WO2025031537A1 - Method for producing an ink for 3d printing using a polypeptide based on human collagen with a reduced hydroxyproline content - Google Patents

Abstract

The invention relates to a method for producing an ink with a polypeptide based on human collagen with a reduced hydroxyproline content, said ink being particularly suitable for 3D printing. The present invention also relates to a corresponding ink for 3D printing and to the use thereof in medicine. The present invention also relates to a method for producing a 3D scaffold by means of a 3D printing method using the corresponding ink, and to a 3D scaffold.

Description

Process for producing an ink for 3D printing using a polypeptide based on human collagen with reduced hydroxyproline content

The present invention relates to a method for producing an ink with a polypeptide based on human collagen with a reduced hydroxyproline content according to one of the claims 1 to 7, which is excellently suited for 3D printing. Furthermore, the present invention relates to a corresponding ink for 3D printing and its use in medicine according to one of the claims 8 to 10. The present invention also relates to a method for producing a 3D framework by means of a 3D printing process using the corresponding ink according to one of the claims 11 to 14 and a 3D framework according to one of the claims 15 to 18.

In recent years, the 3D printing of 3D scaffolds with a matrix of biopolymers has increased significantly. These 3D scaffolds are either used for the subsequent colonization of living cells, or the living cells are incorporated directly into the matrix of the biopolymer when the 3D scaffold is constructed. In addition to other possible applications, living tissues and organs, such as breast implants, are also to be produced from living cells that can be implanted into a living being.

However, not every biopolymer is suitable, as some of these biopolymers contain sequences and/or groups that can trigger an immune reaction because the immune system recognizes them as foreign proteins, or the biopolymers contain sequences of amino acids that the immune system recognizes as foreign. Furthermore, the biopolymers must be able to be processed into a 3D scaffold at a physiologically tolerable temperature. Biological cells must also find a physiological environment in the biopolymer matrix so that they can maintain or develop their biological activity and retain their differentiation status. There is therefore a need for a biopolymer that comes very close to the properties of the natural extracellular matrix. It is therefore the object of the present invention to provide an ink with a polypeptide which can be processed in the working temperature range of 3D printing and in which the polypeptide meets the aforementioned requirements as well as possible.

For this purpose, the present invention provides a method for producing an ink for 3D printing according to claim 1, an ink according to claim 8, a method for producing a 3D framework according to claim 11, and a 3D framework according to claim 15. Subclaims 2 to 7, 9, 10, 12 to 14 and 16 to 18 relate to preferred embodiments.

All preferred features mentioned herein in relation to the ink of the method according to the invention for producing an ink should apply equally to the ink according to the invention, and vice versa. The same also applies to the ink used in the method according to the invention for producing a 3D framework. Likewise, all preferred features mentioned herein in relation to the 3D framework of the method according to the invention for producing a 3D framework should also apply to the 3D framework according to the invention, and vice versa.

In one embodiment of the invention, the present invention relates to a method for producing an ink for 3D printing, wherein a polypeptide is added to a solvent, characterized in that the polypeptide is one based on human collagen which has a hydroxyproline content of a maximum of 5%, based on the total number of amino acids of the polypeptide.

The inventors of the present invention have come to the conclusion that a maximum proportion of hydroxyproline of 5% in the amino acid chain of the polypeptide makes it possible to produce an ink in which the polypeptide is dissolved. The reason is that such a polypeptide based on human collagen does not form consistently triple-helical structures, so that it is in the liquid state in a working temperature range of 3D printing of preferably 12 °C and higher. For the same reason, it is even more preferred that the polypeptide has a proportion of hydroxyproline of a maximum of 4%, more preferably a maximum of 3%, even more preferably a maximum of 2%, even more preferably a maximum of 1% and most preferably no hydroxyproline, in each case based on the total number of amino acids of the Polypeptide. In contrast to naturally occurring collagens, the collagen used according to the invention has a lower content of hydroxyproline and this is preferably present as proline.

The term "polypeptide based on human collagen" is intended to include the following: all collagens, for example fibrillar (I, II, III, V) and non-fibrillar collagens (IV). Polypeptides from or with complete sequences of human collagen, polypeptides from or with partial sequences of human collagen as well as polypeptides that comprise or consist of two or more repeating partial sequences of human collagen, where the amino acid sequence of these polypeptides can have at least 80%, preferably at least 90%, more preferably at least 95% sequence identity with the natural human collagen sequences. This is also intended to include the polypeptides mentioned if they are functionalized with at least one crosslinkable group, as described in more detail below. According to the invention, a polypeptide from or with a partial sequence of human collagen is preferred as a polypeptide based on human collagen.

Preferably, the human collagen-based polypeptide is a complete sequence or a partial sequence of collagen I, more preferably the collagen I a-1 chain.

"Sequence identity" is to be understood here as meaning that the sequence or order of the amino acids in the polypeptide is identical. "X% sequence identity" is to be understood as meaning that the sequence or order of the amino acid sequence is X% identical.

