U.S. patent application number 15/846534 was filed with the patent office on 2018-05-03 for hydrogel precursor formulation and production process thereof.
The applicant listed for this patent is QGEL SA. Invention is credited to Matthias LUTOLF, Simone RIZZI.
Application Number | 20180119092 15/846534 |
Document ID | / |
Family ID | 42224660 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180119092 |
Kind Code |
A1 |
RIZZI; Simone ; et
al. |
May 3, 2018 |
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.
Inventors: |
RIZZI; Simone; (Novazzano,
CH) ; LUTOLF; Matthias; (Tolochenaz, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QGEL SA |
Lausanne |
|
CH |
|
|
Family ID: |
42224660 |
Appl. No.: |
15/846534 |
Filed: |
December 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14684490 |
Apr 13, 2015 |
9850461 |
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15846534 |
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13640141 |
Oct 9, 2012 |
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PCT/EP2011/056187 |
Apr 19, 2011 |
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14684490 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08G 65/3344 20130101;
C12N 5/0068 20130101; C12N 2533/30 20130101; C12N 2533/50
20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; C08G 65/334 20060101 C08G065/334; C08G 75/04 20060101
C08G075/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2010 |
EP |
10160796.8 |
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.degree. C.
39. A hydrogel precursor formulation obtainable by the process
according to claim 23.
Description
[0001] The present invention relates to a hydrogel precursor
formulation, its process of production as well as a kit comprising
said formulation and a method of production of a hydrogel using
said formulation.
[0002] Three dimensional ceil culture scaffolds have been
recognized to allow patterns of gene expression arid other cellular
activities that more closely mimic living organisms than the
conventional two dimensional ceil cultures in dishes.
[0003] This has led to the development of novel families of
synthetic polymer hydrogels, which are often termed artificial ECMs
(aECM), since they mimic many aspects of the extracellular matrix.
One major challenge is to provide a chemistry which allows
cross-linking of the matrix in the presence of cells or
biomolecules or well as stable tethering of biomolecules to the
matrix itself.
[0004] In recent years, different, mechanisms were developed
allowing the formation of gels in the presence of cells or
biomolecules. For example, mechanisms based on the self-assembly of
low molecular weight building blocks such as peptides (Estroff et
al.: Water gelation by small organic molecules; Chem. Rev. 2004;
104(3); 1201-18) or ueidopyrmimidinone (Zhang S.: Fabrication of
novel biomaterials through molecular self-assembly; Nat.
Biotechnol. 2003; 21(10); 1171-8) and moderate molecular weight
amphiphilic block copolymers (e.g. see Hartgerink et al.:
Peptide-amphiphile nonofibers: A versatile scaffold for the
preparation of self-assembling materials; Proc. Nat. Acad. Sci.
U.S.A. 2002; 99 (8); 5133-8) were proposed.
[0005] WO 00/454808 describes novel biomaterials, especially for
the formation of hydrogels, having a cross-linking chemistry based
on a Michael-type addition reaction between a nucleophile and a
conjugated unsaturated bond or group, which allows the formation of
the gel in the presence of cells or biomolecules. Moreover,
specific signal molecules may be integrated into the gel matrix by
a specific reaction.
[0006] One major drawback of this system is that it relies on
manually mixing at least, two precursor components together prior
to gelation. In practice, the use of multiple component solutions
may be a source of error due to unintentional variations of i) the
re-suspension conditions of the different components in powder
form, ii) the mixing ratios of the precursor solutions, thus
leading to more complex use and reproducibility problems of the gel
compositions. Additionally, the upscale of a manufacturing process
of a gel system that requires the mixing of several solutions is
more expensive than the upscale of a manufacturing process of a gel
requiring a single.
[0007] It is therefore an objective of the present invention to
avoid the disadvantages of the known hydrogel formulations and
specifically to provide a hydrogel precursor formulation which is
easy in handling and which enables the production of hydrogels with
highly reproducible compositions. This objective is solved with a
hydrogel precursor according to claim 1.
[0008] The hydrogel precursor formulation according to the present
invention comprises at least one structural compound and at least
one linker compound. The structural and the 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.
