filters (e.g. from Drache, Dietz, FRG), which have a large surface area and are made of ceramic, chemically and thermally highly stressable material can also be used. The liquid flows run past these surfaces and do not penetrate them. Since the liquid flows do not encounter large resistances, large pressure drops can be avoided. This means that pumps and valves can also be used that are sufficient for medium pressure conditions and also less expensive than is the case for high-pressure equipment.

The device for carrying out the method according to the invention is provided with several so-called separation units, in which there are separating materials. The separation units are at least chromatographically essentially the same and preferably essentially identical. The separating materials used are preferably approximately the same in terms of shape, size and type and preferably essentially identical. A plurality of separation units are preferably connected in series in terms of flow and combined to form separation zones.

The inflow and outflow of both the entry and the discharge is done by means of valve units which are controlled by a computer and whose position is changed using a mathematical model, so that the weak separation effect based on the interactions is optimized in such a way that the separation of the substances can be done continuously. The material flows are carried in and out with the aid of several and typically four pumps. The flow and circulatory system, which is maintained by another pump, is designed like SMB chromatography. There are therefore several separation zones, each of which is assigned a certain number of separation units and which are coupled to the corresponding lines by valve circuits.

In the process according to the invention, normally not pure two-substance mixtures are separated, as is common in simulated moving bed chromatography. Rather, more complex mixtures of biomolecules are used according to the invention, in which, however, a separation into two components is usually sufficient. By repeating the separation into two components, it is ultimately also possible to display individual biomolecules, if this is desired. In this case, components are to be understood as meaning more complex mixtures. The design and control of the device for performing the method according to the invention is otherwise known. Such devices are also used in SMB chromatography and TMB chromatography. However, these known devices work with porous separating materials, as described above. The separation method according to the invention, which uses the weak interaction mentioned and in which the separation takes place on non-porous surfaces, can also be carried out as computer-controlled countercurrent chromatography by separation on surfaces or in English “Computer controlled counter current chromatography using separation on surfaces” or abbreviated “ C-5-S-2 ". Only the term "C-5-S-2" is used below.

This C-5-S-2 process is described in more detail with reference to Fig. 1 below. Fig. 1 shows the structure of a device for performing the method according to the invention in a highly simplified and very schematic manner, numerous details being omitted in order to be able to illustrate the principle of the method according to the invention more clearly and simply. However, the structure and the control shown there are - as shown - of a known type. In this regard, reference is made to the publications mentioned above.

The individual separation units A, B, C, D, E, F, G and H of the device shown in Fig. 1 are connected in series in terms of flow. Between these individual separation units there are inflows and outflows 1, 2, 3, 4, 5, 6, 7 and 8 to a large valve unit 10. The inflows and outflows 1 to 8 each have an inflow and an outflow; for the sake of simplicity, the two lines are only shown with a line 1 to 8 in the figure. At the mouths 11 to 18 of these inflows and outflows 1 to 8 into the circulation line 19, three-way valves are arranged, which are not shown. The valve unit 10 is computer-controlled and equipped with two inflows 21 and 22 and two outlets 23 and 24, with one inflow 21 for each Separation medium and an inlet 22 for the mixture to be separated and an outlet 23 for component 1 and an outlet 24 for component 2 are present.

In the embodiment shown in Fig. 1, two separation units A, B; C, D; E, F and G, H connected to separation zones AB, CD, EF and GH. The inflows and outflows are switched in such a way by the valve arrangement that an inflow and outflow always alternate. The first separation zone AB is thus between the inflow of the separation medium and the outflow of component 2 (extract). The second separation zone CD is located between the outflow of component 2 (extract) and the inflow of the mixture to be separated. The third zone EF is here between the supply of the mixture to be separated and the outflow of component 1 (raffinate). The fourth zone GH is located between the outflow of component 1 (raffinate) and the inflow of the separation medium. As explained, two separation units are connected to a separation zone. However, this type of circuit is only one embodiment. Of course, the separation units can also be used alone or connected in groups of three, four, etc.

