EP2731958A1

EP2731958A1 - Ion-exchanger material with high salt-tolerance

Info

Publication number: EP2731958A1
Application number: EP12737260.5A
Authority: EP
Inventors: Markus Arendt; Gerhard Stumm; Martin Welter; Thomas Schwarz
Original assignee: Instraction GmbH
Current assignee: Instraction GmbH
Priority date: 2011-07-13
Filing date: 2012-07-12
Publication date: 2014-05-21
Also published as: CA2841346A1; US20140336355A1; AR087173A1; CN103827135A; JP2014524916A; WO2013007799A1; CN103827135B; KR20140116051A

Abstract

The invention relates to a cross-linked sulfonated polymer, or to a cross-linked sulfonated polymer which is coated with a cross-linked polymer that contains amino groups, for use as an ion-exchanger material with high salt-tolerance for separating macromolecules out from a solution which originates from a biological source.

Description

Ion exchange material with high salt tolerance

The present invention relates to a crosslinked sulfonated polymer or an amino group-containing polymer

crosslinked polymer coated crosslinked sulfonated

Polymer for use as a high salt tolerance ion exchange material for the separation of macromolecules from one

Solution that comes from a biological source.

The Coulomb interaction of ion exchange resins is the most commonly used interaction in chromatographic purification processes. For ion exchange resins

Preferably, ionic groups such as strong acids (e.g., sulfonic acid), strong bases (e.g., quaternary amines), weak acids (e.g., carboxylic acids), and weak bases (e.g., primary or tertiary amines) are covalently attached as groups to a rigid matrix material. These ionic groups interact with complementary functional groups of the molecules to be purified, which are thus bound to the ion exchange resin. The elution of the target molecules bound by ionic interaction is usually achieved by increasing the salt concentration in the eluent so that the target molecule is replaced by one or more corresponding salt ion (s). Relatively low

Salt concentrations of less than 150 mmol / L are

usually sufficient to break the Coulomb interaction and elute the target molecule.

Depending on the origin of the mixture from which the target molecule is to be separated, the salt concentration already higher than concentrations that can be commonly used for elution. This usually has the

Disadvantage that the target molecules in the presence of high

Do not bind salt concentration on the ion exchange resin.

In particular, solutions derived from biological sources, such as

Fermentation fluids, body fluids or

Plant extracts are obtained is the conductivity

(electrical conductivity, a corresponding quantity to

Salt concentration) normally too high for the direct use of ion exchange chromatography. Therefore, an undesirable dilution step is often necessary to prevent the

Reduce conductivity of the mixture (reduction of salt concentration). There are several known and available ion exchange resins that are capable of producing substances at a relatively high level

To bind salt concentration. However, all previously known ion exchange resins are no longer able to bind biological macromolecules, such as insulin, with a sufficient loading capacity at concentrations of more than 250 mmol / L sodium chloride. Sodium chloride should be mentioned here only as an example; in principle, however, other salts may be present in this molar amount. In addition, hitherto known ion exchange resins used are not stable over the entire pH range of pH 1 to 14 and thus not universally applicable.

It was therefore an object of the present invention to provide a method for the separation of macromolecules in which the macromolecules can be separated directly from a solution derived from biological sources. In addition, it is desirable that the ion exchange material used is stable over a pH range of 1 to 14. Due to its high salt tolerance, the ion exchange material is it allow no additional dilution step to be performed to increase the salt concentration

Reduce. Such a method would have the advantage that the cost of solvents for the additional dilution step and for the treatment of waste substances in the

Purification of salt-containing mixtures could be reduced.

To solve the above problem, the present invention

Sign the use of a cross-linked sulfonated

A polymer for separating a macromolecule from a solution derived from a biological source, wherein the crosslinked sulfonated polymer attached to its backbone contains a sulfonated aromatic moiety substituted or unsubstituted with an aliphatic moiety.

In other words, the present application relates to

A method for separating a macromolecule from a solution derived from a biological source using a cross-linked sulfonated polymer containing, attached to its backbone, a sulfonated aromatic moiety substituted or unsubstituted with an aliphatic moiety. According to the invention, macromolecules are understood as meaning molecules which have a molecular weight of greater than or equal to 10,000 g / mol. Most preferably, the macromolecule is a

Biomolecule, such as peptides and proteins, DNA, RNA, polysaccharides and Lipopolysacharide, such as

Endotoxins.

