DE102006020463B4 - Method for removing nucleic acids from a liquid, method for producing adsorber elements and use of membrane adsorber material blend - Google Patents

Abstract

Process for the at least partial removal of nucleic acids (18) from a liquid (12), as occur in the digestion of microorganisms, such as bacteria, yeasts, fungi, protozoa or cell cultures of eukaryotic origin and of tissues of animal or vegetable origin, by selectively binding adsorber elements (20) which float freely in the liquid and are separated from the liquid together with the nucleic acids (18) after an intended incubation time, characterized in that the adsorber elements used are flat, microporous membrane pieces (20) of a size of 1 mm 2 to 10,000 cm 2 which carry groups capable of interacting with the nucleic acids on their inner and outer surfaces.

Description

The invention relates to a method for the at least partial removal of nucleic acids from a liquid, as occur in the digestion of microorganisms such as bacteria, yeasts, fungi, protozoa or cell cultures of eukaryotic origin and of tissues of animal or vegetable origin, by selectively binding adsorber elements which float freely in the fluid and are separated from the fluid along with the nucleic acids after an intended incubation period.

The invention further relates to a process for the preparation of such adsorber elements and to the use of membrane adsorbent waste for the production of such adsorber elements.

For removal of nucleic acids from liquids, column methods are known in which a nucleic acid-containing liquid is passed through a multilayer stack of adsorbent elements in a housing. These retain the nucleic acids and, after fluid passage, i. H. after incubation, removed from the housing and appropriately treated or discarded. This known method has the disadvantage that it is associated with a plurality of handling steps and therefore expensive. In addition, the absorber elements can clog in the housing.

In the filtration technology, the use of free-floating filter aids is known. The basic idea here is to bring special adsorber elements, which bind with a contaminant present in the liquid, into close contact with the contaminant, so that a firm binding of the contaminant to the adsorber elements can take place. In a subsequent process step, the contaminants loaded with the contaminant are then separated from the liquid. The contaminant and / or the purified liquid can then be supplied to further, separate treatment steps.

In the DE 41 10 252 C1 a number of filter aids are described or cited. Specifically named are fibrous or granular materials such. As silica gel or silica fibers, aluminum flake powder, plastic and cellulose fibers and metals, metal oxides, coal and fibrous or granular plastics. In the cited document such filter aids are used in the clarification of beverages. They are used to soak a filter cake, which binds turbidity and forms an easily removable layer on fixed filter elements through which the drinks to be clarified are passed.

The DE 199 17 399 A1 discloses the use of humic acid impregnated paper shreds as filter aids for water softening.

From the EP 0 775 313 B1 whose German translation as DE 695 15 675 T2 is published, it is known to use as adsorber a magnetic particle powder in which the individual powder particles which float freely in the liquid, are coated specifically binding. After binding of the contaminant to the powder particles, the powder can be fixed by magnetic force to a solid support and removed with the contaminant from the liquid.

A similar process using magnetic microparticles and polymer nanoparticles as free-floating adsorption elements is known from US Pat DE 197 36 366 A1 known.

Other free-floating filter aids are, for example, under the trade name DEAE Sephadex gel or DEAE Sepharose gel from General Electric or DEAE cellulose fiber material, which are used specifically for removing DNA from a biological fluid.

Other methods use only fixed filter aids. From the DE 39 30 947 A1 discloses the use of a column having a porous matrix of agarose / polyacrylamide beads or coated glass microbeads over which a liquid to be purified is passed. The particle bed of said beads serves as a filter element to which the contaminant attaches.

Furthermore, non-particulate membrane filters are known, through which a liquid to be purified is passed, the contaminant binding with the filter membrane. Generally speaking, membrane adsorbers may be used, including those membranes which carry on their surface functional groups, ligands or reactants which are capable of interacting with at least one substance of a liquid phase in contact with the membrane adsorber. The transport of the liquid phase through the membrane takes place convectively. The term membrane adsorber is to be understood as a generic term for various types of membrane adsorbers, such as membrane ion exchangers, Ligand membranes and activated membranes, which in turn are divided into different Membranadsorbertypen depending on the functional groups, ligands and reactants.

