DE10149587A1 - Preparation of photoreactive coated polymer membranes by surface functionalization useful for separation of specific substances, e.g. by membrane adsorption, solid phase extraction, membrane chromatography - Google Patents

Abstract

Preparation of photoreactive coated polymer membranes where the between layer structure is formed by a single step functionalization, with direct production of functional composite membranes where the matrix membrane (pore structure, specific surface) and surface functionality (synthetic functional polymers) can be varied independently of each other via a simple, well defined parameter, is new. Coated composite membranes based on separately prepared matrix membranes with surface functionality, obtained so that the reaction mixture contains a functionalized monomer suitable for surface functionalization, which can build up, by using two different sets of reaction conditions, two different polymer layer structures on the matrix membrane, by bringing the matrix membrane into contact with the reaction mixture, and while the surface functionalization is maintained over a long period of contact, and while the reaction mixture is in contact with the matrix membrane. Reaction conditions (I) and then after a time reaction conditions (II) are used, which differ from reaction conditions (I) in that two different layers for surface functionalization are formed successively on the matrix membrane. An Independent claim is included for a process for preparation of membranes as described above.

Description

The invention relates to a coated composite membrane based a separately manufactured matrix membrane, the surface is functionalized, a process for producing this Composite membrane and its use.

In conventional membrane separation processes, the separation is based on the Molecule or particle size. However, to approximately the same size It is necessary to be able to separate molecules or particles from one another different substance-specific interactions (affinities) between the separation media or membranes and those to be separated Fabrics to separate the fabrics from each other. The Separation should be done by reversible substance-specific (affinity) Interactions when flowing through a membrane can be achieved.

Affinity membranes are now known in which one such substance-specific binding to isolated, simple synthetic Functional groups (ionic, hydrophobic, metal chelate) Chains containing functional groups (multipoint binding) or on fixed chains biological or biomimetic receptors (proteins, peptides, DNA) takes place. So such substance-specific interaction forces must occur on the membrane surface Functional groups are available that are either direct or covalent Binding of corresponding receptors become separating. In order to achieve such substance-specific interactions are as pronounced as possible, the unspecific Interactions of substances with the membrane as minimal as possible his.

The affinity membranes described can be obtained directly from polymers are produced, the affine or reactive groups (the latter for Fixation of receptors) included. As examples you can Call cellulose or cellulose derivative membranes, however have the disadvantage that they are mechanically and chemically unstable (E. Klein Affinity Membranes, John Wiley & Sons, New York, 1991). It is already 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 among others in this respect disadvantageous than the pore structure that is typically used in the Phase inversion process for the preparation of these polymers obtained the polymer functionality is determined. They are also Functional groups not only on the surface, but also in the volume of the Membrane present. The distribution and the amount of Functional groups are therefore difficult to control and the stability of these can also affect known membranes negatively.

Because of these disadvantages, affinity Membranes mostly commercially available porous membranes, their Pore diameters typically range from 0.2 to 5 µm, subsequently modified heterogeneously to at least "fine tune" the To achieve specificity. The variability of these membranes can for example, be increased by reactive groups (for example Epoxy groups) for subsequent polymer analogs Generation / fixation of functional groups, for fixation of affine or reactive coatings or for the direct immobilization of Receptors.

It is also known on the inner surface of the Matrix membrane to fix a hydrophilic polymer coating, which then also carries the functional groups or the functional groups, see E. Klein, P.A. Feldhof, U.S. 5,053,133; (E. Klein, D.H. Yeager (Akzo Nobel NV), US-A 5766908; F. B. Anspaach et al., DE-A 196 09 479, WO-A 97/33683). The coating with cellulose derivatives is included so far for the suppression of non-specific interactions prefers.

Of particular interest are modifications, the so-called Introduce tentacle affinity structures into microporous membranes, man see T. Sugo, S. Saito; Japan Atomic Energy Research Institute, U.S. A 5 344 560. These can also be the unspecific adsorption on the solid support surface reduce and also allow better Exchange capacities based on the membrane volume and the Formation of interactions with target molecules (Multi-point binding).

