Rat collagen polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a mouse collagen polypeptide as well as a preparation method and application thereof. The polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide disclosed by the invention can be used for qualitatively and quantitatively detecting the rat type I collagen, monitoring the residue of the rat type I collagen in a cell treatment product, reducing the risk of animal-derived impurity residue and improving the quality and safety of the cell treatment product.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a murine tail collagen polypeptide, a preparation method thereof, and an application thereof. Background Art

[0002] Research has found that most tissues and organs contain type I collagen, which is particularly abundant in tissues such as skin, tendons, and bones. Type I collagen is a commonly used extracellular matrix (ECM) protein for cell culture, which can make the in vitro cell environment closer to the in vivo situation. During cell culture, a thin layer of collagen dilution solution is coated on the cell culture vessel, and a gel that supports 3D cell culture will be formed, which can promote the adhesion, growth, differentiation, migration, and tissue morphogenesis of primary cells (such as hepatocytes).

[0003] With the vigorous development of China's biopharmaceutical technology, more and more cell therapy products have moved from research and development to clinical applications. According to the requirements of relevant regulations such as the current version of the "Chinese Pharmacopoeia" and the "Technical Guidelines for the Pharmaceutical Research and Evaluation of Human Stem Cell Products", during the development of stem cell therapy products, if animal-derived residues must be used, reasonable internal control detection items and acceptance criteria need to be set to ensure the quality and safety of cell therapy products. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiency in the prior art of lacking a method for detecting the residue of rat type I collagen during cell culture, and to provide a collagen characteristic polypeptide, a preparation method thereof, and an application thereof. The characteristic polypeptide can qualitatively and quantitatively detect rat type I collagen well, can monitor the residue of rat type I collagen in cell therapy products, reduce the risk of animal-derived impurity residues, and improve the quality and safety of cell therapy products.

[0005] The present invention solves the above technical problems through the following technical solutions:

[0006] The first aspect of the present invention provides a murine collagen polypeptide, and the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0007] The second aspect of the present invention provides an isolated nucleic acid, and the nucleic acid encodes the polypeptide as described in the first aspect.

[0008] The third aspect of the present invention provides a method for preparing the polypeptide as described in the first aspect, and the method is selected from natural extraction, enzymatic hydrolysis method, fermentation method, genetic engineering expression, and chemical synthesis.

[0009] The fourth aspect of the present invention provides a kit for detecting murine-derived collagen, and the kit comprises the polypeptide as described in the first aspect as a standard product.

[0010] The fifth aspect of the present invention provides a method for detecting murine collagen, which includes the step of preparing a standard curve using the polypeptide as described in the first aspect or the kit as described in the fourth aspect.

[0011] In an example of preparing the standard curve of the present invention, a series of polypeptide standard working solution concentrations (μg / mL) are used as the abscissa, and the peak area of the polypeptide is used as the ordinate to plot the standard curve, obtaining a regression equation and a correlation coefficient.

[0012] Y = aX + b; R 2

[0013] Where: X - the concentration of the characteristic polypeptide, μg / mL;

[0014] Y - the peak area value of the characteristic polypeptide;

[0015] a - a constant;

[0016] b - a constant;

[0017] R 2 - the goodness of fit.

[0018] In some embodiments of the present invention, the method further includes the step of determining the retention times of the protein and the polypeptide in the sample to be tested by chromatography - mass spectrometry.

[0019] In some embodiments of the present invention, when the retention time of the polypeptide is equivalent to the retention time of the protein in the sample to be tested, it is determined that the sample to be tested contains murine collagen; otherwise, the sample to be tested does not contain murine collagen.

[0020] In the present invention, the "equivalent" means that for certain indicators of two or more products, within the error range allowed in experiments or practical applications, those skilled in the art can consider the results indicated by them to be the same.

[0021] In some embodiments of the present invention, the sample to be tested is blood, cell culture, or the supernatant of cell culture.

[0022] The sixth aspect of the present invention provides a system for detecting murine components in a sample to be tested. The system includes an analysis module and a judgment module; the analysis module performs chromatography - mass spectrometry detection on the protein components in the sample to be tested and inputs the obtained retention time of the protein components to the judgment module;

[0023] The judgment module compares the retention time input by the analysis module with the retention time of the standard product. When the retention time of the standard product is equivalent to the retention time of the protein in the sample to be tested, it is determined that the sample to be tested contains murine components; otherwise, it is determined that the sample to be tested does not contain murine components;

[0024] The reference substance is the polypeptide as described in the first aspect.