The term "separation" is intended to mean both the

Recovery / purification of a target molecule from the solution as well as the removal of unwanted macromolecules from the solution Solution understood so that the target molecule remains in the purified solution.

The backbone of the crosslinked sulfonated polymer may be any known polymeric backbone that comprises

hydrocarbon repeating units.

By the backbone of the polymer is meant the main chain of the polymer, to which in the form of side chains

Subgroups, such as the sulfonated aromatic moiety may be bound. In addition to the sulfonated aromatic unit, the polymer may also contain other side chains, which are not to be expected to the backbone, but - as named - are to be counted among the side chains. In other words, the framework includes all the atoms that make up the skin chain of the polymer and at least two others

at least bivalent atoms of the main chain are linked. Single-bonding atoms, such as hydrogen atoms, which bind to the atoms mentioned, are also atoms of the

Backbone. In the case where the crosslinked sulfonated polymer is crosslinked polystyrene, the linked ones would be

Vinyleinheiten the skeleton and the sulfonated

Phenyl groups the side chains. Under hydrocarbon repeating units

one understands all conceivable connections, which are built up predominantly from carbon and hydrogen, in addition, however

May contain heteroatoms. The link of the

Repeating units to a polymer can be made by any known polymerization method. Particularly preferred according to the invention is free-radical, cationic or anionic olefin polymerization. The backbone is particularly preferably a polyvinyl skeleton. The basic structure is

preferably a cross-linked skeleton, so that a networked Polymer is formed. Especially in the case of a

Polyvinyl skeleton results in the crosslinking by

Copolymerization of a vinyl group-containing monomer with a monomer containing two vinyl groups. However, it is also conceivable in principle that initially a polymer

is prepared, which has a linear skeleton. The subsequent crosslinking can then be followed by reaction of functional groups in the side chain with a

Crosslinking carried out.

The crosslinked sulfonated polymer used in the invention preferably contains sulfonic acid groups in the side chain. The side chains are crosslinked in the invention

sulfonated polymer sulfonated aromatic moieties as detailed below. The sulfonated ones

Aromatic units are preferably attached to the backbone by a single covalent bond. The sulfonated aromatic units may also be substituted with an aliphatic radical. It is particularly preferred that the sulfonated aromatic units by a covalent

Single bond are bonded directly to an atom of the backbone.

By an aromatic unit is meant in the

present invention with an aliphatic radical substituted or unsubstituted mono- or polycyclic aromatic ring system. For the purposes of this invention, an aromatic ring system is preferably an aromatic ring system having 6 to 60 carbon atoms, preferably 6 to 30, particularly preferably 6 to 10

Carbon atoms. These aromatic ring systems may be monocyclic or polycyclic, ie they may have one ring (eg phenyl) or two or more rings which may also be condensed (eg naphthyl) or covalently linked can (eg biphenyl), or a combination of

include condensed and linked rings.

Preferred aromatic ring systems are, for example, phenyl, biphenyl, triphenyl, naphthyl, anthracyl, binaphthyl,

Phenanthryl, dihydrophenanthryl, pyrene, dihydropyrene, cryses, perylene, tetracene, pentacene, benzpyrene, fluorene and indene. The aromatic ring systems are particularly preferably phenyl, biphenyl or naphthyl, particularly preferably phenyl.

As already mentioned, the aromatic ring systems may be substituted by an aliphatic group. It is conceivable that the aromatic ring system not only by one but by two or more aliphatic groups

is substituted. An aliphatic radical is preferably a hydrocarbon radical having 1 to 20 or 1 to 10

Carbon atoms. Inventive aliphatic

Hydrocarbon radicals are preferably linear or

branched or cyclic alkyl groups in which also one or more hydrogen atoms may be replaced by fluorine. Examples of the aliphatic hydrocarbon radicals having 1 to 20 carbon atoms include the following:

Methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl (1-methylpropyl), tert-butyl, iso-pentyl, n-pentyl, tert-pentyl (1, 2-dimethylpropyl ), 1,2-dimethylpropyl, 2,2-

Dimethylpropyl (neopentyl), 1-ethylpropyl, 2-methylbutyl, n-hexyl, iso -hexyl, 1,2-dimethylbutyl, 1-ethyl-1-methylpropyl, 2-methylbutyl, 1-ethyl-2-methylpropyl, 1, 1 , 2-trimethylpropyl, 1, 2, 2-trimethylpropyl, 1-ethylbutyl, 1-methylbutyl, 1,1-dimethylbutyl, 2, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl , 2-ethylbutyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-ethylhexyl,

Trifluoromethyl, pentafluoroethyl and 2, 2, 2-trifluoroethyl. Particularly preferred as the aliphatic hydrocarbon radical is methyl or ethyl.

