Liquid chromatography-mass spectrometry detection method for recombinant collagen in collagen product

Abstract

The invention discloses a liquid chromatography-mass spectrometry detection method for recombinant collagen in a collagen product. The detection method is based on liquid chromatography-mass spectrometry, is suitable for qualitative and quantitative detection of recombinant collagen in various collagen products, and is accurate and reliable in detection result. The detection method comprises a pretreatment step, an enzymolysis step, a liquid chromatography-mass spectrometry detection step and a detection result interpretation step. Wherein whether a peak consistent with the retention time of the characteristic polypeptide appears in a detection map of a test sample test solution or not is judged, and whether the test sample test solution contains the specific recombinant collagen or not is judged; and / or according to the proportional relation between the peak area of the peak of the characteristic polypeptide in the detection spectrum of the test sample test solution and the peak area of the peak of the characteristic polypeptide in the detection spectrum of the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample, determining the characteristic polypeptide according to the peak area of the peak of the characteristic polypeptide in the detection spectrum of the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample, and determining the content of the recombinant collagen in the test solution of the test sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological material detection and analysis, and specifically relates to a qualitative identification and quantitative detection method of recombinant collagen in biological materials. Background Art

[0002] Recombinant collagen is prepared by genetic manipulation and / or modification of genes encoding the desired human collagen protein using recombinant DNA technology, using plasmids or viral vectors to bring the target gene into appropriate host cells (bacteria, yeast or other eukaryotic cells, etc.), expressing and translating it into collagen or collagen-like polypeptides, and then extracting and purifying it. Due to its good biocompatibility, biological efficacy and safety, it is widely used in beauty and skin care, tissue engineering and other fields.

[0003] Currently, the recombinant collagen products on the market are in the form of solid, gel, liquid, patch, paste, etc. The recombinant collagen used in the products varies due to the differences in amino acid sequences or expression systems of different manufacturers (such as: different types or different types of combinations, single chain or trimer with triple helix structure, full length, fragment or fragment editing and splicing). Although the existing standards provide a method for determining protein content, it is not specific and cannot distinguish between recombinant collagen and other protein molecules. It cannot accurately determine the content of recombinant collagen in the product, resulting in uneven quality of products on the market, unclear content, and unknown efficacy. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a qualitative and quantitative detection method for recombinant collagen in collagen products, which is applicable to a wide range of sample types and has accurate and reliable detection results.

[0005] The inventors conducted in-depth research to solve the above problems and found that the recombinant collagen raw material used to manufacture the test sample or the characteristic polypeptide derived from the recombinant collagen can be used as a standard sample to qualitatively identify the recombinant collagen in the test sample or quantitatively detect it by the external standard method, thereby completing the present invention.

[0006] That is, the technical solution of the present invention includes:

[0007] 1. A qualitative or quantitative detection method for recombinant collagen in a collagen product, comprising:

[0008] Pretreatment step: if the collagen product contains a liquid or semi-solid component, take the liquid or semi-solid component, remove the solvent component to obtain a dry component, and then redissolve the dry component to obtain a sample re-solution solution; if the collagen product contains a solid component, redissolve the solid component to obtain a sample re-solution solution;

[0009] Enzymatic hydrolysis step: enzymatically hydrolyzing the sample solution with a protease to obtain a test sample solution;

[0010] Liquid chromatography-mass spectrometry detection step: the test sample test solution, and the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample as a standard are respectively loaded on a liquid chromatography-mass spectrometer, and detection and analysis are performed according to the set liquid chromatography-mass spectrometry conditions; and

[0011] The test result interpretation step is as follows: judging whether the test solution of the test sample contains the recombinant collagen protein according to whether a peak with the same retention time as the characteristic polypeptide appears in the test spectrum of the test solution of the test sample; and / or determining the content of the recombinant collagen protein in the test solution of the test sample according to the ratio of the peak area of ​​the characteristic polypeptide peak in the test spectrum of the test solution of the test sample to the peak area of ​​the characteristic polypeptide peak in the test spectrum of the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample.

[0012] In the present invention, the enzymatic hydrolysis should be sufficient, that is, each of the cleavage sites of the protease in the protein treated by the protease is cleaved. Those skilled in the art know how to set the experimental conditions to fully perform the enzymatic hydrolysis.

[0013] In some embodiments, when a synthetic characteristic polypeptide is used for quantitative analysis, the content of the characteristic polypeptide in the test sample (C) is obtained based on the peak area (Y) of the characteristic polypeptide in the test sample using the regression equation of the characteristic polypeptide standard curve. i ), and then calculate the content of recombinant collagen in the test sample according to the molecular weight of the recombinant collagen characteristic polypeptide, the molecular weight of the recombinant collagen, the sampling volume or sampling mass of the test sample, the dilution multiple of the sample after enzymatic hydrolysis, and the volume of the test sample after pre-treatment (see Formula 2 or Formula 3).

