Method for efficiently purifying recombinant collagen and removing endotoxin

Abstract

The invention provides a method for efficiently purifying recombinant collagen and removing endotoxin, and belongs to the technical field of collagen purification. Affinity chromatography, anion exchange chromatography, ultrafiltration concentration and dialysis filtration are combined, the affinity chromatography technology is used for primarily purifying recombinant collagen and removing endotoxin, and the anion exchange chromatography technology is used for further purifying recombinant collagen and removing endotoxin. And finally, carrying out ultrafiltration concentration and dialysis filtration to remove small molecular impurities and residual salts so as to achieve the purposes of efficiently purifying the recombinant collagen and removing endotoxin. The method improves the purification efficiency, has flexibility and expandability, can improve the quality and consistency of the product, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen purification, and specifically relates to a method for efficiently purifying recombinant collagen and removing endotoxin. Background Art

[0002] Collagen is one of the most abundant proteins in the human body. It is widely present in skin, bones, tendons, blood vessels and other tissues, and plays an important role in maintaining tissue structure and function. With the development of biotechnology, the production of recombinant collagen has gradually become a research hotspot, and it has broad application prospects in the fields of medicine, beauty and biomaterials.

[0003] At present, the purification methods of recombinant collagen mainly include precipitation, centrifugation, chromatography, etc. These methods can remove impurities to a certain extent, but the effect of removing endotoxins is not ideal. Precipitation and centrifugation can usually only remove macromolecular impurities, and although chromatography can improve purity, its specific removal ability for endotoxins is limited. In addition, the purification efficiency of recombinant collagen by the above methods is also relatively limited, so it is necessary to develop a new method for efficiently purifying recombinant collagen and removing endotoxins at the same time. Summary of the invention

[0004] In view of some deficiencies in the prior art, the present invention provides a method for efficiently purifying recombinant collagen and removing endotoxins; the present invention combines affinity chromatography, anion exchange chromatography, ultrafiltration concentration and diafiltration, the affinity chromatography technology preliminarily purifies the recombinant collagen and removes endotoxins, the anion exchange chromatography technology further purifies the recombinant collagen and removes endotoxins, and finally ultrafiltration concentration and diafiltration are used to remove small molecule impurities and residual salts, thereby achieving the purpose of efficiently purifying the recombinant collagen and removing endotoxins; the method improves the purification efficiency, has flexibility and scalability, can improve the quality and consistency of the product, and has good practicality.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention provides a method for efficiently purifying recombinant collagen and removing endotoxin, the method comprising:

[0007] (1) Preliminarily filtering the cell culture medium I expressing the recombinant collagen to remove cell debris and large particles to obtain a preliminarily filtered cell culture medium II;

[0008] (2) selecting a specific ligand of the target recombinant collagen and immobilizing the specific ligand on the surface of an affinity chromatography medium to form an affinity chromatography column;

[0009] The obtained cell culture fluid II is pretreated, and then added into an affinity chromatography column for affinity chromatography treatment, and an intermediate product A is obtained after the treatment is completed;

[0010] (3) gradient eluting the intermediate product A, collecting the eluate, and pre-treating the eluate to obtain a pre-treated eluate for later use;

[0011] The pretreated eluate is added to an anion exchange chromatography column for anion exchange chromatography treatment, and an intermediate product B is obtained after the treatment;

[0012] (4) The intermediate product B is subjected to ultrafiltration concentration and diafiltration in sequence to obtain a recombinant collagen solution that is completely free of residual salts and small molecular impurities and has high purity and low endotoxin.

[0013] Preferably, in step (1), the cells in the cell culture medium I expressing recombinant collagen include: a cell line that has been genetically engineered and is capable of stably and efficiently expressing recombinant collagen.

[0014] Preferably, in step (1), the preliminary filtration uses a microporous filter membrane with a pore size range of 0.2 to 1 μm.

