A method for separating and purifying recombinant human collagen

Abstract

The present invention discloses a method for separating and purifying recombinant human collagen, wherein the method first filters the recombinant human collagen fermentation broth, then uses cationic composite chromatography to crudely purify the recombinant human collagen to obtain a cationic composite chromatography eluent, and then uses a hydrophobic chromatography column to purify the purified recombinant human collagen, wherein the cationic composite chromatography column is filled with a high-flow rate agarose matrix containing a hydrophobic group of a phenyl group and an electrostatic group of a carboxyl group as a filler, and the hydrophobic chromatography column is filled with a hydrophilic polymethacrylate matrix containing a phenyl group as a filler. The final target protein purity of the present invention is greater than 99%, the recovery rate is greater than 85%, and the endotoxin content is less than 0.5EU / mg. The process of the present invention is simple and easy to operate, and the purification cost requirement is low. The recombinant human collagen obtained by the method has high purity and low endotoxin content, and meets the requirements of cosmetic raw materials, medical devices, and medical-grade high-purity collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for separating and purifying recombinant human collagen. Background Art

[0002] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% or more in some organisms. The unique triple helix structure of collagen gives it good structural stability, biocompatibility and low immunogenicity. These unique characteristics make it widely used in food, medicine, tissue engineering and cosmetics manufacturing.

[0003] The traditional method of producing collagen is to extract it from animal connective tissue (pigskin, cowhide, donkey, skin, fish, etc.) by acid and alkali hydrolysis, but the resulting product is complex in composition, the collagen aqueous solution is poor, and it is difficult to be compatible with the human body. Therefore, people are constantly trying to use genetic recombination technology to express and produce recombinant human collagen. This recombinant collagen material with a full-length amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombination technology and a triple helix structure can be mass-produced through biological fermentation technology. This type of collagen has a large expression amount, good water solubility, high biological activity, and its performance is better than traditional collagen. Since medical-grade recombinant human collagen has high requirements for purity and endotoxins, the focus is on removing impurities such as host proteins, aggregates, degradation fragments and endotoxins through appropriate separation and purification methods.

[0004] In the prior art, Chinese patent application CN 108070032A uses solid-liquid separation technology, filtration technology and ultrafiltration technology to purify a large amount of recombinant human collagen, and then uses ion exchange chromatography technology to purify the product. This method has many pre-treatment steps, high requirements on equipment and technical capabilities, and one-step ion exchange chromatography cannot effectively remove many impurities such as host proteins, aggregates, degradation fragments and endotoxins. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a separation and purification method for high-purity, low-endotoxin recombinant human collagen that can effectively remove many impurities such as host proteins, aggregates, degradation fragments and endotoxins. The material can then be used as a raw material for medical-grade collagen.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides a method for separating and purifying recombinant human collagen, the method comprises first filtering the fermentation broth of the recombinant human collagen, then using cationic composite chromatography to crudely purify the recombinant human collagen to obtain a cationic composite chromatography eluate, and then using a hydrophobic chromatography column to purify the recombinant human collagen to obtain the purified recombinant human collagen, the cationic composite chromatography column is filled with a high-flow agarose matrix containing hydrophobic groups of phenyl and electrostatic groups of carboxyl as a filler, and the hydrophobic chromatography column is filled with a hydrophilic polymethacrylate matrix containing phenyl as a filler.

[0007] Wherein, the particle size of the filler in the cationic composite chromatography column is 45 to 160 μm, and the average particle size is 90 to 100 μm.

[0008] Wherein, the cationic composite chromatography column is filled with Agarosix MC90-MMC chromatography filler.

[0009] Wherein, the average particle size of the filler in the hydrophobic chromatography column is 30 μm.

[0010] Wherein, the hydrophobic chromatography column is filled with Polar MC30-Phenyl chromatography filler.

[0011] The specific step of filtering the recombinant human collagen fermentation broth is as follows: filtering the recombinant human collagen fermentation broth with a 0.2-0.45 μm membrane package to obtain a protein filtrate.