It is extremely preferable that the with a

aliphatic radical substituted or unsubstituted

sulfonated aromatic moiety of the crosslinked sulfonated polymer is a phenylsulfonic acid group or a derivative thereof. In the case of the derivative of the phenylsulfonic acid group, it is meant derivatives having an aliphatic radical

are substituted. In this case, preferably, a sulfonic acid group is on the phenyl moiety in para position to the backbone binding site on the phenyl ring. Of the

In this case, the aliphatic radical is preferably a methyl or ethyl group which is located in the ortho and / or meta position on the phenyl group to the point on the phenyl ring which binds to the skeleton.

However, it is particularly preferred that the sulfonated aromatic moiety is unsubstituted. Here, in particular, a sulfonated crosslinked polystyrene in

Consideration. The crosslinking of the sulfonated polystyrene is preferably carried out by the copolymerization of styrene with

Divinylbenzene, followed by the sulfonation of the phenyl groups. But any other two vinyl group-containing crosslinking agents are conceivable here for the production of a crosslinked copolymer.

The degree of crosslinking of the crosslinked sulfonated polymer is according to the invention preferably 0.5 to 50%, more preferably 5 to 45% and most preferably 10 to 35%. In the indication of the degree of crosslinking in percent is in the

present invention, the percentage mole fraction of

employed two vinyl group-containing compound to the Total number of monomer units to be polymerized

Understood .

The degree of sulfonation of the crosslinked sulfonated polymer is preferably 1 to 80%, more preferably 3 to 60%, and most preferably 5 to 40%. The indication of the

Percent sulfonation refers to the number of moles of sulfonic acid groups in relation to all

Polymerization used monomer units containing a

have sulfonatable group. The monomer units used for the polymerization, which have a sulfonatable group, are understood as meaning all monomer units which have the

sulfonated aromatic moiety, as well as all the monomer units containing a sulfonatable group, preferably an aromatic moiety, and optionally all

Compounds that cause cross-linking, provided they contain a sulfonatable or sulfonated group. When used as a crosslinked sulfonated polymer sulfonated polystyrene-divinylbenzene copolymer, so refers

Percent sulfonation on the number of sulfonic acid groups in relation to all phenyl or phenylene groups contained in the polymer.

The in the method according to the invention or in the

Crosslinked sulfonated polymer used according to the invention is preferably in the form of regular or irregularly shaped resin particles. In the present invention, the term "regularly shaped" is understood to mean shapes which can be represented by symmetry operations such as surface reflection, point reflection or axes of rotation or combinations thereof

to call spherical shape. The term "spherical" is understood to mean not only purely symmetrical spheres, but also deviating forms such as ellipses but should also be enclosed with two dumbbells connected to spherical bodies in this case. By an irregular form is meant any broken form that has no symmetry. The resin particles have

preferably has an average average diameter of from 1 to 1000 μm, more preferably from 5 to 100 μm, and particularly preferably from 10 to 50 μm.

The in the method according to the invention or in the

Crosslinked sulfonated polymer used according to the invention preferably has pores in which the actual interaction with the substances to be separated takes place. It is thus preferably a porous polymer material. These pores preferably have an average

Diameter of 6 to 400 nm, more preferably from 30 to 100 nm. The pore diameter is determined by an inverse

Size exclusion chromatography determines: The phase material to be tested is packed into a chromatography column and injected with a series of polymer size standards. From the course of the curve in the plot of the logarithm of the molecular weight of the respective standard against the

Elution volume can, according to literature methods, the distribution of the pore diameter and thus the average

Pore diameter can be determined.