[0015] Preferably, in step (2), the specific ligand comprises a collagen-specific antibody fragment; the collagen-specific antibody fragment is a tag added to the carboxyl end of the recombinant collagen; the tag comprises a Strep tag, which is a short peptide consisting of 8 amino acids (tryptophan-serine-histidine-threonine-glutamine-phenylalanine-glutamic acid-lysine);

[0016] The pretreatment step includes: adjusting the pH value and ionic strength of the cell culture fluid II so that it can stably bind to the collagen-specific antibody fragment during affinity chromatography; that is, controlling the pH value of the cell culture fluid II within the range of 7.2 to 7.4 and maintaining the ionic strength within the range of 280 to 320 mOsm.

[0017] Preferably, in step (2), the affinity chromatography treatment step comprises:

[0018] The pretreated cell culture fluid II is added to the affinity chromatography column to allow the recombinant collagen in the cell culture fluid II to fully contact and bind with the specific antibody fragment. After the binding is completed, the affinity chromatography column is washed and balanced.

[0019] Preferably, in step (3), during the gradient elution, the starting pH value is 6.5-7.0, the starting ionic strength is 0-50 mmol / L NaCl concentration; the ending pH value is 5.5-6.0, and the ending ionic strength is 200-300 mmol / L NaCl concentration.

[0020] Preferably, in step (3), the step of pretreating the eluate comprises: adjusting the pH value to 5.5-6.0, and adjusting the ionic strength to a NaCl concentration of 200-300 mmol / L, so as to ensure that the endotoxin can be stably bound to the anion exchange medium during the chromatography process.

[0021] Preferably, in step (3), the anion exchange chromatography step comprises:

[0022] S1. adding the pretreated eluate to an anion chromatography column, so that the endotoxin specifically binds to the anion exchange group in the anion chromatography column;

[0023] S2. Washing and eluting the chromatography column to elute the endotoxin bound to the anion exchange medium from the chromatography column, collecting the recombinant collagen solution after the endotoxin is removed, and obtaining the intermediate product B;

[0024] S3. Regenerate the anion exchange column using a high concentration salt solution to remove residual endotoxins and other impurities and restore the binding capacity of the column.

[0025] Preferably, in step (4), the ultrafiltration concentration uses a membrane material with a molecular weight cutoff slightly larger than that of the recombinant collagen, so as to effectively remove small molecule impurities and solvents while retaining the integrity of the recombinant collagen.

[0026] Preferably, in step (4), the diafiltration uses a buffer system whose pH value and ionic strength match the requirements of the final application to maintain the stability and activity of the recombinant collagen.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The method for highly efficient purification of recombinant collagen and removal of endotoxins of the present invention significantly improves the purification efficiency of recombinant collagen, with a purification efficiency higher than 95%, and even capable of reaching a purification efficiency of more than 99%, and removes endotoxins in a targeted manner while ensuring high purity. The method of the present invention is highly flexible and has strong scalability, and can select the most suitable specific ligand and chromatography conditions according to different recombinant collagen sequences and characteristics to achieve the best purification effect.

[0029] The present invention uses affinity chromatography technology and the combination of specific ligands and recombinant collagen to achieve efficient purification of the target protein and preliminarily remove endotoxins in the target protein; the present invention uses anion exchange chromatography technology based on the negative charge characteristics of endotoxins to specifically capture and remove endotoxins to ensure the safety of the product while further purifying the target protein. In addition, ultrafiltration concentration and diafiltration steps are also used to effectively remove small molecule impurities and residual salts, thereby improving the purity of the recombinant collagen.

