Method for detecting purity of collagen in a composite solution

Abstract

This invention relates to the field of collagen detection technology, specifically to a method for detecting the purity of collagen in a composite solution. The method for detecting the purity of collagen in a composite solution provided by this invention includes the following steps: S1, adding polyethylene glycol solution to a composite solution of recombinant type III human collagen and sodium hyaluronate, adjusting the pH to 6.5-7.0, allowing it to stand at 3-5°C, separating the solid and liquid phases, and collecting the precipitate; S2, washing the precipitate, adding phosphoric acid solution and diluent to obtain a test solution; S3, detecting the test solution using liquid chromatography. This detection method can eliminate the interference of sodium hyaluronate on the detection and accurately determine the purity of recombinant type III human collagen in the composite solution of recombinant type III human collagen and sodium hyaluronate.

Description

Technical Field

[0001] This invention relates to the field of collagen detection technology, and more specifically, to a method for detecting the purity of collagen in a composite solution. Background Technology

[0002] Recombinant type III human collagen has the characteristics of high biocompatibility, low immunogenicity, and tissue repair promotion; sodium hyaluronate (HA) has moisturizing, viscoelastic support, and sustained-release effects. The combination of the two is widely used in medical aesthetic filling, skin repair, and wound care products.

[0003] In the recombinant type III human collagen and sodium hyaluronate complex system, collagen purity is a core quality control indicator. Its aggregates, degradation fragments, and process impurities can affect safety, stability, and efficacy, and must be accurately detected.

[0004] Currently, conventional methods for detecting collagen purity include SEC-HPLC, SDS-PAGE, and capillary electrophoresis. However, for the recombinant type III human collagen and sodium hyaluronate complex system, these methods have the following problems: Sodium hyaluronate co-elution interference: When the molecular weights of the two components are close, the retention times of the large sodium hyaluronate molecules, collagen main peak, and impurity peak are close, causing peak overlap and inability to integrate, resulting in a significantly higher collagen purity result.

[0005] Baseline elevation and background absorption: Sodium hyaluronate has strong absorption at a wavelength of 215nm, which directly raises the baseline and reduces the ability to detect impurities.

[0006] High viscosity damages the chromatographic column: Sodium hyaluronate has high viscosity, and direct injection can easily lead to increased column pressure, peak broadening, and tailing, thus shortening the column life.

[0007] Non-selective pretreatment: Conventional dilution and filtration cannot remove sodium hyaluronate, making it impossible to perform specific measurements of collagen.

[0008] Poor recovery rate and accuracy: Collagen is easily adsorbed or entangled with sodium hyaluronate, resulting in low recovery rates of aggregates and fragments, and unreliable data.

[0009] Existing technologies cannot accurately, specifically, and stably determine the purity of recombinant type III human collagen in the presence of sodium hyaluronate, and therefore cannot meet quality control requirements.

[0010] In view of this, the present invention is hereby proposed. Summary of the Invention

[0011] The purpose of this invention is to provide a method for detecting the purity of collagen in a composite solution, which can eliminate the interference of sodium hyaluronate on the detection and accurately measure the purity of recombinant type III human collagen in a composite solution of recombinant type III human collagen and sodium hyaluronate.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a method for detecting the purity of collagen in a composite solution, comprising the following steps: S1. Add polyethylene glycol solution to the composite solution of recombinant type III human collagen and sodium hyaluronate, adjust the pH to 6.5~7.0, let it stand at 3~5℃, separate the solid and liquid, and collect the precipitate; S2. After washing the precipitate, add phosphoric acid solution and diluent to obtain the test solution; S3. The test solution is detected by liquid chromatography.

[0013] Furthermore, in step S1, the molecular weight of the polyethylene glycol is ≥4000.

[0014] Preferably, in step S1, the molecular weight of the polyethylene glycol is 4000~10000.

[0015] Furthermore, in step S1, the concentration of the polyethylene glycol solution is ≥50wt%.

[0016] Further, in step S1, the volume ratio of the recombinant type III human collagen and sodium hyaluronate composite solution to the polyethylene glycol solution is (1.5~3):1.

