Chromatographic method for the purification of collagen-like protein

Abstract

The present invention refers to a method for the purification of a collagen-like protein mixture, comprising or consisting of the following steps: a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution, followed by heating of the collagen-like protein solution followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reversed-phase chromatography which includes a mobile phase in form of at least one mobile phase solvent and a stationary phase in form of at least one column. Furthermore, the present invention refers to the collagen-like protein obtained by the method according to the invention. In addition, the present invention refers to the use of the collagen-like protein obtained by the method according to the invention for medical applications.

Description

[0001] 202300182 1

[0002] Chromatographic method for the purification of collagen-like protein

[0003] Field of the invention

[0004] The present invention refers to a method for the purification of a collagen-like protein mixture, comprising or consisting of the following steps: a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution followed by heating of the collagen-like protein solution followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reverse phase chromatography which includes a mobile phase in form of at least one mobile phase-solvent and a stationary phase in form of at least one column.

[0005] Furthermore, the present invention refers to the collagen-like protein obtained by the method according to the invention.

[0006] In addition, the present invention refers to the use of the collagen-like protein obtained by the method according to the invention for medical applications.

[0007] Description of the related art

[0008] There are several purification methods for recombinant collagen-like proteins known in the art, for example, affinity chromatography, ion exchange chromatography, purification with aprotic solvents and inorganic salts, and precipitation-methods. A review of common methods has been published from Awang et al. (Awang, Nursyahidatul & Amid, Azura & Arshad, Zatul (2020), Method for purification of collagen: A systematic review. Asia Pacific Journal of Molecular Biology and Biotechnology, 99-112).

[0009] Recombinantly produced collagen-like protein in raw solution has different chain lengths compared to the target product. As a result, the protein folding of the individual strands, building up the triple helix, leads to a statistical distribution with unwanted sequence lengths. The mentioned purification methods of the related art do not describe how high-purity recombinant collagen-like protein can be obtained with homologous single strands so that they have defined properties and therefore are suitable for medical applications.

[0010] The inventors of the present invention surprisingly found that a purification method comprising of a pretreatment of the collagen-like protein in form of heating and allowing to cool, followed by a reversed- phase chromatography solves this problem. Therefore, it was possible to separate collagen-like proteins from a matrix with different chain lengths to obtain high-purity collagen-like proteins which are suitable for medical applications. 202300182 2

[0011] Summary

[0012] In a first aspect the present invention refers to a method for the purification of a collagen-like protein, comprising or consisting of the following steps: a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution followed by heating of the collagen-like protein solution followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reversed-phase chromatography which includes a mobile phase in form of at least one mobile-phase solvent and a stationary phase in form of at least one column.

[0013] In a second aspect the present invention pertains to the collagen-like protein obtained by the method according to the invention.

[0014] In a third aspect the present invention pertains to the use of the collagen-like protein obtained by the method according to the invention for medical applications

[0015] These and other aspects, embodiments, features, and advantages of the invention will become apparent to a person skilled in the art through the study of the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it will readily be understood that the examples contained herein are intended to describe and illustrate the invention but not to limit the invention and that, in particular, the invention is not limited to these examples.

[0016] 202300182 3

[0017] Detailed description

[0018] Numerical ranges that are indicated in the format “from x to y” also include the stated values. If several preferred numerical ranges are indicated in this format, it is self-evident that all ranges that result from the combination of the various endpoints are also included.

[0019] "At least one" means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one", as used herein in relation to any component, refers to the number of chemically different molecules, i.e., to the number of different types of the referenced species, but not to the total number of molecules. For example, "at least one collagen-like protein" means that at least one type of molecule falling within the definition for a collagen-like protein is used but that also two or more different types of collagen-like protein falling within this definition can be present but does not mean that only one or more molecules of one type of collagen- like protein are present.

[0020] All percentages given herein in relation to the compositions or formulations relate to wt.-% relative to the total weight of the respective composition, if not explicitly stated otherwise.

[0021] In the following, “collagen-like protein” is referred to as “CLP” as well.

[0022] In particular the present invention refers to:

[0023] A method for the purification of a CLP comprises or consists of the following steps: a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution followed by heating of the collagen-like protein solution, followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reversed-phase chromatography which includes a mobile phase in form of at least one mobile phase solvent and a stationary phase in form of at least one column.