The central fourfold distributor valve unit 1 0 is controlled with the aid of a computer program and can have different mechanical structures. For example, it can be designed as a rotary multiple valve or can be constructed from several triple or single valves. It is only important that the valves are opened and closed in such a way that the separation zones AB, CD, EF and GH are switched on in the direction of flow of the mobile or liquid phase in such a way that a quasi counterflow to the solid phase is generated. The pumps are preferably controlled by a computer program in such a way that the flow rates in the separation zones are regulated in such a way that only the pure components 1 and 2 are discharged. It is a complex controlled process in which the suitable parameters are expediently determined by computer simulation. The mathematical foundations that have already been developed for simulated moving bed chromatography can be used, namely: Gottschlich, N. et al., J. Chromatogr. 719: 267-274 (1994); Zong, G. et al. AlChEJ. 43, 2960-2969 (1997); Zong, G. and Guiochon, G. Chem. Eng. Be. 52,4403-4418 (1997); Yun, T. et al. AlChEJ 43, 2970-2983 (1997).

The method according to the invention is explained in more detail with reference to the following examples which describe preferred embodiments.

Example 1 :

Separation of peripheral stem cells from a donor's blood

With the help of the C-5-S-2 method, stem cell separation is carried out continuously from the blood of a donor. The separation units are manufactured as follows:

Teflon tubing 8 mm in diameter is terminated at a length of 40 centimeters at the ends with stainless steel nets, and the tapered ends are each connected to pumps or valves with thinner tubing. A wick-like nylon fabric is inserted between the ends as a separating material, on which the glycan sialyl-Lewis A is coupled as an adhesion phase by activation with cyanuric chloride and dicyclohexylcarbodiimide. The group density was chosen so that less than 0.1 μmol of the ligand is bound per gram of nylon. Such a piece of Teflon tubing represents a separation unit. A total of 8 separation units are connected in series, the separation zones each consisting of 2,2,3,1 units. Separation zone 1 (separation medium / stem cell extract) consists of 2 units. Separation zone 2 (stem cell extract / blood flow) consists of 2 units. Separation zone 3 (blood inflow / blood outflow) consists of 3 units. Separation zone 4 (blood drain / separation medium) consists of 1 unit. Isotonic saline is used as the separation medium. The entire apparatus is heated to 37 degrees Celsius.

In this way it is possible to obtain around 10 million stem cells from the peripheral blood of a donor within 2 hours. Example 2:

Separation of lactoferrin from cow whey

Lactoferrin is separated from cow whey using the C-5-S-2 process. The separation units are manufactured as follows. Spacers (nets made of woven cellulose acetate fibers of 0.5 mm diameter) are inserted into tubes of 50 mm diameter made of cellulose acetate and wound flat on cores in lengths of 3 m. The tubing is non-porous and the ends are each sealed and tapered with plastic frit so that the appropriate inflow and outflow tubing can be connected to the units. The cellulose acetate surfaces are activated with the aid of 1.1 carbonylimidazole and chitosans are covalently bound to them. The group density is approximately 1.0 mmol per gram of cellulose acetate. 10 separation units are connected in series, an arrangement 1, 4, 4, 1 being used. Separation zone 1 (separation medium / lactoferrin outflow) is 1 unit, separation zone 2 (lactoferrin outflow / inflow whey) 4 units, separation zone 3 (inflow whey / outflow residual whey) 4 units and separation zone 4 (outflow residual whey / separation medium) ) 1 unit in size.

10 mM saline solution is used as the separation medium. In this way it is possible to produce between 10 and 30 kg of lactoferrin continuously per hour.

Claims

1. Method for the separation of cells and biomolecules from mixtures containing these cells and biomolecules by means of countercurrent chromatography with continuous sample application and removal using solid

Separation materials, characterized in that only a single separation medium is used and thus there is no change of the separation medium, that non-porous separation materials with a modified or unmodified surface are used and that so-called weak interactions are used to separate the cells and biomolecules to be separated, which the cells and biomolecules to be separated enter into with the modified or unmodified surface, weak interactions being understood to mean those in which the bonds have a dissociation constant of greater than or equal to 10 ^"5 M (K _d > 10 ^{" 5} M).

2. The method according to claim 1, characterized in that weak interactions are used, which are based on the cell-cell recognition or correspond to the protein-protein interaction, the protein-carbohydrate interaction or the carbohydrate-carbohydrate interaction.

3. The method according to claim 1 or 2, characterized in that proteins, carbohydrates, nucleic acids, glycoproteins, glycolipids, vitamins, enzymes, lipids, and bioactive substances are used as biomolecules and those from plant, animal or unicellular cell sources or from peripheral blood are used as cells become.

