Method for determining absolute molecular weight of recombinant human thrombopoietin

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining the absolute molecular weight of recombinant human thrombopoietin. According to the method, the absolute molecular weight of the recombinant human thrombopoietin is measured by combining volume exclusion chromatography with a multi-angle laser scattering method. The method is simple and rapid in operation, can reduce the workload of manual operation and data processing, and is high in detection efficiency. The method is suitable for detection or quality control of recombinant human thrombopoietin products.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the absolute molecular weight of recombinant human thrombopoietin. Background Technology

[0002] In the biopharmaceutical field, quality control of recombinant human thrombopoietin (rhTPO) is crucial, and the determination of its absolute molecular weight is an important parameter for assessing its quality and stability. Therefore, developing an accurate, efficient, and convenient method for determining the absolute molecular weight of rhTPO is of great significance for ensuring product quality and promoting clinical applications.

[0003] Existing methods for determining the molecular weight of rhTPO mainly include gel permeation chromatography (GPC / SEC), mass spectrometry (MS), dynamic light scattering (DLS), and circular dichroism spectroscopy (CD). Although existing technologies offer various methods for determining the molecular weight of rhTPO, they still have shortcomings in terms of accuracy, operational complexity, and cost-effectiveness. For example, while GPC / SEC is commonly used, it relies on calibration curves for standard proteins and has high requirements for sample pretreatment; MS can provide accurate quality information, but the equipment is expensive, the operation is complex, and there are resolution limitations for the analysis of large molecules; while DLS is simple to operate, its accuracy is easily affected by sample solution conditions; and CD is mainly used to study the secondary structure of proteins, with limited ability to directly determine molecular weight. Therefore, there is an urgent need to develop a simple, rapid, and accurate method for determining the absolute molecular weight of rhTPO. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the absolute molecular weight of recombinant human thrombopoietin, which is rapid, accurate and requires no standard substances.

[0005] Another objective of this invention is to provide the application of the method in the detection or quality control of recombinant human thrombopoietin products.

[0006] To achieve the objectives of this invention, the following technical solutions are provided:

[0007] The first aspect of the present invention provides a method for determining the absolute molecular weight of recombinant human thrombopoietin, which uses size exclusion chromatography coupled with multi-angle laser scattering to determine the absolute molecular weight of recombinant human thrombopoietin.

[0008] This invention combines multi-angle laser scattering technology with size exclusion high-performance liquid chromatography (HPLC) separation technology to effectively determine the absolute molecular weight of macromolecules. This method utilizes the principle of static light scattering, eliminating the need for standard materials to construct standard curves, thus providing a more direct, simpler, and more accurate measurement of the absolute molecular weight of macromolecules. By simultaneously measuring the change in scattered light intensity with angle and concentration from multiple angles, higher measurement accuracy can be achieved. It is applicable to a wide range of molecular weights, from low to high, meeting the measurement needs of various polymers. The method of this invention can directly measure liquid samples without complex sample pretreatment, is non-destructive to the samples, is reusable, and features automated operation, reducing manual operation and data processing workload, and enabling the measurement of a large number of samples in a short time.

[0009] The method of the present invention specifically includes the following steps: using size exclusion chromatography coupled with a multi-angle laser scattering instrument to detect the test solution containing recombinant human thrombopoietin to obtain a chromatogram, and using Astra analysis software to analyze the chromatogram and calculate the absolute molecular weight of recombinant thrombopoietin.

[0010] The method of this invention is applicable to recombinant human thrombopoietin of different purity levels, including unpurified stock solution and purified stock solution. The advantage of this method is that it is independent of sample purity, thus providing flexibility and convenience for the determination of the molecular weight of recombinant human thrombopoietin.

[0011] Preferably, the concentration of recombinant human thrombopoietin in the test solution is 0.3 mg / mL to 1.0 mg / mL. More preferably, the concentration of recombinant human thrombopoietin in the test solution is 0.5 mg / mL.

[0012] Preferably, when analyzing data using Astra software, the dn / dc (specific refractive index increment, mL / g) is 0.185.

[0013] Preferably, in the size exclusion chromatography coupled with multi-angle laser scattering, the chromatographic column is selected from TSK-gel G3000SW. The column has a size of 7.5 × 600 mm and is branded as TOSOH.

