EP-4739336-A2 - METHOD FOR IMMOBILIZATION OF PROTEINS

Abstract

The present invention relates to a method of producing a composition, the composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also relates to the composition obtainable by the method.

Inventors

• KRONENBERG, Vanessa
• SPECIOSO, Gabriele
• BRIAND, Manon
• LAPRÉVOTTE, Emilie
• DUDAL, Yves Victor René
• SHAHGALDIAN, PATRICK
• WATSON, John Ashleigh

Assignees

• Perseo Pharma AG

Dates

Publication Date

20260513

Application Date

20240704

Claims (15)

1. 1. A method of producing a composition, the composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group, the method comprising the following steps: (a) providing a solid carrier, wherein the solid carrier is provided in suspension; (b) immobilizing a protein or a fragment thereof on the solid carrier, wherein i) a linker is added to the suspension of the solid carrier and ii) a solution of the protein or of a fragment thereof is added to the suspension comprising the solid carrier and the linker, wherein the linker connects the solid carrier with the protein or a fragment thereof; (c) forming a protective layer on the surface of the solid carrier to protect the protein or the fragment thereof immobilized on the solid carrier, wherein the linker which has not connected the solid carrier with the protein or a fragment thereof in step (b), or a part therof, covalently binds the protective layer to the protein or the fragment thereof; and optionally (d) immobilizing a functional constituent on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units, wherein each repeat unit comprises at least one amino group and/or at least one thiol group.
2. 2. The method of claim 1, wherein there is no washing step between adding the linker to the suspension of the solid carrier in (i) of step (b) and adding the protein or a fragment thereof to the suspension comprising the solid carrier and the linker in ii) of step (b).
3. 3. The method of claim 1 or 2, wherein there is no washing step between any of steps (a) to (c).
4. 4. The method of any one of claims 1-3, wherein the solid carrier is provided in suspension in water, buffer or non-ionic surfactants or mixtures thereof.
5. 5. The method of any one of claims 1-3, wherein the solid carrier is provided in suspension in mixtures of water and non-ionic surfactants.
6. 6. The method of any one of claims 1-5, wherein the linker is added to the suspension of the solid carrier in i) of step (b) in a molar excess to the protein or a fragment thereof added to the suspension comprising the solid carrier and the linker in ii) of step (b).
7. 7. The method of any one of claims 1-5, wherein the linker is added to the suspension of the solid carrier in i) of step (b) in a 1 fold to 1000 fold molar excess to the protein or a fragment thereof added to the suspension comprising the solid carrier and the linker in ii) of step (b).
8. 8. The method of any one of claims 1-5, wherein the linker is added to the suspension of the solid carrier in i) of step (b) in a 4 fold to 250 fold molar excess to the protein or a fragment thereof added to the suspension comprising the solid carrier and the linker in ii) of step (b).
9. 9. The method of any one of claims 1-8, wherein the linker which has not connected the solid carrier with the protein or a fragment thereof in step (b), is present during formation of a protective layer on the surface of the solid carrier in step (c).
10. 10. The method of any one of claims 1-9, wherein the amount of the linker which has not connected the solid carrier with the protein or a fragment thereof in step (b) after addition of the protein or a fragment thereof in ii), is between 30% and 70% of the amount of linker added to the suspension of the solid carrier in i) of step (b).
11. 11. The method of any one of claims 1-10, wherein the protein or a fragment thereof is immobilized on the solid carrier by covalent binding via the linker in step (b).
12. 12. The method of any one of claims 1-11, wherein the surface of the solid carrier is at least partly modified before the protein or a fragment thereof is immobilized on the solid carrier in step (b).
13. 13. The method of any one of claims 1-12, wherein the linker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, l,5-difluoro-2,4- dinitrobenzene, BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DSP (Dithiobis[succinimidyl]propionate]), DTSSP (3,3 '- Dithiobis[sulfosuccinimidyl]propi onate]), DTBP (Dimethyl 3,3 '- dithiobispropionimidate-2 HC1), DST (Disuccinimidyl tartarate), BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane).
14. 14. The method of any one of claims 1-12, wherein the linker is glutaraldehyde.
15. 15. A composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group, wherein the composition is obtainable by the method of any one of claims 1-14.

Description

Method for immobilization of proteins The field of the invention The present invention relates to a method of producing a composition, the composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also relates to the composition obtainable by the method. Background of the invention Proteins such as enzymes are frequently needed, e.g. in industrial applications, diagnostics or for therapeutic use. In order to stabilize the proteins and/or to provide resistance to various types of stresses it has been suggested in the prior art to immobilize the proteins on the surface of a carrier and to protect them with a layer of protective material. Such an approach has been described e.g. in WO2015/014888 Al which discloses a biocatalytical composition comprising a solid carrier, a functional constituent like an enzyme and a protective layer for protecting the functional constituent by embedding the functional constituent at least partially and a process to produce such biocatalytical composition. Nevertheless, the method described in WO20 15/014888 Al is difficult to use in large scale as the load of protein per dry weight of particle is low. Thus there is a need for providing improved immobilization methods in order to obtain a high protein load per dry weight of particle, enabling the particles produced to have high enzymatic activity. Summary of the invention The present invention provides a method of producing a composition, the composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group, the method comprising the following steps: (a) providing a solid carrier, wherein the solid carrier is provided in suspension; (b) immobilizing a protein or a fragment thereof on the solid carrier, wherein i) a linker is added to the suspension of the solid carrier and ii) a solution of the protein or of a fragment thereof is added to the suspension comprising the solid carrier and the linker, wherein the linker connects the solid carrier with the protein or a fragment thereof; (c) forming a protective layer on the surface of the solid carrier to protect the protein or the fragment thereof immobilized on the solid carrier, wherein the linker which has not connected the solid carrier with the protein or a fragment thereof in step (b), or a part therof, covalently binds the protective layer to the protein or the fragment thereof; and optionally (d) immobilizing a functional constituent on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units, wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also provides a composition comprising a solid carrier, a protein or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the protein or a fragment thereof by embedding the protein or a fragment thereof, and optionally a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group, wherein the composition is obtainable by the methods described herein. It has been surprisingly found by the inventors of the present application that the methods as provided by the present invention and described herein can be used in large scale protein immobilization as they provide a high load of immobilized protein per dry weight of particle which significantly increases the activity of particles. Brief description of the figures Figure 1) shows a schematic representation of the process for the production of the composition of the invention: a) engineered PAL or fragment thereof is immobilized on the solid carrier; b) and c) a protective layer grows around the immobilized engineered PAL or fragment thereof embedding the immobilized engineered PAL or fragmen