DE10149587B4 - Photoreactive coated composite membrane for affinity separation, process for their preparation and their use - Google Patents

Abstract

Coated composite membrane for affinity separation based on a separately prepared, porous matrix membrane whose surface is functionalized,
obtainable by that
a reaction mixture suitable for a surface functionalization and containing at least one functionalizing, graft-polymerizable monomer is used;
the matrix membrane is contacted with this reaction mixture and remains in continuous contact during surface functionalization, and
the reaction mixture while in contact with the matrix membrane, a reaction condition I corresponding to a primary excited graft polymerization to form crosslinked graft tentacles, and then thereafter a reaction condition II corresponding to a post reaction without primary excitation, graft tentacles in a graft polymerization without extension is continued, so that two different polymeric layers are successively formed on the matrix membrane for surface functionalization.

Description

The The invention relates to a coated composite membrane for affinity separation based on a separately produced, porous matrix membrane whose surface functionalizes is a method for producing this composite membrane and its Use.

At usual Membrane separation processes, the separation is based on the molecule or Particle size. However nearly same size molecules or separate particles from each other, it requires different substance-specific interactions (affinities) between the separation media or membranes and the substances to be separated, around the substances as well to be able to separate from each other. The separation should be characterized by reversible substance-specific (affinity) interactions when flowing through a membrane can be achieved.

It are already affinity membranes known in which such a substance-specific binding to isolated, simple synthetic functional groups (ionic, hydrophobic, metal chelate), chains containing functional groups (Multipoint binding) or fixed biological or biomimetic Receptors (proteins, peptides, DNA) takes place. With such Substance-specific interaction forces may occur the membrane surface Functional groups available standing, either directly or after covalent bonding of appropriate Receptors become effective. To achieve that substance-specific interactions are as pronounced as possible, should the nonspecific interactions of substances with the Membrane as possible be minimal.

The described affinity membranes can can be made directly from polymers that are affine or reactive Groups (the latter for the fixation of receptors) included. As examples can be called membranes made of cellulose or cellulose derivatives, However, they have the disadvantage of being mechanical and chemical unstable (E. Klein Affinity Membrane, John Wiley & Sons, New York, 1991). It is also an aldehyde-reactive homogeneous polymer membrane known, which is described in Wolpert S., Aldehyde activated microporous membranes, Journal of Membrane Science 132 (1997), 23-32.

such known affinity membranes are disadvantageous, inter alia, in that the pore structure, the in the typically used phase inversion process for production of these polymers, which determines polymer functionality. In addition, the functional groups are not only on the surface, but also present in the volume of the membrane. The distribution and the amount The functional groups is therefore difficult to control and can also the stability negatively affect these known membranes.

by virtue of These disadvantages have become mostly commercial for the preparation of affinity membranes available porous Membranes whose pore diameters typically range from 0.2 to 5 μm are enough, later heterogeneously modified to achieve at least a "fine-tuning" of specificity. The variability of this For example, membranes can be increased by reactive groups (For example, epoxy groups) for subsequent polymer-analog generation / fixation of functional groups, for the fixation of affine or reactive Coatings or for direct immobilization of receptors.

It is also already known, on the inner surface of the matrix membrane a To fix hydrophilic polymer coating, which then also carrier the functional groups or functional groups, see E. Klein, P. A. Feldhof, US Pat. No. 5,053,133; (E. Klein, D.H. Yeager (Akzo Nobel NV), US Pat. No. 5,766,908; F. B. Anspaach et al., DE-A 19609479, WO-A 97/33683). The coating with cellulose derivatives is so far unspecific for the suppression Interactions preferred.

From Of particular interest are modifications, the so-called tentacle affinity structures in microporous Introduce membranes, compare T. Sugo, S. Saito; Japan Atomic Energy Research Institute, US Pat. No. 5,344,560. Also these can reduce and enable nonspecific adsorption on the solid support surface also better replacement capacities based on the membrane volume and the formation of interactions with target molecules (Multi-point binding).