[0009] The formulation has the advantage that the powder may simply
be re-suspended, preferably in a buffer, to start the gelling
reaction. No mixing of different components is required, thus
considerably reducing the probability of erroneous ratios between
the at least one structural compound and the at least one linker
compound. Thus, this increases the reproducibility of the hydrogels
produced from these hydrogel precursors. Moreover, the hydrogel
precursor of the present invention provides ease of use.
[0010] The hydrogel precursor formulation of the present invention
is in the form of a powder. The powder may comprise particles
having any size and shape. Alternatively, the powder may also be
provided as pressed tablet or pill. Most preferably, the powder is
provided in the form of a stable compact cake, e.g. at the bottom
or a container.
[0011] The powder is unreacted, meaning that almost none of the at
least one structural compound has reacted with the at least one
linker compound via the selective reaction. Preferably more than
70%, more preferably more than 85%, most preferably more than 95%
of the compounds have not undergone the selective reaction.
[0012] The selective reaction is a reaction between a nucleophile
and a conjugated unsaturated bond or group by nucleophilic
addition. Such reactions are also known as Michael-Type addition
reactions.
[0013] The structural compound has a functionality of at least
three, but most preferably the structural compound has a
functionality of four or more. By "functionality" the number of
reactive sites on a molecule is meant.
[0014] The structural compound is preferably selected from the
group consisting of oligomers, polymers, biosynthetic or natural
proteins or peptides and polysaccharides. Preferably, the
structural compound is a polymer selected from the group consisting
of poly(ethylene glycol), poly(ethylene oxide), poly(vinyl
alcohol), poly(ethylene-co-vinyl alcohol), poly(acrylic acid),
poly(ethylene-co-acrylic acid), poly(ethyloxazoline), poly(vinyl
pyrrolidone), poly(ethylene-co-vinyl pyrrolidone), poly(maleic
acid), poly(ethylene-co-maleic acid), poly(acrylamide), or
poly(ethylene oxide)-co-poly(propylene oxide) block copolymers or
mixtures thereof. More preferably, the structural compound is a
poly(ethylene glycol), most preferably a branched poly(ethylene
glycol) with three, four or more arms.
[0015] The linker compound has a functionality of at least two and
is selected from the group consisting of oligomers, polymers,
biosynthetic or natural proteins or peptides and polysaccharides or
mixtures thereof. Preferably, the linker compound is a peptide
sequence, most preferably containing an adhesion site, a growth
factor binding site, or a protease binding site.
[0016] The nucleophile preferably is a strong nucleophile, such as
a thiol or a thiol containing group. The nucleophile could also be
any other type of nucleophile known in the art, provided that it is
strong enough to undergo the selective reaction, e.g. such as an
amine. Further, the conjugated unsaturated group preferably is an
acrylate, an acrylamide, a quinone or a vinylpyridinium. Most
preferably, the unsaturated group is a vinyl sulfone.
[0017] Additionally, the hydrogel precursor formulation of the
present invention may comprise at least one bioactive compound,
preferably comprising an RGD peptide sequence, which is
conjugatable with the structural compound through a selective
reaction between a nucleophile and a conjugated unsaturated bond or
group.
[0018] The bioactive compound may comprise an adhesion site, such
as the RGD sequence from fibronectin or the YISG sequence from
laminin; a growth factor binding site, such as a heparin binding
site; a protease binding site or a therapeutically active compound.
Preferably, the bioactive compound comprises a cell adhesion site,
most preferably an RGD sequence.
[0019] The bioactive compound comprises at least one active group
capable of undergoing the self selective reaction. More preferably,
the bioactive compound comprises at least one nuclophilic group,
most preferably a thiol group.
[0020] The bioactive compound is conjugatable with the structural
compound through a self selective reaction between a nucleophile
and a conjugated unsaturated bona or group. Most preferably, this
self selective reaction is the same reaction as the self selective
reaction between the structural compound and the linker compound,
especially between the same type of nucleophile and conjugated
unsaturated bond or group. Alternatively, the bioactive compound
may be conjugated to the structural compound through a self
selective reaction prior to the polymerization of the linker
compound with the structural compound.
[0021] The structural compound preferably is a multi-branched
poly(ethylene glycol) (PEG) with functionalized end groups. More
preferably, the end groups are functionalized with vinyl sulfone.