4. The method according to claim 3, characterized in that hormones are used as bioactive substances, those from the fermentation as cells and those from humans as peripheral blood.

5. The method according to any one of the preceding claims, characterized in that biological polymers, modified biological polymers, modified or unmodified plastics, modified or unmodified inorganic matrices are used as separating materials.

6. The method according to claim 5, characterized in that as biological polymers celluloses, chitosans, dextrans, dextrins, cyclodextrins, starches, agaroses, poly-L-leucine and

Poly-DL-methionine, cellulose modified as modified biological polymers such as cellulose acetates, carboxymethyl cellulose, cellulose acetate butyrate, ethyl cellulose, cellulose and other cellulose esters and mixed esters, as modified and unmodified plastics polyethylene, polysulfones, polyether sulfones, polyamides, nylones,

PVC (polyvinyl chloride), polyacrylonitrile, polyaramid, polystyrenes including styrene / divinylbenzene polymers, polyester, polypropylene, teflone, PTFE, FEP, PFA, polyvinyl alcohols,

Polyvinylidene fluoride, polyvinyl difluoride, polyolefins,

Methacrylic polymers, polybenzimidazole, polyfuran,

Polyphenylene oxide, polyethyleneimine, polyacrylates, polybutadienes, polybenzimidazolone, polyether / amide, polyether / urea, polyether-viscose, polydimethylsiloxane, silicone, siloxanes,

Polycarbonate, polyalkyl sulfone, polyisoprene, polyethylene terephthalate and their mixtures and as modified and unmodified inorganic matrices carbon / carbon fiber composites, ceramics (for example: TiO ₂ , Al ₂ O ₃ , ZrSiO ₄ , ZrO ₂ ), silicates, glasses and other matrices (such as: metal or glass mesh with / without modified surface), which are typical for chemical apparatus construction.

7. The method according to any one of the preceding claims, characterized in that separating materials with a modified surface are used, the modification being known per se

Process of chemical linking (cross-linking) was carried out and the groups present on the surface to be modified were activated with activation reagents known per se, and molecules from the group of the polymeric carbohydrates, the glycoproteins, of the polymer, were activated on the surfaces thus activated

Glycopeptides, glycans, lectins, polymerized amino acids, peptides, proteins, nucleotides or biomolecule-binding dyes.

8. The method according to claim 7, characterized in that the groups present are hydroxyl, carboxy, carboxyl, amino, amide, aryl, sulfhydryl, alkyl, methoxy and ethoxy groups the activation reagents for cyanogen bromide, tosyl chloride, N-hydroxy succinimide, carbonyl di-imidazole, carbodiimides, 1, 4-butanediol diglycidol ether,

Glutaraldehyde, 2-fluoro-1-methylpyridine), the molecules to be added in the form of the polymeric carbohydrates are chitosans and dextrans and the molecules to be added in the form of polymerized amino acids are poly-L-leucine.

9. The method according to any one of the preceding claims, characterized in that the separating materials with the modified or unmodified surface are used in separation units, are flushed or flushed with by the liquid to be chromatographed and are designed on the one hand as channels, tubes, flexible hoses or winding units, spacers made of the same separating material in the form of threads, nets, fabrics or wick-like fabrics can be inserted therein, or on the other hand in the lumen of tubes, channels,

Hoses or winding units made of an inert material as threads, nets, fabrics or wick-like fabrics, as well as irregular structures e.g. made of ceramic materials.

10. The method according to claim 9, characterized in that the separation units are at least substantially the same.

1 1. The method according to claim 9 or 10, characterized in that a plurality of separation units are connected in series in terms of flow and are interconnected to form separation zones.

12. The method according to any one of the preceding claims, characterized in that those biomolecules are bound to the surfaces of the separating materials, which can interact with blood cells and peripheral stem cells in the liquid to be chromatographed.

13. The method according to any one of claims 1 to 1 1, characterized in that animal milk as the liquid to be chromatographed, Fermentation supernatants, molasses, plant extracts, residues from plant processing, blood products, wash water from industrial productions and industrial residues are used.

14. Use of the non-porous separating materials described in claims 1 to 13 with a modified or unmodified surface for the separation of cells and biomolecules from mixtures which contain these cells and biomolecules by means of countercurrent chromatography with continuous sample application and removal using so-called weak interactions that the cells and biomolecules to be separated enter into with this surface.