[0012] Wherein, the material of the membrane package includes polyethersulfone.

[0013] Among them, the specific steps of using cationic composite chromatography to purify recombinant human collagen are: use balance liquid A to balance the cationic composite chromatography column, load the protein filtrate into the cationic composite chromatography column, use balance liquid A to balance, then use washing liquid B to rinse, and finally use eluent C to elute to obtain a cationic composite chromatography eluent, wherein the balance liquid A contains 20-50mM CH3COONH4-HAc, pH4.5-5.5; the washing liquid B contains 20-50mM CH3COONH4-HAc, 150-170mM NaCl, pH4.5-5.5; the eluent C contains 20-50mM CH3COONH4-HAc, 500mM NaCl, pH4.5-5.5.

[0014] Among them, the specific steps of using a hydrophobic chromatography column for purification are: using a balancing liquid D to equilibrate the hydrophobic chromatography column, adjusting the final concentration of ammonium sulfate in the obtained cationic composite chromatography eluate to 1.8M and then loading it onto the hydrophobic chromatography column, then balancing with the balancing liquid D, eluting with the eluent E, collecting the eluent, and obtaining the purified recombinant human collagen, wherein the balancing liquid D contains 20-50mM CH3COONH4-HAc, 1.6-2M (NH4)2SO4, pH4.5-5.5; the eluent E contains 20-50mM CH3COONH4-HAc, 0.8-1M (NH4)2SO4, pH4.5-5.5.

[0015] The method for preparing the fermentation broth of the recombinant human collagen comprises: artificially synthesizing a human collagen gene, inserting it into a pPIC9K expression vector, transferring the vector into a Pichia pastoris strain, fermenting and amplifying the Pichia pastoris host cells on a large scale in a fermenter, and performing solid-liquid separation by centrifugation after the expression is completed to obtain a recombinant human collagen fermentation broth.

[0016] Mechanism of the present invention:

[0017] In the crude and pure step of the present invention, cationic composite chromatography has electrostatic action and hydrophobic action at the same time, and utilizes the difference of the point property and hydrophobicity of collagen molecule and impurity, can achieve high resolution separation of higher purity collagen, its principle is that collagen molecule is positively charged in the buffer system of pH4.5-5.5, combined with cationic composite chromatography, and most of host proteins, pigments, endotoxins are negatively charged, not combined with cationic composite chromatography, so as to flow through. Collagen molecule has good hydrophilicity at the same time, weak hydrophobicity, and other miscellaneous proteins are more hydrophobic, so when carrying out different concentrations of salt isocratic elution, this part of the impurities with strong hydrophobicity will be more strongly combined with the phenyl group on the cationic composite chromatography, and retained in the cleaning stage after elution and removed, conventional cation exchange chromatography ligand does not have phenyl group so as to remove this part of impurities poorly. Therefore, by optimizing the salt gradient eluent, the target collagen is eluted to obtain a target collagen solution of higher purity. At the same time, since most of the impurities have been removed in this step, the pressure of the next hydrophobic chromatography is effectively alleviated. In the purification step, the matrix is ​​hydrophilic polymethacrylate, the ligand is phenyl, and the average particle size is 30μm. At the same time, the particle size of the filler is 30μm, compared with the traditional sepharose average particle size of 90μm. The smaller the particle size, the better the separation effect, and more impurities can be removed. At the same time, according to the obvious difference in hydrophobicity between the target collagen molecule and impurities such as aggregates, degradation fragments, and endotoxins, high-purity, low-endotoxin recombinant human collagen can be obtained by salt gradient elution.