[0024] In the method at least one collagen-like protein is used. In general, all collagen-like proteins are suitable.

[0025] In a preferred embodiment of the present invention the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.

[0026] Expression of collagen-like proteins have been attempted in several systems, including Escherichia coli and Saccharomyces cerevisiae. In one embodiment the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0027] In a preferred embodiment the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.

[0028] One particularly suitable collagen-like protein is derivable from following polynucleotide. 202300182 4

[0029] A polynucleotide encoding an amino acid sequence that is at least > 60% identical to the amino acid sequence of SEQ ID NO:1 , wherein the polynucleotide is a replicable polynucleotide encoding a collagen- like protein and wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N- terminus of the amino acid sequence of SEQ ID NO:1.

[0030] It is preferred, when the amino acid sequence comprises a deletion of between 38 and 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1. This includes a complete deletion of the N- terminal V-domain (comprising 74 amino acids) and different truncations of the V-domain of at least 38 amino acids.

[0031] In a preferred embodiment, the amino acid sequence that is at least > 60% identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0032] In a further configuration, the amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or

[0033] > 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0034] In a preferred configuration, the polynucleotide encodes an amino acid sequence that is at least > 90%,

[0035] > 92%, > 94%, > 96%, > 97%, > 98%, > 99% or 100%, preferably > 97%, particularly preferably > 98%, very particularly preferably > 99%, and extremely preferably 100% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0036] In a preferred embodiment of the present invention, the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.

[0037] Polynucleotide and nucleic acid molecules comprising such sequences and encoding polypeptide variants of SEQ ID NO:1 to 4, which contain one or more insertion(s) or deletion(s) are suitable as well. Preferably, the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2 insertions or deletions of amino acids.

[0038] Mixture of polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 9 can be used as well.

[0039] Plasmids and vectors that comprise the nucleotide sequences according to the invention and optionally replicate in microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia are suitable. In a preferred configuration, the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.

[0040] Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well. Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum. 202300182 5

[0041] Microorganism of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention, any of the polypeptides or any of the vectors according to the present invention are suitable.

[0042] The microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.

[0043] Overexpression according to the invention means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain. A starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.

[0044] In the overexpression, the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or like methods.

[0045] The extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity.

[0046] The bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.

[0047] The culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are mutually exchangeable.

[0048] As carbon source, sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.

[0049] As nitrogen source, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture. 202300182 6

[0050] As phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used.

[0051] The culture medium must, in addition, contain salts, for example, in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids, for example, homoserine and vitamins, for example, thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.

[0052] Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.

[0053] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture. The pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8. For control of foam development, antifoams can be used, such as, for example, polyglycol esters of fatty acids. For maintaining the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics, can be added to the medium. The fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The use of liquids that are enriched with hydrogen peroxide is likewise possible. Optionally, the fermentation is carried out at superatmospheric pressure, for example, at a superatmospheric pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C. In the case of batch or fed-batch processes, the culturing is preferably continued until an amount sufficient for the measure of obtaining the desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and / or in the cells of the microorganisms occurs.

[0054] Examples of suitable fermentation media may be found, inter alia, in patent documents US 5,770,409, US 5,990,350, US 5,275,940, WO 2007 / 012078, US 5,827,698, WO 2009 / 043803, US 5,756,345 or US 7,138,266; appropriate modifications may optionally be carried out to the requirements of the strains used.

[0055] The process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process, and continuous process.

[0056] The process may be further characterized by a fine chemical or a liquid, or a solid fine chemical-containing product is obtained from the fine chemical-containing fermentation broth.

[0057] The performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1 %, at least 1 .5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present. 202300182 7

[0058] Owing to the measures of the fermentation, a fermentation broth is obtained which contains the desired collagen-like protein, and preferably amino acid or organic acid.

[0059] Then, a product in liquid or solid form that contains the collagen-like protein is provided or produced or obtained.

[0060] A fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature. The fermentation medium, or the media used during the fermentation, contains / contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and / or viability.

[0061] On completion of the fermentation, the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.

[0062] The organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.

[0063] The fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein. The expression "obtaining the CLP-containing product" is also used therefor. In the simplest case, the CLP- containing fermentation broth withdrawn from the fermentation container is itself the product obtained.

