WO1989011648A1

WO1989011648A1 - Process for separation and recovery of charged molecules

Info

Publication number: WO1989011648A1
Application number: PCT/US1989/002144
Authority: WO
Inventors: Barbara Schneider
Original assignee: Biosyn-R Corporation
Priority date: 1988-05-17
Filing date: 1989-05-17
Publication date: 1989-11-30
Also published as: EP0377016A1

Abstract

A process for the separation of charged molecules from a solution of complex mixtures which compromise the steps of (a) providing a chromatographic apparatus with an electrically conductive polymer; (b) passing an electrical charge through said polymer in an amount sufficient to cause binding of a charged molecule in the mixture on said polymer; (c) treating said charged polymer with a solution containing complex mixtures; and, (d) recovering said charged molecules by varying either the current or potential applied to said polymer.

Description

- I -

PROCESS FOR SEPARATION AND RECOVERY OF CHARGED MOLECULES

Field of the Invention

The present invention relates to a selective method for the purification and recovery of charged molecules from mixtures. More particularly, the present invention is concerned with the purification of horticultural factors or biologically active factors on a chromatographic and/or electrophoresis apparatus through the use of electrical current or voltage. The invention also provides an apparatus for performing electrical chromatography.

Background of the Invention

This application is a continuation-in-part of copending application U.S.S.N. 111,360, entitled

"Method for Producing Cells", filed October 2, 1987, by Barbara Schneider.

Purification of biologically active products has become a major industry. Isolation and purification of complex biological factors such as interleukins, interferon, monoclonal antibodies and hormones represent only a small part of biologically active materials obtained from tissue culture materials.

The article entitled "Large Scale Purification of Monoclonal Antibodies" by Christina Ostlund, Trends In Biotechnology. November 1986, p. 288-293, which is incorporated herein by reference, discloses biological mixtures that can be separated by means of the present invention. The article further discloses the drawbacks of some of the prior art techniques for purifying the monoclonal antibodies.

The article of G. Stewart et al entitled "Transverse Alternating Field Electrophoresis", Biotechniques, Vol. 6, No. 1 (1988) pages 68-75, which is incorporated herein by reference, discloses the use of pulsed-field electrophoresis in the separation of large DNA fragments. Also disclosed is the use of a transverse field wherein long DNA molecules are placed under the influence of two alternating fields.

Many protocols for purifying complex biological factors such as human gamma interferon have been found suitable for laboratory scale separations. However, the degree of purity yielded, the multiple dialyses, buffer exchanges and dilutions needed to make these protocols unacceptable for large scale production.

While volume reduction may not appear relevant at the lab bench, it is essential for production. Process volumes should be reduced as early as possible, using techniques that offer both high capacity and high selectivity. Ion-exchange chromatography is thus a very good first step because of its capacity (approximately 30 mg protein per ml) and relatively low cost. As with other adsorption techniques, ion exchange is relatively independent of feed volume. However, ion exchange chromatography does not provide a high degree of selectivity and much of the active factors remain on the adsorbent. Gel filtration, though a frequent first step at the laboratory scale, is not suitable for handling large volumes. When gel filtration is used to separate molecules of similar molecular weights, sample sizes may range only from 1 percent to 3 percent of the total gel volume. Thus, a 100-liter feed-stream would require a gel filtration column of 10,000-50,000 liters, whole the same 100-liter stream could possibly be applied to a 20-liter ion-exchange column, depending on the sample concentration and process conditions. If one is separating a large molecule from a small molecule—as in buffer exchange (or "desalting")—the applied sample volume may be up to 30 percent of the total gel volume. Affinity chromatography, the most selective technique, may be used fairly early in purification. There are drawbacks to using affinity chromatography if the affinity ligand is expensive and the feedstream fouls the media. The bulk of contaminants, such as lipids, should be removed first. If the ligand is proteinaceous, removing proteases before affinity chromatography is essential to preserving media life. Lipids are usually separated from proteins during an initial precipitation step, and proteases are usually removed during an initial ion exchange or hydrophobic interaction. Therefore, affinity chromatography must also employ the conventional purification techniques when the substrate is complex.