[0014] Preferably, in the size exclusion chromatography coupled with multi-angle laser scattering, the mobile phase is a 9–11 mmol / L phosphate buffer solution containing 140–160 mmol / L NaCl, with a mobile phase pH of 6.7–6.9; the flow rate of the mobile phase is 0.8–1.2 mL / min. More preferably, the mobile phase is a 10 mmol / L phosphate buffer solution containing 150 mmol / L NaCl, with a mobile phase pH of 6.8; the flow rate of the mobile phase is 1 mL / min.

[0015] In the size exclusion chromatography coupled with multi-angle laser scattering method, the detectors include an ultraviolet detector, a differential detector, and a multi-angle laser scatterer.

[0016] Preferably, the wavelength of the ultraviolet detector is 278–280 nm. The optical device temperature of the differential detector is 33–37 °C. More preferably, the wavelength of the ultraviolet detector is 280 nm. The optical device temperature of the differential detector is 35 °C. The multi-angle laser scattering instrument is a Wyatt DAWN HELEOS-II.

[0017] A second aspect of the present invention provides the application of the above method in the detection or quality control of recombinant human thrombopoietin products.

[0018] The main advantages of this invention are as follows: Recombinant human thrombopoietin (TPO) consists of 332 amino acid residues. Mature TPO has two domains: a hydrophobic receptor-binding region (residues 1-153, N-terminus) and a highly glycosylated carboxyl terminus (residues 154-332, C-terminus), which mainly maintain the stability of the protein structure. The carboxyl terminus of rhTPO is a polysaccharide region, including six N-glycosylation sites and several O-glycosylation modification potential sites. Furthermore, the side chain amino groups of the 19 arginine and 7 lysine residues in the theoretical rhTPO sequence, as well as the N-terminal amino group, can undergo complex chemical reactions with aldoses or ketoses under enzyme-free conditions to introduce glycosylation modification. Due to the complex glycosylation of TPO, the determination of its molecular weight is quite difficult. The determination method of this invention is simple and rapid, requires no special pretreatment of the sample, and eliminates the need to establish a standard curve with standard substances, reducing the workload of manual operation and data processing, and achieving high detection efficiency. The method of this invention is applicable to the quality control of the absolute molecular weight of recombinant human thrombopoietin products. Attached Figure Description

[0019] Figure 1 This is the chromatogram from Example 1.

[0020] Figure 2 This is the chromatogram from Example 2. Detailed Implementation

[0021] The present invention will be further explained in detail below with reference to specific embodiments.

[0022] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, all general equipment, materials, and reagents used are commercially available.

[0023] Example 1

[0024] 1.1 Preparation of the test sample

[0025] Transfer 200 μl of the TEBIA S12 gel chromatography stock solution (i.e., the TEBIA stock solution after S12 gel chromatography, in which the concentration of TEBIA is 0.5 mg / ml) into a 2 ml sample vial equipped with a 250 μl inner tube to obtain the test solution.

[0026] 1.2 Preparation of the mobile phase

[0027] Weigh 1.7526 g of disodium hydrogen phosphate dodecahydrate, 0.6911 g of sodium dihydrogen phosphate monohydrate, and 8.7809 g of sodium chloride using a precision balance. Place them in a 1 L beaker, dissolve them in 700 mL of purified water, adjust the pH to 6.8 with 1 mol / L sodium hydroxide solution, and bring the volume to 1 L using a volumetric flask. Filter the solution through a 0.45 μm filter membrane and transfer it to a 1 L reagent bottle.

[0028] 1.3 Testing Instruments and Conditions

[0029] The high-performance liquid chromatography (HPLC) system was an Agilent 1260 Infinity II HPLC system; the column was a TOSOH TSK-gel G3000SW with dimensions of 7.5 × 600 mm; the mobile phase flow rate was 1.0 mL / min; the sample cell temperature was 8 °C; the injection volume was 100 μL; the detection time was 45 min; the UV detector wavelength was 280 nm; the differential detector optical equipment temperature was 35 °C; and the multi-angle laser scattering instrument was a Wyatt DAWN HELEOS-II.

