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EP-4739348-A1 - BIOCATALYTICAL COMPOSITIONS

EP4739348A1EP 4739348 A1EP4739348 A1EP 4739348A1EP-4739348-A1

Abstract

The present invention relates to a composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation. The present invention also relates to methods of producing said composition.

Inventors

  • BRIAND, Manon
  • LAPRÉVOTTE, Emilie
  • DUDAL, Yves Victor René
  • SHAHGALDIAN, PATRICK

Assignees

  • Perseo Pharma AG

Dates

Publication Date
20260513
Application Date
20240704

Claims (20)

  1. 1. A composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation.
  2. 2. The composition according to claim 1, wherein the agent which interacts with the lid domain of the lipase or a fragment thereof is selected from the group consisting of a colipase or a fragment thereof, a colipase-mimicking peptide, and an amphipathic molecule.
  3. 3. The composition according to claim 1 or 2, wherein the agent interacts specifically with the lid domain of the lipase or a fragment thereof so that the lipase or a fragment thereof shifts to and/or maintains the open conformation.
  4. 4. The composition according to anyone of claims 1-3, wherein about 100% of the lipase or a fragment thereof immobilized on the surface of the solid carrier is in the open conformation.
  5. 5. The composition of anyone of claims 1-4, wherein the protective layer embeds the solid carrier and embeds the lipase or a fragment thereof immobilized on the surface of the solid carrier.
  6. 6. The composition of anyone of claims 1-4, wherein the protective layer embeds the solid carrier, embeds the lipase or a fragment thereof immobilized on the surface of the solid carrier and embeds the agent which interacts with the lid domain of the lipase or a fragment thereof.
  7. 7. The composition of anyone of claims 1-5, for use as a medicament.
  8. 8. The composition of anyone of claims 1-5, for use in a method of enzyme replacement therapy (ERT) preferably gastrointestinal enzyme replacement therapy, or for use in a method for the prevention, delay of progression or treatment of exocrine pancreatic insufficiency (EPI).
  9. 9. A method of producing a composition, the composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation , the method comprising the following steps: (a) providing a solid carrier; (b) providing a lipase or a fragment thereof; (c) providing an agent which interacts with the lid domain of a lipase or a fragment thereof; (d) allowing the lipase or a fragment thereof of (b) to interact with the agent of (c); (e) immobilizing the lipase or a fragment thereof on the solid carrier; (f) forming a protective layer on the surface of the solid carrier to protect the lipase or the fragment thereof immobilized on the solid carrier.
  10. 10. A method of producing a composition, the composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation , the method comprising the following steps: (a)providing a solid carrier; (b)providing a lipase or a fragment thereof; (c)immobilizing the lipase or a fragment thereof on the solid carrier; (d) providing an agent which interacts with the lid domain of a lipase or a fragment thereof; (e) allowing the lipase or a fragment thereof of (c) to interact with the agent of (d); (f) forming a protective layer on the surface of the solid carrier to protect the lipase or the fragment thereof immobilized on the solid carrier.
  11. 11. The method of claim 9, wherein i) a linker is added to the solid carrier provided in step (a), and ii) the lipase or a fragment thereof provided in step (b) is added to the solid carrier and the linker, wherein the linker connects the solid carrier with the lipase or a fragment thereof in step (e).
  12. 12. The method of claim 11, wherein the linker which has not connected the solid carrier with the lipase or a fragment thereof in step (e), is present during formation of a protective layer on the surface of the solid carrier in step (f).
  13. 13. The method of claim 11, wherein there is no washing step between adding the linker to the solid carrier provided in step (a) in (i) and adding the lipase or a fragment thereof to the solid carrier and the linker in ii).
  14. 14. The method of any one of claims 9 and 11-13, wherein there is no washing step between any of steps (a) to (f).
  15. 15. The method of any one of claims 9 and 11-14, wherein the linker which has not connected the solid carrier with the lipase or a fragment thereof in step (e), or a part thereof, covalently binds the protective layer to the lipase or a fragment thereof in step (f).
  16. 16. The method of claim 10, wherein i) a linker is added to the solid carrier provided in step (a), and ii) the lipase or a fragment thereof provided in step (b) is added to the solid carrier and the linker, wherein the linker connects the solid carrier with the lipase or a fragment thereof in step (c).
  17. 17. The method of claim 16, wherein the linker which has not connected the solid carrier with the lipase or a fragment thereof in step (c), is present during formation of a protective layer on the surface of the solid carrier in step (f).
  18. 18. The method of claim 16, wherein there is no washing step between adding the linker to the solid carrier provided in step (a) in (i) and adding the lipase or a fragment thereof to the solid carrier and the linker in ii).
  19. 19. The method of any one of claims 10 and 16-18, wherein there is no washing step between any of steps (a) to (f).
  20. 20. The method of any one of claims 10 and 16-19, wherein the linker which has not connected the solid carrier with the lipase or a fragment thereof in step (c), or a part thereof, covalently binds the protective layer to the lipase or a fragment thereof in step (f).