[0017] Furthermore, step S1 includes at least one of the following features (1) to (3); (1) The settling time is 0.5~2h; (2) The temperature for solid-liquid separation is 3~5℃; (3) The solid-liquid separation includes centrifugation.

[0018] Furthermore, in step S2, the washing includes: washing with water 3 to 5 times.

[0019] Further, in step S2, the ratio of the precipitate, the phosphoric acid solution, and the diluent is 1 mg: 0.8~1.2 mL: 0.8~1.2 mL.

[0020] Further, in step S2, the concentration of the phosphoric acid solution is 0.04~0.06 mol / L.

[0021] Further, in steps S2 and S3, the diluent and the mobile phase of the liquid chromatography comprise isopropanol and phosphate buffer in a volume ratio of 1:(8.5~9.5).

[0022] Preferably, the phosphate buffer solution comprises sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride; the pH of the phosphate buffer solution is 6.8 to 7.0.

[0023] Further, in step S3, the conditions for liquid chromatography include: Chromatographic columns include size exclusion columns; The column temperature is 25~35℃; The elution method is isocratic elution; The flow rate is 0.4~0.8 mL / min; The detection wavelength is 213~215nm; The injection volume is 15~25μL; The data collection time is 28-32 minutes.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a pretreatment strategy of selectively precipitating recombinant type III human collagen with polyethylene glycol under specific pH and low temperature conditions to remove interference from sodium hyaluronate. This achieves accurate and stable detection of the purity of recombinant type III human collagen in a composite solution of recombinant type III human collagen and sodium hyaluronate, and has the advantages of high specificity, high accuracy, simple operation, and long column life. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a liquid chromatogram of the test solution in the detection method of Example 1 of the present invention.

[0027] Figure 2 This is a liquid chromatogram of the test solution in the detection methods of Examples 1, 2 and 3 of the present invention.

[0028] Figure 3 This is a liquid chromatogram of the test solution in the detection method of Comparative Example 1 of the present invention.

[0029] Figure 4 This is a liquid chromatogram of the test solution in the detection method of Comparative Example 2 of the present invention.

[0030] Figure 5 This is a liquid chromatogram of the test solution in the detection method of Comparative Example 3 of the present invention.

[0031] Figure 6 This is a liquid chromatogram of the recombinant type III human collagen solution of the present invention. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] In some embodiments of the present invention, a method for detecting the purity of collagen in a composite solution is provided, comprising the following steps: S1. Add polyethylene glycol (PEG) solution to the recombinant type III human collagen and sodium hyaluronate (HA) composite solution, adjust the pH to 6.5~7.0, let stand at 3~5℃, separate the solid and liquid, and collect the precipitate; S2. After washing the precipitate, add phosphoric acid solution and diluent to obtain the test solution; S3. The test solution is detected by liquid chromatography.

[0034] In recombinant type III human collagen and sodium hyaluronate composite solutions, the purity of recombinant type III human collagen is a core quality control indicator. Aggregates primarily affect product safety, rheological stability, and long-lasting shaping and repair efficacy. In terms of safety, they can easily induce skin immune inflammation, redness, swelling, and induration, posing risks of microvascular blockage and abnormal local tissue blood supply. Regarding stability, they can disrupt the homogeneous composite system of collagen and sodium hyaluronate, causing solution turbidity, stratification, and precipitation, accelerating product shelf-life expiration. In terms of efficacy, they block collagen's active binding sites, significantly weakening skin regeneration, repair, and barrier maintenance capabilities, resulting in poor shaping and support effects, short-lasting effects, and uneven treatment outcomes. Degradation fragments primarily affect the user experience, storage stability, and long-lasting skincare efficacy of the product. In terms of safety, small molecule peptides can easily cause injection stinging, burning, and persistent edema, and sensitive individuals are prone to allergic reactions. Long-term use can increase the body's metabolic burden. In terms of stability, they can cause pH shifts and solution discoloration, providing nutrients for microbial growth and accelerating the dual degradation of hyaluronic acid and collagen. In terms of efficacy, they completely lose the regenerative activity of the type III collagen triple helix structure, retaining only short-term basic moisturizing ability, and completely losing their core functions of anti-aging, skin repair, and long-lasting hydration. Processing impurities are the highest-risk impurities, comprehensively damaging product safety, stability, and efficacy, with the most severe and fatal negative impacts. In terms of safety, endotoxins and foreign bacterial proteins can easily trigger severe inflammation, acute allergies, and even systemic adverse reactions. Residual reagents, proteases, and buffer salts can produce cytotoxicity, causing irreversible damage such as skin pigmentation and permanent hardening. In terms of stability, they can continuously catalyze the degradation of raw materials and disrupt colloidal balance, directly leading to a significant reduction in product shelf life and excessive microbial levels. In terms of efficacy, they can damage active skin cells and inhibit collagen regeneration, not only failing to provide any skin care or repair effects but also damaging the original skin condition.