For sensitive proteins, moreover, the tendency to denature on such tentacle structures can be markedly reduced, see M. Kaufmann, Unstable Protein: how to subject them to chromatographic separations for purification processes. J. Chromatogr. B 699 (1997) 347. In addition, variants of the tentacle concept described above can be prepared by means of the electron beam-induced graft polymer modification of polyethylene MF membranes on a laboratory scale (Matoba S., Tsuneda S., Saito K. and Sugo T., Higly Efficient Enzyme Recovery Using a Porous Membrane with Immobili zed Tentacle Polymer Chains, Bio / Technology 13 (1995), 795-797).

These known affinity membranes however, are disadvantageous in many respects. That's the way they are the very uneven pore structure over the Layer thickness of the matrix membrane over a relatively low binding capacity. Further, different ones these known membranes because of the uneven pore structure of the matrix membrane only suitable as a membrane adsorber and thus have only a small chromatographic resolution. In particular, in the tentacle membranes of the type described above is not a steam sterilizability due to the limited stability of the matrix membrane possible. In addition, the surface functionalization (Functional group density, coupling chemistry) not optimal, so that she just over a low activity of covalently linked biological receptors. Further their production is technologically very complicated and only by Multi-step process of polymer modification and polymer surface activation and subsequent further coatings or functionalizations possible.

by virtue of these shortcomings of the known membranes, affinity membrane separation has become a technology not yet generally enforced.

From the DE 196 22 959 A1 a composite membrane is provided comprising a polymeric support membrane and a polymeric release layer applied thereon, which is characterized in that the release layer is applied by a photoinitiated graft polymerization. This composite membrane is in particular obtainable by a photochemical modification of the surface of asymmetric ultrafiltration membranes, preferably of polyacrylonitrile. The membrane described there is a stable, defect-free, thin composite membrane with high separation efficiency, which can be used for selective organics separation by pervaporation.

task The present invention is one for affinity membrane separation suitable coated composite membrane and a method for the production provide the same, on the one hand economically producible and whose properties, on the other hand, are easily adaptable Separation problem can be adjusted.

Is solved This object is achieved by a composite membrane according to the teaching of claim 1 and a method according to the teaching of claim 11. Preferred embodiments are described in the subclaims.

According to the invention of a porous one Matrix membrane, which was prepared separately. As matrix membrane In doing so, preference is given to those which already have regard to Pore structure and specific surface are optimized. According to the invention it can in the matrix membranes also already known, if necessary also commercially available Membranes act, both in terms of mechanical, thermal and chemical stability as well as regarding their pore structure for the intended purpose are suitable. Typically, these exist from relatively hydrophobic polymers with less directly usable functionality or reactivity.

According to the invention is for Production of a Matrix Membrane According to the Invention the previously prepared matrix membrane with a reaction mixture brought into contact. This reaction mixture is one for a surface functionalization suitable reaction mixture containing at least one functionalizing monomer, which is capable of using two different reaction conditions two different polymeric layer structures on the matrix membrane train. In other words, it becomes a polymerizable reaction mixture used in two different reaction conditions for the Polymerization in two different reactions to different results in polymeric product structures.

After the matrix membrane has been brought into contact with this reaction mixture, the two different reaction conditions are applied successively or adjusted from the outside, as it were. While performing these two reactions, the matrix membrane remains in contact with the reaction mixture. The matrix membrane is thus functionalized in one process step. The term "a" process step is intended to describe the fact that the matrix membrane remains in contact with the reaction mixture when the two reactions are carried out and only the reaction conditions specified "from the outside" are changed so that the surface functionalization of the matrix membrane results in a two-layer reaction. Structure arises. Preferably, the reaction mixture contains as functionalizing monomer only a monofunctional monomer or only monofunctional monomers. As a functional sierender monomer is used a capable of graft polymerization monomer. In addition, the reaction condition I is chosen such that it corresponds to a primarily excited, in particular a physically initiated, graft polymerization. The reaction condition II is chosen such that it corresponds to that of an after-reaction without primary excitation.