[0014] For semi-solid and solid samples

[0015] Calculate the recombinant collagen content in the test sample using the system processing software or according to formula (2):

[0016]

[0017] Where:

[0018] X i ——Content of recombinant collagen in the test sample, μg / g;

[0019] C i ——Content of characteristic peptide in the test sample, μg / mL;

[0020] M1 ——Molecular weight of recombinant collagen;

[0021] M 2 ——Molecular weight of characteristic polypeptide of recombinant collagen;

[0022] V——volume of the test sample after pretreatment, mL;

[0023] X——dilution multiple of sample after enzymatic hydrolysis;

[0024] n——the number of repeats of characteristic polypeptides in the theoretical sequence of recombinant collagen;

[0025] m——sampling mass of the test product, g.

[0026] Formula (2) is applicable to the calculation of the recombinant collagen content in semi-solid and solid samples, that is, it is expressed as the amount of recombinant collagen (μg) per gram of sample.

[0027] For liquid samples, the recombinant collagen content in the test sample was calculated according to formula (3):

[0028]

[0029] Where:

[0030] X i ——Content of recombinant collagen in the sample, μg / mL;

[0031] Ci——the content of characteristic polypeptide in the test sample, μg / mL;

[0032] M 1 ——Molecular weight of recombinant collagen;

[0033] M 2 ——Molecular weight of characteristic polypeptide of recombinant collagen;

[0034] V 1 ——Volume of the test sample after pretreatment, mL;

[0035] X——dilution multiple of sample after enzymatic hydrolysis;

[0036] n——the number of repeats of characteristic polypeptides in the theoretical sequence of recombinant collagen;

[0037] V 2 ——Sampling volume of the test sample, mL.

[0038] In other embodiments, when recombinant collagen raw materials are used for quantitative analysis, the regression equation of the recombinant collagen raw material standard curve is used to directly calculate the recombinant collagen content (Ci) in the test sample based on the characteristic polypeptide peak area (Y) in the test sample, and then the recombinant collagen content in the final test sample is calculated based on the sampling volume or sampling mass of the test sample, the dilution multiple of the sample after enzymatic hydrolysis, the volume of the test sample after pretreatment, and the number of characteristic polypeptides in the recombinant collagen (see Formula 4 or Formula 5).

[0039] For semi-solid and solid samples

[0040] Calculate the recombinant collagen content in the test sample according to formula (4):

[0041]

[0042] Where:

[0043] X i ——Content of recombinant collagen in the test sample, μg / g;

[0044] C i ——Recombinant collagen content in the test sample, μg / mL;

[0045] V——volume of the test sample after pretreatment, mL;

[0046] X——dilution multiple of sample after enzymatic hydrolysis;

[0047] n——the number of repeats of characteristic polypeptides in the theoretical sequence of recombinant collagen;

[0048] m——mass of the sample to be tested, g;

[0049] Formula (4) is applicable to the calculation of the content of recombinant collagen in semi-solid and solid samples, that is, it is expressed as the amount of recombinant collagen (μg) per gram of sample.

[0050] For liquid samples

[0051] The recombinant collagen content in the test sample was calculated according to formula (5):

[0052]

[0053] X i ——Content of recombinant collagen in the test sample, μg / mL;

[0054] C i ——Recombinant collagen content in the test sample, μg / mL;

[0055] V 1 ——Volume of the test sample after pretreatment, mL;

[0056] X——dilution multiple of sample after enzymatic hydrolysis;

[0057] n——the number of repeats of characteristic polypeptides in the theoretical sequence of recombinant collagen;

[0058] V——sample volume of the test product, mL;

[0059] 2. The detection method according to claim 1, wherein the solvent is removed by freeze drying or nitrogen blowing in the pretreatment step.

[0060] 3. The detection method according to claim 1, wherein for recombinant collagen having a triple helix structure or a polymer structure, a denaturation step is further included after the pretreatment step and before the enzymatic hydrolysis step:

[0061] The test sample reconstituted solution is heated to denature the recombinant collagen into a single chain.

