Method for detecting molecular weight of small-molecular recombinant collagen peptide

Abstract

The invention discloses a method for detecting molecular weight of small-molecular recombinant collagen peptide. According to the method, four commercial peptides are adopted as standard substances, the same high performance liquid chromatography detection method is adopted to respectively carry out high performance liquid chromatography detection on the standard substances and the small molecule recombinant collagen peptides of a test sample, and the obtained atlas of the test sample and a standard curve constructed by the standard substances are compared, calculated and analyzed. Therefore, the molecular weight of the test sample is determined. The method disclosed by the invention can be used for effectively identifying and analyzing the small-molecule recombinant collagen peptide produced by a heat treatment process, and meets the requirements of daily quality detection of the small-molecule recombinant collagen peptide.

Description

Technical Field

[0001] This invention belongs to the field of protein analysis technology and relates to a method for detecting the molecular weight of small molecule recombinant collagen peptides. Background Technology

[0002] Recombinant collagen products are used in skincare, food, and pharmaceuticals. Small-molecule recombinant collagen peptides (molecular weight 500-2000 Da) can penetrate the dermis more effectively, forming reservoirs in subcutaneous tissue and exerting their effects for up to 24 hours. However, due to variations in expression systems and recombinant processes among different recombinant collagen products, as well as differences in the length of selected nucleic acid sequences and the combination and splicing of fragments, molecular weight determination of recombinant collagen has become a crucial aspect of quality evaluation and control for these products. The structural characteristics of recombinant collagen determine its biological function and product stability. Although the Chinese Pharmacopoeia includes methods for molecular weight determination, conventional testing procedures are not applicable to recombinant collagen with varying structural characteristics.

[0003] Commonly used methods for molecular weight determination include mass spectrometry, size exclusion chromatography, scattered light scattering (SLS), and osmometry. Mass spectrometry requires a mass spectrometer, which, while highly accurate, is expensive and difficult to meet the low-cost, high-accuracy requirements of routine quality monitoring. Osmometry has limitations in its measurable molecular weight range. When the molecular weight is too large, the osmotic pressure decreases due to the reduced number of solutes, increasing the relative error. When the molecular weight is too small, solute molecules can pass through semi-permeable membranes, making the measurement unreliable. Light scattering measures the weight-average molecular weight of the solute. The intensity of the scattered light is proportional to the square of the superimposed amplitude, i.e., Ⅰ∞(∑iAi)2. This results in a decrease in the total scattered light intensity, and the degree of decrease is related to the scattering angle. The measured scattered light intensity is related not only to the molecular weight and the interaction between the polymer segments and the solvent but also to the morphology of the polymer chains, making it unsuitable for molecular weight determination of recombinant collagen. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting the molecular weight of small-molecule recombinant collagen peptides. This method employs high-performance liquid chromatography (HPLC) to obtain a chromatogram of the small-molecule recombinant collagen peptides, thereby determining their molecular weight and meeting the needs of routine product monitoring.

[0005] The technical solution for achieving the objective of this invention is as follows:

[0006] The method for determining the molecular weight of small molecule recombinant collagen peptides includes the following steps:

[0007] (1) Preparation of standard products:

[0008] Cytochrome C, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen were dissolved in water to prepare standard solutions of 1 mg / ml.

[0009] (2) Determination of the standard curve:

[0010] The standard solutions were injected separately and analyzed by high-performance liquid chromatography (HPLC) under the following conditions:

[0011] Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v)

[0012] Flow rate: 0.5 mL / min

[0013] Column temperature: 30 ℃

[0014] Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm.

[0015] Detection wavelength: 220nm

[0016] Injection volume: 10 μL

[0017] Injection time: 30 min;

[0018] Retention times were recorded, and liquid chromatograms of cytochrome C, bacitrazyme, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen were obtained. The standard curve formula was calculated by plotting the logarithm of the relative molecular mass against retention time: y = -0.2294x + 6.7286, where R0... 2 =0.9978;

[0019] (3) Preparation of the test solution:

[0020] The small molecule recombinant collagen peptides were dissolved by ultrasonic oscillation in ultrapure water or sterile purified water, and then filtered through a microporous membrane with a pore size of 0.22 μm to obtain a test solution of 1 mg / mL.

[0021] (4) Molecular weight analysis of the test sample:

[0022] The test solution was injected and analyzed by high-performance liquid chromatography (HPLC) under the following conditions:

[0023] Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v)

[0024] Flow rate: 0.5 mL / min

[0025] Column temperature: 30 ℃

[0026] Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm.

[0027] Detection wavelength: 220nm

[0028] Injection volume: 10 μL

[0029] Injection time: 30 min;

[0030] Record the retention time, obtain the liquid chromatogram of the test sample, and calculate and process the liquid chromatogram and data of the test sample according to the standard curve formula to obtain the relative molecular mass and distribution range of small molecule recombinant collagen peptides in the test sample.