[0035] In summary, collagen aggregates, degradation fragments, and process impurities exhibit a mutually reinforcing but also negatively synergistic effect: process impurities accelerate collagen aggregation and peptide chain degradation, increasing the content of aggregates and degradation fragments; aggregates amplify inflammatory risks and exacerbate the sensitization caused by impurities; degradation fragments disrupt system stability, continuously amplifying the harmful effects of various impurities. Therefore, accurate detection of the purity of recombinant type III human collagen is necessary.

[0036] The detection method of this invention establishes a pretreatment method that selectively retains recombinant type III human collagen and removes sodium hyaluronate, eliminating chromatographic interference of sodium hyaluronate on the purity detection of recombinant type III human collagen, achieving accurate quantification of the main peak, aggregates, and degradation fragments of recombinant type III human collagen, and accurately measuring the purity of recombinant type III human collagen in the composite solution of recombinant type III human collagen and sodium hyaluronate. This detection method can be used for routine quality control.

[0037] Conventional pretreatment methods, such as enzymatic hydrolysis, precipitation, ultrafiltration centrifugation, and dialysis, are ineffective in removing sodium hyaluronate from the composite solution of recombinant type III human collagen and sodium hyaluronate. Enzymatic hydrolysis, while eliminating sodium hyaluronate, damages the structure of recombinant type III human collagen, leading to distorted purity test results. Precipitation, due to its high viscosity and interaction with recombinant type III human collagen, cannot completely remove sodium hyaluronate, leaving residual sodium hyaluronate to significantly interfere with detection. Ultrafiltration centrifugation or dialysis is difficult to achieve effective separation because sodium hyaluronate and recombinant type III human collagen have similar molecular weights and high viscosity. This invention employs a pretreatment method using polyethylene glycol selective precipitation of recombinant type III human collagen. This method can completely remove the interference of sodium hyaluronate without damaging the native conformation of recombinant type III human collagen, offering advantages such as high selectivity, high recovery rate, good chromatographic behavior, and accurate and reliable detection results.

[0038] Polyethylene glycol (PEG) is a nonionic, water-soluble polymer that does not chemically react with proteins or alter their primary structure. It causes proteins to dehydrate, aggregate, and precipitate through physical exclusion. The precipitation principle is as follows: Water molecule competition (hydration competition): PEG molecules have numerous hydroxyl groups and are strongly hydrophilic, binding large amounts of free water in the system and competing for the hydration membrane on the protein surface. After losing the hydration membrane, the intermolecular distance shortens, exposing hydrophobic regions and leading to hydrophobic aggregation. Volume repulsion effect (steric hindrance): PEG macromolecules form random coils in solution, occupying a large amount of space, squeezing protein molecules together, reducing their degrees of freedom of movement, and promoting intermolecular collisions and aggregation. No protein structure damage: It is a mild precipitation process that does not change the protein conformation, degrade, or modify it. The precipitated protein can be redissolved, maintaining its original structure and chromatographic behavior.

[0039] This invention uses polyethylene glycol to precipitate only recombinant type III human collagen, without precipitating sodium hyaluronate. This is determined by the differences in the structure, molecular weight, surface properties, and charge of the two molecules. Recombinant type III human collagen has a triple helix structure, with rigid rod-shaped molecules, many hydrophobic regions on its surface, and is prone to intermolecular entanglement, making it highly susceptible to aggregation and precipitation induced by polyethylene glycol. Sodium hyaluronate, on the other hand, is a linear polysaccharide, highly hydrophilic, carrying a large number of negative charges, with highly extended molecules, an extremely thick hydration layer, and strong steric repulsion. Under the conditions of this invention, it does not aggregate and remains in the supernatant.