In a further embodiment of the invention of the above-mentioned method, it is preferred that the polypeptide is one which is present in the liquid state in an aqueous solution at concentrations of 1 to 10 percent by weight per volume (% w/v) at temperatures of 18 °C and above, preferably 16 °C and above, more preferably 14 °C and above, most preferably 12 °C and above. By "X% w/v" is meant herein that X g of polypeptide are contained in 100 ml of water. In this way, it is ensured that the ink in the processing area of 3D printing.

In a further embodiment of the above-mentioned method according to the invention, it is preferred that the polypeptide is functionalized with at least one crosslinkable group. The crosslinkable group is preferably a photocrosslinkable group. "Photocrosslinkable" is to be understood here as meaning that the functionalized polypeptide can be crosslinked by the action of (electromagnetic) radiation and optionally the presence of a photoinitiator.

In a further embodiment of the above-mentioned method according to the invention, it is preferred that the polypeptide has a molecular weight in the range of 20 to 200 Daltons, more preferably in the range of 25 to 150 Daltons, even more preferably 30 to 100 Daltons, based on the amino acids of the amino acid sequence.

In a further embodiment of the above-mentioned method according to the invention, it is preferred that the ink contains biological cells.

A further embodiment of the invention relates to an ink for 3D printing with a polypeptide based on human collagen, which has a hydroxyproline content of a maximum of 5%, based on the total number of amino acids of the polypeptide. The ink according to the invention is preferably obtainable by the above-mentioned method according to the invention. The method for determining the hydroxyproline content is carried out using a Model 835 High-Speed Amino Acid Analyzer from Hitachi, as described below.

The ink is preferably one in which the human collagen-based polypeptide is dissolved in water, preferably in a concentration range as specified above with the method according to the invention.

In a further embodiment of the ink mentioned above according to the invention, it is preferred that the ink is one for use in medicine, preferably in clinical medicine. A further embodiment of the present invention relates to a method for producing a 3D framework using the ink according to the invention, wherein the polypeptide is cured by means of a 3D printing method. The polypeptide is preferably functionalized with at least one crosslinkable group. In the 3D printing method, the curing of the polypeptide preferably takes place by light-based shaping, preferably by light-induced chemical reaction, in which the crosslinkable groups of the polypeptide are chemically crosslinked with one another.

Furthermore, the ink in the process for producing a 3D scaffold can contain biological cells. Any biological cells that are required to replicate a complex biological tissue can be used.

A further embodiment of the present invention relates to a 3D scaffold that has cross-linked polypeptides, the polypeptides being polypeptides based on human collagen, each of which has a hydroxyproline content of a maximum of 5%, based on the total number of amino acids of the respective polypeptide. Here too, the 3D scaffold according to the invention can have biological cells. The cross-linked polypeptides preferably represent an extracellular matrix for the biological cells, in which the biological cells are embedded. The 3D scaffold according to the invention is preferably one that is obtainable by the method according to the invention for producing a 3D scaffold. The 3D scaffold according to the invention can be used in medicine, for example for building replacement tissue structures.

Water or water-based buffer systems from cell culture or cell culture media can be used as solvents for the peptides in the ink according to the invention or used according to the invention.

In a particularly preferred embodiment, the polypeptide used according to the invention is a polypeptide which comprises a first amino acid sequence with at least 70% sequence identity with SEQ ID No. 2, wherein the polypeptide has a further amino acid sequence with a length of 0 to 200 amino acids. The use of the first amino acid sequence makes it possible to provide a polypeptide which is in the liquid state in an aqueous solution at concentrations of 10 weight percent per volume (% w/v) at temperatures of 12 °C and more, so that the polypeptide is well suited for use in bioinks for 3D printing. The polypeptide used according to the invention particularly preferably consists of the first amino acid sequence and the respective further amino acid sequence at the N-terminal end and the C-terminal end of the first amino acid sequence. The further amino acid sequence at the C-terminal end of the first amino acid sequence can be the same or different from the further amino acid sequence at the N-terminal end of the first amino acid sequence. As already mentioned, the polypeptides used according to the invention are preferably functionalized with at least one crosslinkable group, and light-based shaping is preferably used in the 3D printing.

"Sequence identity" is to be understood here as meaning that the sequence or order of the amino acids in the polypeptide is identical. "X% sequence identity" is to be understood as meaning that the sequence or order of the amino acid sequence is X% identical.

The term “the polypeptide has a further amino acid sequence with a length of 0 to Y amino acids at the first amino acid sequence both at its N-terminal end and at its C-terminal end” is to be understood here as meaning that the first amino acid sequence with at least X% sequence identity with SEQ ID No. 2 is linked at both ends via a peptide bond to a further (peptide) sequence with a length of 0 to Y amino acids.

In one embodiment of the first embodiment, the first amino acid sequence of the polypeptide according to the invention preferably has at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 93%, even more preferably at least 95%, even more preferably at least 98% and most preferably 100% sequence identity.