From the DE 199 00 681 A1 For example, a method for purifying nucleic acids from an aqueous solution is known in which the solution is filtered through a stack of membranes so that the nucleic acids as well as endotoxins contained in the solution are adsorbed on the individual membrane layers. Subsequent passage of high ionic strength neutral salt solution elutes only the nucleic acids while the endotoxins remain bound in the membrane stack. This method has the disadvantages mentioned above in connection with general column methods.

The DE 102 36 664 A1 discloses a filtering method in which a filter cartridge with a housing through which the liquid to be filtered flows, in which an adsorber membrane is arranged in a stationary manner parallel to the flow direction, is used. In this system, which dispenses with a forced flow through the stationary membrane, although there is no risk of clogging of the filter; However, it must be expected that the filtration efficiency decreases with increasing loading of the membrane, so that only by a complex and difficult to implement control of the flow velocity of the liquid, a uniform filtration yield can be achieved.

The DE 697 18 752 T2 discloses a process for the isolation of nucleic acids from a liquid in which a sol is produced in the liquid by polymerization of water-soluble monomers, which adsorbs the nucleic acids to be isolated.

The DE 692 32 677 T2 discloses a chromatographic method of separating double-stranded DNA from single-stranded DNA or RNA by selectively adsorbing the duplex DNA to an acrylic membrane having aralkylamine molecules covalently attached thereto.

From Rodney JY Ho and Milo Gibaldi, Biotechnology and Biopharmaceuticals, Transforming Proteins and Genes into Drugs, John Wiley & Sons, 2003, page 72, it is known to remove cell membranes from cells which have been destroyed for the purpose of extracting a desired substance from a medium in which the medium is passed through membrane filter of defined pore size. This can be done by flow or overflow. The term membrane filter here also includes fixed hollow fiber systems and membrane systems with precision pore size.

In Gaey Walsh, Proteins Biochemistry and Biotechnology, John Wiley & Sons, 2002, pages 110-112 various concentration methods are explained, including concentration by ion exchange and concentration by ultrafiltration using membrane filters.

In "Commercial Production of Monoclonal Antibodies, A Guide for Scale-Up," edited by Sally S. Seaver, Decker Publishing, New York 1987, pp. 304-309, there is disclosed a method for obtaining monoclonal antibodies, wherein column purification done with particulate matrix.

All the above-mentioned processes with particulate filter aids have the disadvantage that they are difficult to remove from the liquid after loading with the contaminant. This is usually done by filtration through suitable filter media, preferably layer filters, or by centrifugation. This is very expensive and has the further disadvantage that particulate, d. H. granular or fibrous filter aids are very susceptible to shear and therefore tend to break into even smaller particles. These are all the more difficult to remove from the liquid to be cleaned.

From the DE 29 14 203 A1 For example, a method is known which addresses this problem. The cited document discloses large adsorber body, which consist of a stable, mechanically protective frame whose inner surfaces are coated or covered with adsorbent material. These adsorber bodies, which can be used to extract uranium from seawater, have a lower density than water, so that when they are discharged at a defined depth into a sea current, they automatically float to the surface, passing through various layers of water they can bind the contaminant and then be fished off the surface. Although these known adsorber solve the problem of easy separability of the adsorber of the liquid; they are expensive to manufacture and transport because of the rigid frame and can not be used in industrial applications in closed or open vessels, since the rigid frames colliding with agitators and / or container walls can cause considerable damage.

It is the object of the present invention to develop the known methods for removing nucleic acids from liquids in such a way that an uncomplicated use under industrial conditions becomes possible. It is a further object of the invention to provide a low cost manufacturing process for such adsorber elements.