[0064] By way of one or more of the measures selected from the group a) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the water, b) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the biomass, wherein this is optionally inactivated before the removal, c) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the organic by-products formed in the course of the fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the components ofthe fermentation medium used orthe starting materials that are not consumed by the fermentation, a concentration or purification of the desired collagen-like protein is achieved from the fermentation broth. In this manner, products are isolated that have a desired content of the compound. 202300182 8

[0065] The partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80% to < 100%) removal of the water (measure a)) is also termed drying.

[0066] In a variant of the process, by complete or virtually complete removal of the water, the biomass, the organic by-products and the non-consumed components of the fermentation medium used, pure (> 80% by weight, > 90% by weight) or high-purity (> 95% by weight, > 97% by weight, > 99% by weight) product forms of the desired collagen-like protein, preferably bacterial collagen-like protein, are successfully arrived at. For the measures according to a), b), c) or d), a great variety of technical instructions are available in the prior art.

[0067] In the case of processes for producing bacterial collagen-like protein, processes are preferred in which products are obtained that do not contain any components of the fermentation broth. These products are used, in particular, in human medicine, in the pharmaceuticals industry, and in the food industry.

[0068] In an embodiment the CLP mixture in step a) comprises CLP of different chain lengths.

[0069] In an embodiment in step a) a1) the CLP-solution is provided with a concentration of 1 to 25 g / L, preferably 10 to 25 g / L, more preferably 15 to 20 g / L, most preferably 20 g / L and / or a2) the CLP-solution is heated to 30°C to 50°C, preferably 30°C to 45°C, more preferably 30°C to 40°C, most preferably 37°C to 40°C and / or a3) the CLP-solution is allowed to cool to 20°C to 30°C, preferably 22°C to 28°C, more preferably 23°C to 27°C, most preferably 25°C.

[0070] In an embodiment in step b) b1) the CLP-solution is adjusted to pH-value of 3 to 5, preferably 3.5 to 4.5, more preferably

[0071] 3.5 to 4, most preferably 4, b2) the column is equilibrated, b3) the temperature of the at least one mobile phase-solvent is adjusted to 20°C to 40°C, preferably 20 to 30°C, more preferably 20 to 25 °C. b4) the column is loaded with the CLP-solution, b5) the CLP is eluted using the at least one mobile phase solvent and b6) fractions of the purified CLP are collected.

[0072] In an embodiment the solvent of the CLP-solution is selected from an aqueous ammonium acetate solution or water.

[0073] In an embodiment the solvent for equilibration in b2) is an aqueous ammonium acetate solution / alcohol- mixture, wherein the alcohol is selected from methanol, ethanol or mixtures thereof, preferably isopropyl alcohol. 202300182 9

[0074] In an embodiment the ratio of the aqueous ammonium acetate solution to alcohol for equilibration in b2) is 50 to 100 vol% aqueous ammonium acetate solution to 0 to 50 vol% alcohol, preferably 70 to 90 vol% aqueous ammonium acetate solution to 10 to 30 vol% alcohol, more preferably 83 to 84 vol% aqueous ammonium acetate solution to 16 to 17 vol% alcohol, wherein the total volume is 100 vol%.

[0075] In an embodiment the solvent for equilibration in b2) has a pH-value of 3 to 5, preferably 3.5 to 4.5, more preferably 3.5 to 4, most preferably 4.

[0076] In an embodiment the solvent for equilibration in b2) has a linear velocity of 30 to 300 cm / h, preferably 50 to 250 cm / h, more preferably 100 to 200 cm / h, most preferably 150 to 168 cm / h.

[0077] In an embodiment 0.01 to 0.25 bed volumes, preferably 0.05 to 0.2 bed volumes, more preferably 0.1 to 0.15 bed volumes, most preferably 0.124 bed volumes of the CLP-solution are injected with an injection speed of 30 to 300 cm / h, preferably 30 to 200 cm / h, more preferably 30 to 100 cm / h, most preferably 30 cm / h in b4).

[0078] In an embodiment the solvent for the elution in b5) is an aqueous ammonium acetate solution / alcohol- mixture, wherein the alcohol is selected from methanol, ethanol or mixtures thereof, preferably isopropyl alcohol.

[0079] In an embodiment the ratio of the aqueous ammonium acetate solution to the alcohol for the elution in b5) is 50 to 90 vol% aqueous ammonium acetate solution to 10 to 50 vol% alcohol, preferably 75 to 88 vol% aqueous ammonium acetate solution to 12 to 25 vol% alcohol, more preferably 83 to 84 vol% aqueous ammonium acetate solution to 16 to 17 vol% alcohol, wherein the total volume is 100 vol%.