[0018] Beneficial effects: Compared with the prior art, the method provided by the present invention only needs to use a one-step 0.2μm membrane package for filtration treatment, which reduces the use of equipment and material consumption, and saves costs. The present invention first filters the recombinant human collagen fermentation broth, and then uses cationic composite chromatography to roughly purify a large amount of recombinant human collagen, and then uses hydrophobic chromatography for fine purification. The final target protein purity is greater than 99%, the recovery rate is greater than 85%, and the endotoxin content is less than 0.5EU / mg, effectively removing impurities such as host proteins, polymers and degradation fragments. The process of the present invention disclosed by the present invention is simpler and easier to operate, and has low requirements for purification costs. The recombinant human collagen prepared by the method has high purity and low endotoxin content, meeting the requirements of cosmetic raw materials, medical devices and medical-grade high-purity collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the cationic composite chromatography purification profile in Example 1;

[0020] Figure 2 is the hydrophobic chromatography purification profile in Example 1;

[0021] Figure 3 It is the non-reducing SDS-PAGE gel electrophoresis result of the recombinant human collagen in Example 1 and Example 2; Lane 1 is the protein Maker, with a molecular weight range of 15-245 kDa, and the molecular weight of the recombinant human collagen is about 44 KDa, Lane 2 is the protein filtrate in step (1) described in Example 1, Lane 3 is the cationic composite chromatography washing solution in step (2) described in Example 1, Lane 4 is the cationic composite chromatography elution solution in step (2) described in Example 1, Lane 5 is the purified recombinant human collagen solution in step (3) described in Example 1, and Lane 6 is the recombinant human collagen solution in step (3) described in Example 2;

[0022] Figure 4 is the HPLC spectrum of the recombinant human collagen solution in Example 1;

[0023] Figure 5 This is the HPLC spectrum of the recombinant human collagen solution in Example 2. DETAILED DESCRIPTION

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0026] The detection method related to recombinant human collagen of the present invention specifically includes:

[0027] 1. The non-reducing SDS-PAGE electrophoresis method used is to separate the proteins in the solution in the electrophoresis gel according to their different molecular weights. It is usually used to detect protein expression and semi-quantitatively analyze the purity of the target protein.

[0028] 2. Size exclusion chromatography is a method of separation based on the molecular weight of proteins in a solution by using a porous high-resolution medium loaded in a chromatographic column. With the help of a high-performance liquid chromatography instrument, quantitative purity analysis of the target protein can be achieved.

[0029] 3. The BCA method used in the present invention is currently a widely used method for determining protein concentration. It is based on the biuret reaction, that is, in an alkaline environment, protein converts Cu 2+ Reduction to Cu + , producing a purple-blue complex with a high absorbance at 562nm. The amount of the reaction product is proportional to the protein concentration, and the protein concentration can be determined by combining it with the standard curve.

[0030] 4. The horseshoe crab reagent colorimetric method is a commonly used kit based on the principle of horseshoe crab reagent enzymatic reaction. It is a highly sensitive, highly stable, and highly anti-interference test kit for quantitative detection of bacterial endotoxin (also known as lipopolysaccharide, LPS) by colorimetry. The detection sensitivity can be as low as 0.01EU / ml. Compared with the gel method horseshoe crab reagent, it has stronger anti-interference ability and higher sensitivity. Example 1

[0031] 1. Reagents and fillers:

[0032] Balance solution A: 20 mM CH4COONH4-HAc aqueous solution, pH 4.5;

[0033] Washing solution B: 20 ​​mM CH3COONH4-Hac and 160 mM NaCl aqueous solution, pH 4.5;

[0034] Eluent C: 20 mM CH3COONH4-HAc and 500 mM NaCl aqueous solution, pH 4.5;

[0035] Balance solution D: 20 mM CH3COONH4-HAc and 1.8 M (NH4)2SO4 aqueous solution, pH 4.5;

[0036] Eluent E: 20 mM CH3COONH4-HAc and 0.9 M (NH4)2SO4 aqueous solution, pH 4.5;

[0037] Chromatographic column: Saifen Technology Agarosix MC90-MMC chromatography packing column, 50*250mm; Saifen Technology Polar MC30-Phenyl chromatography packing column, 50*250mm.