In the case of sensitive proteins, the tendency to Denaturation on such tentacle structures can be significantly reduced, one see M. Kaufmann, Unstable Proteins: how to subject them to chromatographic separations for purification processes. J. Chromatogr. B 699 (1997) 347. Variants of the above can also be used Tentacle concept with the help of electron beam induced Graft polymer modification of MF membranes made of polyethylene in Laboratory scale (Matoba S., Tsuneda S., Saito K. and Sugo T., Higly Efficient Enzyme Recovery Using a Porous Membrane with Immobilized Tentacle Polymer Chains, Bio / Technology 13 (1995), 795-797).

However, these known affinity membranes are numerous Disadvantageous. So they dispose of the very uneven Pore structure over the layer thickness of the matrix membrane over a relatively low binding capacity. Furthermore, several of these are known membranes because of the uneven pore structure of the Matrix membrane only suitable as membrane adsorber and therefore also have only a low chromatographic resolution. Especially with the Tentacle membranes of the type described above are not Steam sterilization because of the limited stability of the Matrix membrane possible. In addition, the surface functionalization (Functional group density, coupling chemistry) not optimal, so they only about a low activity of covalently bound biological Have receptors. Furthermore, their manufacture is very technological complex and only by multi-step process Polymer modification and polymer surface activation and subsequent further coatings or functionalizations possible.

Because of these shortcomings of the known membranes So far, the affinity membrane separation as a technology has not generally enforced.

The object of the present invention is one for Affinity membrane separation suitable coated composite membrane To provide which is economically producible on the one hand and their Properties, on the other hand, easily match the separation problem that arises can be customized.

This task is solved by a composite membrane according to the Teaching of claim 1. Preferred embodiments are in the Subclaims described.

According to the invention, a matrix membrane is assumed which was made separately. The matrix membrane is used preferably those that already have a pore structure and specific surface are optimized. According to the invention, it can in the case of the matrix membranes, also known, if appropriate commercially available membranes act both with regard to mechanical, thermal and chemical stability as well as regarding their pore structure are suitable for the intended purpose. Typically, these consist of relatively hydrophobic polymers less directly usable functionality or reactivity.

According to the invention is used to produce an inventive Matrix membrane the previously made matrix membrane with a Contacted reaction mixture. With this reaction mixture is one for a surface functionalization suitable ones containing at least one functionalizing monomer Reaction mixture capable of using two different reaction conditions two different to form polymer layer structures on the matrix membrane. With in other words, it becomes a polymerizable reaction mixture used for two different reaction conditions Polymerization in two different reactions too leads to different polymeric product structures.

After the matrix membrane is in contact with this reaction mixture the two are different Reaction conditions applied in succession or virtually from the outside set. During the implementation of these two reactions remains the matrix membrane is in contact with the reaction mixture. The The matrix membrane is thus functionalized in one process step. With the expression "a" process step is intended to describe the fact that the matrix membrane when performing the two Reactions remain in contact with the reaction mixture and only the "externally" specified reaction conditions are changed, so that through the surface functionalization of the matrix membrane a two-layer structure is created. Preferably, the Reaction mixture as a functionalizing monomer only one monofunctional monomer or only monofunctional monomers. Preferably used as a functionalizing monomer Graft polymerized monomer. You also choose the Reaction condition I in such a way that it corresponds to a primarily excited especially a physically initiated graft polymerization equivalent. The reaction condition II is preferably chosen such that that it corresponds to that of a post-reaction without primary excitation.

If, for example, you choose one that is only monofunctional Graft polymerization capable monomers containing Reaction mixture the reaction condition I such that a photoinitiated graft polymerization takes place, then form in the Initiation on the matrix membrane, so-called graft tentacles. You irradiate now simultaneously or simultaneously with UV light, then networking takes place the graft tentacle instead. In other words, the Reaction condition I is such that a photo-initiated Graft polymerization with simultaneous UV crosslinking of the Graft tentacles running on the surface of the matrix membrane.