In one embodiment, the polypeptide used according to the invention has at the first Amino acid sequence preferably has at both its N-terminal end and its C-terminal end a further amino acid sequence with a length of 0 to 150 amino acids, more preferably 0 to 100 amino acids, even more preferably 0 to 80 amino acids, even more preferably 0 to 60 amino acids, even more preferably 0 to 50 amino acids, even more preferably 0 to 40 amino acids, even more preferably 0 to 30 amino acids, even more preferably 0 to 20 amino acids and most preferably 0 to 10 amino acids.

Furthermore, the polypeptide used according to the invention can also be one with a sequence according to SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, or result from these by cleavage of a starting or ending sequence.

The polypeptide used according to the invention is preferably encoded by a polynucleotide. For this purpose, it is preferred that the polynucleotide comprises a nucleotide sequence according to SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8. For this purpose, a host cell is preferably used which expresses the polypeptide used according to the invention. The host cell is preferably one which is free of an enzyme complex which can convert proline to hydroxyproline, as mentioned above. The host cell can be a mammalian cell or a microorganism, for example yeast, such as Pichia Pastoris. The enzyme complex is preferably prolyl-4-hydroxylase. The polypeptide used according to the invention is therefore preferably a recombinantly produced polypeptide.

3D printing or 3D printing processes are understood to mean all 3D printing processes that are known in the prior art, such as extrusion printing processes, inkjet printing processes or printing processes using light-based shaping, such as stereolithography or volumetric/holographic 3D printing, with printing processes using light-based shaping being preferred according to the invention.

Light-based shaping is understood here to mean that the functionalized polypeptide from the ink is cured by light irradiation, whereby the crosslinkable groups are linked together, preferably by chemical reaction. It is therefore only cured in those places where sufficient light energy is irradiated (structuring curing). The polypeptides are cross-linked with one another and the polypeptide hardens in the area in which sufficient light energy is irradiated. An exposure process with structuring curing of the photocross-linkable polypeptide to form a biocompatible cross-linked polymer - as used according to the invention - has the advantage of superior printing speed compared to other biological printing processes, such as the extrusion process, the inkjet process and the laser-assisted forward transfer process, i.e. a large volume of the object to be printed can be built up per unit of time. The combination of high printing speed and high spatial resolution is unique to light-based structured curing. The performance of the process is based on optical principles, and by using commercially available optics, superior resolution down to the submicrometer range is possible.

In a volumetric/holographic 3D printing process, a 3D structure is simultaneously cured by light irradiation, for example by projecting light onto a rotating cylinder, or by crossing several laser beams, whereby the light intensity at the points where the laser beams cross is high enough for light-induced cross-linking.

A stereolithographic 3D printing process is particularly preferably used as the 3D printing process. A stereolithographic 3D printing process is one in which the structure of the 3D framework is gradually produced by point-by-point, layer-by-layer (in one layer) or volumetric/holographic curing, with curing within a layer being preferred according to the invention. A laser is preferably used for point-by-point curing. For layer-by-layer curing, irradiation is preferably projection-based or area-wide, with so-called “digital light processing” preferably being used. For reasons of speed, a projection-based exposure process is preferred according to the invention.

The term “photocrosslinkable” is understood here to mean that the functionalized

Polypeptide by exposure to electromagnetic radiation and, if necessary, presence of a photoinitiator.

In the methods according to the invention for producing 3D scaffolds, it is preferred that a 3D scaffold is constructed from the polypeptide by the following step:

(i) curing the functionalized polypeptide from the ink by irradiating electromagnetic radiation (exposure) pointwise, in a layer or volumetrically/holographically, in the region of the ink in which curing or the construction of the 3D framework is desired, wherein irradiation in a layer is preferred according to the invention.

By irradiation, the functionalized polypeptide is converted into a cross-linked polypeptide by cross-linking the functional groups, which provides a spatial structure of the 3D scaffold.

Irradiation in a layer is to be understood here in particular as meaning that the 3D framework according to the invention to be built up is irradiated over its entire surface while it is in the ink. In this case, only those areas in the surface in which spatial curing is desired are irradiated. Areas of the surface in which curing is not desired can also not be irradiated. In a further step (i), however, an area with a different structure than in the first step (i) can then also be produced in the same layer of the 3D framework according to the invention to be built up by introducing the layer into another ink (the same or different to the ink according to the invention) and corresponding irradiation into the desired area of the layer. Furthermore, the 3D framework according to the invention can also be built up by applying further cured areas of the same or different structure and/or composition to the already cured areas by repeating step (i). In this way, a complex 3D framework with different structures in each layer can be obtained.

In one embodiment of the method according to the invention for producing a 3D framework, it is preferred that the construction of the 3D framework is carried out by the following steps (preferably in the order given): (I) introducing said ink containing said polypeptide into a reaction vessel;

(II) immersion or presence of a support plate on which the 3D scaffold is to be built in said ink;

(III) irradiating electromagnetic radiation pointwise, in a layer or volumetrically/holographically in the areas in which curing of said polypeptide is desired;

(IV) generating a cross-linked structure of the polypeptide by the electromagnetic radiation,

(V) immersion or presence of the structure produced in step (IV) on the carrier plate in a further ink (said ink or another photocrosslinkable or photopolymerizable ink);

(VI) Irradiation of electromagnetic radiation selectively, in a layer or volumetrically/holographically in the areas where curing of the further ink is desired,

(VII) producing a further polymerized or cross-linked structure by the electromagnetic radiation in step (VI),

(VIII) Repeating steps (V) to (VII) with one ink each (either said ink or another photocrosslinkable or photopolymerizable ink) until the 3D scaffold is produced.