[0038] 2. Specific methods:

[0039] The preparation method of recombinant human collagen fermentation broth is as follows:

[0040] A) The gene sequence of human type III collagen α1 chain (GenBank: AB384877.1) was obtained from the NCBI website, and the 1983-3512 bases (shown in SEQ ID NO.1) were intercepted as the expression fragment, i.e., the 661-1170 amino acid sequence (SEQID NO.2). The restriction site at the 5' end of the gene was XhoI, and the restriction site at the 3' end was SacII. The gene fragment was synthesized by a company, and then the gene fragment was introduced into the linearized pPIC9K expression vector that had also been subjected to restriction digestion to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Pichia pastoris GS115 competent cells to obtain recombinant human type III collagen engineering bacteria.

[0041] The sequence of SEQ ID NO.1 is as follows:

[0042]

[0043]

[0044] The sequence of SEQ ID NO.2 is as follows: The theoretical molecular weight of the protein is about 44KDa.

[0045]

[0046]

[0047] B) The recombinant human type III collagen engineering bacteria were inoculated into 200 mL YPD liquid culture medium, and cultured at 29° C. and 200 r / min for 20 h to obtain a seed solution of the recombinant human collagen engineering bacteria, wherein the components of the YPD liquid culture medium include: 1% yeast powder, 2% peptone, and 2% glucose.

[0048] C) The recombinant human collagen engineering bacteria seed liquid was inoculated into 2.5L BMGY liquid culture medium at a volume ratio of 1:12.5, and cultured at 29°C and 200 r / min for 20 hours to obtain the recombinant human collagen engineering bacteria activation liquid. The components of the BMGY liquid culture medium include: 1% yeast powder, 2% peptone, 1.34% YNB, 0.3% K2HPO4, 1.18% KH2PO4, and 10% glycerol.

[0049] D) The recombinant human collagen engineering bacteria activation liquid was inoculated into 50L BMGY liquid culture medium at a volume ratio of 1:20. During this process, methanol was continuously added to a final concentration of 0.5% to induce fermentation. The fermentation was induced at 29°C and 200 r / min for 80 h. After the fermentation was completed, the supernatant was collected by centrifugation at 8000 r / min for 20 min to obtain the fermentation liquid containing recombinant human type III collagen.

[0050] A method for separating and purifying recombinant human collagen, the specific steps are as follows:

[0051] (1) Fermentation broth filtration: 50 L of recombinant human collagen fermentation broth was filtered using a 0.2 μm membrane package to remove insoluble impurities and obtain a protein filtrate.

[0052] (2) Cationic composite chromatography purification: Use balancing solution A to equilibrate a cationic composite chromatography column for 5 column volumes, wherein the cationic composite chromatography column is filled with Agarosix MC90-MMC chromatography filler of Saifen Technology, with a flow rate of 50 mL / min and a residence time of 10 min. Load 22.5 L of the protein filtrate from step (1) into the cationic composite chromatography column, then use balancing solution A to equilibrate for 5 column volumes, use washing solution B to rinse for 5 column volumes, and finally use eluent C to elute for 5 column volumes to collect the cationic composite chromatography eluate.

[0053] (3) Purification by hydrophobic chromatography: Use balancing liquid D to equilibrate a hydrophobic chromatography column, wherein the hydrophobic chromatography column is filled with Polar MC30-Phenyl chromatography filler produced by Saifen Technology, with a flow rate of 50 mL / min and a residence time of 10 min. Adjust the final ammonium sulfate concentration of the cationic composite chromatography eluate obtained in step (2) to 1.8 M, and then load 6.5 L onto the hydrophobic chromatography column. Use balancing liquid D to equilibrate for 5 column volumes, use eluent E to elute for 5 column volumes, collect the eluate, and obtain the purified recombinant human collagen solution.