Electrophoresis provides an effective method for adsorbing or desorbing bound material. It is very useful for desorbing tightly bound material from affinity adsorbents and is especially useful for desorbing tightly bound substances such as antibodies and hormone-finding proteins. However, electrophoresis is dependent upon the selectiveness of the adsorbent utilized and the fluid medium through which the electrical current is passed.

The prior art procedures of separation do not provide a means for separating complex biological factors without rendering a significant portion of the molecule to be separated inactive.

In a paper entitled "Electrochemically Prepared Polypyrroles from Aqueous Solutions" by Qian, et al, Polymer Journal. Vol. 19, No.l, pp. 157-172 (1987), which is herein incorporated by reference, there is disclosed electrically conductive polypyrroles as well as other electrically conductive polymers which may be utilized in the process of the present invention.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and a method for the separation and recovery of commercially useful products from a substrate. More particularly, the invention provides a means for the separation of biologically inert and/or biologically active factors from a substrate wherein the biologically active factors may be in the form of cell homogenates of organ tissues,- or microorganisms, of plant material and/or of body liquids. In accordance with one embodiment of the invention, a chromatographic column is filled with an electrically conductive polymer and an electrical current sufficient to cause binding is passed through the polymer. The substance of interest in a suitable solvent is then eluted by altering the charges on the polymer. The amount and type of current used can readily be determined beforehand by preliminary runs.

Separation and purification utilizing alternating current, preferably 1-60 herz, has been found suitable for tumor cells, lymphocytes and epithelial cells. Direct current (0.5 to 5 milliampε) has been found to be advantageous for use with fibroblasts, lymphocytes, epithelial cells, yeast, reticular endothelial cells, and the like. It is also possible to achieve a desirable separation with these cell types.

In accordance with another embodiment of the invention, separation is achieved when a biospecific ligand is ionically attached to the chromatographic bed material by means of an electrical current or voltage while retaining a specific binding affinity for a factor of interest. The fact is then passed through the column and the factor bound to the ligand. After removal of impurities by eluting with a proper solvent, the factor alone or together with the ligand to which it is bound may be detached by a change of electrical charge. The selection of the ligand for the electrical chromatograph is influenced by two factors. First, the ligand should exhibit specific and reversible binding affinity for the factor to be purified. Second, it should have groups which allow it to covalently attach to the electrically charged matrix without destroying its binding activity.

It is possible in some cases to bind a ligand to the polymer to prevent binding of some charged molecules to the polymer. It is therefore an object of the invention to provide a means for the selective separation of charged molecules by an electrical charge which causes binding on the charged molecules to an electrically conductive matrix. It is a further object of the invention to provide for the selective separation of biologically active factors by binding the factors to electrically conductive polymers.

It is still a further object of the invention to provide a method for isolating enzymes and other proteins from microorganisms without deactivating the pharmacologically active factors.

It is yet a further object of the invention to provide a means for simultaneously separating cells and stimulating cell proliferation.

It is still another object of the invention to provide a low cost process for purifying biologically active factors which can be utilized in large scale operations.

DETAILED DESCRIPTION OF THE INVENTION The biological factors which can be separated in the process of the present invention may be animal, plant or microorganism. The animal factors include those derived from mammalian, avian and amphibian or bacterial, fungal or viral origin. Those of fungal origin include aspergillus and rhizopus. Bacterial microorganisms include and are not limited to Bacillus and Clostridium genera. The method of the invention is especially useful in the separation and purification of chemical products derived in the cultivation of E. Coli and yeast through biotechnological procedures. That is, not only can the biologically active factors from naturally occurring microorganisms and cells be separated, but also factors from microorganisms and cells which have been modified by genetic engineering techniques, such as transformation, DNA insertions, transduction, fusion and the like.