The former object is achieved in conjunction with the preamble of claim 1, characterized in that as adsorber elements areal microporous membrane pieces of a size of 1 mm ² to 10,000 cm ² are used, which carry on its inner and outer surfaces capable of interacting with the nucleic acids groups.

According to the invention, nucleic acids, such as oligomeric and polymeric deoxyribonucleic acid and / or ribonucleic acid, can be removed from solutions and suspensions which occur during the digestion of microorganisms such as bacteria, yeasts, fungi, protozoa and cell cultures of eukaryotic origin and tissues of animal and plant origin. For this purpose, surface adsorber elements are used which consist of microporous membranes which carry groups capable of reversible or irreversible interaction with the nucleic acids on their inner and outer surfaces. Preferably, strong or weak basic ion exchange groups are used. Especially preferred are those of the secondary amine or quaternary ammonium type.

One advantage is that the solutions or suspensions can be brought into direct contact with the adsorber elements without any pretreatments.

While there are useful effective solutions for the removal of other contaminants, such as endotoxins, proteins or dyes, there are no such for the removal of nucleic acids. Thus, endotoxins can be removed by filtration, proteins by precipitation or ion exchange and dyes adsorptively. So far, nucleic acids can be isolated only time consuming filter columns or centrifuged by ultracentrifuges on an analytical scale. The precipitation of nucleic acids with alcohols would also denature the proteins present in the fluid, which could then no longer be recovered in pure form.

A further advantage of the method according to the invention is that the liquid or suspension treated according to the invention can be added directly to further processing filter modules, including adsorber modules, without the risk of blockage of the modules by the often threadlike present nucleic acids.

Furthermore, any type of stabilization frame or housing is dispensed with. Rather, the flexible properties of membrane adsorbers are exploited. The membrane pieces not only follow in their direction of movement the flows in a liquid, as caused for example by agitators, but also adapt in shape to the local conditions flexible. As they move, they will, over time, contact a larger volume of fluid than rigid adsorbent bodies, making the cleaning of the fluid more efficient. In collisions with container walls, agitators or the like, no damage is to be feared.

The above second object is achieved by the features of claim 8 and claim 13. Thereafter, it is envisaged to use as the starting material for the adsorber elements according to the invention Membranadsorbermaterial blend a size of 1 mm ² to 10,000 cm ² from the production of large-area Membranadsorber in the form of flat membranes. In the production of such adsorbents in the form of flat membranes usually roll material is used. The waste usually has to be disposed of expensively. According to the invention, it is therefore provided that the waste is suitable, namely to be comminuted to a size of 1 mm ² to 10,000 cm ² .

Membrane pieces of a size of 10 mm ² to 100 cm ² and particularly preferably a size of approximately 100 mm ^{2 have} proved to be particularly favorable. The special of the size is to be made by the expert in view of the specific application, in particular also the container sizes.

Accordingly, the use of such shredded waste from the Membranadsorberherstellung for the process according to the invention is an independent invention, which not only ensures a particularly cost-effective production of the membrane pieces according to the invention, but also significantly cheaper the production of large adsorber due to the reduction of disposal costs.

Preferably, the membrane pieces are made of a porous membrane adsorbent material. As a result, the contaminant is (also) bound inside the membranes, so that a mechanical protection against abrasion in collision with agitators, container walls or other membrane pieces is guaranteed. In addition, this increases the adsorbing surface, which leads to greater efficiency of the process. In particular, in the application for the removal of DNA from a liquid, it is favorable due to the polyelectrolytic, highly ionic character of DNA, when the membrane pieces have a basic character.

Preference is given to using membrane materials which lead to membrane pieces which, in the floating state, have a density similar to the density of the liquid. This means that the membrane pieces float in the liquid and neither sink nor float, so that an efficient cleaning can take place in all layer depths of the liquid.

Further features and advantages of the invention will become apparent from the following, specific description and the drawings.

Show it:

1 : a schematic representation of steps of the method according to the invention,

2 a schematic representation of steps of the manufacturing method according to the invention and

3 : a diagram to illustrate the efficiency of the method according to the invention.