Most preferably, the structural compound is PEG-tri(vinyl sulfone)
or PEG-tetra(vinyl sulfone). Vinyl sulfone functionalization of the
alcohol groups of PEG may be carried out with any suitable reaction
known in the art. By using branched PEG with three, four or more
arms it is possible to produce structural compounds with a
functionality of three, four or more.
[0022] Preferably, the linker comprises at least two nucleophilic
groups, preferably thiol groups. Thiols are strong nucleophiles
readily undergoing Michael-Type addition reactions with unsaturated
bonds or groups at physiological pH. Moreover, thiols are commonly
found in biological systems, so that their use poses no issue in
view of toxicity.
[0023] The linker compound preferably is a peptide comprising at
least two cysteines, preferably located near the N- and C-terminus
of the peptide. Synthesizing peptides with two or more cysteine
residues is straightforward. It is further possible to introduce
specific protease sites in the peptide such as to produce
degradable hydrogels, e.g. for in-vivo use. Further, by varying the
amino acids neighbouring the cysteines, it is possible to change
the pKa value of the thiol group.
[0024] Preferably, the cysteines are located at the N- and
C-terminus of the peptide, resulting in peptides with the structure
CXXXXXXXXC-COOH (SEQ ID NO:1), preferably with an acetylated
N-terminus, Ac; and. an amidated C-terminus, NH2; where C is the
one letter representation of cysteine and X represents any amino
acid except cysteine. The peptide may be of any length, thus the
number of X (X.sub.n) may be any number. Preferably, the peptide
has a length of 16 amino acids. Alternatively, the cysteines might
be located one or more ammo acids away from the N- or C-terminus,
e.g. resulting in peptides with the general structure
H.sub.2N--X.sub.mCX.sub.nCX.sub.p--COOH (SEQ ID NO:2), where m, n
and p may be any integer, including zero.
[0025] Most, preferably, the linker compound is a peptide with the
sequence NH.sub.2-GCRE-XXXXXXXX-ERGG-COOH (SEQ ID NO:3). The
Glycine (G) serves as spacer, the Arginin (R) increases the
reactivity of the thiol group of the neighbouring cysteine, and the
glutamic acid (E) enhances the solubility of the peptide in aqueous
solutions.
[0026] Most preferably, the sequence of the linker compound is
NH.sub.2-GCREGPQGIWGQERCG-COOH [SEQ ID NO:4] or
NH.sub.2-GCREGDQGIAGFERCG-COOH [SEQ ID NO: 5], again preferably
with an acetylated N-terminus and an amidated C-terminus.
[0027] Peptides for the linker and bioactive compounds should be
synthesized and processed in acidic solvents, most preferably in
solutions containing trifluoroacetate (TFA). Residual TFA bound to
the peptide powder after peptide synthesis has the effect to lower
the pH of a water suspension containing the respective peptide
below 4.
[0028] The structural compound and/or the linker compound are
selected such that the reaction rate of the selective reaction
between the structural compound and the linker compound is hindered
or highly reduced in the mixing conditions. Preferably, the
reaction rate is highly reduced at or below pH 4 compared to at or
above pH 7.
[0029] A selection of the compounds in this way allows to provide a
precursor formulation which will readily undergo gelation reaction
at physiological conditions, but which allows its preparation under
conditions such that no or almost no selective reaction occurs.
[0030] Preferably, the structural and/or the linker compound are
selected such that the reaction rate of the self selective reaction
is at least twice as fast at pH 7.5 compared to pH 7.0.
[0031] Another objective of the present invention is to provide a
production process for a hydrogel precursor formulation. This
problem is solved by a process as claimed in claim 8.
[0032] The process comprises the steps of:
[0033] providing a first solution A comprising at least one
structural compound;
[0034] providing a second solution B comprising at least one linker
compound;
[0035] mixing of the solutions A and B; and
[0036] lyophilization of the resulting precursor solution
[0037] The at least one structural compound and the at least one
linker compound are polymerizable by a selective reaction between a
nucleophile and a conjugated unsaturated bond or group. Both
solutions A and B are mixed under conditions which hinder the
selective reaction.