The invention is especially useful in the separation of active factors derived from human . diploid cells. The invention is adaptable to the separation and purification of all types of biological factors including, for example, the factors derived from mammalian, avian amphibian cells. Factors derived from embryonic, adult or tumor tissues as well as from established cell lines can thus be separated. Examples of typical cells from which the factors may be separated are primary rhesus monkey kidney cells, baby hamster kidney cells, pig kidney cells, mouse embryo fibroblasts, normal human lung embryo fibroblasts, HeLa cells, primary and secondary chick fibroblasts, and various cells transformed with SV-40 polyoma virus. Separation of the charged particles utilizing DC current is usually performed with about 0.5 μ amps to 15 amps. Alternatively the charged particles can be separated with the use of constant current, constant potential, alternating current alone or pulsed with direct current, transversed alone or in combination with direct current or together with electrophoresis.

For the AC current there is normally utilized 0.1 Hz to 60 Hz.

DC current is preferred for use in the separation of amino acids, proteins, glycoproteins, yeast, bacteria cells, nucleic acids, sugards, lipids, glycolipids, organelles, chromagens and inorganic charged molecules.

After suitable growth of the cells, the cells can be harvested and further treated for the production of desired products by various means. For example, human diploid foreskin fibroblasts cultured by the method of this invention, for example, see copending application U.S.S.N. 111,360 entitled "Method for Producing Cells" filed October 2, 1987 can be treated for the production of angiogenic factor, plas inogen activator and interferόn. The angiogenic factor alone can be isolated from the growth medium or from the cells. Plasminogen activator can be separated from a serum-free maintenance medium during a period of aging after the cells have reached their maximum density.

The electrically conductive materials which may be utilized in the present invention are the natural and synthetic polymers including gums which are normally utilized in the cultivation of cells and which are are electrically conductive or have been made electrically conductive by incorporation or intercalation of other polymers or materials. The aforementioned article of Qian, et al discloses such materials and how the material can be made electrically conductive. One way of preparing conductive polymers is by polymerizing in situ acetylene, pyrrole, and thiophene in a flexible matrix as described by Qian, et al. Molecular composites with polyacetylene, polypyrrole, polythiophene, polystyrene, and the like may be prepared with nylon, polyvinyl chloride, polyvinyl alcohol, polyvinyl acetate, polyvinyl butyral, polyethylene glycol, gelatin, collagen, guar gum, elastin, glycoproteins carotenoids, hemins, diazobenzyloxymethyl, nitrocellulose, paper, agarose, sepharose, sephadex, carbohydrate containing substances, and the like. The following examples are illustrative of the present invention. It will be apparent to those skilled in the art that many modifications, both of materials and methods, can be made without departing from the spirit and scope of the invention. EXAMPLE 1

Formation of Polypyrrole and Cellulose

Conducting Polymer

A polysaccharide matrix, cellulose dialysis membrane, is suspended in a solution prepared by dissolving approximately 54 ml of pyrrole in 2000 ml of 0.10 M NaCl. The duration of the suspension (from seconds to days) and the temperature of the solution (from 0° to 20°C.) do not significantly affect the subsequent formation of the conducting polymer film. The oxidizing solution is prepared by dissolving 46 g of ammonium persulfate ((NH4)₂S₂0₈) in 2000 ml of 0.10 M NaCl. The oxidizing solution is then added to the solution containing the monomer and the matrix. The solution changes color from clear to aqua to black. At this time the matrix is removed and washed with copious quantities of 0.10 M NaCl. The wash process removes excess conducting polymer from the conducting polymer film. The resulting material is dried and ground to form a powder. Conductivity was measured and found to be 0.1 to 1 S/cm. The polymer powder is suitable for chro atographing animal factors.

It is understood that other oxidants, for example, ferric chloride, may be used.