[0062] 4. The detection method according to claim 1, wherein the preparation of the recombinant collagen raw material standard working solution as a standard product comprises the following steps:

[0063] (1) Weighing a certain amount of recombinant collagen raw material used to produce the collagen product, and preparing it into a solution of a certain concentration as a standard sample stock solution;

[0064] (2) For recombinant collagen having a triple helix structure or a polymer structure, heating the standard sample mother solution to denature the recombinant collagen into a single chain;

[0065] (3) enzymatically hydrolyzing the standard sample mother solution with the protease to obtain a recombinant collagen raw material enzymatic hydrolyzate; and

[0066] (4) diluting the recombinant collagen raw material enzymatic hydrolysate into a series of concentrations to obtain a recombinant collagen raw material standard working solution.

[0067] 5. The detection method according to claim 1, wherein the preparation of the characteristic polypeptide standard working solution as a standard comprises the following steps: weighing a certain amount of the characteristic polypeptide, preparing it into a series of concentrations, and obtaining the characteristic polypeptide standard working solution.

[0068] 6. The detection method according to claim 1, wherein the liquid chromatography-mass spectrometry conditions are as follows:

[0069] (1) Chromatographic conditions

[0070] Mobile phase A: 0.1 vol% formic acid in water;

[0071] Mobile phase B: acetonitrile solution containing 0.1 vol% formic acid, or 60 vol% acetonitrile aqueous solution containing 0.1 vol% formic acid;

[0072] Flow rate: 0.2mL / min~0.6mL / min;

[0073] Column temperature: 25℃~30℃;

[0074] Injection volume: 2μL~10μL;

[0075] Chromatographic column: octadecylsilane bonded silica gel column, filler particle size 3μm or 5μm, column length 100mm or 150mm, column diameter 2.1mm or 2.0mm; and

[0076] (2) Mass spectrometry conditions

[0077] Ion source: electrospray ion source;

[0078] Scanning mode: positive ion scanning;

[0079] Spray voltage: 3.5 kV;

[0080] Ion source evaporation temperature: 300°C.

[0081] 7. The detection method according to claim 1, wherein the recombinant collagen is recombinant human type III collagen, and its amino acid sequence is shown in SEQ ID No: 1.

[0082] SEQ ID No:1

[0083] QYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPP

[0084] GEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPA

[0085] GIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPR

[0086] GAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKG

[0087] EVGPAGSPSGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGP

[0088] AGIPAGAPGLMGARGPPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGER

[0089] GEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNG

[0090] IPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGS

[0091] DGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGER

[0092] GGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPG

[0093] TGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGGERGPPGLAGAPG

[0094] LRGGAGPPGPEGGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGD

[0095] KGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAG

[0096] PRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVA

[0097] GPPGGSGPAGPPGPQGVKGERGSPGGGPGAAGFPGARGLPGPPGSNGNPGP

[0098] PGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPP

[0099] GAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGE

[0100] RGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSP

[0101] GAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGP

[0102] RGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRG

[0103] PVGPPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPG

[0104] APGPCCGGVGAAAIAAGIGGEKAGGFAPYYG

[0105] The detection method according to claim 1 , wherein the protease used in the enzymatic hydrolysis step is trypsin.

[0106] 9. The detection method according to claim 1, wherein the amino acid sequence of the characteristic polypeptide is as shown in SEQ ID No: 2.

[0107] SEQ ID No:2

[0108] GEAGIPGVPGAK

[0109] In the protein qualitative and quantitative analysis method using characteristic polypeptides, there are the following difficulties: a. characteristic polypeptides that meet the requirements cannot be found; b. a combination of multiple characteristic polypeptides is required to obtain accurate and reliable test results. However, in this embodiment, only one characteristic polypeptide is used to obtain accurate and reliable test results for various forms of the recombinant type III collagen dressing.

[0110] 10. The detection method according to claim 1, wherein the collagen product is in a solid, liquid, cream or gel form, and can be, for example, freeze-dried fibers or dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 This is the total ion current diagram of the enzymatic hydrolysis products of recombinant type III collagen raw materials.

[0112] Figure 2 This is the primary mass spectrum of m / z 526.79042 in the enzymatic hydrolysis product of recombinant type III collagen raw material.

[0113] Figure 3 This is the secondary mass spectrum of m / z 526.79042 in the enzymatic hydrolysis product of recombinant type III collagen raw material.

[0114] Figure 4The characteristic peptide mass spectra of the recombinant type III collagen raw material in the spray dressing with a final concentration of 0.5 ng / mL (A) and 1 ng / mL (B).

[0115] Figure 5 The characteristic peptide mass spectra of the recombinant type III collagen raw material in the gel dressing with a final concentration of 0.5 ng / g (A) and 1 ng / g (B).

[0116] Figure 6 These are the characteristic peptide mass spectra of the recombinant type III collagen raw material in the repair dressing with a final concentration of 1 ng / g (A) and 2 ng / g (B).