[0025] The seventh aspect of the present invention provides an application of the polypeptide as described in the first aspect, the nucleic acid as described in the second aspect, or the kit as described in the fourth aspect in the preparation of a reagent for detecting murine components.

[0026] In some specific embodiments of the present invention, the murine component is murine collagen.

[0027] On the basis of conforming to common general knowledge in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0028] The reagents and raw materials used in the present invention are all commercially available.

[0029] The positive and progressive effects of the present invention are as follows: Rat type I collagen, as a key raw material in the production process of certain cell therapy products, belongs to animal-derived materials and has a relatively high risk level. It is necessary to develop a quantitative method for detecting rat type I collagen with high sensitivity and good accuracy to monitor the residual rat type I collagen in cell therapy products, reduce the risk of residual animal-derived impurities, and improve the quality and safety of cell therapy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a photograph of the chromatogram-mass spectrum of the characteristic polypeptide.

[0031] Figure 2 It is a standard curve graph. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present invention will be further illustrated below by way of examples, but the present invention is not limited thereto within the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0033] The detection-related materials and reagents used in the examples are shown in Table 1:

[0034] Table 1 Detection-related materials and reagents

[0035]

[0036] Preparation according to the characteristic polypeptide sequence of rat type I collagen:

[0037] Design principle of characteristic polypeptide sequence of rat type I collagen: Use BLAST to search the rat type I collagen sequence to ensure that the peptide segment only appears in rat-derived type I collagen. The finally determined characteristic polypeptide sequence of rat type I collagen is: GETGPAGPAGPIGPAGAR (SEQ ID NO: 1). The polypeptide was synthesized by a biological company, purity: 98 ± 2%.

[0038] Key instruments: Centrifuge (3K-15); Constant temperature reactor; Liquid chromatography-mass spectrometry combined system (HPLC-MS, LCMS-8060); Water bath.

[0039] Example 1 Detection of residual amount of rat type I collagen during cell culture

[0040] 1. Sample treatment

[0041] 1.1 Sample dilution

[0042] Test sample: Cell culture supernatant;

[0043] Positive control: Rat type I collagen

[0044] Accurately pipette the test sample and positive control, boil in a water bath for 30 min and then cool to room temperature, dilute with an equal volume of 0.10 mol / L ammonium bicarbonate solution and shake well;

[0045] 1.2 Sample enzymolysis

[0046] Add trypsin to the test sample and positive control sample solution at a protein:enzyme ratio of approximately 20:1 to 100:1 (V / V), in a 37 °C constant temperature shaking reactor, react for 17 hours, boil for 5 minutes to inactivate trypsin and then cool to room temperature, for later use.

[0047] 1.3 Preparation of sample solution

[0048] 1.3.1 Standard

[0049] Dilute the characteristic polypeptide (GETGPAGPAGPIGPAGAR, SEQ ID NO: 1) to 0.050 μg / mL, 0.075 μg / mL, 0.100 μg / mL, 0.200 μg / mL, 0.500 μg / mL with 0.05 mol / L ammonium bicarbonate. Centrifuge (300 g, 10 minutes), take the supernatant and place it in an injection vial for later use.

[0050] 1.3.2 Preparation of sample solution

[0051] (1) Basal medium (negative control), cell culture supernatant (test sample), rat type I rat tail collagen (positive control);

[0052] (2) Dilute the negative control, test sample, and positive control with 0.05 mol / L ammonium bicarbonate to an appropriate concentration such that the diluted concentration is within the range of the characteristic polypeptide standard curve. Centrifuge (3000 g, 10 min), and take the supernatant into a sample vial for later use.

[0053] (3) Spiked sample: Take 8 μl of the diluted characteristic polypeptide standard solution (concentration 10 μg / mL) and mix well with 4 μL of the sample as the spiked sample for later use.

[0054] (4) Limit of detection (LOD) sample: Dilute the positive control with 0.05 mol / L ammonium bicarbonate to near the lowest point of the standard curve to determine the content of the characteristic polypeptide in the sample. Then dilute the sample 10-fold with 0.05 mol / L ammonium bicarbonate as the LOD sample for later use.