[0031] Furthermore, in step (3), the ultrasonic oscillation time is 10~15 min.

[0032] Further, in step (3), the small molecule recombinant collagen peptide is prepared by the following steps: an aqueous solution of recombinant collagen is put into a reaction vessel, the temperature of the reaction vessel is controlled at 121~125℃ by steam heating and pressure, and heat treatment is carried out for a set time to obtain the small molecule recombinant collagen peptide. The recombinant collagen is produced by fermentation of Pichia pastoris with preservation number CGMCC No. 5021.

[0033] Further, in step (3) or (4), the mobile phase is prepared by mixing acetonitrile, water and trifluoroacetic acid in a volume ratio of 40:60:0.05, and then filtering it with a microporous membrane with a pore size of 0.22 μm to obtain the mobile phase.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] This invention uses cytochrome C, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen as standards to determine the chromatographic conditions required for the molecular weight detection of small molecule recombinant collagen peptides. The detection time for small molecule recombinant collagen peptides is determined to be 30 minutes, supporting the needs of convenient routine laboratory testing. The standard curve determination of the four standards reduces the cost of the standards and improves the speed and accuracy of small molecule recombinant collagen peptide molecular weight detection. It allows for precise control of the molecular weight of small molecule recombinant collagen peptides generated during the processing and provides an effective method for the daily monitoring of the molecular weight of the finished small molecule recombinant collagen peptide product. Attached Figure Description

[0036] Figure 1 The image shows the liquid chromatogram of small molecule recombinant collagen peptides obtained by heat treatment for 40 min.

[0037] Figure 2 The image shows the liquid chromatogram of small molecule recombinant collagen peptides obtained by heat treatment for 80 min.

[0038] Figure 3 The image shows a liquid chromatogram of small molecule recombinant collagen peptides obtained after a heat treatment time of 120 min.

[0039] Figure 4 The image shows a liquid chromatogram of small molecule recombinant collagen peptides obtained by heat treatment for 150 min.

[0040] Figure 5 The image shows the liquid chromatogram of small molecule recombinant collagen peptides obtained by heat treatment for 198 min.

[0041] Figure 6 The image shows a liquid chromatogram of small molecule recombinant collagen peptides obtained by heat treatment for 230 min.

[0042] Figure 7 This is a liquid chromatogram of the small molecule recombinant collagen peptide RA2400S001.

[0043] Figure 8 This is a liquid chromatogram of cytochrome C.

[0044] Figure 9 This is a liquid chromatogram of bacitracin.

[0045] Figure 10 This is a liquid chromatogram of acetic acid-acetic acid-tyrosine-arginine.

[0046] Figure 11 This is the liquid chromatogram of acetic acid-acetic acid-acetic acid. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0048] In the following embodiments, the preparation of the small molecule recombinant collagen peptides was carried out with reference to Chinese Patent ZL2022115798485. Specifically, an aqueous solution of recombinant collagen was added to a reaction vessel, and the temperature of the reaction vessel was controlled at 121~125℃ by steam heating and pressure control. The reaction vessel was heat-treated for a set time to obtain small molecule recombinant collagen peptides. The recombinant collagen was produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021.

[0049] All standards used were commercially purchased. The molecular weight of cytochrome C was 12,500 D, the molecular weight of bacitrazyme was 1,422 D, the molecular weight of acetyl-acetyl-tyrosine-arginine was 451 D, and the molecular weight of acetyl-acetyl-acetyl was 189 D.

[0050] Example 1

[0051] 1. Preparation of standard solutions:

[0052] Accurately weigh 2 mg each of cytochrome C, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen and place them in centrifuge tubes. Add 2 ml of purified water filtered through a 0.22 μm filter membrane and dissolve to obtain a standard solution with a concentration of 1 mg / ml.

[0053] 2. Determination of the standard curve:

[0054] Take 10 µL of each standard solution and inject it for high-performance liquid chromatography (HPLC) analysis. The HPLC conditions are as follows:

[0055] Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v);

[0056] Flow rate: 0.5 mL / min;

[0057] Column temperature: 30 ℃;

[0058] Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm.

[0059] Detection wavelength: 220nm;

[0060] Injection volume: 10 μL;

[0061] Injection time: 30 min.

[0062] Record the retention time and obtain liquid chromatograms for cytochrome C, bacitrazyme, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen, respectively, as follows: Figures 8-11 As shown, the standard curve formula is obtained by plotting the logarithm of the relative molecular mass against the retention time: y = -0.2294x + 6.7286, where R0... 2 =0.9978, indicating a good linear relationship.

[0063] 3. Obtaining the test sample:

[0064] Small molecule recombinant collagen peptides obtained by heat treatment for 40 min, 80 min, 120 min, 150 min, 198 min and 230 min were used as test samples.