[0080] In an embodiment the solvent for the elution in b5) has a pH-value of 3 to 5, preferably 3.5 to 4.5, more preferably 3.5 to 4, most preferably 4.

[0081] In an embodiment the solvent for the elution in b5) has a linear velocity of 30 to 300 cm / h, preferably 30 to 200 cm / h, more preferably 30 to 100 cm / h, most preferably 30 to 33.6 cm / h.

[0082] In an embodiment the stationary phase is a chromatography column.

[0083] In an embodiment the chromatography column contains modified silica gel and / or polymer-based adsorbents, preferably an octadecyl modified silica gel and / or an octyl modified silica gel and / or a polydivinylbenzene adsorbent.

[0084] In an embodiment an UV-detector can be used for collecting the purified fractions in step b6).

[0085] Furthermore, the present invention provides a CLP obtained by the method according to the invention. 202300182 10

[0086] In an embodiment the CLP obtained by the method according to the invention has a purity of > 95%, preferably > 96%, more preferably > 97%, most preferably > 99% related to a homologous single strand. The purity was measured via HPLC using the area% of the respective peak.. In an embodiment the CLP obtained by the method according to the invention is suitable for medical applications.

[0087] 202300182 11

[0088] Examples

[0089] Example 1 : purification of a collagen-like protein mixture obtained from pichia pastoris

[0090] The purpose of this purification is to separate the CLP (SEQ ID No. 4) from other CLP fragments.

[0091] The CLP-mixture to be purified was diluted with a 0,9 molar aqueous ammonium acetate solution to provide a concentration of 20 g / L. Before purification, this diluted CLP-mixture was heated to 37-40°C for at least 10 minutes.

[0092] An empty column (commercially available by Cytiva; model: XK16 / 40) was filled with 40 mL of the polydivinylbenzene absorbent (commercially available by Purolite under the name PCG1200M). For this purpose, a 50% suspension of the absorbent in 20% isopropyl alcohol was prepared, which then was filled in the column.

[0093] The column was equilibrated with 3 bed volumes of a mixture of a 20 mM aqueous ammonium acetate solution with a pH value of 4 (83 vol%) and isopropyl alcohol (17 vol%) at a flow velocity of 2.5 cm / min.. This was followed by the introduction of 10 mL of the diluted CLP-mixture at a flow velocity of 0.5 cm / min.. Subsequently, the elution of the product was started by using 10 bed volumes of a mixture of a 20 mM aqueous ammonium acetate solution with an pH value of 4 (83 vol%) and isopropyl alcohol (17 vol%) at a flow velocity of 0.5 cm / min.. Fractionation was performed automatically in 5 mL fractions starting at a signal value of 100 mAU using an UV-detector at 214 nm.

[0094] With this purification, a purity of 99.7% was achieved.

[0095] Example 2: purification of a collagen-like protein mixture obtained from corynebacterium

[0096] The purpose of this purification is to separate the CLP (SEQ ID No. 4) from other CLP fragments.

[0097] The CLP-mixture to be purified was diluted with 1 .24 mL of a 0.92 molar aqueous ammonium acetate solution and 73 mL water to provide a concentration of 20 g / L. Before purification, this diluted CLP- mixture was heated to 37-40°C for at least 10 minutes.

[0098] An empty column (commercially available by YMC Europe GmbH; model: ECO Gias) was filled with 650 mL of the polydivinylbenzene absorbent (commercially available by Purolite under the name PCG1200M). For this purpose, a 50% suspension of the absorbent in 20% isopropyl alcohol was prepared, which then was filled in the column.

[0099] The column was equilibrated with 2 bed volumes of a mixture of a 20 mM aqueous ammonium acetate solution with a pH value of 4 (84 vol%) and isopropyl alcohol (16 vol%) at a flow velocity of 2.8 cm / min.. This was followed by the introduction of 81 mL of the diluted CLP-mixture at a flow velocity of 0.56 cm / min.. Subsequently, the elution of the product was started by using 2.81 bed volumes of mixture of a 20 mM aqueous ammonium acetate solution with a pH value of 4 (84 vol%) and isopropyl alcohol (16 vol%) at a flow velocity of 0.56 cm / min.. Fractionation took place manually in 15 mL fractions using an UV-detector at 214 nm.