[0030] 1.4 Testing and Result Calculation

[0031] Inject the test solution from the sample vial in section 1.1 into a high-performance liquid chromatography-multi-angle laser scattering system and record the chromatogram, as shown below. Figure 1 As shown in the figure, red represents the multi-angle laser scattering spectrum, green represents the ultraviolet spectrum, and blue represents the differential spectrum. Data analysis using Astra software showed that the dn / dc (specific refractive index increment, mL / g) was 0.185. The absolute molecular weight of the main peak of the recombinant human thrombopoietin intermediate was 80290 Da.

[0032] Example 2

[0033] 2.1 Preparation of the test sample

[0034] Transfer 200 μl of the purified terbinafine stock solution (concentration of 0.5 mg / ml) into a 2 ml sample vial equipped with a 250 μl inner tube to obtain the test solution.

[0035] 2.2 Preparation of the mobile phase

[0036] Weigh 1.7526 g of disodium hydrogen phosphate dodecahydrate, 0.6911 g of sodium dihydrogen phosphate monohydrate, and 8.7809 g of sodium chloride using a precision balance. Place them in a 1 L beaker, dissolve them in 700 mL of purified water, adjust the pH to 6.8 with 1 mol / L sodium hydroxide solution, and bring the volume to 1 L using a volumetric flask. Filter the solution through a 0.45 μm filter membrane and transfer it to a 1 L reagent bottle.

[0037] 2.3 Testing Instruments and Conditions

[0038] The high-performance liquid chromatography (HPLC) system was an Agilent 1260 Infinity II HPLC system; the chromatographic column was a TOSOH TSK-gel G3000SW with dimensions of 7.5 × 600 mm; the mobile phase flow rate was 1.0 mL / min; the sample cell temperature was 8 °C; the injection volume was 100 μL; the detection time was 45 min; the ultraviolet detector wavelength was 280 nm; the differential detector optical equipment temperature was 35 °C; and the multi-angle laser scattering instrument was a Wyatt DAWN HELEOS-II.

[0039] 2.4 Testing and Result Calculation

[0040] Inject the test solution from the sample vial in section 2.1 into a high-performance liquid chromatography-multi-angle laser scattering system and record the chromatogram, as shown below. Figure 2 As shown in the figure, red represents the multi-angle laser scattering spectrum, green represents the ultraviolet spectrum, and blue represents the differential spectrum. Data analysis using Astra software showed that the dn / dc (specific refractive index increment, mL / g) ratio was 0.185. The absolute molecular weight of the main peak of the recombinant human thrombopoietin stock solution was 69400 Da.

[0041] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention, but it should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, any simple changes or substitutions made without departing from the inventive concept should be considered within the scope of protection of the present invention.

Claims

1. A method for determining the absolute molecular weight of recombinant human thrombopoietin, characterized in that, The absolute molecular weight of recombinant human thrombopoietin was determined by size exclusion chromatography coupled with multi-angle laser scattering.

2. The method according to claim 1, characterized in that, Includes the following steps: Chromatograms of test solutions containing recombinant human thrombopoietin were obtained by size exclusion chromatography coupled with multi-angle laser scattering. The absolute molecular weight of recombinant thrombopoietin was calculated by Astra analysis software.

3. The method according to claim 2, characterized in that, The concentration of recombinant human thrombopoietin in the test solution was 0.3 mg / mL to 1.0 mg / mL.

4. The method according to claim 1 or 2, characterized in that, In the size exclusion chromatography coupled with multi-angle laser scattering method, the chromatographic column is selected from TSK-gelG3000SW.

5. The method according to claim 1 or 2, characterized in that, In the size exclusion chromatography coupled with multi-angle laser scattering method, the mobile phase is a 9-11 mmol / L phosphate buffer solution containing 140-160 mmol / L NaCl, and the pH of the mobile phase is 6.7-6.9; the flow rate of the mobile phase is 0.8-1.2 mL / min.

6. The method according to claim 1 or 2, characterized in that, In the size exclusion chromatography coupled with multi-angle laser scattering method, the detectors include an ultraviolet detector, a differential detector, and a multi-angle laser scatterer.

7. The method according to claim 6, characterized in that, The wavelength of the ultraviolet detector is 278–280 nm.

8. The method according to claim 6, characterized in that, The temperature of the optical device of the differential detector is 33-37℃.

9. The method according to claim 6, characterized in that, The multi-angle laser scattering instrument is Wyatt DAWNHELEOS-II.

10. The application of the method as described in any one of claims 1 to 9 in the detection or quality control of recombinant human thrombopoietin products.

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