[0054] (4) The purified recombinant human collagen solution obtained in step (3) was detected by non-reducing SDS-PAGE electrophoresis. The detection results are shown in Figure 3It can be seen that there are a lot of host proteins, aggregates, degradation fragments and other impurities in the collagen fermentation broth. The recombinant human collagen liquid purified after two steps of chromatography removes these impurities. In particular, the purification step in step 3) also removes the impurities and obtains the target protein with higher purity. The purity measured by size exclusion chromatography is 99.57%, and the HPLC test results are shown in Figure 4 The protein content of the recombinant human collagen solution was determined by the BCA method, and the protein recovery rate was calculated to be 85.6%. The endotoxin content was determined by the horseshoe crab reagent colorimetric method, and the content was 0.36EU / mg.

[0055] Example 2

[0056] 1. Reagents and fillers:

[0057] Balance solution A: 20 mM CH3COONH4-Hac aqueous solution, pH 5.5;

[0058] Washing solution B: 20 ​​mM CH3COONH4-HAc and 160 mM NaCl aqueous solution, pH 5.5;

[0059] Eluent C: 20 mM CH3COONH4-HAc and 500 mM NaCl aqueous solution, pH 4.5;

[0060] Balance solution D: 20 mM CH4COONH4-HAc and 1.8 M (NH4)2SO4 aqueous solution, pH 5.5;

[0061] Eluent E: 20 mM CH3COONH4-HAc and 0.9 M (NH4)2SO4 aqueous solution, pH 5.5;

[0062] Chromatographic column: Saifen Technology Agarosix MC90-MMC chromatography packing column, 50*250mm; Saifen Technology Polar MC30-Phenyl chromatography packing column, 50*250mm.

[0063] 2. Specific methods

[0064] (1) Fermentation broth filtration: The recombinant human collagen fermentation broth was filtered using a 0.2 μm membrane package to remove insoluble impurities and obtain a protein filtrate.

[0065] (2) Cationic composite chromatography purification: Use balancing solution A to equilibrate a cationic composite chromatography column for 5 column volumes, wherein the cationic composite chromatography column is filled with Agarosix MC90-MMC chromatography filler of Saifen Technology, with a flow rate of 50 mL / min and a residence time of 10 min. Load the protein filtrate from step (1) onto the cationic composite chromatography column, then use balancing solution A to equilibrate for 5 column volumes, use washing solution B to rinse for 5 column volumes, and finally use eluent C to elute for 5 column volumes to collect the cationic composite chromatography eluate.

[0066] (3) Purification by hydrophobic chromatography: The hydrophobic chromatography column was equilibrated with equilibration solution D, wherein the hydrophobic chromatography column was filled with Polar MC30-Phenyl chromatography filler produced by Saifen Technology at a flow rate of 50 mL / min and a residence time of 10 min. The cationic composite chromatography eluate obtained in step (2) was adjusted to a final concentration of 1.8 M of ammonium sulfate and then loaded onto the hydrophobic chromatography column. The equilibration solution D was then used for 5 column volumes, and the eluate was eluted for 5 column volumes using eluent E. The eluate was collected to obtain a recombinant human collagen solution.

[0067] (4) The recombinant human collagen solution obtained in step (3) was detected by non-reducing SDS-PAGE electrophoresis. The detection results are shown in Figure 3 It can be seen that there are a lot of host proteins, aggregates, degradation fragments and other impurities in the collagen fermentation broth. The recombinant human collagen liquid purified after two steps of chromatography has removed these impurities. The purity was measured by size exclusion chromatography and was 99.13%. The test results are shown in Figure 5 The protein content of the recombinant human collagen solution was determined by the BCA method, and the protein recovery rate was calculated to be 86.1%. The endotoxin content was determined by the Limulus amebocyte lysate colorimetric method, and the content was less than 0.43EU / mg.