Description

Biocatalytical compositions The field of the invention The present invention relates to a composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation. The present invention also relates to methods of producing said composition. 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 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, there is a need for providing enzymes like lipases which are protected and at the same time have high enzymatic activity. Summary of the invention The present invention provides a composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation. The present invention provides also a method of producing a composition, the composition comprising a solid carrier, a lipase or a fragment thereof immobilized on the surface of the solid carrier, an agent which interacts with the lid domain of the lipase or a fragment thereof, and a protective layer to protect the lipase or the fragment thereof by embedding the lipase or the fragment thereof, wherein the lipase or a fragment thereof is in the open conformation , the method comprising the following steps: (a) providing a solid carrier; (b) providing a lipase or a fragment thereof; (c) providing an agent which interacts with the lid domain of a lipase or a fragment thereof; (d) allowing the lipase or a fragment thereof of (b) to interact with the agent of (c); (e) immobilizing the lipase or a fragment thereof on the solid carrier; (f) forming a protective layer on the surface of the solid carrier to protect the lipase or the fragment thereof immobilized on the solid carrier. It has been surprisingly found that the activity of a lipase immobilized and protected as described in WO2015/014888 can be significantly increased by an agent which interacts with the lid domain of the lipase. Brief description of the figures Figure 1) shows a schematic representation of the process for the production of the composition of the invention: a) to a solid carrier, a lipase or a fragment thereof with closed lid and an agent (displayed as round circle) which interacts with the lid domain of the lipase or a fragment thereof is provided and the lipase or a fragment thereof with an open lid is immobilized on the solid carrier; b) and c) a protective layer grows around the immobilized lipase or the fragment with an open lid thereof embedding the immobilized lipase or the fragment thereof. Figure 2) shows the 3D structure of pancreatic lipase in a) its inactive conformation (with closed lid) and b) active conformation (with opened lid). The active site of pancreatic lipase is covered by a lid that prevents substrates from reaching the enzyme active site (Figure 2a). The opening of the lipase lid is induced by interactions with an agent which interacts with the lid domain of the lipase e.g. bile salts and/or a protein cofactor called colipase, allowing the stabilization of the active conformation of pancreatic lipase (Figure 2b). Figure 3) shows the kinetics of lipase substrate hydrolysis by recombinant human pancreatic lipase (HRL) with or without colipase (CLPS): a) in its free form; b) immobilized at the surface of silica nanoparticles (SNPs) and protected in an organosilca layer made of APTES, TEOS and Benzyltriethoxysilane (ATB). Figure 4) shows the kinetics of lipase substrate hydrolysis by porcine pancreatic lipase (PL) with or without colipase (CLPS) immobilized at the surface of silica nanoparticles (SNPs) and protected in an organosilca layer made of APTES, TEOS and Benzyltriethoxysilane (ATB). Figure 5) shows the kinetics of lipase substrate hydrolysis by free human recombinant lipase (HRL) using increasing concentrations of sodium taurocholate (NaTc). Figure 6) shows th