[0100] With this purification, a purity of 99.9% was achieved. 202300182 12

[0101] 1 : of a i-like mixture obtained from without heating of the collagen-like protein mixture

[0102] Without the heating step of the collagen-like protein mixture mentioned in examples 1 and 2, it was not possible to perform the purification according to example 1 successfully. It could be shown that no binding between the CLP and the polydivinylbenzene absorbent took place. in mixture obtained from pi an ion exchanger

[0103] The purpose of this purification is to separate the CLP (SEQ ID No. 4) from other CLP fragments.

[0104] 3 experiments have been performed with the respective buffer systems:

[0105] • Buffer 1 -a: 20 mM TRIS, pH 8

[0106] • Buffer 1-b: 20 mM TRIS, pH 8 + 1 M NaCI

[0107] • Buffer 2-a :20 mM glycine, pH 10

[0108] • Buffer 2-b: 20 mM glycine, pH 10 + 1 M NaCI

[0109] • Buffer 3-a: 20 mM phosphate, pH 6

[0110] • Buffer 3-b: 20 mM phosphate, pH 6 + 1 M NaCI

[0111] The CLP-mixture to be purified was diluted with buffer a of the respective buffer system at a volume ratio of 1 :10. The column (commercially available by YMC Europe GmbH; model: BioPro Q) was eguilibrated with buffer a of the respective buffer system at a flow velocity of 2.5 cm / min. for each experiment. This was followed by the introduction of 5 mL of the respective diluted CLP-mixture. The elution was performed at a flow velocity of 2.5 cm / min using a gradient of 0-100 vol% of buffer b of the respective buffer system. For fractionation, an UV-detector at 214 nm was used.

[0112] The obtained purity of each of the experiments was below 99.0% and therefore below the purity of experiment 1 .

Claims

202300182 13Claims1. Method for the purification of a collagen-like protein mixture, comprising or consisting of the following steps a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution, followed by heating of the collagen-like protein solution, followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reversed-phase chromatography which includes a mobile phase in form of at least one mobile phase solvent and a stationary phase in form of at least one column.

2. Method according to claim 1 , wherein in step a) a1) the collagen-like protein solution is provided with a concentration of 1 to 25 g / L and / or a2) the collagen-like protein solution is heated to 30 °C to 50 °C and / or a3) the collagen-like protein solution is allowed to cool to 20 °C to 30 °C.

3. Method according to claim 1 and 2, wherein in step b) b1) the collagen-like protein solution is adjusted to pH-value 3 to 5, b2) the column is equilibrated, b3) the temperature of the at least one mobile phase solvent is adjusted to 20°C to 40°C, b4) the column is loaded with the collagen-like protein solution, b5) the collagen-like protein is eluted using the at least one mobile phase solvent and b6) fractions of the purified collagen-like protein are collected.

4. Method according to claim 3, wherein in b2) an aqueous ammonium acetate solution / alcohol- mixture with a pH-value of 3 to 5 and / or a linear velocity of 30 to 300 cm / h is used for equilibrating the column.

5. Method according to claim 3, wherein in b4) 0.01 to 0.25 bed volume of the collagen-like protein solution are injected with an injection speed of 30 to 300 cm / h.

6. Method according to claim 3, wherein in b5) an aqueous ammonium acetate solution / alcohol- mixture with a pH-value of 3 to 5 and / or a linear velocity of 30 to 300 cm / h is used.

7. Method according to any of the preceding claims, wherein the collagen-like protein is a bacterial collagen-like protein produced by fermentation.

8. Method according to claim 7, wherein the collagen-like protein is obtained from pichia pastoris, escherichia coli or corynebacterium.202300182 149. Method according to claim 7, wherein the collagen-like protein is derivable from a polynucleotide encoding an amino acid sequence that is at least > 60% identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

10. Method according to any of the preceding claims, wherein the solvent of the collagen-like protein solution is selected from water or an aqueous ammonium acetate solution.

11. Method according to any of the preceding claims, wherein the stationary phase is selected from modified silica gel and / or polymer-based adsorbents.

12. Method according to claim 11 , wherein the stationary phase is an octadecyl modified silica gel and / or an octyl modified silica gel and / or a polydivinylbenzene adsorbent.

13. The collagen-like protein obtained by the method according to any of the preceding claims.

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