[0038] This process allows the production of a hydrogel precursor
formulation in form of a powder comprising both the structural
compound as well as the linker compound. The mixing conditions have
to be selected in such a way, that the self selective reaction is
hindered. This means that the reaction rate is sufficiently low,
that a very large fraction of the compounds of both solutions A and
B do not react through the self selective reaction prior to
lyophilization. Preferably, more than 70%, more preferably more
than 85%, most preferably more than 90% of the molecules in both
solutions have not undergone the selective reaction prior to the
lyophilization step.
[0039] The mixing conditions may be selected by adjustment of pH,
concentration of the different compounds, process time, temperature
or solvent condition. Most preferably, the mixing is carried out at
or below pH 4. Especially when a thiol is used as nucleophile, a pH
at or below 4 sufficiently hinders the self selective reaction.
Mixing is preferably performed at room temperature.
[0040] It is important that solution A is added to solution B,
since the pH of solution A is around 7, whereas the pH of solution
B is below 4. If solution B was added to solution A, tune self
selective polymerization reaction would start during the mixing
step. When adding solution A to solution B, the pH of the resulting
solution will always be below pH 4, therefore hindering the
reaction.
[0041] Solution A preferably comprises 5-10% w/v of the at least
one structural compound. Most preferably, solution A comprises 7.5%
w/v of the at least one structural compound.
[0042] Further, solution B preferably comprises 0.1-2% w/v of the
at least one linker compound. Most preferably, solution B comprises
1% w/v of the at least one linker compound. This concentration of
the linker compound provides for good solubility of the compound in
the solution.
[0043] Using the concentrations of the structural and the linker
compounds as mentioned above for both solutions A and B leads to
the formation of a compact powder after the lyophilisation step.
This compact powder will form a cake-like layer on the bottom of a
container, which is favourable. Additionally, using these
relatively low concentrations of both compounds during the
production process additionally reduces the probability of unwanted
pre-mature reactions between the structural and the linker
compound. Further, material losses in subsequent production steps
are reduced with these concentrations compared with higher
concentrations.
[0044] Further, solution A and/or solution B preferably are
solutions of the at least one structural compound or the at least
one linker compound, respectively, in distilled water. Thus, both
compounds are present in an un-buffered solution. Due to the
trifluoro acetic acid adhering to the peptide linker compound of
solution B, the pH of this solution will be reduced. This leads to
a pH which is preferably under 4 for the resulting mixture of
solutions A and B. More preferably, the pH of the resulting
solution is around 3.5.
[0045] Alternatively, prior to mixing with solution B, solution A)
may be mixed with an additional solution C comprising a
biologically active compound which is dimerizable with the
structural compound by a selective reaction between a nucleophile
and a conjugated unsaturated bond or group. This bioactive compound
may comprise an adhesion site, such as the RGD sequence from
fibronectin or the YISG sequence from laminin; a growth factor
binding site, such as a heparin binding site; a protease binding
site or a therapeutically active compound. Preferably, the
bioactive compound comprises a cell adhesion site, most preferably
an RGD sequence.
[0046] Preferably, solution C comprises 0.1 to 10% w/v of the
biologically active compound. More preferably, solution C comprises
between 0.1 and 5%, most preferably between 0.1 and 2% of the
biologically active compound.
[0047] Variation of the respective amounts of the structural
compound in solution A and the biologically active compound in the
optional solution C as well as the concentration and nature (e.g.
amino acid sequence) of the linker compound in solution B allows
the production of hydrogel precursor formulations with different
characteristics.
[0048] Although the possibilities of variation of the concentration
of the compounds in each of solution A and B are many, it is
preferred that the concentration of the compounds is selected such
that the molar ratio of the nucleophile to the conjugated
unsaturated bond or group results in optimal physicochemical
properties, like maximum shear modulus and minimal swelling
characteristics of the final gels. Normally the optimal ratio of
the nucleophile to the conjugated unsaturated bond or group is
within the range of 0.8:1 to 1.3:1. This ensures the formation of a
hydrogel where almost all active groups have undergone the
selective reaction, so that side reactions of any of the reactive
groups are considerably reduced.