[0040] In some embodiments of the present invention, in step S1, the molecular weight of polyethylene glycol is ≥4000; preferably, the molecular weight of polyethylene glycol is 4000~10000; typically, but not limitingly, for example, the molecular weight of polyethylene glycol can be 4000, 5000, 6000, 7000, 8000, 9000, 10000 and any value between any two of them; more preferably 10000.

[0041] Polyethylene glycol (PEG) with a molecular weight ≥ 4000 can selectively precipitate recombinant type III human collagen; if the molecular weight of PEG is too low, the precipitation of recombinant type III human collagen will be incomplete; PEG with a molecular weight of 4000~10000 can effectively precipitate recombinant type III human collagen, allowing sodium hyaluronate to be removed while remaining in the supernatant; among them, PEG with a molecular weight of 10000 can achieve a better precipitation effect.

[0042] Pre-precipitation pretreatment with polyethylene glycol of molecular weight ≥4000 can selectively precipitate recombinant type III human collagen, while sodium hyaluronate is retained in the supernatant and removed; thus eliminating interference from sodium hyaluronate co-elution and background absorption, exhibiting extremely high specificity.

[0043] In some embodiments of the present invention, in step S1, the concentration of the polyethylene glycol solution is ≥50 wt%; preferably, the concentration of the polyethylene glycol solution is 50 wt% to 55 wt%; typically, but not limitingly, for example, the concentration of the polyethylene glycol solution can be 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, and any value between any two thereof; more preferably, it is 50 wt%. The polyethylene glycol solution is an aqueous solution of polyethylene glycol. If the concentration of the polyethylene glycol solution is too low, the recombinant type III human collagen will not precipitate completely.

[0044] In some embodiments of the present invention, in step S1, the volume ratio of the recombinant type III human collagen and sodium hyaluronate composite solution to the polyethylene glycol solution is (1.5~3):1; typically, but not limitingly, for example, the volume ratio of the recombinant type III human collagen and sodium hyaluronate composite solution to the polyethylene glycol solution can be 1.5:1, 2:1, 2.5:1, 3:1 and any value between any two of them; preferably 2:1.

[0045] In some embodiments of the present invention, in step S1, NaOH is used to adjust the pH to 6.5-7.0; typically, but not limitingly, for example, the pH can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and any value between any two of them.

[0046] Adjusting the pH of the solution system to 6.5-7.0 can enhance selectivity; this pH value is close to the isoelectric point of recombinant type III human collagen, which has less surface charge and is more prone to dehydration and aggregation; while sodium hyaluronate maintains a strong negative charge at this pH value, with strong charge repulsion, thus maintaining its solubility.

[0047] Maintaining a pH of 6.5–7.0 helps preserve the native conformation of recombinant type III human collagen, preventing degradation and ensuring complete precipitation. Under these pH conditions, no false impurity peaks appear in the liquid chromatogram, and the peak shapes are symmetrical.

[0048] In some embodiments of the present invention, in step S1, the settling time is 0.5 to 2 hours; settling includes: settling at 3 to 5°C for 0.5 to 2 hours; typically, but not limitingly, for example, the settling temperature can be 3°C, 4°C, 5°C and any value between any two of them, preferably 4°C; the settling temperature and time can be 0.5 hours, 1 hour, 1.5 hours, 2 hours and any value between any two of them, preferably 1 hour.

[0049] In some embodiments of the present invention, in step S1, the temperature for solid-liquid separation is 3~5°C; typically, but not limitingly, for example, the temperature for solid-liquid separation can be 3°C, 4°C, 5°C and any value between any two thereof, preferably 4°C.

[0050] In some embodiments of the present invention, in step S1, solid-liquid separation includes centrifugation; preferably, the centrifugation time is 10-20 min; and the relative centrifugal force is 10000-15000 g.

[0051] Low temperatures of 3-5°C can promote selective precipitation; low temperatures reduce molecular thermal motion, promoting dense precipitation of recombinant type III human collagen, while reducing random entanglement of sodium hyaluronate molecules to avoid co-precipitation. At the same time, low temperatures protect recombinant type III human collagen from thermal damage.