In the example chosen, switch to the one described above Reaction I removes the UV radiation, then graft polymerization takes place without UV radiation and thus without cross-linking "surviving" radicals take place after the reaction condition I was "switched off". Graft polymerization without UV radiation then represents the reaction condition II, in which predominantly one Extension of polymer chains without further crosslinking takes place.

According to the invention, reaction conditions I and II are selected in such a way that that the layer first formed in reaction condition I the Shielding matrix membrane, for example, for minimization to take care of non-specific interactions. The Reaction condition II is then chosen so that after the first formed layer and thus the layer formed thereafter as functional layer. The reaction condition II is thus preferably formed a functional layer, the z. B. for the Affinity attachment can serve.

It is therefore expedient to use a first layer on the Surface "started" with the most complete coverage possible this surface takes place. Then another layer can be applied be built up step by step and in layers, so that almost a Growth "outside" takes place. It is also possible according to the invention a structure of more than two layers of this type build up by changing the reaction conditions accordingly changes so that in the course of surface functionalization different layers arise.

The surface functionalization of the matrix membrane takes place in a process step instead, since the matrix membrane with the Reaction mixture stays in contact and all layers with the help of this Reaction mixture can be prepared.

The surface functionalization according to the invention results in only one technological step to a structure that is preferably both a effective shielding of the matrix membrane and minimal reduction ensures membrane permeability as well as functional groups in adjustable amount and accessibility (relatively open and flexible Layer with partial "tentacle" character) generated.

In other words, membranes are available according to the invention at which the named two-layer structure by means of a one-stage Functionalization method is trained. That way are direct functional composite membranes available, in which both the Matrix membrane (pore structure, specific surface) as well as their Surface functionality (synthetic functional polymer) varied independently of each other by simple, clear parameters can be.

All of this is easy to do, reproducible One-step process possible, which not only makes the economy of the required functionalization is significantly improved, but a method arsenal is also available, with which the Development of powerful affinity membranes in the first place is possible. The invention thus also relates to a method for Production of the coated composite membranes according to the invention.

The graft polymerization can be carried out by primary excitation with plasma, Electron beam and gamma ray can be initiated or induced. The graft polymerization is preferably a photoinitiated copolymerization and especially a heterogeneous, partially crosslinking graft copolymerization.

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

By setting the reaction parameters in the manufacture of the Membranes obtainable according to the invention are formed at Polymerization a so-called two-layer structure from a relatively compact hydrophilic "shielding" layer, for example an unspecific Prevents protein adsorption, and a relatively open one "Affinity binding" layer.

The membranes obtainable according to the invention are suitable in excellent for substance-specific separations, e.g. B. Membrane adsorber solid phase extracts, membrane chromatography and Membrane reactor.

The membranes obtainable according to the invention are preferably around photoreactive coated composite membranes, which by Photo-initiated polymerization with preferably UV light are available. After the UV excitation, a dark reaction preferably closes on.

This preferred embodiment of those obtainable according to the invention Composite membrane is explained in more detail below. For that are the Test conditions of the composite membrane obtainable according to the invention spelled out in more detail. It also explains which parameters (e.g. UV intensity, monomer concentration, exposure time, etc.) the various properties of those obtainable according to the invention Affect composite membrane.

1. General test 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 the investigation of 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 (pre-treatment of the matrix membrane)
  • - extraction in methanol
  • - Drying to constant weight
    Coating with photoinitiator
  • - Application of the reaction mixture (monomer solution) to the matrix membrane
  • - UV exposure (= reaction condition 1)
  • - Dark reaction (without UV; = reaction condition II)
  optional (after-treatment of the composite membrane)
  • - extract with 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
  • - (non-specific) 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 the reaction mixture (monomer solution) to the matrix membrane, 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:
    Substrate 2% maltose in citric acid phosphate buffer pH = 4.6, T = 40 ° C
    Relative activity:
    Bound enzyme activity / Dissolved enzyme activity * 100%

Results Table 1

Influence of UV intensity on degree of modification (DG) and permeability with 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 the exposure time on DG and permeability with 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 the enzyme immobilization to Table 1

Table 4 Results of the enzyme immobilization to Table 2

discussion

With a higher UV intensity, a higher degree of grafting and thus one lower permeability of the membranes obtained (see Table 1). As the post-reaction time increases, the Degree of grafting and thus a reduction in the permeability (see Table 2).