[0117] Figure 7 This is the standard curve of recombinant type III collagen in the spray dressing.

[0118] Figure 8 Mass spectra of characteristic peptides added to the recombinant type III collagen raw material at concentrations of 100 μg / mL and 200 μg / mL in the spray dressing matrix control.

[0119] Fig. 9 Mass spectra of characteristic peptides added to the spray dressing matrix control at concentrations of 10 μg / mL and 50 μg / mL of recombinant type III collagen raw material.

[0120] Fig.10 This is the standard curve of recombinant type III collagen raw material in gel dressing.

[0121] Fig.11 Mass spectra of characteristic peptides added to the blank matrix of gel dressing with recombinant type III collagen raw material at concentrations of 100 μg / g and 200 μg / g.

[0122] Fig.12 Mass spectra of characteristic peptides added to the blank matrix of gel dressing with recombinant type III collagen raw material at concentrations of 10μg / g and 50μg / g.

[0123] Fig.13 This is the standard curve of the characteristic peptide of recombinant type III collagen in the repair dressing.

[0124] Fig.14 Mass spectra of characteristic peptides added to the recombinant type III collagen raw material in the repair dressing at concentrations of 100μg / g and 200μg / g.

[0125] Fig.15 Mass spectra of characteristic peptides added to the recombinant type III collagen raw material in the repair dressing at concentrations of 10μg / g and 50μg / g.

[0126] Fig.16This is the characteristic peptide mass spectrum of the recombinant type III collagen raw material in the spray dressing product.

[0127] Fig.17 This is the characteristic peptide mass spectrum of the medical type III recombinant collagen gel dressing product.

[0128] Fig.18 This is the mass spectrum of the characteristic peptides of recombinant type III collagen in the repair dressing product. DETAILED DESCRIPTION

[0129] The present invention is described in more detail below by taking the qualitative and quantitative detection of recombinant type III collagen in three dressings as an example. The amino acid sequence of the recombinant type III collagen is shown in SEQ ID No: 1. Example 1 Qualitative and quantitative analysis of recombinant collagen in spray dressing (liquid), repair dressing (cream) and medical gel dressing (gel).

[0130] 1. Instruments and reagents

[0131] 1. Instruments and equipment

[0132] Liquid chromatography column: octadecylsilane bonded silica gel column (filler particle size 5 μm, 150 mm×2.1 mm).

[0133] 2 Test products, reference products and reagents

[0134] Test samples: medical recombinant type III collagen spray dressing (hereinafter referred to as spray dressing, liquid form), medical recombinant type III collagen repair dressing (hereinafter referred to as repair dressing, cream form), medical recombinant type III collagen gel dressing (hereinafter referred to as gel dressing, gel form).

[0135] Reference substance: Recombinant type III collagen raw material, which is the functional component of the test substance, lyophilized powder, purity greater than 95%, standard sample used for qualitative identification and quantitative detection.

[0136] Matrix control samples: The matrix of the test products corresponding to the three dressings (all components except the recombinant type III collagen raw material).

[0137] Methodology confirmation samples: Recombinant type III collagen raw material, which is the functional component of the test article, lyophilized powder, with a purity greater than 95%, used for methodology confirmation, including samples for detection limit and quantification limit confirmation and samples for detection recovery analysis.

[0138] 2. Qualitative identification and quantitative detection operation

[0139] 1. Sample Pretreatment

[0140] a) Test sample

[0141] Accurately measure 2 mL of the spray dressing (liquid) into a 10 mL centrifuge tube; accurately weigh 2 g each of the repair dressing (cream) and medical gel dressing (gel), place them in separate 10 mL centrifuge tubes, and freeze-dry.

[0142] Add 2 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution to the freeze-dried spray dressing and stir magnetically for 20 to 30 min to fully dissolve the dressing matrix and mix it evenly for use; add 4 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution to the repair dressing and gel dressing respectively, stir magnetically for 20 to 30 min to fully mix the dressing matrix and set it aside.

[0143] b) Standard sample stock solution

[0144] Accurately weigh 5.0 mg of recombinant type III collagen raw material and place it in a 5 mL centrifuge tube. Add 2 mL of 0.1

[0145] mol / L Tris-HCl (pH 8.0), transfer to a 10mL volumetric flask, and wash with 0.1mol / L Tris-HCl

[0146] (pH 8.0) and dilute to 10 mL.

[0147] c) Matrix control sample

[0148] 1) Spray dressing matrix control sample solution: accurately measure 5 mL of spray dressing matrix control sample, freeze-dry, add 5 mL of 0.1 mol / L Tris-HCl (pH 8.0) to dissolve, and stir magnetically for 20 to 30 min to fully dissolve the dressing components and mix them evenly for use.