[0055] (5) System suitability sample: Dilute the characteristic polypeptide to 0.2 μg / mL with 0.05 mol / L ammonium bicarbonate, and use the LOD sample for later use.

[0056] (6) Blank control: Use 0.05 mol / L ammonium bicarbonate solution as the blank control.

[0057] 1.4 Chromatographic conditions

[0058] The reference conditions for liquid chromatography are shown in Table 2:

[0059] Table 2 Liquid chromatography conditions

[0060]

[0061]

[0062] 1.5 Mass spectrometry conditions

[0063] Set the corresponding parameters according to different mass spectrometers. The reference conditions are shown in Table 3:

[0064] Table 3 Mass spectrometry conditions

[0065]

[0066] 2. Experimental method

[0067] (1) Perform MS / MS analysis based on the ion charge ratio of the characteristic polypeptide. Select m / z 767.10 as the precursor ion and monitor m / z 1020.35 as the quantitative ion.

[0068] (2) Measure the prepared sample according to the liquid chromatography - mass spectrometry conditions. The retention time of the sample is consistent with that of the reference substance. When the retention time of the protein in the test sample is 8.362 min ± 5%, the test sample contains murine collagen, and the chromatogram - mass spectrometry spectrum of the characteristic polypeptide is as shown in Figure 1 shown.

[0069] (3) Prepare the standard working curve

[0070] Taking the concentrations (μg / mL) of a series of standard working solutions of the characteristic polypeptide as the abscissa and the peak area of the characteristic polypeptide as the ordinate, plot the standard curve to obtain the regression equation and the correlation coefficient.

[0071] Y = aX + b; R 2 …………………(1)

[0072] Where: X - the concentration of the characteristic polypeptide, μg / mL;

[0073] Y - the peak area value of the characteristic polypeptide;

[0074] a - constant;

[0075] b - constant;

[0076] R 2 - goodness of fit.

[0077] (4) Result calculation

[0078] Calculate the concentration Ci of the characteristic polypeptide in the test sample using the regression equation of the standard curve (Ci is the X in the regression equation, μg / mL).

[0079] Calculate the content of the collagen characteristic polypeptide in the test sample based on the content of the characteristic polypeptide in the test sample (Equation 2). Calculate the concentration of the characteristic polypeptide in the test sample according to Equation (2):

[0080] Ct = Ci × D × V1 / V2………………(2)

[0081] Where: Ct - the concentration of the characteristic polypeptide of murine type I collagen in the test sample, μg / mL;

[0082] Ci - the concentration of the characteristic polypeptide of murine type I collagen obtained from the standard curve, μg / mL;

[0083] D - the total dilution factor of the test sample;

[0084] V1 - the final volume of the test sample after dilution, mL;

[0085] V2 - the volume of the test sample in the final test solution, mL.

[0086] Acceptance criteria for the standard curve: R 2≥0.98.

[0087] (5) Method system suitability result determination

[0088] The characteristic polypeptide reference substance at 0.2 μg / mL was repeatedly determined 6 times, and the recovery rate was 80 - 120%, RSD ≤ 20%.

[0089] (6) Sample result determination

[0090] The recovery rate of the spiked sample was 80 - 120%.

[0091] 3. Test results

[0092] 3.1 Standard curve

[0093] The quantitative data of the reference substance - characteristic polypeptide is shown in Table 4, and the standard curve is as Figure 2 shown; the standard curve R 2 0.9999 meets the requirements.

[0094] Table 4 Quantitative data of the reference substance - characteristic polypeptide

[0095]

[0096]

[0097] 3.2 Method suitability test results

[0098] The system suitability test results show that the suitability samples were continuously injected 6 times, and the results are shown in Table 5:

[0099] Table 5 System suitability test results

[0100]

[0101] The results show that the recovery rate of the system suitability sample was 101% and met the requirements (acceptance criteria: 80 - 120%), and the relative standard deviation RSD was 0.85% and met the requirements (acceptance criteria: < 20%), and the method system suitability passed.