With lower UV intensity there is an open, uncrosslinked, with tentacles receive equipped layer (see 3.2), which is necessary for a protein (BSA) or enzyme binding is more easily accessible. Therefore, more enzyme can be bound, but not everything is active (lower rel. activity, see table 3); that becomes with the influence of the membrane pore structure (Type 1 → small pore size → hindrance to substrate transport) explained.

With increasing post-reaction time, the degree of grafting increases during the bound protein remains unchanged, this results in a Increase in specific activity (see Table 4) since the mean Pore size of the membrane (type 2) is significantly larger than that of type 1.

Example B - composite membrane based on an MF membrane

• a) Matrix membrane
  MF membrane: Accurel PP 2E HF (AkzoNobel Wuppertal), polypropylene (PP)
• b) functional monomers
  2-Aminoethyl methacrylate (for the investigation of 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
  • - Application of the reaction mixture (monomer solution) to the matrix membrane, under nitrogen
  • - UV exposure, variable UV intensity I (here by exposure of two membranes one above the 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) Applications of the composite membrane as an affinity carrier
  • - (Non-specific) binding of protein by ion exchange (→ protein purification) → Tables 6, 7.
  • - Covalent immobilization of protein → Table 8

Results and discussion a. Degree of modification (DG) of the composite membranes Table 5 Degree of modification 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 with the preparations with identical monomer solutions can be interpreted as follows:

• - A higher intensity (with constant exposure time) for the 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 support);
• - A thermal after-reaction has a significant influence on DG, especially after exposure to high intensity in membranes 4/1 and 4/2; at high intensity there is a high radical concentration, which in turn causes a significant post-reaction.
• - At low intensity, the effect of the after-reaction on membranes 9/1 and 9/2 is slight.

b. Protein binding to composite membranes ion exchange

Protein: bovine serum albumin (BSA), 1 g / l in 0.066 M phosphate buffer (pH 5, pH 7)
Membrane samples (water-moist, A = 0.785 cm ² ) in protein solution were shaken for 14 h, then washed as follows:
Shaken vigorously with buffer (pH 5 or pH 7) for 1 h, buffer changed twice
Shaken intensively for 1 h with Tween solution (1 g / l Tween 20 + 9 g / l NaCl), Tween solution changed twice 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-moist, A = 0.785 cm ² ) activated with glutaraldehyde (GA); shaken in 10% GA for 5 h at RT, then washed 3 times for 1 min each with water and once with buffer pH 5 or pH 7; Treatment with protein solution for 14 h and washed as follows:
Shaken vigorously with buffer (pH 5 or pH 7) for 1 h, buffer changed twice
shaken intensively for 1 h with Tween solution (1 g / l Tween 20 + 9 g / l NaCl), Tween solution changed twice Table 8 Covalent protein binding at pH = 7.0

• - bei hoher Intensität kompakte, vernetzte Schicht (C) mit geringer Bindungskapazität (absolut & spezifisch: BSA & BSA/DG) insbesondere bei pH = 7
• - bei geringer Intensität offene, unvernetzte Schicht (Tentakeln, T) mit höherer spezifischer Bindungskapazität (BSA & BSA/DG) insbesondere bei pH = 7

Einfluß der thermischen Nachreaktion (vgl. a.) → Vereinzelte, unvernetzte Ketten, (ΔBSA/ΔDG >> BSA/DG der "Vorstufen" ohne thermische Nachreaktion) entweder

• - auf vernetzter Pfropfschicht (Ct, → Erhöhung von absoluter und spezifischer Bindungskapazität) oder
• - in wenig vernetzter Pfropfschicht (Tt, → Erhöhung von absoluter und spezifischer Bindungskapazität).