Furthermore, it is preferred if the crosslinked sulfonated polymer has a pore volume in the range of 1 to 3 mL / g. The pore volume is determined by measuring the

Water absorption capacity determined: The phase material whose weight was determined in the dry state is mixed with the solvent for which the pore volume is to be determined (different solvents can show different results due to different wettability). For the purposes of the present invention, water used as solvent. Excess solvent is filtered off and the phase material in the centrifuge is freed from further solvent in the intermediate grain volume. Then the material is re-weighed. Only the pores should still be filled with the solvent. About the mass difference between filled and empty pores and the density of the solvent, the pore volume can be calculated. The in the process of the invention or in the

Crosslinked sulfonated polymer used according to the invention has the advantage that it also contains ionizable groups such as sulfonic acid groups in addition to the lipophilic skeleton with the aromatic units in the side chain. In this way it is suitable, both by ionic

Interactions as well as lipophilic interactions interact with the macromolecule. Here, the sulfonic acid groups are preferably used as anionic -SÜ ₃ ^~ groups, which are capable of ionic interactions with cations of the

To enter the macromolecule. In addition, macromolecules from biological sources such as proteins, DNA or RNA also have lipophilic regions which can interact with the aromatic units of the crosslinked sulfonated polymer as a lipophilic matrix. In this way it is possible to use solutions derived from a biological source,

which can contain a very high salt content of up to 1 mol / L salt, without causing elution of the macromolecules from the ion exchange material. The crosslinked sulfonated polymer used according to the invention is preferably used for the recovery or purification of macromolecules containing cation groups. The

Macromolecule is preferably a biological macromolecule. The biological macromolecule is preferably a peptide. All particularly preferred is the peptide insulin. In other words, the present invention thus preferably relates to a use of the crosslinked sulfonated polymer for purifying insulin from a solution derived from a biological source.

The preparation of the crosslinked sulfonated polymer is preferably carried out by sulfonation of an already crosslinked

Polymers by using sulfuric acid and the like

Materials, such as in the manufacture of sulfonated crosslinked polystyrene from the British

Patents GB 1116800 and GB 1483587 is known. The preparation of crosslinked polymers is state of the art and can be carried out by any person skilled in the art of polymer chemistry without inventive step.

However, the sulfonation is particularly preferred

carried out as follows: depending on the desired

Sulfonierungsgrad is stirred, for example, a polystyrene-divinylbenzene polymer in a mixture of sulfuric acid and water with a water content of 2 to 15% at temperatures of 20 ° C to 80 ° C for 1 to 6 hours. The increase of the sulfuric acid content, the temperature and the

Reaction time each leads to an increase in the degree of sulfonation. By setting all three parameters, the desired degree of sulfonation can be achieved relatively accurately. After the reaction, the polymer is rinsed with dilute sulfuric acid and water. In the invention, it is also preferable that the crosslinked sulfonated polymer is coated with an amino group-containing crosslinked polymer. The backbone of the amino group-containing crosslinked polymer is preferably the same as mentioned above for the crosslinked sulfonated polymer. Thus, the backbone is particularly preferably a polyvinyl skeleton. To this polyvinyl skeleton are preferably by covalent

Single bonds amino groups directly to atoms of

Basic skeleton knotted.

According to the invention, amino groups are understood as meaning primary, secondary tertiary or quaternary amino groups as well as amidine or guanidine groups. This is the amino groups

however, the crosslinked polymer containing is more preferably a crosslinked polyvinylamine. The crosslinking of the amino group-containing crosslinked

Polymer is preferably carried out by reacting a linear polymer containing primary or secondary amino groups with a cross-linking reagent capable of covalent bonding with the amino groups at two ends.

In principle, any conceivable crosslinking reagent can be used for this purpose. However, particularly preferred according to the invention, crosslinking reagents are used in which all of them are suitable for the

Crosslinking used amino groups after crosslinking still be present in the form of an amino group. In this way it is ensured that the amino groups through

Protonation / alkylation are still able to act as cationic ion exchange groups. This leads to a high density of ion exchange groups on the otherwise lipophilic matrix. After crosslinking, the previously primary or secondary amino groups are then considered as

secondary or tertiary amino groups.