You choose now, for example, in a monofunctional, to a graft polymerization capable of monomers containing reaction mixture I such, that a photoinitiated graft polymerization takes place, then so-called Pfropfententakel form during initiation on the matrix membrane. irradiated you now simultaneously or simultaneously with UV light, then finds one Networking the Pfropfententakel instead. In other words, the reaction condition I is such that a photoinitiated graft polymerization with simultaneous UV crosslinking of the graft tentacles on the surface of the Matrix membrane expires.

switches one at the chosen one Example according to the above-described reaction I the UV irradiation, then a graft polymerization takes place without UV irradiation and thus even without networking the still "surviving" radicals, after the reaction condition I was "turned off". The graft polymerization without UV irradiation then represents reaction condition II, in which predominantly an extension of Polymer chains without further crosslinking takes place.

According to select one the reaction conditions I and II such that in the reaction condition I first formed layer shields the matrix membrane, for example for one Minimize unspecific interactions. The Reaction condition II selects one then such that after the first formed layer and Thus, the layer formed thereafter serves as a functional layer. In the reaction condition II is thus preferably a functional Layer formed z. For example the affinity binding can serve.

Conveniently, is thus "started" with a first layer on the surface, in which a possible full Covering this surface takes place. After that, another layer can be gradual and be built up in layers, so that a quasi-growth takes place "outwards". It is the rest also possible, According to the invention, a structure build from more than two such layers by the Reaction conditions change accordingly, so that in the course the surface functionalization different layers are created.

The surface functionalization the matrix membrane takes place in one process step, because the matrix membrane remains in contact with the reaction mixture and all layers made using this reaction mixture become.

The Surface functionalization according to the invention leads in only a technological step to a structure, preferably Both effective shielding of the matrix membrane and minimal Reduction of membrane permeability ensures as well as functional groups in adjustable quantity and accessibility (relatively open and flexible layer with partial "tentacle" character) generated.

With In other words, according to the invention are membranes available, in which said two-layer structure by means of a single-stage Functionalization method is formed. This way are direct functional composite membranes are available in which both the Matrix membrane (pore structure, specific surface) as well as their surface functionality (synthetic functional polymer) independently be varied from each other by simple, unique parameters can.

Alles this is with an easy to perform well reproducible One-step procedure possible, whereby not only the cost-effectiveness of the required functionalization is significantly improved, but also a method arsenal to disposal stands with the development of powerful affinity membranes at all only possible is. The invention thus also relates to a process for the preparation the coated invention Composite membranes.

The Graft polymerization can be achieved by a primary excitation with plasma, electron beam and gamma-ray initiated or induced. Preferably it is in the graft polymerization to a photoinitiated copolymerization and in particular a heterogeneous, partially crosslinking graft copolymerization.

Benzophenone is preferably used as the photoinitiator for the graft polymerization. As a functionalizing monomer is preferably 2-aminoethyl methacrylate and / or glycidyl methacrylate Applic dung.

By the adjustment of the reaction parameters in the preparation of the membranes obtainable according to the invention arises during the polymerization, a so-called two-layer structure from a relatively compact hydrophilic "shield" layer, for example, a non-specific Prevents protein adsorption, and a relatively open "affinity binding" layer.

The available according to the invention Membranes are eminently suitable for substance-specific separations, z. B. membrane adsorber solid phase extracts, membrane chromatography and membrane reactor.

at the inventively available Membranes are preferably photoreactively coated Composite membranes produced by photoinitiated polymerization preferably UV light are available. After the UV excitation closes preferably a dark reaction.

These preferred embodiment the inventively available Composite membrane is explained in more detail below. These are the experimental conditions the inventively available Composite membrane in more detail explained. It also explains which parameters (for example, UV intensity, monomer concentration, exposure time etc.) the various properties of the invention available Influence composite membrane.

These preferred embodiment the inventively available Composite membrane is explained in more detail below. These are the experimental conditions the inventively available Composite membrane in more detail explained. It also explains which parameters (for example, UV intensity, monomer concentration, exposure time etc.) the various properties of the invention available Influence composite membrane.