[0149] 2) Repair dressing matrix control sample solution: accurately measure 2 g of repair dressing matrix control sample, freeze-dry, add 4 mL of 0.1 mol / L Tris-HCl (pH 8.0) to dissolve, and stir magnetically for 20 to 30 minutes to fully mix the dressing components for use.

[0150] 3) Gel dressing matrix control sample solution: accurately measure 2 g of gel dressing matrix control sample, freeze-dry it, add 4 mL of 0.1 mol / L Tris-HCl (pH 8.0) to dissolve it, and stir it magnetically for 20 to 30 minutes to fully mix the dressing components for use.

[0151] d) Detection limit and quantification limit confirmation samples

[0152] Different amounts of low-concentration recombinant type III collagen raw material solution were added to the matrix control samples of the spray dressing, repair dressing and gel dressing, and then the samples were treated in the same pretreatment method as the test sample.

[0153] 1) Add recombinant type III collagen raw material to the spray dressing matrix control sample to a final concentration of 0.1 ng / mL.

[0154] 0.5ng / mL, 1ng / mL, 2ng / mL, 4ng / mL;

[0155] 2) Add recombinant type III collagen raw material to the repair dressing matrix control sample to a final concentration of 0.1 ng / g.

[0156] 0.5ng / g, 1ng / g, 2ng / g, 4ng / g;

[0157] 3) Add recombinant type III collagen raw material to the gel dressing matrix control sample to a final concentration of 0.1 ng / g.

[0158] 0.5ng / g, 1ng / g, 2ng / g, 4ng / g.

[0159] e) Detection of recovery analysis samples

[0160] (1) Add high, medium and low doses of recombinant type III collagen raw materials to the matrix control sample of the spray dressing, such as 10 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, and treat it according to the same pretreatment method as the test sample.

[0161] (2) Add high, medium and low doses of recombinant type III collagen raw materials to the matrix control samples of the repair dressing and gel dressing, such as 10 μg / g, 50 μg / g, 100 μg / g and 200 μg / g, respectively, and treat them according to the same pretreatment method as the test sample.

[0162] 2. Denaturation and Enzymatic Hydrolysis

[0163] (1) Since the recombinant type III collagen contained in the test sample is a single chain and does not contain a triple helix structure or polymer, no denaturation treatment is required. For samples that need to be denatured, they can be heated in a boiling water bath for 30 minutes and then cooled to room temperature.

[0164] (2) Enzymatic hydrolysis

[0165] a) Enzymatic hydrolysis of standard samples and preparation of standard working solution

[0166] 1) Take 1 mL of recombinant type III collagen standard sample stock solution, mix it with 40 μg of trypsin, react in a 37°C constant temperature shaking reactor for 24 hours, and add 100 μL of 10% formic acid solution to terminate the enzymatic reaction;

[0167] 2) Accurately pipette an appropriate amount of recombinant type III collagen raw material enzymatic hydrolysate and dilute it with the corresponding matrix control sample solution to a series of standard working solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL for drawing the quantitative standard curve.

[0168] 3) The standard working solution was further diluted with the matrix control sample solution to a series of solutions with lower concentrations for confirmation of the quantification limit and detection limit.

[0169] b) Enzymatic hydrolysis of test sample

[0170] Take 500 μL of the pre-treated sample solution and add it to an enzymatic hydrolysis vial (containing 20 μg of trypsin), react in a 37°C constant temperature shaking reactor for 24 h, add 50 μL of 10% formic acid solution to terminate the reaction, centrifuge (3000 g, 10 min), and take the supernatant for use.

[0171] c) Detection limit and quantification limit confirm the sample enzymatic hydrolysis

[0172] Take the detection limit and quantification limit confirmation samples after pretreatment and perform enzymatic hydrolysis according to the above b).

[0173] d) Detection of recovery analysis samples

[0174] Take the sample for recovery analysis after pretreatment and perform enzymatic hydrolysis according to the above b).

[0175] 3. On-machine testing

[0176] Chromatographic conditions

[0177] Mobile phase A is an aqueous solution containing 0.1% formic acid, phase B is a 60% acetonitrile aqueous solution (containing 0.1% formic acid), the flow rate is 0.2 mL / min, the column temperature is 30°C, the injection volume is 5 μL, the chromatographic column is an octadecylsilane bonded silica column (filler particle size 5 μm, 150 mm×2.1 mm), and the elution conditions are shown in Table A.1.