EXAMPLE 2

Formation of Polyaniline and Cellulose Conducting Polymer

A polysaccharide matrix, cellulose dialysis membrane, is suspended in a solution prepared by dissolving approximately 200 ml of aniline in 3000 ml of 1.0 M HC1. The oxidizing solution is prepared by dissolving 111 g of ammonium persulfate ((NH₄)₂S2θ₈) in 2000 ml of 1.0 M HC1. The oxidizing solution is then added to the solution containing the monomer and the matrix. Over a period of several seconds (less than 1 minute) , the solution changes color from clear to aqua to green. At this time the cellulose matrix is removed and washed with copious quantities of 1.0 M HcL. The wash process removes excess conducting polymer from the conducting polymer film. The resulting material is allowed to dry and ground to a powder. The conductivity is measured and found to be 0.1 to 1 S/cm. This polymer has been used for separating factors derived from fungal microorganisms.

EXAMPLE 3 Preparation of Polypyrrole - Polyvinyl

Alcohol Grafted Polymers

12 g of polyvinyl alcohol (Aldrich #18,966-a) average molecular wt. 88,000 was dissolved in 2000 ml of 0.1 M NaCl and then heated at 65°C. When all the PVA granules dissolve. The solution is then cooled to room temperature, and 108 ml of pyrrole is added while stirring the solution. After the pyrrole dissolved, the solution was left overnight at 4'C. A 2000 ml portion of 0.2 M ammonium persulfate, cooled at 4°C., was added slowly to the PVA/Pyrrole solution with stirring. The color of the solution turned from light green to dark green to dark brown and was left stirring for 15-30 minutes at room temperature. . The solution was then filtered through glass wool. The dark filtrate contained polypyrrole polyvinyl alcohol (grafted) polymer in solution. The filtrate was then poured slowly into 5 liters of acetonitrile and left undisturbed for 1-2 hours. The casting of polymer on the organic solvent resulted in thin film of the polymer (black in color) . The solvent was discarded and the black film was washed three times with 1 liter acetonitrile, dried and ground.

In a similar manner there may be prepared conductive polymers comprising polystyrene-pyrrole, polyvinylbutyral-pyrrole, polyethylene-pyrrole and polypropylene-pyrrole.

EXAMPLE 4

Preparation of DEAE - Sephadex Attached to Polypyrrole

40 g of DEAE-Cellulose (Cellex™ from BioRad) was added to 2 liters of 0.1 M NaCl, after which 108 ml of pyrrole was added. The solution was stirred gently for 1 1/2 hours and left overnight at 4°C. A 2 liter portion of 0.2 M ammonium persulfate, in 0.1 M NaCl solution was added to the Cellex - Monomer solution while rapidly stirring. The resulting solution was further stirred for 2 hours at room temperature. The solution was then filtered and the precipitate washed several times with 0.1 m NaCl and then with water. A black powder resulted which was air dried. The resistance of the powder, when measured by a two prong probe attached to a multimeter, measured 0.25 - 0.3 ohms when the prongs were placed - 1 cm apart. EXAMPLE 5 Attachment of Polypyrrole to Microgranular Cellulose

100 g of microgranular cellulose powder (Whatman, cc41, cat #4061 - 050) was suspended in 2 liters of monomer solution(108 ml of pyrrole/0.1 M NaCl) overnight at 4^CC. The suspension of cellulose powder was then stirred gently for 2 hours, after which 2 liters of 0.2 M ammonium persulfate in 0.1 M NaCl was added in a dropwise manner, with stirring. Stirring was continued for 1 hour, then the powder was washed three times with saline solution and then several times with water, till the supernatant was free of chloride ions. The aqueous suspension of cellulose was filtered and dried. The resistance of the cellulose was measured by two pin probes attached to a multimeter. The average resistance of the material was 0.1 to 0.7 k ohms, when the probes were placed about 1 to 2 cms apart.

EXAMPLE 6 Preparation of Polyvinyl Alcohol - Pyrrole Latex

Pyrrole (Aldrich Chemicals) was vacuum-distilled and stored under argon in a refrigerate at 4°C. Water was doubled-distilled from an all-Pyrex apparatus. Ferric chloride (FeCl₃ • 6H₂0; BDH) , potassium iodide (BDH; "analaR" grade, iodine (BDH) , and boric acid (BDH, "AnalaR grade") were all sued as received.