1. General experimental conditions

Surface functionalization - photoinitiated Polymerization and subsequent Dark reaction after UV excitation

• a) matrix membranes MF membrane: Accurel PP 2E HF (AkzoNobel Wuppertal), polypropylene (PP) UF membrane: Polyacrylonitrile (PAN)
• b) functional monomers 2-aminoethyl methacrylate (for investigation the non-specific protein adsorption by ion exchange and, after Glutaraldehyde activation, for covalent protein binding) glycidyl methacrylate (for direct covalent protein binding)
• c) photoinitiator Benzophenone (BP)
• d) surface functionalization optional (Pretreatment of the matrix membrane) • Extraction in methanol • drying to constant weight coating with photoinitiator • loading the matrix membrane with reaction mixture (monomer solution) • UV exposure (= Reaction condition I) • dark reaction (without UV; = reaction condition II) optional (post-treatment the composite membrane) • With Extract water or methanol
• e) Characterization of the composite membrane • Gravimetric Determination of the degree of modification (DG) after membrane drying • Determination of water permeability and filtrate current density
• f) Applications of the composite membrane as an affinity carrier • (unspecific) Binding of proteins by ion exchange (→ protein purification) • covalent Immobilization of proteins or enzymes (→ enzyme reactor)

2. Examples

Example A - Composite membrane based on a UF membrane

• a) matrix membrane UF membrane: polyacrylonitrile (PAN)
• b) functional monomer Glycidyl methacrylate (for direct covalent protein binding)
• c) photoinitiator Benzophenone (BP)
• d) Surface functionalization • Coating with photoinitiator • Application of matrix membrane with reaction mixture (monomer solution), under nitrogen • UV exposure, variable UV intensity I, variable time t _B , under nitrogen (= reaction condition I) • dark reaction (without UV), variable Time t _NR , under nitrogen (= reaction condition II) • Extract with water or methanol
• e) Characterization of the composite membrane • Gravimetric Determination of the degree of modification (DG) after membrane drying → Tables 1-3 • Determination of water permeability and filtrate current density → Tables 1, 2
• f) Applications of the composite membrane as an affinity carrier • Covalent Immobilization of enzyme (→ enzyme reactor) → tables 3, 4 Enzyme: Amyloglucosidase from Aspergillus niger Activity tests: substratum 2% maltose in citric acid-phosphate buffer pH = 4.6, T = 40 ° C relative Activity: Activity of the bound Enzyme / Activity of the solved Enzyme 100%

Results

Table 1: Influence of UV intensity on degree of modification (DG) and permeability on membrane type I c _GMA = 15 μg / ml, 10 min. Exposure time (t _B ), 15 min. Post-reaction time (t _NR), permeability (J _W) of the starting membrane: J _W = 500 l / hm ² bar ± 10%

Table 2: Influence of exposure time on DG and permeability on membrane type 2 c _GMA = 15 μg / ml, UV intensity = 48 mW / cm ² Permeability (J _W ) of the starting membrane: J _W = 1680 l / hm ² bar ± 10%

Table 3: Results of Enzyme Immobilization to Table 1:

Table 4: Enzyme immobilization results to Table 2:

discussion

at higher UV intensity become a higher one Grafting and thus a lower permeability of the membranes obtained (see Table 1).

With renewal the post-reaction time occurs an increase in the degree of grafting and thus a reduction in permeability (see Table 2).

at lower UV intensity becomes an open, uncrosslinked layer with tentacles obtained (see 3.2) for a protein (BSA) or Enzymbindung is more easily accessible. Therefore, more enzyme can be bound, but not everything is active (lower relative activity, see Table 3); this becomes with the influence of the membrane pore structure (Type 1 → small Pore size → disability substrate transport).

As the postreaction time increases, the degree of grafting increases while the amount of bound protein remains unchanged, resulting in an increase in the specific activity (see Table 4), since the mean Pore size of the membrane (type 2) is much larger than in type 1.