The structures created in the repeated steps are preferably connected to one another by covalent bonds. However, non-covalent bonds, for example based on physical interactions, are also possible. The entire 3D framework to be constructed can be built on a carrier plate, which can be moved in such a way that the 3D framework is moved one after the other into the same or different reaction vessels filled with ink, where the light-based, structuring curing is carried out, as already described above. All of the processes mentioned in EP 3 018 531 B1 can, for example, be used according to the invention.

By repeating the process steps in which cross-linking or polymerization of the inks takes place, a layered, point-by-point or volumetric/holographic structure of the 3D framework is achieved. It is possible to build a 3D framework with a complex structure. Undercuts and overhanging structures can also be formed, since cross-linking or polymerization of the ink in a certain layer or at a certain point can occur during point-by-point irradiation even if there is no already cross-linked or polymerized material underneath, but only non-cross-linked or non-polymerized liquid. Polymerization or cross-linking of an ink/polypeptide located outside the layer or point does not occur; instead, only the ink/polypeptide that is inside the layer or point is cross-linked or polymerized.

To use the 3D scaffold according to the invention in medicine, it is preferred that the 3D scaffold is used as a matrix for living cells. The 3D scaffold can either be populated with living cells after it has been produced, or the living cells are in the ink from which the 3D scaffold is produced. In this way, tissue or in vitro models can be constructed, which can then be used in research or implanted.

The ink according to the invention or used according to the invention can therefore also contain living cells. It is also possible to use different inks with the polypeptide mentioned, but these differ in terms of the cell types used. The different cell types used can be located at different, desired positions within the 3D framework.

The inks used preferably contain one or more biological cell types. If cross-linking occurs as a result of the exposure to electromagnetic radiation, the cells contained in the liquid are embedded in the 3D framework. By using several inks, preferably with a or several different biological cell types, a complex 3D scaffold can be constructed that also has a blood vessel structure network. The use of a matrix made of the cross-linked polypeptide enables the directed construction of the 3D scaffold according to the invention.

The polypeptide used according to the invention is preferably functionalized with a crosslinkable group, which is a photoreactive group. In one variant, the crosslinkable group is an acrylic group, by means of which the crosslinking of the polypeptide used according to the invention is achieved. For this purpose, the primary amines in the side chains of the polypeptide used according to the invention are preferably reacted via a nucleophilic substitution reaction with an anhydride or an acid chloride, the latter having a polymerizable double bond. An anhydride or acid chloride of methacrylic acid or carboxynorbornene is particularly preferably used here.

By introducing a crosslinkable group, e.g. an acrylic group, into the polypeptide used according to the invention, the latter is made photocrosslinkable. A crosslinked matrix is produced by the radiation-induced coupling of the acrylic residues between different molecules of the polypeptide used according to the invention.

In order to achieve photocrosslinking via the crosslinkable group, a radical former (a so-called photoinitiator) is preferably used which forms radicals at a selected wavelength of the electromagnetic radiation used in the process. Suitable radical formers are, for example, anthrone derivatives, such as violanthrone or isoviolanthrone, fluorescein, rubrene, anthrazine derivatives, tetrazene derivatives, benzanthrone, benzanthronil, eosin, levolinic acid derivatives, phosphine derivatives, mono- and bis-acyl phosphines, in particular lithium phenyl-2,4,6-trimethylbenzoylphosphinate, metallocenes, acetophenones, benzophenones, xanthones, quinones, ketone derivatives, hydroxyketones, aminoketones, benzoyl peroxides, pyridine salts, phenylglyoxylates and/or iodonium salts.

In addition, a substance can be added to the ink used according to the invention that prevents photocrosslinking of deeper, liquid layers. This way, liquid solution outside the focal plane remains liquid, even if it is in the The radiation area of the focal plane above it is located. This works through absorption of the substance at the wavelength at which cross-linking takes place (cross-linking wavelength). The interception takes place in the focal plane so that the cross-linking wavelength cannot penetrate into deeper layers. All substances that absorb at the desired wavelength, such as dyes, are suitable.

In addition, in one variant it is possible for the ink used according to the invention to have a temperature-sensitive gelling agent. In particular, the use of an inverse temperature-sensitive (also referred to as reverse temperature-sensitive) gelling agent is provided. Such a gelling agent becomes more solid as the temperature rises. By heating the reaction vessel, the reaction liquid solidifies and initially forms a gel that is only metastable. If the ink is not photo-crosslinked at the same time, the metastable gel can be liquefied again and pumped out by subsequently cooling the 3D framework. With conventional temperature-sensitive gelling agents, the temperature conditions to be used are exactly the opposite. For example, a support structure can be created if required so that hanging structures can be created. If, on the other hand, the metastable gel is at least partially irradiated with electromagnetic radiation of a suitable wavelength, photo-crosslinking occurs so that the metastable gel in these areas is converted into a stable gel or polymer network.