To give the amino groups a positive charge, they can be protonated. Alternatively, but also primary, secondary or tertiary amino groups are converted by tri-, bi- or monoalkylation with an alkylating reagent in quaternary ammonium ions. The degree of crosslinking of the amino group-containing crosslinked polymer is preferably in the range of 5 to 80%, more preferably in the range of 6 to 60%, and on

most preferably in the range of 10 to 40%. The percentage refers to the number of crosslinks

used amino groups in relation to all amino groups of the uncrosslinked polymer.

It is particularly preferable that the mass ratio of the amino group-containing crosslinked polymer to the

crosslinked sulfonated polymer ranges from 0.05 to 0.3, more preferably from 0.08 to 0.25, and most preferably from 0.11 to 0.20.

The crosslinked amino-containing polymer is

preferably in the form of a layer / coating on the crosslinked sulfonated polymer. Here is the

crosslinked sulfonated polymer is preferably used in the form of resin particles and coated with the uncrosslinked amino group-containing polymer and then crosslinked with the crosslinking agent. This allows a high concentration of amino groups on the surface

be realized without being affected by this procedure

completely lose the lipophilic properties of the matrix. Thus, there is provided an ion exchange resin capable of anionic groups of the macromolecule by protonation / alkylation of the amino groups

interact. In addition, the lipophilic matrix can also undergo lipophilic interactions with the macromolecule. The amino group-containing crosslinked polymer contained on the surface of the sulfonated polymer is preferably deposited in the pores of the resin particles of the sulfonated polymer, that is, it is preferably in the pores of the sulfonated polymer.

The amino group-containing crosslinked polymer has

preferably an average molecular weight in the range of 20,000 to 50,000 g / mol, more preferably 30,000 to 46,000 g / mol.

Particularly preferred are by this cationic

Ion exchange resin macromolecules such as DNA or RNA removed from the solutions, so that the solution is purified from this, and desired target molecules can be obtained without DNA or RNA from the solution.

Likewise preferred by the inventive

Ion exchange resin (anion exchanger) macromolecules such as endotoxins removed from the solutions, so that these at the

Ion exchange resin initially bound and the solution is largely free of the endotoxins. Either the original solution can thus be freed from the endotoxins and used further, or the endotoxins can be removed by elution from the ion exchange resin with a suitable one

Solution can be won. Endotoxins are a class of biochemicals. They are decay products of bacteria that are numerous physiological in humans

Can trigger reactions. Endotoxins are part of the outer cell membrane (OM) of Gram-negative

Bacteria or blue-green algae. Chemically, they are lipopolysaccharides (LPS), which are composed of a hydrophilic polysaccharide and a lipophilic lipid portion. Unlike the bacteria from which they are derived, endotoxins are very heat stable and even survive the sterilization. The currently most sensitive method of measuring endotoxins is by activating the coagulation cascade in the lysate of amebocytes isolated from horseshoe crabs (Limulus polyphemus). This test is commonly known as the LAL test.

As already mentioned, the invention come

Macromolecules from biological sources. Here, the macromolecules preferably have a molecular weight in the range of 1000 to 0.2 kDa, more preferably 500 to 1 kDa, and most preferably 300 to 5 kDa.

By solution derived from a biological source is meant solutions obtained, for example, by fermentation or fermentation processes, body fluids or plant extracts which preferably have an ionic conductivity in the range of 0.1 mS / cm to 120 mS / cm, more preferably in the Range from 1 to 60 mS / cm, and most preferably from 10 to 20 mS / cm. These solutions are preferably aqueous solutions. They preferably have a salt content of up to 1.2 mol / L. More preferably, their salt content is in the range of 0.01 to 1.2 mol / L, stronger

preferably in the range of 0.05 to 1.0 mol / L, and most preferably in the range of 0.25 to 0.6 mol / L. A salt in the present invention means any salt, such as inorganic and organic salts, which are preferably present in

biological fluids are present. Here, these solutions are not only solutions that are obtained and used directly from the biological sources, but also solutions that have already been processed in some way. By "processed" it is meant that the solutions have been pretreated in some way, for example the Change in the pH or the separation of substances before the use according to the invention.

The ionic conductivity is inventively with a

Conductivity meter of the company Greisinger type GMH 3430 determined.