[0068] Example 3

[0069] This embodiment provides a method for separating and purifying recombinant human collagen. The specific steps and parameters are the same as those in Example 2, except that: balance solution A: 20mM CH3COONH4-HAc aqueous solution, pH 5.0; washing solution B: 20mM CH3COONH4-Hac and 160mM NaCl aqueous solution, pH 5.0; eluent C: 20mM CH3COONH4-Hac and 500mM NaCl aqueous solution, pH 5.0; balance solution D: 20mM CH3COONH4-Hac and 1.8M (NH4)2SO4 aqueous solution, pH 5.0; eluent E: 20mM CH3COONH4-Hac and 0.9M (NH4)2SO4 aqueous solution, pH 5.0.

[0070] Comparative Example 1

[0071] This comparative example provides a method for separating and purifying recombinant human collagen, the specific steps and parameters are the same as those in Example 2, except that the buffer system of the buffer used is replaced by a CH3COONH4-Hac aqueous solution with a Na2HPO4-NaH2PO4 aqueous solution, specifically, balance solution A: 20mM Na2HPO4-NaH2PO4 aqueous solution, pH 5.5; washing solution B: 20mMNa2HPO4-NaH2PO4 and 160mM NaCl aqueous solution, pH 5.5; eluent C: 20mM Na2HPO4-NaH2PO4 and 500mM NaCl aqueous solution, pH 5.5; balance solution D: 20mM Na2HPO4-NaH2PO4 and 1.8M (NH4)2SO4 aqueous solution, pH 5.5; eluent E: 20mM Na2HPO4-NaH2PO4 and 0.9M (NH4)2SO4 aqueous solution, pH 5.5.

[0072] Comparative Example 2

[0073] This comparative example provides a method for separating and purifying recombinant human collagen. The specific steps and parameters are the same as those in Example 2, except that the buffer system of the buffer used is replaced by a CH4COONH4-HAc aqueous solution with a NaAc-HAc aqueous solution, specifically, balance solution A: 20mM NaAc-HAc aqueous solution, pH 5.5; washing solution B: 20mM NaAc-HAc and 160mM NaCl aqueous solution, pH 5.5; eluent C: 20mM NaAc-HAc and 500mM NaCl aqueous solution, pH 5.5; balance solution D: 20mM NaAc-HAc and 1.8M (NH4)2SO4 aqueous solution, pH 5.5; eluent E: 20mM NaAc-HAc and 0.9M (NH4)2SO4 aqueous solution, pH 5.5.

[0074] Comparative Example 3

[0075] This comparative example provides a method for separating and purifying recombinant human collagen. The specific steps and parameters are the same as those in Example 2, except that the filler in the cationic composite chromatography crude purification in step 2) is replaced with Cytiva's high-flow agarose cation exchange filler SP Sepharose FF.

[0076] Comparative Example 4 This comparative example provides a method for separating and purifying recombinant human collagen. The specific steps and parameters are the same as those in Example 2, except that the filler in the cationic composite chromatography crude purification in step 2) is replaced with a hydrophilic polymethacrylate cation exchange filler Monomix HC60-SP of Saifen Technology.

[0077] The recombinant human collagen solution obtained after the final purification of Example 1-3 and Comparative Example 1-4 was tested using a recombinant human collagen-related detection method to obtain the purity, recovery rate and endotoxin content of the recombinant human collagen. The test results are shown in Table 1.

[0078] Table 1 Purity, recovery rate and endotoxin content of recombinant human collagen solution

[0079]