[0178] Table A.1 Gradient elution program

[0179]

[0180] Mass spectrometry conditions

[0181] Ion source: electrospray ion source;

[0182] Scanning mode: positive ion scanning;

[0183] Spray voltage: 3.5 kV;

[0184] Ion source evaporation temperature: 300°C;

[0185] Table A.2 Monitoring ion channels

[0186]

[0187] Note 1: The m / z 526.71→625.21 of the test substance can be used as the quantitative ion channel.

[0188] Appropriate volumes of each sample were injected into the liquid chromatography-mass spectrometer, and the detection operation was performed according to the set chromatography-mass spectrometry conditions.

[0189] 3. Test results

[0190] 1. Confirmation of characteristic peptides

[0191] Total ion current of trypsin hydrolysis product of recombinant type III collagen raw material sample ( Figure 1 ) After analysis by BioFinder software, a peptide with the amino acid sequence of GEAGIPGVPGAK was retrieved, and the primary mass spectrum of m / z 526.79042 was obtained ( Figure 2 ) indicates that the ion is doubly charged and its molecular weight is consistent with the theoretical molecular weight of the polypeptide GEAGIPGVPGAK. The secondary mass spectrum of m / z 526.79042 ( Figure 3 ) is highly matched with the theoretical fragment ion of the polypeptide GEAGIPGVPGAK. Through Blast multiple sequence alignment and Uniprot website search, this polypeptide does not exist in other types of human collagen and other common mammalian (cow, pig, rat, mouse, horse, rabbit, etc.) collagen amino acid sequences. Therefore, the polypeptide GEAGIPGVPGAK can be used as a specific characteristic polypeptide of the recombinant type III collagen raw material of the test sample in this implementation, and is used for qualitative identification and quantitative detection of the test sample.

[0192] It should also be noted that in the protein qualitative and quantitative analysis method using characteristic polypeptides, there are the following difficulties: a. characteristic polypeptides that meet the requirements cannot be found; b. a combination of multiple characteristic polypeptides is required to obtain accurate and reliable test results. However, this embodiment only uses the above-mentioned one characteristic polypeptide to obtain accurate and reliable test results for various forms of the recombinant type III collagen dressing.

[0193] 2. Methodological Validation

[0194] 2.1 Confirmation of detection limit and quantification limit

[0195] a) The mass spectra of the spray dressing with recombinant type III collagen raw material added at a final concentration of 0.5 ng / mL and 1 ng / mL are shown in Figure 4 The mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 1 ng / mL was 10, and the mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 0.5 ng / mL was 3. Therefore, the detection limit of the recombinant type III collagen raw material of the spray dressing product was determined to be 0.5 ng / mL and the quantification limit was 1 ng / mL.

[0196] b) The mass spectra of the gel dressing with recombinant type III collagen raw material added at a final concentration of 0.5 ng / g and 1 ng / g are shown in Figure 5 The mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 1 ng / g was 10, and the mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 0.5 ng / g was 3. Therefore, the detection limit of the recombinant type III collagen raw material of the gel dressing was determined to be 0.5 ng / g and the quantification limit was 1 ng / g.

[0197] c) The mass spectra of the recombinant type III collagen raw material added to the repair dressing at a final concentration of 1 ng / g and 2 ng / g are shown in Figure 6 The mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 2 ng / g was 10, and the mass spectrometry signal-to-noise ratio (S / N) of the characteristic peptide with a final concentration of 1 ng / g was 3. Therefore, the detection limit of the recombinant type III collagen raw material for the repair dressing product was determined to be 1 ng / g and the quantification limit was 2 ng / g.

[0198] 2.2 Addition recovery rate confirmation

[0199] a) Spray dressing

[0200] The control sample of the spray dressing matrix was used as a solution to prepare standard solutions with different concentrations of recombinant type III collagen raw material, and HPLC-MS / MS analysis was performed. The obtained standard curve is shown in the figure. Figure 7 .

[0201] The characteristic peptide mass spectra of the medical recombinant type III collagen spray dressing with added concentrations of 100 μg / mL and 200 μg / mL are shown in Figure 8 ; The characteristic peptide mass spectra of the medical recombinant type III collagen spray dressing with added concentrations of 10μg / mL and 50μg / mL are shown in Fig. 9 ;

[0202] The recovery rate of recombinant type III collagen added in the pre-treatment of the medical recombinant type III collagen spray dressing matrix control sample was calculated using the standard curve and is shown in Table A.3.

[0203] The results showed that the recovery rates of recombinant type III collagen raw materials added to the spray dressing matrix at concentrations of 10μg / mL, 50μg / mL, 100μg / mL and 200μg / mL were all between 79% and 88%, and the average recovery rates were all greater than 80%, meeting the requirements for added recovery rates.