[0065] 4. Preparation of the test solution:

[0066] Small molecule recombinant collagen peptides obtained by different heat treatment times were dissolved in purified water filtered through a 0.22 μm filter membrane, sonicated for 10 min, and then filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain a test solution of 1 mg / ml.

[0067] 5. Determination of molecular weight of the test sample:

[0068] Inject 10 µL of the test solution and perform high-performance liquid chromatography (HPLC) analysis. The HPLC conditions are as follows:

[0069] Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v);

[0070] Flow rate: 0.5 mL / min;

[0071] Column temperature: 30 ℃;

[0072] Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm.

[0073] Detection wavelength: 220nm;

[0074] Injection volume: 10 μL;

[0075] Injection time: 30 min.

[0076] Record the retention time and obtain liquid chromatograms of small molecule recombinant collagen peptides obtained at different heat treatment times, such as... Figures 1-6 As shown. Based on the standard curve formula, the liquid chromatograms and data of the test samples were calculated and processed to obtain the relative molecular mass and distribution range of the small molecule recombinant collagen peptides of each test sample.

[0077] like Figure 1 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 40 min is 8505D.

[0078] like Figure 2 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 80 min is 6089D.

[0079] like Figure 3 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 120 min is 3685D.

[0080] like Figure 4 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 150 min is 2875D.

[0081] like Figure 5 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 198 min is 2126D.

[0082] like Figure 6 As shown, the molecular weight of the small molecule recombinant collagen peptide obtained by heat treatment for 230 min is 1683D.

[0083] Example 2

[0084] The same method as in Example 1 was used to test the small molecule recombinant collagen peptide product RA2400S001.

[0085] The results are as follows Figure 7 As shown, the molecular weight of the small molecule recombinant collagen peptide RA2400S001 is 1698D.

[0086] In summary, high-performance liquid chromatography (HPLC) is a cost-effective method for routine monitoring of the molecular weight of small-molecule recombinant collagen peptides. This allows for the monitoring of the structural characteristics and product stability of small-molecule recombinant collagen peptides, as well as the monitoring of molecular weight during the processing and preparation process.

Claims

1. A method for determining the molecular weight of small molecule recombinant collagen peptides, characterized in that, Includes the following steps: (1) Preparation of standard products: Cytochrome C, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen were dissolved in water to prepare standard solutions of 1 mg / ml. (2) Determination of the standard curve: The standard solutions were injected separately and analyzed by high-performance liquid chromatography (HPLC) under the following conditions: Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v) Flow rate: 0.5 mL / min Column temperature: 30 ℃ Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm. Detection wavelength: 220nm Injection volume: 10 μL Injection time: 30 min; Retention times were recorded, and liquid chromatograms of cytochrome C, bacitrazyme, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen were obtained. The standard curve formula was calculated by plotting the logarithm of the relative molecular mass against retention time: y = -0.2294x + 6.7286, where R0... 2 =0.9978; (3) Preparation of the test solution: The small molecule recombinant collagen peptides were dissolved by ultrasonic oscillation in ultrapure water or sterile purified water, and then filtered through a microporous membrane with a pore size of 0.22 μm to obtain a test solution of 1 mg / mL. (4) Molecular weight analysis of the test sample: The test solution was injected and analyzed by high-performance liquid chromatography (HPLC) under the following conditions: Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 40:60:0.05 (v:v:v) Flow rate: 0.5 mL / min Column temperature: 30 ℃ Chromatographic column: TSKgel G2000SWxl, packing material particle size 5μm, dimensions 7.8*300mm. Detection wavelength: 220nm Injection volume: 10 μL Injection time: 30 min; Record the retention time, obtain the liquid chromatogram of the test sample, and calculate and process the liquid chromatogram and data of the test sample according to the standard curve formula to obtain the relative molecular mass and distribution range of small molecule recombinant collagen peptides in the test sample.

2. The detection method according to claim 1, characterized in that, In step (3), the ultrasonic oscillation time is 10~15 min.

3. The detection method according to claim 1, characterized in that, In step (3), the small molecule recombinant collagen peptide is prepared by the following steps: an aqueous solution of recombinant collagen is put into a reaction vessel, the temperature of the reaction vessel is controlled at 121~125℃ by steam heating and pressure, and heat treatment is carried out for a set time to obtain the small molecule recombinant collagen peptide. The recombinant collagen is produced by fermentation of Pichia pastoris with preservation number CGMCC No. 5021.

4. The detection method according to claim 1, characterized in that, In step (3) or (4), the mobile phase is prepared by mixing acetonitrile, water and trifluoroacetic acid in a volume ratio of 40:60:0.05, and then filtering it through a microporous membrane with a pore size of 0.22 μm to obtain the mobile phase.

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