[0030] The present invention uses a genetically engineered cell line to stably and efficiently express recombinant collagen, and the selection of this cell line significantly improves the yield and quality of collagen. The method of the present invention can significantly improve the quality and consistency of the product by using a genetically engineered cell line to stably and efficiently express recombinant collagen, and adopting strict purification steps and quality control measures, which is crucial for applications in the fields of medical care, beauty and biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of the method for efficient purification of recombinant collagen and removal of endotoxins according to the present invention;

[0032] Figure 2 Flow chart for affinity chromatography purification;

[0033] Figure 3 Flow chart for gradient elution;

[0034] Figure 4 Flow chart for ultrafiltration concentration;

[0035] Figure 5 This is the electropherogram after gradient elution. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0037] Embodiment 1:

[0038] according to Figure 1 As shown in the flowchart, this embodiment takes recombinant type III collagen as an example to illustrate the steps of efficiently purifying recombinant collagen and endotoxin. The recombinant type III collagen can be any one of the prior art. The specific steps are as follows:

[0039] (1) Filtering:

[0040] The cell culture medium I expressing recombinant collagen was initially filtered to remove cell debris and large particles using a microporous filter membrane with a pore size range of 0.2 to 1 μm to obtain the initially filtered cell culture medium II. The pore size range of 0.2 to 1 μm was selected to ensure effective removal of large particle impurities while allowing recombinant collagen molecules to pass through, avoiding the loss of target protein in the initial filtration stage.

[0041] (2) Affinity chromatography treatment:

[0042] according to Figure 2 The affinity chromatography purification flow chart further purifies the recombinant collagen, and the specific steps are as follows:

[0043] S21. Selection of specific ligand and ligand immobilization:

[0044] A collagen-specific antibody fragment of the target recombinant collagen is selected, wherein the collagen-specific antibody fragment is a Strep tag added to the carboxyl end of the recombinant collagen, wherein the Strep tag is a short peptide composed of 8 amino acids (tryptophan-serine-histidine-threonine-glutamine-phenylalanine-glutamic acid-lysine), which exhibits a high specific affinity to a specially engineered streptavidin (Strep-tactin), and a recombinant type III collagen sample containing the Strep tag is applied to a chromatography column with immobilized Strep-tactin to form an affinity chromatography;

[0045] S22. Pretreatment of the obtained cell culture fluid II:

[0046] The pH value of the cell culture medium II is adjusted to 7.2 with a 2% sodium hydroxide solution, and the ionic strength is 280 mOsm, so that it can stably bind to the collagen-specific fragment during the affinity chromatography process to obtain the pretreated cell culture medium II.

[0047] S23. Loading and Binding:

[0048] The pretreated cell culture fluid II is slowly added to the affinity chromatography column to allow the recombinant collagen to fully contact and bind with the collagen-specific antibody fragment. The collagen-specific antibody fragment is used to bind to the recombinant collagen to achieve preliminary purification and obtain the intermediate product A.

[0049] S24. Washing and balancing

[0050] First, wash the column with an appropriate amount of phosphate buffer at a pH of 7.4 to remove unbound impurities and interferences, and then balance the column with the same buffer used for washing to ensure that the column is in a stable state and prepare for the subsequent elution step.

[0051] (3) Gradient elution:

[0052] according to Figure 3 The gradient elution flow chart further purifies the recombinant collagen, and the specific steps are as follows:

[0053] S31. Prepare a series of eluents with gradually changing pH values, starting from pH 7.0 and gradually decreasing to pH 5.0, with each pH value interval being 0.5 or 1 unit. On the basis of pH gradient elution, the NaCl concentration in the eluent of each pH value can be gradually increased from 50mmol / L to 500mmol / L, with each concentration interval being 50 or 100mmol / L.

[0054] S32. Connect the chromatographic column containing the collagen-specific antibody fragment of recombinant collagen and the recombinant collagen to the elution system, and use the pre-prepared pH gradient eluent for elution, using a sufficient volume of eluent at each pH value to ensure sufficient elution. After elution at each pH value, switch to the next ionic strength gradient eluent, repeat the above process, collect the effluent of each elution peak, mark and save them separately.

[0055] S33.SDS-PAGE electrophoresis combined with density scanning analysis:

[0056] The collected eluate is concentrated or dialyzed to remove excess salts and buffers, and then the protein concentration of each sample is determined using a BCA protein quantification kit to ensure consistent loading amounts. The samples are then mixed with SDS-PAGE loading buffer and heated for denaturation before loading.