Example B - composite membrane based on a MF membrane

• a) matrix membrane MF membrane: Accurel PP 2E HF (AkzoNobel Wuppertal), polypropylene (PP)
• b) functional monomers 2-aminoethyl methacrylate (for investigation the non-specific protein adsorption by ion exchange and, after Glutaraldehyde activation, for covalent protein binding).
• c) photoinitiator Benzophenone (BP)
• d) Surface functionalization • Coating with photoinitiator • Applying the matrix membrane with reaction mixture (monomer solution), under nitrogen • UV exposure, variable UV intensity I (here by exposure of two membranes on top of each other → lower intensity for lower membrane), variable time t _B , under Nitrogen (= reaction condition I) • Dark reaction (without UV), variable time t _NR , under nitrogen (= reaction condition II) • Extract with water or methanol
• e) Characterization of the composite membrane • Gravimetric Determination of the degree of modification (DG) → Table 5
• f) Composite membrane applications as affinity carriers • (unspecific) Binding of protein by ion exchange (→ protein purification) → Tables 6, 7. • Covalent Immobilization of protein → table 8th

Results and discussion

a. degrees of modification (DG) of the composite membranes

Table 5: Modification degrees depending on the reaction conditions

• • Eine höhere Intensität (bei konstanter Belichtungszeit) bei den Membranen 3/1 und 3/2 sowie 4/1 und 4/2 führt zu höheren Modifizierungsgraden, d.h. die Effektivität der Initiierung wirkt sich direkt auf den Bruttoeffekt (Bedeckung des Trägers) aus;
• • eine thermische Nachreaktion hat vor allem nach Belichtung hoher Intensität bei den Membranen 4/1 und 4/2 einen deutlichen Einfluß auf DG; bei hoher Intensität entsteht eine hohe Radikalkonzentration, die wiederum eine signifikante Nachreaktion bewirkt.
• • bei niedriger Intensität ist der Effekt der Nachreaktion bei den Membranen 9/1 und 9/2 gering.

The degrees of modification (DG) obtained in the preparations with identical monomer solutions can be interpreted as follows:

• • A higher intensity (with constant exposure time) for membranes 3/1 and 3/2 as well as 4/1 and 4/2 leads to higher degrees of modification, ie the effectiveness of the initiation has a direct effect on the gross effect (coverage of the carrier);
• • a thermal after-reaction has a marked influence on DG especially after exposure to high intensity in the membranes 4/1 and 4/2; At high intensity, a high radical concentration is generated, which in turn causes a significant post-reaction.
• • At low intensity the effect of after-reaction is low at membranes 9/1 and 9/2.

b protein binding on composite membranes

ion exchange

• Protein: bovine serum albumin (BSA), 1 g / L in 0.066 M phosphate buffer (pH 5, pH 7)
• Slightly shaken membrane samples (water-moist, A = 0.785 cm ² ) in protein solution for 14 h, then washed as follows: shaken vigorously for 1 h with buffer (pH 5 or pH 7), buffer changed twice for 1 h with Tween solution (1 g / l Tween 20 + 9 g / l NaCl) shaken vigorously, Tween solution 2 times changed

Table 6: Protein binding by ion exchange at pH = 5.0

Table 7: Protein binding by ion exchange at pH = 7.0

Covalent immobilization

• Membrane samples (water-wet, A = 0.785 cm ² ) activated with glutaraldehyde (GA); in 10% GA shaken for 5 h at RT, then washed 3 times for 1 min with water and 1 time with buffer pH 5 or pH 7;
• Treatment with protein solution 14 h and washed as follows: for 1 h with buffer (pH 5 or pH 7) shaken intensively, Buffer changed twice For 1 h with Tween solution (1 g / l Tween 20 + 9 g / l NaCl) shaken vigorously, Tween solution 2 times changed

Table 8: Covalent protein binding at pH = 7.0

The Results can be interpreted as follows:

Influence of the UV reaction (see a.)

