HYDROGEL PRECURSOR FORMULATION AND PRODUCTION PROCESS THEREOF

Abstract

A hydrogel precursor formulation, its process of production as well as a kit comprising the formulation and a method of production of a hydrogel using the formulation. The precursor formulation comprises at least one structural compound, preferably vinyl sulfone (acrylated branched) poly(ethylene glycol), and at least one linker compound, preferably a peptide with two cysteines. The structural compound and the linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group. The precursor formulation is in the form of a powder.

Claims

1-22. (canceled)

23. A process for production of a hydrogel precursor formulation in form of a powder, the process comprising: providing a first solution A of at least one structural compound, wherein the structural compound has conjugated unsaturated bond or groups; providing a second solution B comprising at least one linker compound, wherein the linker compound comprises at least two nucleophilic groups; mixing of the solutions A and B under conditions which hinder a selective reaction between the unsaturated bond or group and the nucleophile resulting in a precursor solution; and lyophilization of the resulting precursor solution to an unreacted powder, wherein the unreacted powder comprises the at least one structural compound and the at least one linker compound, which are polymerizable by a selective reaction between the nucleophile and the conjugated unsaturated bond or group.

24. The process according to claim 23, further comprising mixing the solutions A and B at a pH of 4.0 or below.

25. The process according to claim 23, further comprising forming the solution A to comprise 5-10% w/v of the at least one structural compound.

26. The process according to claim 23, further comprising forming the solution B to comprise 0.1-2% w/v of the at least one linker compound.

27. The process according to claim 23, further comprising forming a solution C to comprise at least one biologically active compound which is dimerizable with the structural compound by the selective reaction between the nucleophile and the conjugated unsaturated bond or group is added to the solution A prior to the mixing of the solutions A and B.

28. The process according to claim 27, further comprising forming the solution C to comprise 0.1-10% w/v of the at least one active compound.

29. The process according to claim 27, further comprising forming at least one of the solution A, the solution B and the solution C as a solution of the at least one structural compound, the at least one linker compound or the at least one biologically active compound in distilled water.

30. The process according to claim 23, further comprising selecting the concentration of the at least one structural compound and the at least one linker compounds such that a molar ratio of the nucleophile to the conjugated unsaturated bond or group is in a range of 0.8:1 to 1.3:1.

31. The process according to claim 23, further comprising subjecting the precursor solution to filtration prior to the lyophilization step.

32. The process according to claim 23, further comprising aliquoting the precursor solution and filling into containers before the lyophilization step.

33. The process according to claim 32, further comprising filling the containers with sterile nitrogen gas and immediately capping after the lyophilization step.

34. A kit of parts comprising at least one container filled with a hydrogel precursor formulation comprising at least one structural compound and at least one linker compound, wherein said structural compound and said linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated unsaturated bond or group, the hydrogel precursor formulation is in the form of an unreacted powder, said unreacted powder is in the form of a stable compact cake, and a container with a reaction buffer.

35. The kit of parts according to claim 34, wherein the reaction buffer has a pH of at least 7.

38. A method of production of a hydrogel comprising: providing the hydrogel precursor formulation of claim 23 comprising the at least one structural compound and the at least one linker compound in at least one container, wherein said structural compound and said linker compound are polymerizable by a selective reaction between a nucleophile and a conjugated bond or group, and the hydrogel precursor formulation is in the form of an unreacted powder; re-suspending the hydrogel precursor formulation in a buffer having a pH of between 7 and 8; casting a gel precursor with the hydrogel precursor formulation re-suspension; and allowing polymerization of the gel precursor for at least 30 minutes, to form a hydrogel.

37. The method of production of a hydrogel according to claim 36 further comprising the step of adding a cell culture suspension to the precursor suspension.

38. The method of production of a hydrogel according to claim 36, further comprising the step of performing polymerization in an incubator at 37 C.

39. A hydrogel precursor formulation obtainable by the process according to claim 23.

Description

[0063] Further details and benefits of tune present invention will be apparent from the following figures and examples:

[0064] FIG. 1: Schematic representation of an embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

[0065] FIG. 2: Schematic representation of a second embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

[0066] FIG. 3: Schematic representation of a third embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention

[0067] FIG. 1 shows a schematic representation of an embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention. Solution A comprising 7.5% w/v of branched PEG with 4 arms functionalized with vinyl sulfone is added to solution B comprising 1% w/v of a peptide sequence with two cysteines, one near the C- and the other near the N-terminus, in mixing step 1.

[0068] For example, 275 mL of solution A comprising 7.5% w/v of functionalized PEG is added to 425 mL of solution B comprising 1% w/v of a linker peptide.

[0069] Solutions A and B are prepared by suspending the respective compounds in distilled water. For solution B, the peptide linker compound is preferably added to the water in small portions. Mixing is carried out under continuous stirring with a magnetic stirrer at 400 PPM. The so obtained precursor solution 4 is subsequently subjected to a sterile filtration step 5, e.g. using a Mini Kleenpak filter (PALL Corp.) with a PTFE membrane with an absolute rating of 0.2 m, yielding the filtrated precursor solution 6. This solution is then subjected to lyophilization step 7, resulting in the hydrogel precursor formulation 8 in form of a powder. The resulting powder is in the form of a stable compact cake.

[0070] Lyophylization step 7 may be carried out by first freezing the solution a shelf at 50 C. for 150 min, followed by a first drying step at 10 C. for 570 min at a pressure of 0.26 mbar. A second drying step follows at a temperature of 20 C. for 180 min at a pressure of 0.02 mbar.

[0071] On FIG. 2 a second embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention is schematically represented. In this embodiment Solution A comprising 7.5% w/v of a branched PEG with 4 arms functionalized with vinyl sulfone is mixed with Solution C comprising 2% w/v of a peptide comprising an RGD sequence in mixing step 2.

[0072] For example, 275 mL of Solution A comprising 7.5% w/v of a functionalized four branched PEG is mixed with 5 mL of Solution C comprising 2% w/v of a bioactive compound. This solution is then subsequently mixed with solution B comprising 1% w/v of a peptide linker comprising two cysteines, one near the C- and the other near the N-terminus, in mixing step 1.

[0073] The so obtained precursor solution 4 is subsequently subjected to a sterile filtration step 5, yielding the filtrated precursor solution 6. This solution is then subjected to lyophilization step 7, resulting in the hydrogel precursor formulation 8 in form of a powder.

[0074] FIG. 3 shows a third embodiment of a manufacturing process of a hydrogel precursor formulation according to the present invention. Solution A comprising 7.5% w/v of branched PEG with 4 arms functionalized with vinyl sulfone is mixed with Solution B comprising 1% w/v of a linker peptide sequence with a cysteine near the C- and N-terminus, in mixing step 1. Solutions A and B are prepared by suspending the respective compounds in distilled water. The mixing is carried cut under continuous stirring with a magnetic stirrer, preferably at 400 RPM. The so obtained precursor solution 4 is subsequently subjected to a sterile filtration step 5, yielding the filtrated precursor solution 6. This solution is then aliquoted into containers in aliqoting step 3. Each container may contain only a small amount, preferably 0.3-0.4 mL. The containers are scalable and are preferably made of glass. The aliquoted precursor solution 10 is the lyophilized in lyophilization step 7 to yield the precursor formulation powder 8.