The crosslinked sulfonated polymers used in the present invention or the crosslinked one coated with a layer of an amino group-containing crosslinked polymer

sulfonated polymers can thus be bound biological macromolecules from solutions with an extremely high salt content, without the solutions previously by additional

Dilution steps or dialysis must be diluted. In this way, the present invention stops

inexpensive method / inexpensive use for the purification of biological macromolecules, preferably

Insulin, monoclonal antibodies, DNA or RNA ready.

In addition, the ion exchange materials used have the advantage that they can be used in the entire pH range of 1 to 14, as it occurs in biological source fluids.

The present invention also relates to the further embodiments:

(i) Method for separating a macromolecule from a

Solution derived from a biological source using a cross-linked sulfonated polymer bonded to its backbone a sulfonated

contains aromatic moiety which is substituted or unsubstituted with an aliphatic radical. (ii) The method of embodiment (i), wherein the backbone is a crosslinked polyvinyl backbone.

(iii) The process of embodiment (i) or (ii) wherein the aromatic moiety is a phenylsulfonic acid group.

(iv) Method according to any of embodiments (i) to

(iii) wherein the crosslinked sulfonated polymer is a sulfonated polystyrene-divinylbenzene copolymer.

(v) The method according to any one of embodiments (i) to (iv), wherein the crosslinking degree of the crosslinked sulfonated polymer is 0.5 to 50%. (vi) The method according to any one of embodiments (i) to (v), wherein the degree of sulfonation is 1 to 80% based on the number of moles of sulfonic acid groups relative to all the sulfonatable monomer units used for the polymerization.

(vii) A method according to any one of embodiments (i) to (vi), wherein the crosslinked sulfonated polymer is in the form of resin particles. (viii) Method according to embodiment (vii), wherein the

Resin particles a mean average

Have diameter from 1 to 1000 pm.

(ix) The method of embodiment (vii) or (viii) wherein the resin particles have pores with an average diameter in the range of 10 to 400 nm.

(x) The method of any one of embodiments (i) to (ix), wherein the macromolecule is a peptide. (xi) The method of embodiment (x), wherein the peptide is insulin. (xü) A method according to any one of embodiments (i) to (ix), wherein the crosslinked sulfonated polymer is coated with an amino group-containing crosslinked polymer.

(xiii) Method according to embodiment (xii), wherein the

Degree of crosslinking of the amino group-containing polymer is in the range of 5 to 80%.

(xiv) The method of embodiment (xii) or (xiii) wherein the amino group-containing crosslinked polymer

crosslinked polyvinylamine is.

(xv) Method according to one of the embodiments (xii) to

(xiv), wherein all the amino groups used for crosslinking after crosslinking in the form of an amine

available.

(xvi) Method according to one of the embodiments (xii) to

(xv), wherein the mass ratio of the crosslinked

sulfonated polymer to the amino group-containing crosslinked polymer is in the range of 3 to 20.

(xvii) Method according to one of the embodiments (xii) to

(xvi) wherein the macromolecule is an endotoxin, DNA or RNA.

In the following, the invention will be explained with reference to figures and examples, which are not to be understood as limiting the scope of protection. Characters :

Figure 1: Comparison of a used in the invention

Ion exchanger with the non-inventive uses of two ion exchangers according to the prior art by measuring the loading capacity with insulin as a function of the salt concentration.

Figure 2: Order of the extinction of the eluate against time

Flow of a fermentation solution through an anion exchanger material used according to the invention.

Figure 3: Comparison of a used in the invention

Ion exchanger with the non-inventive uses of two ion exchangers according to the prior art by measuring the loading capacity with DNA as a function of the salt concentration.

Examples:

Example 1: Preparation of a cation exchange resin based on a crosslinked sulfonated polymer

Aim of the approach: Sulfonation of the polystyrene carrier

Amberchrom XT 30 (commercially available from The Dow Chemical Company, formerly Rohm & Haas) at 20 ° C.

It was 165 mL conc. Place H ₂ SO ₄ in a temperature-controlled 250 mL reactor. To the sulfuric acid was added 30.0 g of the

Carrier material is added and the weighing container three times with 20 mL conc. Rinsed sulfuric acid. After the addition of the support material, the suspension was stirred and heated to 20 ° C. After 2 h reaction time, the suspension was drained from the reactor and distributed to two 150 mL syringes. The sulfuric acid was filtered off with suction and the phase was rinsed successively with 200 ml of dilute (62% strength) sulfuric acid, 125 ml of water, 175 ml of methanol, 125 ml of water and finally with 175 ml of methanol. The phase was sucked dry and

then dried at 50 ° C in a vacuum.