In other words, the temperature-sensitive, particularly inverse-temperature-sensitive, gelling agent and temperature control of the reaction chamber make it even easier to work with hanging sections and undercuts or cavities. In this variant, too, it is still possible to work with liquid structures as a support.

It is also possible to provide a temperature gradient so that a metastable gel does not form in all areas of the liquid mixed with the temperature-sensitive, in particular inverse-temperature-sensitive, gelling agent. With the help of such a gradient, even more complex structures can be created.

Alternatively or in addition to the formation of cavities via the use of a gelling agent, enzymes can also be used to digest parts of the formed 3D scaffold The principle is as follows: A 3D scaffold with cavities/undercuts (e.g. a channel system) is printed as a solid body by filling all cavities with a sacrificial material during printing, which can later (i.e. after printing is complete) be dissolved by adding the right enzyme. The sacrificial material is, for example, a digestible polymer that is digested by adding a digesting enzyme. This is an elegant strategy for creating cavities using stereolithographic printing processes. For example, a hyaluronidase (digesting enzyme) can digest hyaluronic acid (sacrificial material), so that a cavity is created by hyaluronidase digestion at the point in the 3D scaffold where hyaluronic acid was printed.

Alternatively, a photoblocker can be used in the ink according to the invention or the ink used according to the invention to create a cavity/undercut, whereby the photoblocker limits the curing depth of the ink. In this way, curing can also take place by irradiation in one layer without curing all areas in the irradiation depth that lie in the beam path. The possibility of layer-by-layer curing increases the printing speed immensely compared to point-by-point processes.

In one variant, an optical system is arranged between a source for the electromagnetic radiation (radiation source), which serves to generate one and/or the other electromagnetic radiation, and the reaction vessel, which serves to focus the electromagnetic radiation on the respective focal plane in the reaction vessel. In one variant, it is provided that this optical system can be refocused in order to change the layer of curing within the reaction vessel. Such refocusing can be achieved, for example, by changing the distance of the optical system from the radiation source. A computer-controlled stepper motor can be provided to impart a corresponding movement of the optical system. The optical system can, for example, be a system of optical lenses or - in a case with a particularly simple design - a single focusing lens. Alternatively, the point or layer of curing can also be static and the 3D framework to be built can be moved relative to the electromagnetic radiation. The irradiation pattern selected for producing the cross-linked 3D framework can be provided by a computer program, for example. It is conceivable that a user creates the 3D framework to be produced using a CAD program. The digital object created in this way is then cut into individual irradiation planes by a suitable computer program. Control information is created for a printer using this information, which is used to carry out the described process. This control information specifies when which photo-cross-linkable ink/liquid must be introduced into the reaction vessel. This control information also specifies when which image of an irradiation plane should be projected onto the respective focal plane in the reaction vessel. In this way, the 3D framework previously created on the computer can then be converted into a real 3D framework.

In one variant, the electromagnetic radiation used in the method according to the invention has a wavelength in the range from 200 nm to 1000 nm (i.e. a wavelength that lies between the UV range and the infrared range), more preferably in the range from 350 nm to 800 nm. With such wavelengths, the substances preferably used as radical formers can be particularly well excited, so that radicals are formed in order to enable cross-linking of the polypeptide carrying the cross-linkable groups.

Other suitable wavelengths of the electromagnetic radiation used are in the range from 250 nm to 950 nm, in particular from 250 nm to 850 nm, in particular from 300 nm to 800 nm, in particular from 300 nm to 750 nm, in particular from 300 nm to 700 nm, in particular from 350 nm to 650 nm and very particularly from 350 nm to 410 nm.

As can be seen from the previous description of the production of the 3D framework according to the invention, the step of 3D printing the 3D framework can be carried out completely automatically, so that user intervention is not required. This further facilitates the application of the method.

If the 3D framework is created on a carrier plate, this carrier plate can be completely removed from the remaining ink in the reaction vessel. The generated 3D scaffold can then be removed from the carrier plate by the user.

The present invention will now be explained in more detail using the following examples:

Images of the figures:

Fig. 1 shows temperature-dependent gelling behaviour of the inventive

polypeptide (CoIMP) compared to gelatin;

Fig. 2 shows a ¹ H-NMR of the polypeptide according to the invention (CoIMP) in unmodified form;

Fig. 3 shows a ¹ H-NMR of the polypeptide (CoIMP) according to the invention modified with methacryloyl groups;

Fig. 4 shows a CD spectrum of the polypeptide of the invention (CoIMP) at 5 °C, 20 °C and 37 °C;

Fig. 5 shows a CD spectrum of gelatin at 5 °C, 20 °C and 37 °C.