[0102] 3.3 Limit of quantitation & limit of detection test results

[0103] The limit of detection was calculated by the signal - to - noise ratio method. When the signal - to - noise ratio was 3:1, the corresponding concentration or the amount injected into the instrument was determined as the limit of detection; when the signal - to - noise ratio was 10:1, the corresponding concentration or the amount injected into the instrument was determined as the limit of quantitation; the limit of detection & limit of quantitation results in this test are shown in Table 6:

[0104] Table 6 Limit of detection & limit of quantitation results

[0105] Name Results of detection limit & quantification limit Original concentration of sample characteristic polypeptide (μg / mL) 0.052 Dilution factor 10 Concentration of characteristic polypeptide in diluted sample (μg / mL) 0.0052 Peak area of diluted sample (μV·s) 3655 S / N of diluted sample 322.81 Coefficient of minimum detection limit 3 Coefficient of minimum detectable amount 10 Injection volume (μL) 20 Injection volume (μL) 0.05 Detection limit (ng) (amount injected into the instrument) 1.00 Quantification limit (ng / mL) (concentration) 0.16 Quantification limit (ng) (amount injected into the instrument) 3.20

[0106] The results showed that the detection limit of this method was 0.05 ng / mL (or 1.00 ng), and the quantification limit was 0.16 ng / mL (or 3.20 ng).

[0107] 3.4 Detection Results of Positive Control

[0108] Taking rat type I collagen as the positive control and 0.05 mol / L ammonium bicarbonate solution as the blank control. See Table 7 for details:

[0109] Table 7 Detection Results of Positive Control

[0110]

[0111]

[0112] Note: NA means not applicable.

[0113] The results showed that the concentration of the characteristic polypeptide of the positive control (rat type I collagen) was (0.332 ± 0.002) μg / mL, and the result of the blank control was below the detection limit (<0.05 ng / mL).

[0114] 3.5 Detection Results of Spiked Samples

[0115] 0.08 μg of the characteristic polypeptide of rat type I collagen was added to each of the 8 test samples, and the detection results are shown in Table 8:

[0116] Table 8 Detection Results of Spiked Samples

[0117]

[0118]

[0119] The results showed that the range of the spike recovery rate of the test samples was 113 - 120%, all within the range of 80 - 120%. The detection results indicated that the cell culture supernatant samples had no interference on the detection of rat tail type I collagen.

[0120] The determination results of all 8 test samples were not detected, being lower than the method detection limit of 0.05 ng / mL.

Claims

1. A murine collagen polypeptide, characterized in that the polypeptide comprises the amino acid sequence shown in SEQ ID NO:

1.

2. An isolated nucleic acid, characterized in that the nucleic acid encodes the polypeptide according to claim 1.

3. A method for preparing the polypeptide according to claim 1, characterized in that the method is selected from natural extraction, enzymatic hydrolysis, fermentation, genetic engineering expression and chemical synthesis.

4. A kit for detecting murine collagen, characterized in that the kit comprises the polypeptide according to claim 1 as a standard.

5. A method for detecting murine collagen, characterized in that it includes the step of making a standard curve using the polypeptide according to claim 1 or the kit according to claim 4.

6. The method according to claim 5, characterized in that the method further includes the step of determining the retention times of the protein and the polypeptide in the sample to be tested by chromatography-mass spectrometry.

7. The method according to claim 6, characterized in that the method further includes: when the retention time of the polypeptide is the same as that of the protein in the sample to be tested, it is determined that the sample to be tested contains murine collagen; otherwise, the sample to be tested does not contain murine collagen.

8. The method according to claim 6, characterized in that the sample to be tested is blood, cell culture or the supernatant of cell culture.

9. A system for detecting murine components in a sample to be tested, characterized in that the system includes an analysis module and a judgment module; the analysis module performs chromatography-mass spectrometry detection on the protein components in the sample to be tested and inputs the obtained retention times of the protein components into the judgment module; the judgment module compares the retention time input by the analysis module with the retention time of the standard, and when the retention time of the standard is the same as that of the protein in the sample to be tested, it is determined that the sample to be tested contains murine components; otherwise, it is determined that the sample to be tested does not contain murine components; the standard is the polypeptide according to claim 1.

10. Use of the polypeptide according to claim 1, the nucleic acid according to claim 2, or the kit according to claim 4 in the preparation of a reagent for detecting murine components; Preferably, the murine component is murine collagen.

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