[0052] This invention employs a combination of low-temperature static incubation at 3-4°C and centrifugation, which effectively inhibits the degradation of recombinant type III human collagen, reduces conformational changes, and simultaneously makes the precipitate structure more compact and easier to separate. Under these conditions, there is no significant loss of aggregates and degradation fragments of recombinant type III human collagen, accurately reflecting the distribution of each component in the sample, resulting in accurate and reliable detection results.

[0053] In some embodiments of the present invention, step S2 includes washing with water 3 to 5 times. After each water wash, the supernatant is discarded by centrifugation.

[0054] This invention involves multiple water washes of the precipitate, which can thoroughly remove residual sodium hyaluronate and polyethylene glycol from the precipitate, preventing these impurities from entering the chromatographic system. This ensures stable column pressure and prevents column contamination during detection, resulting in a flat baseline without drift. It significantly extends the lifespan of the chromatographic column while guaranteeing the stability and reproducibility of the detection results.

[0055] In some embodiments of the present invention, in step S2, the volume ratio of the precipitate, phosphoric acid solution, and diluent is 1 mg: 0.8~1.2 mL: 0.8~1.2 mL; typically, but not limitingly, for example, the volume ratio of the precipitate, phosphoric acid solution, and diluent is 1 mg: 0.8 mL: 0.8 mL, 1 mg: 1.2 mL: 1.2 mL, 1 mg: 0.8 mL: 1.2 mL, 1 mg: 1.2 mL: 0.8 mL, and any value between any two.

[0056] In some embodiments of the present invention, in step S2, the concentration of the phosphoric acid solution is 0.04~0.06 mol / L; typically, but not limitingly, for example, the concentration of the phosphoric acid solution can be 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, and any value between any two thereof; preferably 0.05 mol / L. The phosphoric acid solution is an aqueous phosphoric acid solution.

[0057] In some embodiments of the present invention, in step S2, the diluent comprises isopropanol and phosphate buffer in a volume ratio of 1:(8.5~9.5); typically, but not limitingly, for example, the volume ratio of isopropanol and phosphate buffer can be 8.5:1, 9.0:1, 9.5:1 and any value between any two thereof; preferably 1:9.

[0058] In some embodiments of the present invention, in step S2, the phosphate buffer includes sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride; preferably, the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride is (4~5):25:(8~9); more preferably, it is 4.6:25:8.8; the phosphate buffer also includes water.

[0059] In some embodiments of the present invention, in step S2, the pH of the phosphate buffer solution is 6.8 to 7.0; preferably 7.0.

[0060] The precipitate is reconstituted with phosphoric acid solution and then diluted. The diluted solution is the same as the mobile phase of the liquid chromatography. This improves solubility, matches the mobile phase system, makes the peaks sharper and eliminates tailing, and improves resolution.

[0061] In some embodiments of the present invention, in step S2, after washing the precipitate, phosphoric acid solution is added, followed by the addition of diluent and mixing. The mixture is then centrifuged, and the supernatant is collected to obtain the test solution.

[0062] In some embodiments of the present invention, the conditions for liquid chromatography in step S3 include: The chromatographic column includes a size exclusion column; preferably, the column is a BioCore SEC-300 5μm, 7.8×300mm; The column temperature is 25~35°C; typically, but not limitingly, the column temperature can be 25°C, 30°C, 35°C, or any value between any two of these; preferably 30°C; Mobile phase: isopropanol and phosphate buffer at a volume ratio of 1:(8.5~9.5); phosphate buffer consists of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water; the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride is (4~5):25:(8~9); the pH of phosphate buffer is 6.8~7.0; The elution method is isocratic elution; The flow rate is 0.4 to 0.8 mL / min; typically, but not limitingly, the flow rate can be 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, and any value between any two thereof; preferably 0.6 mL / min; The detection wavelength is 213~215nm; preferably 215nm. The injection volume is 15~25 μL; typically, but not limitingly, the injection volume can be 15 μL, 20 μL, 25 μL and any value between any two of them; preferably 20 μL; The collection time is 28-32 minutes; preferably 30 minutes.