[0204] Table A.3 Recovery results of recombinant type III collagen added to spray dressing

[0205]

[0206] b) Gel dressing

[0207] The gel dressing matrix control sample was used as a solution to prepare standard solutions of recombinant type III collagen raw materials with different concentrations, and HPLC-MS / MS analysis was performed. The obtained standard curve is shown in the figure. Fig.10 .

[0208] The characteristic peptide mass spectra of the recombinant type III collagen raw material added to the gel dressing at a concentration of 100 μg / g and 200 μg / g are shown in Fig.11 ; The characteristic peptide mass spectra of the recombinant type III collagen raw material with added concentrations of 10μg / g and 50μg / g are shown in Fig.12 ;

[0209] The recovery rates of recombinant type III collagen raw materials added to the gel dressing samples before treatment were calculated using the standard curve and are shown in Table A.4. The results show that the recovery rates of recombinant type III collagen raw materials added to the gel dressing matrix at concentrations of 10 μg / g, 50 μg / g, 100 μg / g and 200 μg / g were all between 80% and 112%, meeting the requirements for the recovery rates of addition.

[0210] Table A.4 Recovery results of recombinant type III collagen added to gel dressing

[0211]

[0212] c) Repair dressing

[0213] The repair dressing matrix control sample was used as a solution to prepare recombinant type III collagen raw material standard solutions of different concentrations, and HPLC-MS / MS analysis was performed. The obtained standard curve is shown in Fig.13 .

[0214] The characteristic peptide mass spectra of the recombinant type III collagen raw material added to the repair dressing at a concentration of 100 μg / g and 200 μg / g are shown in Fig.14 ; The characteristic peptide mass spectra of the recombinant type III collagen raw material with added concentrations of 10μg / g and 50μg / g are shown in Fig.15 ;

[0215] The recovery rates of recombinant type III collagen raw materials added to the repair dressing samples calculated using the standard curve are shown in Table A.5. The results show that the recovery rates of recombinant type III collagen raw materials added to the repair dressing matrix at concentrations of 50 μg / g, 100 μg / g and 200 μg / g are all between 77% and 88%, and the average recovery rates are all greater than 80%, which meets the requirements for the added recovery rate; the recovery rates of recombinant type III collagen raw materials added to the repair dressing matrix at a concentration of 10 μg / g are all between 67% and 74%, and the average recovery rate is 70.46%, which is lower than the requirements for the added recovery rate, which may be caused by the large effect of the matrix on the quantitative detection results under low concentration conditions.

[0216] Table A.5 Recovery results of recombinant type III collagen added to repair dressings

[0217]

[0218] 3. Test results of test products

[0219] 3.1 Medical recombinant type III collagen spray dressing

[0220] The characteristic peptide mass spectrum of the product is shown in Fig.16 The characteristic polypeptide mass spectrum is similar to the characteristic polypeptide mass spectrum of the corresponding recombinant type III collagen raw material standard sample (see Figure 8 , Fig. 9 ) are consistent.

[0221] The content of recombinant type III collagen in the product is shown in Table A.7. The average content of recombinant type III collagen raw materials in the medical recombinant type III collagen spray dressing product was tested to be 106 μg / mL, with a test recovery rate of 100%, which met the methodological requirements. The theoretical addition amount of recombinant type III collagen in this product is 100 μg / mL, and the test results are basically consistent with the theoretical addition amount.

[0222] Table A.6 Content of recombinant type III collagen in spray dressing products

[0223]

[0224]

[0225] 3.2 Medical recombinant type III collagen gel dressing

[0226] The characteristic peptide mass spectrum of the product is shown in Fig.17 , its characteristic polypeptide mass spectrum is similar to the characteristic polypeptide mass spectrum of the corresponding recombinant type III collagen raw material standard sample (see Fig.11 , Fig.12 ) are consistent.

[0227] The content of recombinant type III collagen in the gel dressing product is shown in Table A.7. The results show that the average content of recombinant type III collagen in the gel dressing product is 92μg / g, and the detection recovery rate is greater than 90%, which meets the methodological requirements. The theoretical addition amount of recombinant type III collagen in this product is 100μg / g, and the detection results are basically consistent with the theoretical addition amount.

[0228] Table A.7 Content of recombinant type III collagen raw material in gel dressing products

[0229]

[0230] 3.3 Medical recombinant type III collagen repair dressing

[0231] The characteristic peptide mass spectrum of the product is shown in Fig.18 , its characteristic polypeptide mass spectrum is similar to the characteristic polypeptide mass spectrum of the corresponding recombinant type III collagen raw material standard sample (see Fig.14 , Fig.15 ) are consistent.