[0057] Use pre-made SDS-PAGE gel and perform electrophoresis according to standard electrophoresis conditions. After the electrophoresis, use Coomassie Brilliant Blue to stain the gel, and then use destaining solution to destain it. Use gel imaging system to scan the stained gel, obtain the image, use image analysis software to perform density scanning and analysis on the gel image, and calculate the purity of the recombinant collagen.

[0058] Through the electrophoresis image and density scanning analysis results, it can be observed that the main bands of the recombinant collagen are clear, with no or very few impurities, indicating that the purification process is effective and the purity of the recombinant collagen is high ( Figure 5 ).

[0059] S34. Limulus amebocyte lysate (LAL) test:

[0060] The collected eluate and final product were diluted to ensure that the endotoxin concentration was within the detection range of the horseshoe crab reagent, and then the samples were mixed with the horseshoe crab reagent and incubated for a certain period of time according to the instructions of the horseshoe crab reagent kit, the reaction results were observed and recorded, and the endotoxin content in the samples was calculated according to the standard curve. The test results showed that the endotoxin content in the eluate and the final product was lower than the set safety limit (such as 0.1EU / mL or lower), indicating that the product safety met the requirements.

[0061] (4) Anion exchange chromatography:

[0062] S41. Prepare anion exchange chromatography column:

[0063] Select a resin with anion exchange groups as the chromatography medium. These media can specifically bind to negatively charged molecules, such as endotoxins. Load the anion exchange chromatography medium into the chromatography column and balance it with phosphate buffered saline (PBS), acetate buffer or citrate buffer to ensure that the chromatography column is in a stable state and ready to receive the sample.

[0064] S42. Loading and binding:

[0065] The eluate of high-purity recombinant collagen collected in step (3) is subjected to necessary pretreatment; the pretreated sample is slowly added to the anion exchange chromatography column to allow the endotoxin to specifically bind to the anion exchange group on the chromatography medium. The pretreatment is: adding an appropriate amount of inorganic salts (such as sodium chloride, ammonium sulfate, etc.) to the eluate to gradually increase the ionic strength of the eluate; using a buffer (such as phosphate buffer, acetate buffer, etc.) to adjust the pH value of the eluate. The step of pretreating the eluate includes: adjusting the pH value to 5.5-6.0, and adjusting the ionic strength to a NaCl concentration of 200-300 mmol / L to ensure that the endotoxin can stably bind to the anion exchange medium during the chromatography process.

[0066] S43. Washing and removing impurities:

[0067] The column was washed with Tris-HCl buffer, pH 7.0, to remove unbound, weakly bound impurities and interferences.

[0068] S44. Elution and collection:

[0069] The endotoxin bound to the anion exchange medium is eluted from the chromatography column; the recombinant collagen solution after the endotoxin is removed is collected and prepared for subsequent purification steps and directly used in subsequent applications to obtain intermediate product B.

[0070] S45. Column regeneration and storage:

[0071] After elution is completed, the anion exchange column is regenerated using a high concentration salt solution to remove residual endotoxins and other impurities and restore the binding capacity of the column; the regenerated column is stored in an appropriate buffer to prevent drying and contamination and is prepared for next use.

[0072] (5) Ultrafiltration concentration and diafiltration:

[0073] according to Figure 4 The ultrafiltration concentration flow chart further purifies the recombinant collagen, and the specific steps are as follows:

[0074] S51.Ultrafiltration concentration:

[0075] Ultrafiltration concentration is performed using cellulose acetate or polyethersulfone membrane materials with a molecular weight cutoff slightly larger than that of recombinant collagen, so as to effectively remove small molecule impurities and solvents while retaining the integrity of the recombinant collagen; wherein, appropriate membrane materials and operating conditions are selected to ensure that the recombinant collagen is not filtered out, while small molecule impurities and solvents are effectively removed.