The determination of the sulfonic acid groups is carried out in an HPLC column by loading with ammonium acetate, followed by

Elution of bound ammonium and detection over

Indophenol blue. The result was a sulfonic acid content of 375 pmol / mL. This corresponds to a degree of sulfonation of

approximately 13%. The particle size is on average 30 pm. The particles are spherical with a medium

Pore diameter of 22 nm and a mean pore volume of 1.25 mL / g.

Example 2: Preparation of an anion exchanger based on a cross-linked sulfonated polymer coated with an amino group-containing crosslinked polymer

The basis for the ion exchange material is Amberchrom

CG1000S used by Rohm & Haas. This will, as in

Example 1, sulfonated at 80 ° C with 98% sulfuric acid for 3 hours. This gives particles with a mean average size of 30 pm and a

average pore diameter of 22 to 25 nm. Of the

resulting sulfonated polystyrene becomes the

Water absorption capacity, or the pore volume determined by weighing the dried, sulfonated polystyrene, with the same volume of water is added and then

Excess water is centrifuged off. The water in the pores remains in place. To

Weighing again can turn the weighing difference into a dry one Polystyrene, the pore volume to about 1.2 to 1.3 mL / g are determined.

To coat the polystyrene is an aqueous

Polyvinylamine solution which consists of polyvinylamine having an average molecular weight of 35000 g / mol. The pH is adjusted to 9.5. The amount of polyvinylamine is 15% of the polystyrene to be coated, and the volume of the solution is 95% of the determined pore volume of the polystyrene. The polyvinylamine solution is used together with the

Put polystyrene in a tightly closed PE bottle and shake for 6 hours on a high speed sieve shaker. Care must be taken to ensure adequate mixing. After the procedure, the

Polyvinylamine solution worked into the pores of polystyrene. The polystyrene is then at 50 ° C in

Vacuum drying oven dried to constant weight.

To crosslink the polyvinylamine is the coated

Polystyrene taken in three volumes of isopropanol and with 5% diethylene glycol diglycidyl ether, based on the

Amino group number of polyvinylamine, added. The

Reaction mixture is stirred for six hours in the reactor at 55 ° C. It is then transferred to a glass suction chute and rinsed with 2 bed volumes of isopropanol, 3 bed volumes of 0.5 M TFA solution, 2 bed volumes of water, 4 bed volumes of sodium hydroxide solution and finally 8 bed volumes of water.

Example 3: Purification of Insulin by the Cat ion Exchanger Prepared in Example 1

The determination of the loading capacity with insulin of the salt-tolerant ion exchanger prepared in Example 1 is carried out with a solution of 10 mg / ml insulin in 30% isopropanol with 50 mM

Lactic acid at pH 3.5 and different concentrations NaCl. Loadability was determined at 10% breakthrough and compared to two competing materials. The results are shown in FIG. The commercially available ion exchange materials "Eshumo S" from Merck (polyvinyl ether, ionic capacity 50-100 pmol / ml, particle size 75-95 μm) and "Source 30S" from GE Healthcare (polystyrene / divinylbenzene, particle size 30 μm ) used.

While the comparison materials at 250 mM NaCl content in the eluent show only very low capacity, which is no longer measurable at higher salt levels, the

Ion exchangers used in the invention still have a significant capacity to 1 M NaCl. This is clear from FIG. 1

to read.

Example 4 Separation of DNA by Using the Anion Exchange Resin Prepared in Example 2

The first step in the process of purification of monoclonal antibodies from fermentation solutions is the depletion of the contained DNA. This works by "filtering" the fermentation solution through a phase of the anion exchanger prepared in Example 2. The DNA binds to the phase in this step, and the quantitatively run through fermentation solution is thus nearly freed from the DNA.