1 Figure 3 shows a sequence of process steps in the exemplary application of the method according to the invention for obtaining biomolecules of cellular origin, such as a protein from an inclusion body within a bacterial cell, such as Escherichia coli. In a container 10 with a nutrient medium 12 Float bacterial cells 14 adjacent to the inclusion body of interest 16 especially DNA 18 contain. In a first process step, the cell membranes of the bacterial cells 14 decomposes and the inclusion bodies 16 as well as the DNA 18 into the nutrient medium 12 released. The decomposition of the cell membranes of the membrane fragments 17 can be done by known techniques, such as ultrasound, chemical lysing, etc. By releasing the DNA 18 increases the viscosity of the liquid 12 clear. This hinders subsequent filtration processes, since it reduces the flow of the liquid 12 through a filter with pores that are small enough to contain the inclusion body 16 or the membrane fragments 17 withhold, significantly slows or even stops. According to the invention, in the step of the liquid shown in sub-figure c, membrane adsorber pieces are obtained 20 of a few cm ² size added. They remain in the liquid for a given incubation time 12 , wherein for improving the contact, for example, a stirrer 22 can be used. For example, become basic membrane pieces 20 used, binds the free DNA 18 to the membrane pieces, whereas the inclusion bodies 16 and the cell membrane fragments 17 in the liquid 12 remain. In a subsequent process step (d), the membrane pieces 20 due to their size easily from the liquid 12 be removed, for. B. by fishing or passing the liquid 12 through rough sieves. In this way, the DNA content in the fluid 12 be easily and inexpensively reduced.

2 schematically shows process steps for the production of the membrane pieces according to the invention. In subfigure a is a roll material 30 from Membranadsorbermaterial shown. For this, for example, by a punch 32 , large membrane adsorber filter 34 punched out. In addition to the adsorber filters 34 falls off 36 (see sub-figure b).

The blend 36 is in a subsequent process step (c), for example by means of a cutting unit 38 crushed. Depending on the setting of the cutting unit with regard to knife size and density, running speed, etc., the size of the shredder can be influenced. As a result, membrane adsorber pieces according to the invention are produced 20 which are suitable for use in the method according to the invention.

3 shows in the form of a diagram the course of the inventive method in the purification of DNA from phosphate buffered saline (PBS). Pieces of about 1 mm ² (called small pieces below) or 9 cm ² (hereinafter called large pieces) of a total of 190 cm ² membrane area were cut from the blend of a commercially available membrane adsorber material with the trade name SARTOBIND Q. 90 mg DNA from fish sperm were dissolved in 200 ml PBS with stirring. The solution was divided into two large fractions, with one fraction containing the large and the other small pieces of membrane added to the fraction. Both suspensions were agitated on an orbital shaker at 60 rpm so that the membrane pieces floated freely. After predetermined time intervals, samples were taken and their DNA content, after appropriate dilution, was measured by spectrophotometry at 260 nm against PBS as a control. The following table shows the result that is shown in 3 is shown graphically. Time [min] Percent DNA (small pieces) Percent DNA (large pieces) 0 100 100 15 58 70.2 30 45.8 54.5 45 38.9 45.4 60 34.2 40.3 75 30.2 34.0 90 31.5 34.8 105 30 33.2

As from the table and 3 easily seen, the DNA levels run asymptotically to about one third of the original DNA content. Two thirds of the DNA could be removed from the liquid in a comparatively short time simply and inexpensively. The comparison of the curves with the use of large (40) or smaller (50) membrane pieces shows that the use of smaller membrane pieces is slightly more efficient than the use of large membrane pieces. This can be explained by the larger total surface area of the membrane adsorbers that results at the cutting edges. However, it is noticeable that the efficiency advantage is very low, so that even larger pieces, which show an even better separability from the liquid, can be used without great losses.