Two samples of partially hydrolyzed poly(vinyl acetate) were used as received: (i) 88% hydrolyzed material (BDH) , having a nominal relative molar mass of 125,000 (PVA-88) ; (ii) 96% hydrolyzed material (Aldrich Chemicals) , having a nominal relative molar mass of 96,000 polyvinyl alcohol (PVA-96) .

Dispersion polymerization reactions were carried out in 5 1 flasks. Initially x g of PVA, plus 88.3 g (0.033 moles) of FeCl₃ • 6H₂ was dissolved in 1 of water. To this solution was added 10 ml (0.014 moles) of purified pyrrole monomer, and the mixture was stirred (magnetically) at 20^βC for 18-24 h. It was shown, in all cases, that greater than 95% conversion of monomer was achieved within this time. Within a few seconds of adding the pyrrole to the reaction mixture, the color changed from orange to brown-black, indicative of the onset of polymerization, but not precipitate was formed. At the end of the reaction period, the dispersions formed were centrifuged at 15,000 rpm for about 1 h. This lead to a black sediment and a pale green, but transparent supernatant. Indeed, it was subsequently shown by visible absorption spectroscopy that the supernatant contained no polypyrrole. The sediment could be readily redispersed by shaking in pure water. The dispersions formed in this way showed no tendency to aggregate on standing. The aqueous dispersion were freeze-dried to yield a fine, black powder.

EXAMPLE 7

Separation of Crude Yeast Extract

220 mg of crude yeast extract was dissolved in a 10 ml buffer (0.02 M Tris/HCl, pH 6.4 containing 5 M Mg Cl₂ 0.4 M EDTA and 2 mM 2-mercaptoethanol. A 1.6 x 5 cm column (bed volume 10 ml) having electrical leads at its base and top was filled with the electrically conductive polymer of Example 4 and washed with starting buffer. An electrical current of 5 illiamps of direct current was applied. The solution of yeast extract was then added to the column. The column was eluted several times with buffer and the fractions were saved (I) .

The current was changed to 3 milliamps and the column was eluted with buffer (30 ml) and the fractions were saved (II) .

The current was changed to 1.5 milliamps and the column was eluted with buffer (30 ml) and the fractions saved (III) .

The current was removed and the column was eluted with buffer (50 ml) and the fractions saved.

RESULTS Fraction No. Enzyme %.

Recovery

I Alcohol dehydrogenase 85

II Glucose-6-phosphate dehydrogenase 90

III Hexokinase 87 IV Glyceraldehyde-3-phoεphate dehydrogenase 92

The separation can also be performed with alternating current utilizing 0.5 μA - 10 A (1 - 60 Hz) . In addition, there may be utilized a constant potential or a potential gradient down the column.

EXAMPLE 8 The procedure of Example 7 was followed except that a constant potential of 3 V is applied to the column. The mixture of yeast is added to the column and eluted with buffer and the first fraction saved. The voltage was changed to 1.5 V and the column was further eluted with buffer and these fractions were saved.

The voltage was changed to 0.1 V and the remainder of the column was eluted.

Similar to Example 7, the various enzymes were separated and recovered.

EXAMPLE 9 A chromatographic column is filled with electrically conductive polypyrrole-agarose. Separate electrode sets were positioned to provide an angle of 115° between the fields. A programmed regulator was utilized for switching of fields so as to provide pulsed electrical fields alternating between AC and DC current. 100 volt potential was applied to each field and pulsed alternately every two seconds. The AC current applied was 20 Hz.

DNA and rRNA in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.01 MgCl₂ was passed on the buffer treated column.

Separation on the column was achieved after 2 hrs. The voltage was reduced to 50 volts and the DNA was eluted from the column with buffer. The voltage was removed and the RNA was eluted from the column with buffer.