For this purpose, the anion exchanger prepared in Example 2 is packed into a 270 × 10 mm column with a bed volume of 21.2 ml and equilibrated with first 500 mM NaKPO ₄ pH 7.0 and then with 50 mM NaKPO ₄ pH 7.0. The fermentation solution is filtered through a 0.45 μm filter and filtered from

Precipitation freed. 300 mL of the fermentation solution is added to the column via an external pump. It is the flow, the eluate with 1 M NaCl pH 6.5 and the rinse step with 1 M NaOH collected. The part designated flow in FIG. 2 contains almost exclusively the monoclonal antibody and no DNA. However, elution of the DNA takes place only by the application of NaOH.

The content of DNA in the flow and in the fermentation solution is determined by a Picogreen assay according to the manufacturer

certainly .

Table 1:

dsDNA

Fermentation solution dsDNA

(filtered) flow

4767 μ9 30 μ9

100% 0.7%

9046 ppm 57 ppm

From Table 1 shows that 99.3% of the DNA in the

Filtration could be removed via the phase material. The bound DNA does not elute in the 1 M NaCl step, but only by rinsing with 1 M NaOH, since here the amino groups of the phase are deprotonated and no binding to the DNA is present.

Alternatively to the one prepared in Example 2

Anion exchangers were also commercially available

Materials for Q Sepharose FF from Amersham Biosciences and Fractogel TMAE from Merck as release agent as used in Example 4. Determining the static capacity at different salt contents compared to Q Sepharose FF and Fractogel TMAE results in a higher loadability of the developed ion exchanger even at high salt contents.

Example 5: Separation of endotoxins

Fermentation solutions by using the in Example 2

An anion exchange resin prepared: A fermentation solution containing endotoxins is "filtered" through a phase of the anion exchanger prepared in Example 2. The endotoxins bind in this step to the Phase, and the quantitatively running fermentation solution is thus released from the endotoxins approximately.

For this purpose, the anion exchanger prepared in Example 2 is packed in a 270 × 10 mm column with a bed volume of 21.2 ml. The fermentation solution is filtered through a 0.45 pm filter and freed of precipitates. 300 mL of the fermentation solution is added to the column via an external pump. The column effluent contains at least 90% less endotoxin than the fermentation solution. The LAL test was used to detect the endotoxin content. In this way, the fermentation solution of a large part of the

Endotoxins are freed. The endotoxins were then washed with a suitable eluate from the ion exchanger.

Claims

claims

Use of a crosslinked sulfonated polymer for separating a macromolecule from a solution derived from a biological source, wherein the crosslinked sulfonated polymer attached to its backbone contains a sulfonated aromatic moiety substituted or unsubstituted with an aliphatic radical.

2. Use according to claim 1, wherein the backbone is a crosslinked polyvinyl skeleton.

Use according to claim 1 or 2, wherein the sulfonated aromatic moiety is a phenylsulfonic acid group.

4. Use according to any one of claims 1 to 3, wherein the crosslinked sulfonated polymer is a sulfonated

Polystyrene-divinylbenzene copolymer.

Use according to any one of claims 1 to 4, wherein the degree of crosslinking of the crosslinked sulfonated polymer is 0.5 to 50%.

6. Use according to one of claims 1 to 5, wherein the degree of sulfonation is 1 to 80%, based on the number of moles of sulfonic acid groups in relation to all sulfonated used for the polymerization

Monomer units.

Use according to any one of claims 1 to 6, wherein the crosslinked sulfonated polymer is in the form of resin particles.

8. Use according to claim 7, wherein the resin particles have an average average diameter of 1 to 1000 pm.

Use according to claim 7 or 8, wherein the

Resin particles pores with an average

Have diameters in the range of 10 to 400 nm.

Use according to any one of claims 1 to 9, wherein the macromolecule is a peptide.

Use according to claim 10, wherein the peptide is insulin.

Use according to any one of claims 1 to 9, wherein the crosslinked sulfonated polymer is coated with an amino group-containing crosslinked polymer.

13. Use according to claim 12, wherein the degree of crosslinking of the amino group-containing crosslinked polymer is in the range of 5 to 80%.

Use according to claim 12 or 13, wherein the amino group-containing crosslinked polymer is crosslinked

Polyvinylamine is.

Use according to any one of claims 12 to 14, wherein

all amino groups used for crosslinking are in the form of an amine after crosslinking.

Use according to any one of claims 12 to 15, wherein the macromolecule is an endotoxin, DNA or RNA.