Amino acid analysis:

2 to 12 mg of polypeptide and 200 pL of a mixture of concentrated hydrochloric acid and trifluoroacetic acid (volume ratio 2:1) are added to a glass sample tube. The sample tube is sealed and hydrolyzed for 25 minutes at 166°C. It is then completely dried under vacuum. The dry product is dissolved in 1000 pL of demineralized water. The resulting solution is called the hydrolyzate.

For amino acid analysis, a portion of the hydrolysate is diluted so that the diluted solution corresponds to a concentration of 8 to 18 pg of the outgoing, now hydrolysed, polypeptide (depending on the instrument). Amino acid analysis is performed using a Hitachi Model 835 High-Speed Amino Acid Analyzer under standard conditions for protein hydrolysates. A cation exchange column is used for separation. Amino acids are detected spectroscopically using a subsequent ninhydrin reaction. The absorption maximum of the colored hydroxyproline is at 440 nm. An amino acid standard was used to calibrate the instrument.

Example 1: Cloning work to produce an expression construct for recombinant human polypeptides according to the invention:

The gene for recombinant production of the polypeptide of the invention (SEQ ID No. 2) is one that encodes a portion of the human collagen I alpha 1 protein. The coding DNA sequence was codon optimized for expression in yeast (or general organism) and ordered as a product of artificial gene synthesis (Invitrogen). The coding sequence was then cloned by restriction digestion followed by in-frame ligation behind the α-secretion signal of the pPIC9K vector (Thermo Fisher Scientific). (SEQ ID No. 5) Correct integration of the target gene was confirmed by DNA sequencing.

For purification by immobilized metal ion chromatography (IMAC), a His-6 tag was inserted into the construct described above (SEQ ID No. 4). To do this, the His-tag coding sequence was inserted between the polypeptide coding sequence and the stop codon by PCR with a primer encoding the His-6 tag on a non-binding overhang. The correct integration of the His-6 tag was confirmed by DNA sequencing (SEQ ID No. 7).

Example 2: Transformation of the expression construct into Komagataella phaffir.

For transformation, the previously generated DNA construct was linearized by restriction digestion and then introduced into the GS115 Komagataella phaffii strain (Thermo Fisher Scientific) by electroporation. Positive clones were identified by growth on selection medium. Example 3: Expression of the polypeptide according to the invention in a laboratory scale bioreactor:

The recombinant polypeptide is produced in a fed-batch process on a 30 L scale. For this purpose, a 30 L stainless steel bioreactor (Sartorius) is equipped with all the necessary sensors (DO, pH, temperature). In addition, a methanol sensor (Raven Biotech) is installed in the reactor. The fermentation is started by inoculation with an optical density at 600 nm (ODeoo) of 0.5 from a pre-culture. During fermentation, the temperature and pH are kept constant at 30 °C and 5, respectively. The dissolved oxygen is kept at values above 30% by varying the stirrer speed, varying the gassing rate, and adding oxygen to the supply air.

The end of the batch phase is detected by a sudden spike in the dissolved oxygen reading when the C source in the fermentation medium is used up. In the subsequent fed phase, the methanol concentration is kept constant at 0.5% by automated addition of methanol based on the reading of the methanol sensor. Fermentation as described above is typically terminated after 50-60 hours.

Example 4: Purification of the polypeptide according to the invention from the fermentation broth:

For purification, the cells are first removed by centrifugation. Coarse impurities are then separated by filtration with a 1 pm filter, before smaller particles and remaining cells are removed by filtration through a 0.45 pm membrane. The filtrate is then dialyzed against ultrapure water by tangential flow filtration with a 30 kDa membrane until a constant conductivity is achieved. The volume is then reduced by concentration in the tangential filtration process and the concentrate is dried by lyophilization.

For chromatographic purification, the dry product is dissolved in a suitable running buffer and further purified using either cation exchange chromatography or IMAC. The eluate from both chromatographic processes is then dialyzed against ultrapure water using tangential flow filtration and a 30 kDa membrane until a constant conductivity is achieved and then dried by lyophilization.

Example 5: Biocompatibility tests:

The purified polypeptide is tested for its biocompatibility using the MTT test. For this, L929 cells are cultivated in a 96-well plate for 24 hours after seeding. The recombinant polypeptide is then added to the cells in concentrations of 1% and 0.1% (w/v) dissolved in cell culture medium and the cells are cultivated for a further 24 hours. For the analysis, thiazolyl blue (MTT) is added to the cells in a concentration of 1 mg/mL in cell culture medium and incubated for 2 hours at 37 °C. The staining solution is then removed and the formazan formed is dissolved in isopropanol. The metabolic activity of the cells is determined by measuring the absorption of the isopropanol solution at 570 nm and 650 nm. The measured values are normalized to a control group without the addition of any substance. Cells treated with a 1% solution of the recombinant material typically show a metabolic activity of at least 70% compared to the control group.