[0049] Further the precursor solution may be subjected to
filtration prior to the lyophilization step. The filtration
preferably is a sterile filtration. Any undissolved compounds as
well as bacterial contaminations may be removed from the mixture
prior to the lyophilization step.
[0050] Preferably, the pre-mixed precursor solution is aliquoted
and filled into containers, preferably sterile containers, before
the lyophilization step. This allows the production of single
containers containing a defined amount of a hydrogel precursor
formulation. The containers may be of any suitable material, such
as plastic or glass. Preferably the containers are vials.
[0051] The containers are preferably filled with sterile nitrogen
gas and capped immediately after the lyophilization step. This
protects the hydrogel precursor powder from contact with moisture
and/or oxygen, which may lead to premature polymerization or
oxidation of the nucleophiles.
[0052] Another object of the present invention is the use of a
hydrogel precursor formulation as described herein for the
manufacture of a hydrogel.
[0053] A further object of the present invention is to provide a
simple to use system for the production of hydrogels with highly
reproducible results. This problem is solved with a kit according
to claim 20.
[0054] The kit of the present invention comprises at least one
container filled with a hydrogel precursor formulation as described
herein and a container with a reaction buffer. The container
contains preferably an amount of precursor formulation powder,
which will result in a gel with predefined characteristics when
re-suspended with a defined amount of reaction buffer.
[0055] The reaction buffer preferably has a pH above 7. More
preferably the reaction buffer has a pH between 7 and 8. The buffer
preferably comprises HEPES, preferably at 0.3M concentration with
the pH adjusted to a value between 7 and 8. This allows for a
sufficiently fast polymerization reaction.
[0056] A further object of the present invention is to provide an
easy to use method to produce a hydrogel. This objective is
achieved with a method according to claim 22.
[0057] The method of production of a hydrogel comprises the steps
of:
[0058] Re-suspending a hydrogel precursor formulation as described
herein in a buffer having pH 7, more preferably with a buffer
having a pH between 7 and 8;
[0059] Optionally adding a cell culture suspension to the precursor
suspension;
[0060] Casting of a gel precursor with the precursor suspension;
and
[0061] Polymerization of the gel precursor for at least 30 minutes,
preferably for 30 to 45 minutes, preferably in an incubator at
37.degree. C.
[0062] The hydrogel precursor formulation of the present invention
is polymerizable under physiological conditions, which allows the
addition of a cell culture to the precursor suspension so that the
cells may be evenly distributed in the suspension prior to
gelation. This would not readily be possible with any other
precursor system.
[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 .mu.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.degree. C. for 150 min, followed by a
first drying step at -10.degree. C. for 570 min at a pressure of
0.26 mbar. A second drying step follows at a temperature of
20.degree. 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.
Sequence CWU 1
1
51302PRTArtificial SequenceDesigned peptide used as linker
compoundVARIANT(2)..(301)Xaa = any amino acid; any one or all of
amino acids 2-301 can either be present or absent 1Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165
170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 245 250 255 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 290 295 300
2302PRTArtificial SequenceDesigned peptide used as linker
compoundVARIANT(1)..(100)Xaa = any amino acid; any one or all of
amino acids 1-100 can either be present or
absentVARIANT(102)..(201)Xaa = any amino acid; any one or all of
amino acids 102-201 can either be present or
absentVARIANT(203)..(302)Xaa = any amino acid; any one or all of
amino acids 203-302 can either be present or absent 2Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155
160 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 245 250 255 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280
285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295
300 316PRTArtificial SequenceDesigned peptide used as linker
compoundVARIANT(5)..(12)Xaa = any amino acid; any or all of amino
acids 5-12 can either be present or absent 3Gly Cys Arg Glu Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Glu Arg Cys Gly 1 5 10 15 416PRTArtificial
SequenceDesigned peptide used as linker
compoundPEPTIDE(1)..(16)Designed peptide 4Gly Cys Arg Glu Gly Pro
Gln Gly Ile Trp Gly Gln Glu Arg Cys Gly 1 5 10 15 516PRTArtificial
SequenceDesigned peptide used as linker
compoundPEPTIDE(1)..(16)Designed peptide 5Gly Cys Arg Glu Gly Asp
Gln Gly Ile Ala Gly Phe Glu Arg Cys Gly 1 5 10 15
* * * * *