# US 2019/0167860 A1 · Method for producing a protein-functionalized film as well as protein functionalized film

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- Number: US 2019/0167860 A1
- Document ID: US20190167860A1
- Status: Published application
- Kind: A1
- Application number: 16/324,105
- Filed: 2017-05-24
- Published: 2019-06-06
- Earliest claimed: 2016-08-12
- Assignees: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.
- Applicants: Fraunhofer-Gesellschaft Zur FöRderung Der Angewandten Forschung E. V.
- Inventors: Stefan REINICKE; Alexander BÖKER
- Web page: https://patentgrep.com/patent/US20190167860A1

## CPC

- A61L 31/16: Biologically active materials, e.g. therapeutic substances {(A61L31/047 takes precedence)}
- A61L 31/043: {Proteins; Polypeptides; Degradation products thereof}
- A61L 31/08: Materials for coatings
- B05D 1/28: performed by transfer from the surfaces of elements carrying the liquid or other fluent material, e.g. brushes, pads, rollers
- C12N 11/087: Acrylic polymers
- C12N 11/089: obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- B05D: PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL (conveying articles or workpieces through baths of liquid B65G, e.g. B65G49/02)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to a method for producing a protein-functionalized film, in which a protein is bound to a copolymer or a polymer having an unhydrolyzed or hydrolyzed thiolactone functionalization is bound to the film by means of the existing functionalization. The present invention also relates to a correspondingly produced film.

## Claims

### Claim at position 1

1-15. (canceled)

### Claim 16

A method of producing a protein-functionalized film in which a film comprising
at least one copolymer or polymer having an unhydrolyzed and/or hydrolyzed thiolactone functionalization and at least one protein are produced or provided, and
the at least one protein is covalently bonded to the at least one copolymer or polymer via the unhydrolyzed or hydrolyzed thiolactone functionalization.

### Claim 17 (depends on 16)

The method in accordance with claim 16, wherein the protein-functionalized film is produced directly on a substrate or by means of a Langmuir-Schaefer technique.

### Claim 18 (depends on 17)

The method in accordance with claim 17, wherein the production of the protein-functionalized film takes place
on a substrate by coating with a doctor knife, spin coating and/or spray application from a solution of the at least one copolymer and/or polymer, and/or
by means of a Langmuir-Schaefer technique by spreading a solution of the at least one copolymer and/or polymer in a volatile solvent not miscible with water over the surface of water or of an aqueous solution (aqueous subphase).

### Claim 19 (depends on 18)

The method in accordance with claim 18, wherein the substrate is selected from the group of polymer films and polymer membranes.

### Claim 20 (depends on 16)

The method in accordance with claim 16, wherein the at least one protein
is applied to the surface of the film; and/or
is worked into the film of the copolymer and/or polymer during the production of the film by mixing the at least one protein with the at least one copolymer and/or polymer; and/or
in the case of the production of the film of the at least one copolymer and/or polymer by means of the Langmuir-Schaefer technique by adding the protein into the aqueous subphase before, during and/or after the production of the film of the at least one copolymer and/or polymer and the adsorption and/or absorption of the at least one protein on and/or in the film.

### Claim 21 (depends on 16)

The method in accordance with claim 16, wherein the unhydrolyzed thiolactone functionalization is selected from the group consisting of residues having the general formula I shown below and the hydrolyzed thiolactone functionalization is selected from the group of residues having the general formula II shown below
with the symbols meaning, respectively independently of one another
R hydrogen or a linear or branched alkyl residue having 1 to 8 carbon atoms; and
x 1 to 6.

### Claim 22 (depends on 16)

The method in accordance with claim 16, wherein the copolymer contains repeat units or that polymer is formed from repeat units having the following general formula III (unhydrolyzed) or IV (hydrolyzed)
with the symbols meaning, respectively independently of one another
R hydrogen or a linear or branched alkyl residue having 1 to 8 carbon atoms; and
x 1 to 6.

### Claim 23 (depends on 16)

The method in accordance with claim 16, wherein the copolymer contains repeat units of the general formula shown below
with the symbols meaning, respectively independently of one another
X NH, O or NR1 and
R1 a linear or branched alkyl residue having 1 to 8 carbon atoms or hydrogen.

### Claim 24 (depends on 16)

The method in accordance with claim 16, wherein the copolymer is formed from the two following repeat units a) and b):
with the symbols meaning, respectively independently of one another
X NH;
R2 one of the following residues
R hydrogen or a linear or branched alkyl residue having 1 to 8 carbon atoms; and
x 1 to 6;
and
with the symbols meaning, respectively independently of one another
X NH, 0 or NR′ and
R1 a linear or branched alkyl residue having 1 to 8 carbon atoms or hydrogen,
with the repeat units a) and b) being present in statistically distributed form in the copolymer.

### Claim 25 (depends on 16)

The method in accordance with claim 16, wherein the protein is an enzyme.

### Claim 26 (depends on 16)

The method in accordance with claim 16, wherein the film is charged with 0.01 to 50 wt % of the at least one protein.

### Claim 27 (depends on 16)

The method in accordance with claim 16, wherein the film containing protein produced in the first step is stored over 0.1 to 24 h for the covalent bonding of the protein to the at least one copolymer or polymer having an unhydrolyzed thiolactone functionalization.

### Claim 28 (depends on 27)

The method in accordance with claim 27, wherein the storage takes place
at temperatures from 0 to 30° C.; and/or
at a pH of 7.5 to 12; and/or
under the effect of an oxidation agent.

### Claim 29 (depends on 16)

The method in accordance with claim 16, wherein the film containing a protein produced in the first step is treated under oxidizing conditions for the covalent bonding of the protein to the at least one copolymer or polymer having a hydrolyzed thiolactone functionalization, with disulfide bridges being produced between the protein's own thiol groups and the thiol groups of the hydrolyzed thiolactone.

### Claim 30

A protein-functionalized film, containing at least one copolymer and/or polymer, to which at least one protein is covalently bonded via a spacer selected from the general formulas V and VI shown below
with the symbols meaning, respectively independently of one another
R hydrogen or a linear or branched alkyl residue having 1 to 8 carbon atoms; and
x 1 to 6.