Example 6: Functionalization of the purified product:

For use in 3D printing, the purified polypeptide is functionalized by adding methacryloyl groups. For this purpose, the purified protein is dissolved in a concentration of 10% (w/v) in 0.25 M CB buffer (pH 9) at 25 °C. The pH is then adjusted to 9. To start the reaction, 0.1 mL methacrylic anhydride (MAA) (Sigma-Aldrich) per gram of dry protein is added to the reaction with constant stirring. From the addition of MAA, work is carried out avoiding direct light. The reaction is then carried out under reflux cooling for 3 h. After 3 h, the reaction is terminated by diluting the reaction solution with CB buffer. The reaction solution is transferred to a dialysis tube with a MWCO of 14 kDa and dialyzed against H2O for 3 days. The dialysis water is changed twice a day. After dialysis, the contents of the dialysis tube are filtered through a 0.45 pm filter and stored under sterile conditions. A comparison material (GelMA) is produced analogously from gelatin (Medella Pro, Gelita).

Example 7: Rheological analysis of crosslinking properties:

To analyze the crosslinking properties, the lyophilized and functionalized polypeptide is dissolved in a concentration of 10% (w/v) in H2O and a final concentration of 0.1% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The crosslinking properties through radical chain reaction of the inserted methacryloyl groups (loss and storage modulus) before, during and after UV exposure (365 nm, 19.95 mW/cm ² ) are measured on a rheometer (Anton-Paar) at 1 Hz and 0.1% deformation at a 0.5 mm measuring gap and 37 °C. To cure the material during the measurement, it is irradiated with UV light for 180 s. GelMA is measured as a comparison material using the same method. When irradiated with UV light, both materials show a rapid increase in the storage modulus (G') as a result of the polymerization reaction.

Example 8: Determination of functionalization using ¹ H-NMR:

To determine the degree of functionalization, 20 mg of unfunctionalized and 20 mg of functionalized polypeptide (from examples 4 and 6) are dissolved in 1 mL D2O and measured by ¹ H-NMR on an Ascend 400 (Bruker) at 400 MHz. The degree of functionalization is determined by the ratios of the integral of the lysine peak in the unmodified and modified recombinant material normalized to the phenylalanine peak. In contrast to the modified polypeptide (see Fig. 3), the spectrum of the unmodified polypeptide (see Fig. 2) shows a higher lysine peak at approx. 2.9 ppm. In addition, the peaks typical for the methacryloyl group at approx. 5.3 ppm and approx. 5.6 ppm are missing for the unmodified polypeptide.

Example 9: Cell adhesion test with cross-linked functionalized polypeptide according to the invention:

To test cell adhesion, the functionalized polypeptide is dissolved in a concentration of 10% (w/v) in phosphate buffered saline (PBS) at a concentration of 0.1% LAP. The bioink is placed in a 96-well ultra-low attachment (ULA) plate and completely polymerized by exposure. An 8% GelMA ink is used as a positive control, and an uncoated well of the plate serves as a negative control.

For colonization, primary human fibroblasts from the foreskin (FS fibroblasts) are each placed on the well with polymerized GelMA and cross-linked, functionalized polypeptide according to the invention (Example 7) as well as the uncoated plate and incubated for 24 hours at 37 °C.

The cells are then examined by live-dead staining with Hoechst 33342, Calcein Red-Oragne AM and CellTox Green and fluorescence microscopy.

FS fibroblasts on the recombinant polypeptide show a lower proportion of dead cells compared to GelMA and also better cell adhesion with a more even distribution. FS fibroblasts on the untreated plate form spheroids with a high proportion of dead cells.

Example 10: 3D bioprinting of the functionalized polypeptide according to the invention

(according to example 6):

For 3D bioprinting, the functionalized polypeptide is dissolved in 10% and 5% (w/v) concentrations in PBS with 0.1% LAP as previously described. The bioink produced is then used to print a cylinder with a height of 1 mm and a radius of 1 mm on a proprietary multi-material stereolithograph (Cellbricks). The material shows good printability with polymerization in the irradiated area within a few seconds and clearly defined contours. After 24 h, FS fibroblasts (20,000 cells/construct) in the construct were examined by fluorescence microscopy using live-dead staining as already described in the adhesion test. FS fibroblasts show adhesion to the material of the printed scaffold over a period of several days with migration of the cells through the material of the sample body. Example 11: Thermal gelling behaviour of the unfunctionalised polypeptide according to the invention (according to Example 4):

Since experience has shown that the functionalized polypeptide according to the invention has a lower gelling point, the unfunctionalized polypeptide according to the invention is used to measure the thermal gelling behavior. In this way, it can be ensured that if the thermal gelling behavior of the unfunctionalized polypeptide is suitable, the functionalized polypeptide is also suitable. To measure the thermal gelling behavior, the unfunctionalized polypeptide is dissolved in 10% (w/v) H2O and loss and storage modulus are measured at decreasing temperature between 37 °C and 4 °C with a cooling rate of 0.5 °C/min. A 10% gelatin solution is used for comparison. In contrast to gelatin, the recombinant peptide does not show any thermal gelling behavior in the ambient temperature range (see Fig. 1). This can be observed by an increase in the storage modulus when the temperature is reduced. While an increase in the storage modulus is observed for gelatine already below approx. 27 °C, the recombinant polypeptide only shows such an increase below approx. 12 °C and is therefore, in contrast to gelatine, in liquid form at the same concentration at working temperatures typical for 3D bioprinting (room temperature approx. 18-22 °C).