[0063] In some embodiments of the present invention, after detecting the test solution by liquid chromatography, a liquid chromatogram is obtained, and the purity and content of impurity proteins of recombinant type III human collagen are calculated by peak area normalization method.

[0064] Example 1 The method for detecting collagen purity in a composite solution of recombinant type III human collagen and sodium hyaluronate provided in this embodiment includes the following steps: A method for preparing a composite solution of recombinant type III human collagen and sodium hyaluronate includes the following steps: Recombinant Type III human collagen: molecular weight 300,000-400,000, water content 12.3%, Beijing Jingyu Yimei Biotechnology Co., Ltd. Sodium hyaluronate: Bloomage Biotechnology Co., Ltd. Sodium hydroxide solution with a concentration of 0.15 mol / L: Weigh 0.300 g of sodium hydroxide, dissolve it in water for injection, make up to 50 mL, and filter it into a sterile container through a 0.22 μm filter. Buffer solution with pH 2.9~3.1: Weigh 0.288g of phosphoric acid, add 0.9g of sodium chloride, dilute with water to a final volume of 100mL, then pour into a beaker, add 15.2mL of 0.15mol / L sodium hydroxide solution to adjust the pH to 2.9~3.1; Recombinant type III human collagen solution: Take 70 mL of the above buffer solution with a pH of 2.9~3.1, add 344.81 mg of weighed recombinant type III human collagen, seal and stir magnetically to swell and dissolve (2~8℃) until completely dissolved (6~12 h, no obvious small gel lumps observed in the glass bottle). Sodium hyaluronate solution: Weigh 400.0 mg of the above sodium hyaluronate and dissolve it in 100 mL of 0.9 wt% sodium chloride solution by stirring and swelling. Then dispense it into 50 mL injection bottles, 50 ± 5 g per bottle, and sterilize by moist heat (121℃, 30 min). Composite solution: In a clean bench, the recombinant type III human collagen solution was filtered through 0.45μm and 0.22μm filter membranes into a sterile container. Then, sterilized sodium hyaluronate solution was added, sealed, and shaken by hand for 5 minutes. Subsequently, the above-mentioned 0.15mol / L sodium hydroxide solution (the volumes of the recombinant type III human collagen solution, sodium hyaluronate solution, and sodium hydroxide solution were 50mL, 54mL, and 4mL respectively, with a volume ratio of 12.5:13.5:1) was added. The mixture was then shaken by hand for 5-10 minutes, dispensed, and capped to obtain the recombinant type III human collagen and sodium hyaluronate composite solution. S1. Take 1g of PEG (molecular weight 10000), add water to make up to 2g, dissolve and shake well to obtain a PEG solution with a concentration of 50wt%; take 0.5mL of the above recombinant type III human collagen and sodium hyaluronate composite solution into a centrifuge tube, add 0.25mL of PEG solution, and mix gently; then adjust the pH to 6.8 with NaOH; let stand at 4℃ for 1h; centrifuge at 12000g and 4℃ for 15min, discard the supernatant, and obtain 0.6mg of precipitate; S2. Wash the precipitate with 0.5 mL of water, centrifuge at 12000 g and 4℃ for 15 min, and discard the supernatant; repeat the above washing 3 times. Then add 0.5 mL of 0.05 mol / L phosphoric acid solution to dissolve the precipitate; then add 0.5 mL of diluent and mix well. Centrifuge at 12000 g and 4℃ for 15 min, and take the supernatant as the test solution. The preparation method of the diluent includes: weighing 4.6g of sodium dihydrogen phosphate dihydrate, 25g of disodium hydrogen phosphate dodecahydrate and 8.8g of sodium chloride, adding 1000mL of water to dissolve and shake well to obtain a phosphate buffer with a pH of 7.0; after filtering the phosphate buffer through a 0.45μm filter membrane, measure 900mL of the phosphate buffer, add 100mL of isopropanol, mix well, and degas by sonication; S3. The test solution was detected by liquid chromatography to obtain the liquid chromatogram. The purity and content of impurities of recombinant type III human collagen were calculated by peak area normalization method. The conditions for liquid chromatography are as follows: Column: BioCore SEC-300 5μm, 7.8×300mm; Column temperature: 30℃; Mobile phase: Weigh 4.6 g of sodium dihydrogen phosphate dihydrate, 25 g of disodium hydrogen phosphate dodecahydrate and 8.8 g of sodium chloride, add 1000 mL of water to dissolve and shake well to obtain a phosphate buffer solution with a pH of 7.0; after filtering the phosphate buffer solution through a 0.45 μm filter membrane, measure 900 mL of the phosphate buffer solution, add 100 mL of isopropanol, mix well, and degas by sonication to obtain the mobile phase; The elution method is isocratic elution; Flow rate: 0.6 mL / min; Detection wavelength: 215nm; Injection volume: 20 μL; Data collection time: 30 minutes.