[0232] The content of recombinant type III collagen in the repair dressing product is shown in Table A.8. The results show that the average content of recombinant type III collagen in the repair dressing product is 83μg / g, and the detection recovery rate is greater than 80%, which meets the methodological requirements. The theoretical addition amount of recombinant type III collagen in this product is 100μg / g, and the detection results are basically consistent with the theoretical addition amount.

[0233] Table A.8 Content of recombinant type III collagen in repair dressing products

[0234]

Claims

1. A qualitative or quantitative detection method for recombinant collagen in a collagen product, comprising: Pre-treatment step: if the collagen product contains a liquid or semi-solid component, take the liquid or semi-solid component, remove the solvent component to obtain a dry component, and then redissolve the dry component to obtain a sample solution; If the collagen product contains a solid component, the solid component is redissolved to obtain a sample solution of the test product; Enzymatic hydrolysis step: enzymatically hydrolyzing the sample solution with a protease to obtain a test sample solution; Liquid chromatography-mass spectrometry detection step: the test sample test solution, and the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample as a standard are respectively loaded on a liquid chromatography-mass spectrometer, and detection and analysis are performed according to the set liquid chromatography-mass spectrometry conditions; and The test result interpretation step is as follows: judging whether the test solution of the test sample contains the recombinant collagen protein according to whether a peak with the same retention time as the characteristic polypeptide appears in the test spectrum of the test solution of the test sample; and / or determining the content of the recombinant collagen protein in the test solution of the test sample according to the ratio of the peak area of ​​the characteristic polypeptide peak in the test spectrum of the test solution of the test sample to the peak area of ​​the characteristic polypeptide peak in the test spectrum of the recombinant collagen raw material standard working solution sample or the characteristic polypeptide standard working solution sample.

2. The detection method according to claim 1, wherein: In the pre-treatment step, the solvent is removed by freeze drying or nitrogen blowing.

3. The detection method according to claim 1, wherein: For recombinant collagen with a triple helix structure or a polymer structure, a denaturation step is further included after the pretreatment step and before the enzymatic hydrolysis step: The test sample reconstituted solution is heated to denature the recombinant collagen into a single chain.

4. The detection method according to claim 1, wherein: The preparation of the recombinant collagen raw material standard working solution as a standard product comprises the following steps: (1) Weighing a certain amount of recombinant collagen raw material used to produce the collagen product, and preparing it into a solution of a certain concentration as a standard sample stock solution; (2) For recombinant collagen having a triple helix structure or a polymer structure, heating the standard sample mother solution to denature the recombinant collagen into a single chain; (3) enzymatically hydrolyzing the standard sample mother solution with the protease to obtain a recombinant collagen raw material enzymatic hydrolyzate; and (4) diluting the recombinant collagen raw material enzymatic hydrolysate into a series of concentrations to obtain a recombinant collagen raw material standard working solution.

5. The detection method according to claim 1, wherein: The preparation of the characteristic polypeptide standard working solution as a standard product comprises the following steps: weighing a certain amount of the characteristic polypeptide, and preparing it into a series of concentrations to obtain the characteristic polypeptide standard working solution.

6. The detection method according to claim 1, wherein: The liquid chromatography-mass spectrometry conditions are as follows: (1) Chromatographic conditions Mobile phase A: 0.1 vol% formic acid in water; Mobile phase B: acetonitrile solution containing 0.1 vol% formic acid, or 60 vol% acetonitrile aqueous solution containing 0.1 vol% formic acid; Flow rate: 0.2mL / min~0.6mL / min; Column temperature: 25℃~30℃; Injection volume: 2μL~10μL; Chromatographic column: octadecylsilane bonded silica gel column, filler particle size 3μm or 5μm, column length 100mm or 150mm, column diameter 2.1mm or 2.0mm; and (2) Mass spectrometry conditions Ion source: electrospray ion source; Scanning mode: positive ion scanning; Spray voltage: 3.5 kV; Ion source evaporation temperature: 300°C.

7. The detection method according to claim 1, wherein: The recombinant collagen is recombinant human type III collagen, and its amino acid sequence is shown in SEQ ID No:

1.

8. The detection method according to claim 1, wherein: The protease used in the enzymatic hydrolysis step is trypsin.

9. The detection method according to claim 1, wherein: The amino acid sequence of the characteristic polypeptide is shown in SEQ ID No:

2.

10. The detection method according to claim 1, wherein: The collagen product is in solid, liquid, cream or gel form; the collagen product may be, for example, freeze-dried fibers or dressings.

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