[0080] It can be seen from the data in Table 1 that in Examples 1 to 3 of the present invention, a cationic composite chromatography filler is used as the filler for crude chromatography, and a CH3COONH4-Hac aqueous solution is selected as the buffer system. After purification, the purity of the recombinant human collagen solution obtained is greater than 99%, the recovery rate is greater than 85%, and the endotoxin content is less than 0.5EU / mL. In Comparative Examples 1 and 2, the buffer system is replaced with a Na2HPO4-NaH2PO4 aqueous solution or a NaAc-HAc aqueous solution. Compared with Examples 1-3, the purity, recovery rate and endotoxin of the recombinant human collagen solution obtained are not as good. In Comparative Examples 3 and 4, the cationic composite chromatography filler is replaced with a high-flow agarose cation exchange filler and a hydrophilic polymethacrylate cation exchange filler to purify the recombinant human collagen. Compared with Examples 1-3, the purity, recovery rate and endotoxin of the recombinant human collagen solution obtained are not as good. The cationic composite chromatography filler used in the present invention has both electrostatic and hydrophobic effects. It utilizes the differences in charge and hydrophobicity between collagen molecules and impurities and uses a buffer system such as a CH3COONH4-HAc aqueous solution to separate high-purity recombinant human collagen with a high recovery rate and low endotoxin content.

Claims

1. A method for separating and purifying recombinant human collagen, characterized in that: The method comprises the steps of first filtering the fermentation broth of the recombinant human collagen, then crudely purifying the recombinant human collagen by cationic composite chromatography to obtain a cationic composite chromatography eluate, and then finely purifying the recombinant human collagen by using a hydrophobic chromatography column to obtain the purified recombinant human collagen, wherein the cationic composite chromatography column is filled with a high-flow rate agarose matrix containing a hydrophobic group of phenyl and an electrostatic group of carboxyl as a filler, and the hydrophobic chromatography column is filled with a hydrophilic polymethacrylate matrix containing a phenyl as a filler.

2. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The particle size of the filler in the cationic composite chromatography column is 45 to 160 μm, and the average particle size is 90 to 100 μm.

3. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The cationic composite chromatography column is filled with Agarosix MC90-MMC chromatography filler.

4. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The average particle size of the filler in the hydrophobic chromatography column is 30 μm.

5. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The hydrophobic chromatography column is filled with Polar MC30-Phenyl chromatography filler.

6. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The specific step of filtering the recombinant human collagen fermentation broth is as follows: filtering the recombinant human collagen fermentation broth with a 0.2-0.45 μm membrane package to obtain a protein filtrate.

7. The method for separating and purifying recombinant human collagen according to claim 6, characterized in that: The material of the membrane package includes polyethersulfone.

8. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The specific steps of using cationic composite chromatography to purify recombinant human collagen are as follows: using balancing solution A to equilibrate a cationic composite chromatography column, loading the protein filtrate onto the cationic composite chromatography column, balancing using balancing solution A, then washing using washing solution B, and finally eluting using eluent C to obtain a cationic composite chromatography eluent, wherein the balancing solution A contains 20-50 mM CH3COONH4-HAc, pH 4.5-5.5; the washing solution B contains 20-50 mM CH3COONH4-HAc, 150-170 mM NaCl, pH 4.5-5.5; and the eluent C contains 20-50 mM CH3COONH4-HAc, 500 mM NaCl, pH 4.5-5.

5.

9. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The specific steps of using a hydrophobic chromatography column for purification are: using a balancing liquid D to equilibrate the hydrophobic chromatography column, adjusting the final concentration of ammonium sulfate in the obtained cationic composite chromatography eluate to 1.6-2M and then loading it onto the hydrophobic chromatography column, then balancing with the balancing liquid D, eluting with the eluent E, collecting the eluent, and obtaining the purified recombinant human collagen, wherein the balancing liquid D contains 20-50mM CH3COONH4-HAc, 1.8M (NH4)2SO4, and pH4.5-5.5; the eluent E contains 20-50mM CH3COONH4-HAc, 0.8-1M (NH4)2SO4, and pH4.5-5.

5.

10. The method for separating and purifying recombinant human collagen according to claim 1, characterized in that: The preparation process of the recombinant human collagen fermentation broth is as follows: synthesizing a human collagen gene, inserting it into a pPIC9K expression vector, transferring the vector into a Pichia pastoris strain, fermenting and amplifying the Pichia pastoris host cells on a large scale in a fermenter, and performing solid-liquid separation by centrifugation after the expression is completed to obtain the recombinant human collagen fermentation broth.

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