[0065] Example 2 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this embodiment is the same as in Example 1, except that the molecular weight of PEG is 8000 in step S1.

[0066] Example 3 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this embodiment is the same as in Example 1, except that the molecular weight of PEG is 4000 in step S1.

[0067] Comparative Example 1 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this comparative example is the same as in Example 1, except that step S1 is omitted. Take 0.5 mL of the recombinant type III human collagen and sodium hyaluronate composite solution and place it in a centrifuge tube. Add 0.5 mL of 0.05 mol / L phosphate solution and mix gently. Then add 1 mL of diluent and mix well to obtain the test solution.

[0068] Comparative Example 2 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this comparative example is the same as in Example 1, except that in step S1, the step of adjusting the pH to 6.8 with NaOH is omitted, and in step S2, the pH of the test solution is 2. Take 0.5 mL of the recombinant type III human collagen and sodium hyaluronate composite solution and place it in a centrifuge tube. Add 0.25 mL of PEG solution and mix gently. After standing at 4°C for 1 h, centrifuge at 12000 g and 4°C for 15 min, discard the supernatant, and obtain 0.3 mg of precipitate. Add 0.5 mL of water to the precipitate and centrifuge at 12000 g and 4°C for 15 min, discard the supernatant. Repeat the above washing 3 times. Then add 0.5 mL of 0.05 mol / L phosphoric acid solution to dissolve the precipitate. Add 0.5 mL of diluent and mix well. Centrifuge at 12000 g and 4°C for 15 min, and take the supernatant as the test solution.

[0069] Comparative Example 3 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this comparative example is the same as in Example 1, except that in step S1, after standing at 25°C for 1 hour, the sample is centrifuged at 12000g and 25°C for 15 minutes; in step S2, the precipitate is washed with 0.5mL of water, centrifuged at 12000g and 25°C for 15 minutes, and the supernatant is discarded; the above washing is repeated 3 times; then 0.5mL of 0.05mol / L phosphoric acid solution is added to dissolve the precipitate; then 0.5mL of diluent is added and mixed well, centrifuged at 12000g and 25°C for 15 minutes, and the supernatant is taken as the test solution.

[0070] Comparative Example 4 The method for detecting the purity of collagen in the recombinant type III human collagen and sodium hyaluronate composite solution provided in this comparative example is the same as that in Example 1, the only difference is that in step S1, after centrifugation at 8000g and 4℃ for 15min, the supernatant was discarded and no precipitate was obtained; therefore, the test solution could not be prepared.

[0071] Test case The recombinant type III human collagen solution was detected by liquid chromatography. Recombinant type III human collagen solid was dissolved and diluted with 0.05 mol / L phosphoric acid solution to prepare a solution containing 1 mg of recombinant type III human collagen per milliliter. 0.5 mL of this solution was taken, and 0.5 mL of diluent (same as in Example 1) was added and mixed well. The mixture was then centrifuged at 12000 g and 25 °C for 15 min. The supernatant was used as the test solution. The liquid chromatography conditions were the same as in Example 1. The liquid chromatogram of the recombinant type III human collagen solution is shown below. Figure 6As shown, the liquid chromatogram of the recombinant type III human collagen solution (without sodium hyaluronate) shows sharp peaks and analyzable impurities, proving that the purity of recombinant type III human collagen can be normally detected using the liquid chromatographic conditions of this invention, even with sodium hyaluronate-free solution.

[0072] The liquid chromatogram of the test solution in the detection method of Example 1 is as follows: Figure 1 As shown.