Of course, the embodiments and examples illustrated in the specific description and drawings represent only exemplary applications and embodiments of the present invention, which should by no means be construed in a limiting sense. In particular, the invention is open to use in other biological or nonbiological applications. Also, the specific choice of the adsorbent material to vote on the specific application.

Claims (15)

Process for the at least partial removal of nucleic acids ( 18 ) from a liquid ( 12 ), as they occur in the digestion of microorganisms such as bacteria, yeasts, fungi, protozoa or cell cultures of eukaryotic origin and of tissues of animal or vegetable origin, by selectively binding adsorber elements ( 20 ) floating freely in the fluid and after a designated incubation time together with the nucleic acids ( 18 ) are separated from the liquid, characterized in that as adsorber flat, microporous membrane pieces ( 20 ) of a size of 1 mm ² to 10,000 cm ² , carrying groups capable of interacting with the nucleic acids on their inner and outer surfaces. A method according to claim 1, characterized in that are used as groups of basic ion exchange groups. A method according to claim 2, characterized in that are used as ion exchange groups of the type of secondary amines or quaternary ammonium compounds. Method according to one of the preceding claims, characterized in that the membrane pieces ( 20 ) from a porous membrane adsorber material ( 30 ) are made. Method according to one of the preceding claims, characterized in that the membrane pieces ( 20 ) have a size of 10 mm ² to 100 cm ² and especially a size of about 100 mm ² . Method according to one of the preceding claims, characterized in that the floating membrane pieces ( 20 ) one of the density of the liquid ( 12 ) have similar density. Method according to one of the preceding claims, characterized in that the membrane pieces ( 20 ) by comminution of Membranadsorbermaterial blend ( 36 ) in the production of large-area membrane adsorbers ( 34 ) are made. Use of membrane adsorber material blend ( 36 ) of a size of 1 mm ² to 10,000 cm ² from the production of large-area membrane adsorbers ( 34 ) on its inner and outer surface to interact with nucleic acids ( 18 ), as they occur in the digestion of microorganisms such as bacteria, yeasts, fungi, protozoa or of cell cultures of eukaryotic origin and of tissues of animal or vegetable origin, carry groups capable of acting as adsorber elements ( 20 ) for the at least partial removal of nucleic acids ( 18 ) from a liquid ( 12 ) wherein the nucleic acids selectively binding adsorber elements ( 20 float freely in the liquid and after a designated incubation time together with the nucleic acids ( 18 ) are separated from the liquid. Use according to claim 8, characterized in that basic groups of ion exchange groups are used as groups. Use according to Claim 9, characterized in that the ion exchange groups used are those of the type of secondary amines or quaternary ammonium compounds. Use according to any one of claims 8 to 10, characterized in that the membrane adsorbent material is porous. Use according to claim 8 to 11, characterized in that the adsorber elements ( 20 ) have a size of 10 mm ² to 100 cm ² and especially have a size of about 100 mm ² . Process for the production of adsorber elements ( 20 ) suitable for use in a method for at least partially removing nucleic acids ( 18 ) from a liquid ( 12 ), as they occur in the digestion of microorganisms such as bacteria, yeasts, fungi, protozoa or cell cultures of eukaryotic origin and of tissues of animal or vegetable origin, by freely floating in the liquid, selectively binding, surface, microporous adsorber elements ( 20 ) which carry groups capable of interacting with the nucleic acids on their inner and outer surfaces and which, after a designated incubation time, together with the nucleic acids ( 18 ) are separated from the liquid, wherein Membranadsorbermaterial-Verschnitt ( 36 ) from the production of large-area membrane adsorbers ( 34 ) to membrane pieces ( 20 ) is crushed from a size of 1 mm ² to 10,000 cm ² . Method according to claim 13, characterized in that the waste ( 36 ) to membrane pieces ( 20 ) is crushed from a size of 10 mm ² to 100 cm ² and especially a size of about 100 mm ² . Method according to one of claims 13 or 14, characterized in that the membrane adsorbent material is porous.

Images