• → graft layer with intensity-dependent degree of crosslinking - at high intensity compact, cross-linked layer (C) with low binding capacity (absolute & specific: BSA & BSA / DG) in particular at pH = 7 - at low intensity open, uncrosslinked layer (tentacles, T) with higher specific binding capacity (BSA & BSA / DG) in particular at pH = 7

Influence of the thermal after-reaction (see a.)

• → isolated, uncrosslinked chains, (ΔBSA / ΔDG >> BSA / DG of the "precursors" without thermal post-reaction) either - on networked Graft layer (Ct, → increase of absolute and specific binding capacity) or - in little crosslinked graft layer (Tt, → increase of absolute and specific binding capacity.

The above with C, T, Ct and Tt properties and conditions are in the accompanying single figure schematically and not to scale for explanatory purposes shown.

Specific Effects on covalent bonding

• → correlation with total amount of bound protein; i.e. both unspecific as well as the ionically bound protein becomes after activation covalently fixed by glutaraldehyde.
• → afterreaction with less influence (analogous to UF membrane, example A!)
• → for BSA / DG significant dependence from the intensity (analogous to UF membrane, example A!)
• → clearer Trend: higher specific binding capacity with less networking.

Claims (14)

Coated composite membrane for affinity separation based on a separately prepared, poro sen matrix membrane whose surface is functionalized, obtainable by using a suitable for a surface functionalization, at least one functionalizing, graft polymerization capable monomer containing reaction mixture, the matrix membrane is brought into contact with this reaction mixture and during the surface functionalization in continuous contact, and the reaction mixture while in contact with the matrix membrane, a reaction condition I corresponding to a primary excited graft polymerization to form crosslinked graft tentacles, and then thereafter a reaction condition II corresponding to a post reaction without primary excitation, graft tentacles in a graft polymerization without extension is continued, so that two different polymeric layers are successively formed on the matrix membrane for surface functionalization. Composite membrane according to claim 1, obtainable thereby, that as a functionalizing monomer is a monofunctional monomer is used. Composite membrane according to claim 1 or 2, obtainable thereby, that the reaction condition I that of a physically initiated Graft polymerization corresponds. Composite membrane according to claim 3, obtainable thereby, that the graft polymerization by a primary excitation with plasma, electron beam, Gamma ray or UV exposure is initiated. Composite membrane according to claim 3 or 4, obtainable thereby, that an initiator for the physical initiation is used. Composite membrane according to claim 5, obtainable thereby, that benzophenone is used as a photoinitiator. Composite membrane according to one of claims 2 to 5, available through that as a functionalizing monomer 2-aminoethyl methacrylate or Glycidyl methacrylate can be used. Composite membrane according to one of the preceding claims, characterized available, that the matrix membrane with a solution of a photoinitiator coated with the functionalizing monomer containing Reaction mixture is applied and irradiated with UV light. Composite membrane according to one of the preceding claims, characterized characterized in that obtained in the reaction condition I. Layer shields the matrix membrane and the reaction condition II layer is formed as a functional layer. Composite membrane according to one of the preceding claims, characterized characterized in that the surface with covalently fixed, biological or biomimetic receptors functionalized. Process for the preparation of a coated composite membrane for the affinity separation based on a separately produced, porous matrix membrane whose surface functionalizes is characterized in that one for a surface functionalization suitable, at least one functionalizing, for graft polymerization UNTRAINED Monomer-containing reaction mixture is used, the matrix membrane is contacted with this reaction mixture and during the Surface functionalization permanently remains in contact, and the reaction mixture, while working with the matrix membrane is in contact, a reaction condition I, which is a primary excited Graft polymerization, formed in the crosslinked Pfropfententakel be, corresponds, and then temporally thereafter a reaction condition II, the post-reaction without primary excitation, at the Pfropfententakel be extended in a Pfropfolymerisation without further networking, equivalent, is exposed, leaving on the matrix membrane for surface functionalization successively formed two different polymeric layers become. Use of the coated composite membrane after one of the claims 1 to 10 as an affinity membrane. Method according to claim 11, characterized in that that at least one or more of the in one or more of claims 2 to 8 steps described. Method according to claim 11 or 12, characterized that under flushing working with a protective gas.