[0073] from Figure 1 As can be seen, the liquid chromatogram of the recombinant type III human collagen and sodium hyaluronate composite solution after sodium hyaluronate removal by PEG shows sharp peaks and analyzable impurities. The results show no decrease or increase in impurities compared to the recombinant type III human collagen solution, proving that the main peak, aggregates, and degradation fragments of recombinant type III human collagen are accurately quantified, and the method can be used for routine quality control.

[0074] The liquid chromatograms of the test solutions in the detection methods of Examples 1, 2, and 3 are shown below. Figure 2 As shown.

[0075] from Figure 2 It can be seen that all three molecular weights of PEG can meet the detection requirements.

[0076] The liquid chromatograms of the test solutions in the detection methods of Comparative Examples 1, 2, and 3 are shown below. Figure 3 , Figure 4 and Figure 5 As shown.

[0077] from Figure 3 As can be seen from the liquid chromatogram of recombinant type III human collagen and sodium hyaluronate complex solution without sodium hyaluronate removal, sodium hyaluronate interferes with the collagen peak shape and causes tailing, and the baseline is raised, making purity detection impossible. This proves that the purity of recombinant type III human collagen cannot be detected without removing sodium hyaluronate.

[0078] from Figure 4 and Figure 5 It can be seen that in Comparative Example 2, the pH was not adjusted to 6.5~7.0 without the use of sodium hydroxide, so the impurities could not be detected; in Comparative Example 3, the standing temperature and centrifugation temperature were room temperature, so the impurities were detected at a magnified scale.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the purity of collagen in a composite solution, characterized in that, Includes the following steps: S1. Add polyethylene glycol solution to the composite solution of recombinant type III human collagen and sodium hyaluronate, adjust the pH to 6.5~7.0, let it stand at 3~5℃, separate the solid and liquid, and collect the precipitate; S2. After washing the precipitate, add phosphoric acid solution and diluent to obtain the test solution; S3. The test solution is detected by liquid chromatography.

2. The method for detecting the purity of collagen in a composite solution according to claim 1, characterized in that, In step S1, the molecular weight of the polyethylene glycol is ≥4000; Preferably, in step S1, the molecular weight of the polyethylene glycol is 4000~10000.

3. The method for detecting the purity of collagen in a composite solution according to claim 2, characterized in that, In step S1, the concentration of the polyethylene glycol solution is ≥50wt%.

4. The method for detecting the purity of collagen in a composite solution according to claim 3, characterized in that, In step S1, the volume ratio of the recombinant type III human collagen and sodium hyaluronate composite solution to the polyethylene glycol solution is (1.5~3):

1.

5. The method for detecting the purity of collagen in a composite solution according to claim 4, characterized in that, Step S1 includes at least one of the following features (1) to (3); (1) The settling time is 0.5~2h; (2) The temperature for solid-liquid separation is 3~5℃; (3) The solid-liquid separation includes centrifugation.

6. The method for detecting the purity of collagen in a composite solution according to claim 5, characterized in that, In step S2, the washing includes washing with water 3 to 5 times.

7. The method for detecting the purity of collagen in a composite solution according to claim 6, characterized in that, In step S2, the ratio of the precipitate, the phosphoric acid solution, and the diluent is 1 mg: 0.8~1.2 mL: 0.8~1.2 mL.

8. The method for detecting the purity of collagen in a composite solution according to claim 6, characterized in that, In step S2, the concentration of the phosphoric acid solution is 0.04~0.06 mol / L.

9. The method for detecting the purity of collagen in a composite solution according to claim 6, characterized in that, In steps S2 and S3, the diluent and the mobile phase of the liquid chromatography consist of isopropanol and phosphate buffer in a volume ratio of 1:(8.5~9.5). Preferably, the phosphate buffer solution comprises sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride; the pH of the phosphate buffer solution is 6.8 to 7.

0.

10. The method for detecting the purity of collagen in a composite solution according to claim 6, characterized in that, In step S3, the conditions for liquid chromatography include: Chromatographic columns include size exclusion columns; The column temperature is 25~35℃; The elution method is isocratic elution; The flow rate is 0.4~0.8 mL / min; The detection wavelength is 213~215nm; The injection volume is 15~25μL; The data collection time is 28-32 minutes.

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