[0076] S52. Dialysis filtration:

[0077] Dialysis filtration uses phosphate buffer or Tris-HCl buffer, whose pH and ionic strength match the final application requirements to maintain the stability and activity of the recombinant collagen; among them, the appropriate buffer and dialysis conditions are selected to ensure that the recombinant collagen remains stable during the dialysis process, while removing unnecessary salts and other small molecules to obtain a high-purity, low-endotoxin recombinant collagen solution.

[0078] Embodiment 2:

[0079] It is basically the same as Example 1, with the following differences:

[0080] The cells used in step (1) are cell lines that have been genetically engineered to stably and efficiently express recombinant collagen. The purpose of using genetically engineered cell lines is to ensure that these cells can stably and efficiently express recombinant collagen. This modification can increase the production of collagen while maintaining its biological activity and structural consistency, which is crucial for subsequent purification steps and the quality of the final product.

[0081] Embodiment 3:

[0082] In this example, the type of specific ligand is adjusted on the basis of Example 1 to examine the effect of selecting different ligands on the removal of endotoxin and the efficiency of collagen pruning during affinity chromatography. The types of specific ligands include collagen-specific fragments, metal chelate ligands, and metal chelate ligands + collagen-specific fragments. The metal chelate ligand is ethylenediaminetetraacetic acid, which can provide 2 nitrogen atoms and four hydroxyl oxygen atoms to coordinate with metals, and can form stable chelates with metal ions (iron ions, copper ions) other than alkali metals. The results of the investigation are shown in Table 1.

[0083] Table 1. Endotoxin content and purification efficiency under different specific ligands

[0084]

[0085] Note: EU / mL means endotoxin units per milliliter.

[0086] Purification efficiency = [(endotoxin content before purification - endotoxin content after purification) / endotoxin content before purification] × 100%

[0087] Table 1 shows the endotoxin content and purification efficiency under different specific ligands. It can be seen from Table 1 that all experiments successfully reduced the endotoxin content to a very low level (<5.0EU / mL, even lower to <0.5EU / mL), which shows that the use of specific ligands combined with anion exchange chromatography technology can efficiently capture and remove endotoxins. Using metal chelating ligands and collagen-specific fragments for affinity chromatography treatment together achieved an endotoxin removal efficiency of more than 99.5%, which shows the excellent performance of this method in removing endotoxins.

[0088] In addition, the purification efficiency of all experiments exceeded 95%, and the purification efficiency of Experiment 1 and Experiment 4 reached more than 99%. This shows that the improved method can significantly improve the purification efficiency of recombinant collagen.

[0089] Embodiment 4:

[0090] In this example, based on Example 1, the endotoxin content and purification efficiency of affinity chromatography alone, affinity chromatography + anion exchange chromatography, centrifugation, and centrifugation + anion exchange chromatography were investigated by adjusting the purification steps. The investigation results are shown in Table 2.

[0091] Table 2. Purification efficiency and endotoxin removal under different purification methods

[0092]

[0093]

[0094] As can be seen from Table 2, only affinity chromatography purification was performed, and the endotoxin content was reduced to 20EU / mL, with a purification efficiency of 80%. After the affinity chromatography, an anion exchange chromatography step was added, and the endotoxin content was significantly reduced to <1.0EU / mL, with a synergistic efficiency of more than 99.5%. This shows that there is a significant synergistic effect between the purification and endotoxin removal steps, and the safety and purity of the product can be greatly improved by using them in combination.

[0095] The endotoxin content was reduced to 60EU / mL by centrifugation purification, and the purification efficiency was low, only 40%; after centrifugation purification, an anion exchange chromatography step was added, and the endotoxin content was further reduced to 5.0EU / mL, and the purification efficiency was increased to 95%. This shows that compared with the centrifugation method, the affinity purification of the present invention has higher selectivity, can eliminate the interference of other non-target proteins, and thus improve the purification efficiency.