Claims

What is Claimed is:

1. A process for the separation of charged molecules from a solution which comprises the steps of:

(a) providing a chromatographic apparatus including an electrically conductive polymeric material;

(b) treating said polymeric material with a solution containing a mixture of molecules;

(c) passing an electrical current or voltage through said polymeric material to alter the binding of the molecule in the mixture; and

(d) recovering said molecules.

2. The process of Claim 1 wherein alternating current is applied to said conductive polymer.

3. The process of Claim 1 wherein direct current is applied to said polymer.

4. The process of Claim 3 wherein a current of about 0.1 to 5 milliamps is applied to said polymer.

5. The process of Claim 1 wherein current is varied to separate molecules.

6. The process of Claim 1 wherein said molecules are biologically active factors.

7. The process of Claim 6 wherein said factors are directly bound to said polymer.

8. The process of Claim 6 wherein said factors are bound to said polymer through a ligand.

9. The process of Claim 8 wherein said ligand has a specific affinity for said factor.

10. The process of Claim 8 wherein said ligand and factor are bound through a functional group.

11. The process of Claim 6 wherein said factor is derived from a member of the group consisting of mammalian cells, avian cells, amphibian cells, microorganism and plant cells.

12. The process of Claim 11 wherein said cells are bacterial.

13. The process of Claim 1 wherein said electroconductive polymer comprises a polymer selected from the group consisting of collagen, elastin, guar gum, gelatin and polysaccharide.

14. The process of Claim 13 wherein said polymer has been made electrolytically conductive by the incorporation of an electrically conductive synthetic polymer.

15. The process of Claim 14 wherein said synthetic polymer comprises polyacetylene.

16. The process of Claim 1 wherein said electrically conductive polymer comprises a polypyrrole.

17. The process of Claim 14 wherein said electrically conductive polymer is a copolymer selected from the group consisting of polypyrrole- cellulose, polyaniline-cellulose, polypyrrole- polyvinyl alcohol and polypyrrole-collagen.

18. The process of Claim 1 wherein uniform pulsed fields are used.

19. The process of Claim 1 wherein a constant potential is applied to said polymer.

20. The process of Claim 1 wherein alternating pulsed electric fields are applied to said polymer.

21. The process of Claim 1 wherein an electrically conductive polymer is cast into a membrane and the pore size of the membrane is changed by the current applied.

22. The process of Claim 1 wherein a potential is applied to said conductive polymer.

23. The process of Claim 22 wherein said potential is varied and a charged molecule is as a result of the varied potential.

24. The process of Claim 1 wherein the electrical charge is removed to separate and remove different charged molecules.

25. The process of Claim 1 wherein a ligand is bound to the polymer to prevent binding of a select charged molecule.

26. An apparatus to separate and recover molecules in a mixture comprising: a chromatographic column including an electricially conductive material; and means for passing an electrical current or voltage through the material.

27. The apparatus of Claim 26 wherein a ligand which binds the molecule to be separated is attached to the electrically conductive material.

28. The apparatus of Claim 27 wherein the ligand is ionically attached to the electrically conducting material.

29. The apparatus of Claim 27 wherein the ligand is detachable from the polymer by a change in the electrical charge of the polymer.

30. The apparatus of Claim 26 wherein the electrically conductive material comprises a conducting polymer formed on a flexible matrix.

31. The apparatus of Claim 30 wherein the conducting polymer is selected from the group consisting of polyacetylene, polypyrrole, polythiophene, and polystyrene.

32. The apparatus of Claim 30 wherein the flexible matrix is selected from the gorup consisting of nylon, polyvinyl chloride, pol (vinyl)alcohol, polyvinyl acetate, polyvinyl butyral, polyethylene glycol, gelatin, collagen, guar gum, elastin, glycoproteins, carotenoids, hemins, diazobenzyloxymethyl, nitrocellulose, paper, agarose, sepharose, sefadex, and carbohydrate-containing substances.

33. The apparatus of Claim 26 wherein then electrically conductive material is cast into a membrane and the pore size of the membrane is changeable by application of current or voltage.