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) especially at pH = 7
• - At low intensity, open, uncrosslinked layer (tentacles, T) with higher specific binding capacity (BSA & BSA / DG), especially at pH = 7

Influence of the thermal post-reaction (cf. a.) → Scattered, uncrosslinked chains, (ΔBSA / ΔDG >> BSA / DG of the "preliminary stages" without thermal post-reaction) either

• - on cross-linked graft layer (Ct, → increase in absolute and specific binding capacity) or
• - in a slightly cross-linked graft layer (Tt, → increase in absolute and specific binding capacity).

The properties or states designated above with C, T, Ct and Tt are shown schematically in the accompanying single figure and are not drawn to scale for the purpose of explanation. Special effects with covalent binding → correlation with total amount of bound protein; ie both the non-specific and the ionically bound protein are covalently fixed after preactivation by glutaraldehyde.
→ Post-reaction with less influence (analogous to UF membrane, example A!)
→ for BSA / DG significant dependence on the intensity (analogous to UF membrane, example A!)
→ clear trend: higher specific binding capacity with less networking.

Claims (14)

1. Coated composite membrane based on a separately manufactured matrix membrane, the surface of which is functionalized, available in that
a reaction mixture which is suitable for surface functionalization and contains at least one functionalizing monomer and is capable of forming two different polymeric layer structures on the matrix membrane when using two different reaction conditions,
the matrix membrane is brought into contact with this reaction mixture and remains in constant contact during the surface functionalization, and
the reaction mixture, while in contact with the matrix membrane, is exposed to a reaction condition I and then afterwards to a reaction condition II which differs from the reaction condition I in such a way that two different layers are successively formed on the matrix membrane for surface functionalization. 2. composite membrane according to claim 1, available through that as a functionalizing monomer is a monofunctional Monomer is used. 3. composite membrane according to claim 1 or 2, thereby obtainable,
that a monomer capable of graft polymerization is used as the functionalizing monomer and
the reaction condition I corresponds to that of a primary excited, in particular a physically initiated, graft polymerization, while the reaction condition II corresponds to that of a post-reaction without primary excitation. 4. composite membrane according to claim 3, available through that the graft polymerization by a primary excitation with plasma, Electron beam, gamma ray or UV exposure is initiated. 5. composite membrane according to claim 3 or 4, available through that an initiator is used for the physical initiation. 6. composite membrane according to claim 5, available through that benzophenone is used as a photoinitiator. 7. 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. 8. composite membrane according to one of the preceding claims, available through that the matrix membrane with a solution of a photoinitiator coated with the containing the functionalizing monomer Reaction mixture is applied and irradiated with UV light. 9. composite membrane according to one of the preceding claims, characterized, that the layer obtained in reaction condition I the Shielding the matrix membrane and the reaction condition II obtained layer is formed as a functional layer. 10. composite membrane according to one of the preceding claims, characterized, that the surface with covalently fixed, biological or biomimetic receptors is functionalized. 11. A method for producing a coated composite membrane based on a separately produced matrix membrane, the surface of which is functionalized, characterized in that
that a reaction mixture which is suitable for surface functionalization and contains at least one functionalizing monomer and is capable of forming two different polymeric layer structures on the matrix membrane when using two different reaction conditions is used,
the matrix membrane is brought into contact with this reaction mixture and remains in constant contact during the surface functionalization,
the reaction mixture, while in contact with the matrix membrane, is exposed to a reaction condition I and then afterwards to a reaction condition II which differs from the reaction condition I in such a way that two different layers are successively formed on the matrix membrane for surface functionalization. 12. The method according to claim 11, characterized, that at least one or more of the in one or more of the Claims 2 to 8 steps described is performed. 13. The method according to claim 11 or 12, characterized, that a protective gas is used while purging. 14. Use of the coated composite membranes according to one of the Claims 1 to 10 for substance-specific separations.