[0096] In summary, the present invention combines affinity chromatography, anion exchange chromatography, ultrafiltration concentration and diafiltration. The affinity chromatography technology preliminarily purifies recombinant collagen and removes endotoxins. The anion exchange chromatography technology further purifies the recombinant collagen and removes endotoxins. Finally, small molecule impurities and residual salts are removed by ultrafiltration concentration and diafiltration, thereby achieving the purpose of efficiently purifying recombinant collagen and removing endotoxins. The method improves the purification efficiency, has flexibility and scalability, can improve the quality and consistency of the product, and has good practicality.

[0097] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A method for efficiently purifying recombinant collagen and removing endotoxin, characterized in that: The method comprises: (1) Preliminarily filtering the cell culture fluid I expressing the recombinant collagen to obtain a preliminarily filtered cell culture fluid II; (2) selecting a specific ligand of the target recombinant collagen and immobilizing the specific ligand on the surface of an affinity chromatography medium to form an affinity chromatography column; The obtained cell culture fluid II is pretreated, and then added into an affinity chromatography column for affinity chromatography treatment, and an intermediate product A is obtained after the treatment is completed; (3) gradient eluting the intermediate product A, collecting the eluate, and pre-treating the eluate to obtain a pre-treated eluate for later use; The pretreated eluate is added to an anion exchange chromatography column for anion exchange chromatography treatment, and an intermediate product B is obtained after the treatment; (4) The intermediate product B is subjected to ultrafiltration concentration and diafiltration in sequence to obtain a recombinant collagen solution that is completely free of residual salts and small molecular impurities and has high purity and low endotoxin.

2. The method according to claim 1, characterized in that In step (1), the cells in the cell culture medium I expressing recombinant collagen include: a cell line that has been genetically engineered and is capable of stably and efficiently expressing recombinant collagen.

3. The method according to claim 1, characterized in that In step (1), the preliminary filtration uses a microporous filter membrane with a pore size range of 0.2 to 1 μm.

4. The method according to claim 1, characterized in that In step (2), the specific ligand comprises a collagen-specific antibody fragment; The collagen-specific antibody fragment is a tag added to the carboxyl end of the recombinant collagen; the tag includes a Strep tag; The pre-processing steps include: The pH value of the cell culture medium II is controlled within the range of 7.2 to 7.4, and the ionic strength is maintained within the range of 280 to 320 mOsm.

5. The method according to claim 1, characterized in that In step (2), the affinity chromatography treatment step comprises: The pretreated cell culture fluid II is added to the affinity chromatography column to allow the recombinant collagen in the cell culture fluid II to fully contact and bind with the specific antibody fragment. After the binding is completed, the affinity chromatography column is washed and balanced.

6. The method according to claim 1, characterized in that In step (3), during the gradient elution, the initial pH value is 6.5-7.0, the initial ionic strength is 0-50 mmol / L NaCl concentration; the ending pH value is 5.5-6.0, and the ending ionic strength is 200-300 mmol / L NaCl concentration.

7. The method according to claim 1, characterized in that In step (3), the step of pre-treating the eluate includes: adjusting the pH value to 5.5-6.0, and adjusting the NaCl concentration and ionic strength to 200-300 mmol / L, so as to ensure that the endotoxin can be stably bound to the anion exchange medium during the chromatography process.

8. The method according to claim 1, characterized in that In step (3), the anion exchange chromatography step comprises: S1. adding the pretreated eluate to an anion chromatography column; S2. Washing and eluting the chromatography column, collecting the recombinant collagen solution after removing endotoxin, and obtaining the intermediate product B; S3. Use a high concentration salt solution to regenerate the anion exchange chromatography column to restore the binding capacity of the chromatography column.

9. The method according to claim 1, characterized in that: In step (4), the ultrafiltration concentration uses a membrane material with a molecular weight cutoff slightly larger than that of the recombinant collagen.

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