Example 13: Measurement of the circular dichroism of the unfunctionalized polypeptide according to the invention (according to Example 4):

To measure circular dichroism, the recombinant polypeptide is dissolved in ultrapure water at a concentration of 1 mg/mL and then filtered through a 0.22 pm syringe filter. As a comparison solution, gelatin is dissolved at a concentration of 1 mg/mL and filtered through a 0.22 pm syringe filter. The circular dichroism of the two solutions is measured in a J-815 CD spectrometer (Jasco) in the wavelength range from 190 to 290 nm. The mean values of five consecutive measurements are plotted. The measurements were carried out for both solutions at 5 °C, 20 °C and 37 °C (see Fig. 4 and Fig. 5). For gelatin, a peak typical for triple helices is seen at approx. 220 nm at 5 °C and 20 °C, but not at 37 °C. For the recombinant polypeptide, a positive signal typical for triple helices cannot be observed at any of the temperature ranges investigated. Peak at 220 nm can be observed. This is due to the lack or lower prolyl hydroxylation of the polypeptide according to the invention compared to the naturally occurring polypeptide fragment.

The following tables list the sequence lists (SEQ ID No. 1 to 8) starting with the N-terminal end and ending with the C-terminal end:

Tabelle 2: Proteinsequenzen SEQ ID Nr. 3 und 4

Tabelle 3: SEQ ID Nr. 5: Nucleotidsequenz codierend für Proteinsequenz SEQ ID Nr. 1

Tabelle 4: SEQ ID Nr. 6: Nucleotidsequenz codierend für Proteinsequenz SEQ ID Nr. 2

Tabelle 5: SEQ ID Nr. 7: Nucleotidsequenz codierend für Proteinsequenz SEQ ID Nr. 3

Tabelle 6: SEQ ID Nr. 8: Nucleotidsequenz codierend für Proteinsequenz SEQ ID Nr. 4

Table 1: Protein sequences SEQ ID No. 1 and 2

Table 2: Protein sequences SEQ ID No. 3 and 4

Table 3: SEQ ID No. 5: Nucleotide sequence encoding protein sequence SEQ ID No. 1

Table 4: SEQ ID No. 6: Nucleotide sequence encoding protein sequence SEQ ID No. 2

Table 5: SEQ ID No. 7: Nucleotide sequence encoding protein sequence SEQ ID No. 3

Table 6: SEQ ID No. 8: Nucleotide sequence encoding protein sequence SEQ ID No. 4

Claims

patent claims 1. A method for producing an ink for 3D printing, wherein a polypeptide is added to a solvent, characterized in that the polypeptide is one based on human collagen having a hydroxyproline content of maximum 5%, based on the total number of amino acids of the polypeptide. 2. The method according to claim 1, wherein the polypeptide has a hydroxyproline content of maximum 4%, preferably maximum 3%, based on the total number of amino acids of the polypeptide. 3. The method of claim 1 or 2, wherein the polypeptide is functionalized with at least one crosslinkable group. 4. A process according to any one of claims 1 to 3, wherein the crosslinkable group is a photocrosslinkable group. 5. A method according to any one of claims 1 to 4, wherein the polypeptide is one which is in the liquid state in an aqueous solution at concentrations of 10 weight percent per volume (% w/v) at temperatures of 18°C and above. 6. The method of any one of claims 1 to 5, wherein the polypeptide has a molecular weight in the range of 20 to 200 Daltons. 7. The method of any one of claims 1 to 6, wherein the ink contains biological cells. 8. Ink for 3D printing containing a human collagen-based polypeptide containing a maximum of 5% hydroxyproline based on the total number of amino acids of the polypeptide. 9. Ink according to claim 8, obtainable by a process according to one of the Claims 1 to 7. 10. Ink according to claim 8 or 9 for use in medicine. 11. A method for producing a 3D scaffold using an ink according to any one of claims 8 to 10, wherein the polypeptide is cured by means of a 3D printing process. 12. The method of claim 11, wherein the polypeptide is functionalized with at least one crosslinkable group. 13. The method according to claim 11 or 12, wherein the 3D printing process uses light-based shaping, preferably by light-induced chemical reaction. 14. The method of any one of claims 11 to 13, wherein the ink contains biological cells. 15. A 3D scaffold comprising cross-linked polypeptides, wherein the polypeptides are human collagen-based polypeptides each containing a maximum of 5% hydroxyproline based on the total number of amino acids of the respective polypeptide. 16. 3D scaffold according to claim 15, additionally comprising biological cells. 17. 3D scaffold according to claim 15 or 16, obtainable by a process according to one of claims 11 to 14. 18. 3D framework according to one of claims 15 to 17 for use in medicine.