Method for detecting poloxamer 188 in pegylated human erythropoietin

Abstract

< TableDetails number = '0001' > < Title / > < tbl: tgroup cool = '3' > < tbl: colspec cool = 'c001' cool width = '35%' / > < tbl: colspec cool width = 'c002' cool width = '32%' / > < tbl: tbody > < tbl: start = '1' > time < / The column temperature is 60-70 DEG C; the temperature of the sample chamber is 2-8 DEG C; and (3) detecting the sample to be detected. According to the method, the P188 is effectively separated, and the P188 is accurately and quantitatively analyzed.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and more specifically, to a method for detecting and quantifying poloxamer 188. Background Art

[0002] PEGylated recombinant human erythropoietin (PEG-EPO) is a long-acting erythropoietin developed by modifying recombinant human erythropoietin (EPO) with polyethylene glycol. It is mainly used to treat anemia in patients with renal insufficiency. The solubility and stability of recombinant human erythropoietin are improved by modification with polyethylene glycol (PEG) and the addition of the excipient poloxamer 188 (abbreviated as P188).

[0003] Polyethylene glycol (PEG) is a class of compounds formed by the polymerization of ethylene oxide, with a certain molecular weight distribution and hydrophobic characteristics, and its physical and chemical properties are similar to those of P188. PEG-EPO is modified by covalent reaction between the active group of the polyethylene glycol terminal group and the amino acid side chain group and the amino group at the N-terminus of recombinant human erythropoietin.

[0004] Poloxamer (P188) is a non-ionic surfactant. When added to the preparation of protein drugs, it can reduce the adsorption of proteins on the container surface, reduce the surface tension of liquids, prevent protein denaturation, and reduce the formation of polymers. In view of the important role of P188 in protein stability, in order to ensure the safety of medication, the content of P188 must be accurately controlled and its content changes over time during the shelf life must be detected to ensure that P188 can play the expected protective effect. Therefore, it is necessary to develop appropriate test methods to detect the content of P188.

[0005] Common methods for detecting the content of P188 in biological drugs mainly use mixed-mode chromatographic columns, size exclusion chromatography (SEC) columns, etc. to separate and detect P188 from other components based on the difference in hydrophilicity, surface charge, or solvation hydration radius between different components. However, for PEG-EPO products, due to their molecular characteristics and the particularity of the production process, the hydrophilicity of P188 and the residual process-related impurities, i.e., free PEG, and the main component of the product, PEGylated human erythropoietin, are very close. The currently known chromatographic methods are difficult to completely and effectively separate P188 from other components, and thus it is difficult to accurately quantify the chromatographic methods. Currently, there is no report on the detection method of P188 content in PEGylated human erythropoietin.

[0006] The contents of the background technology section are merely the technologies known to the inventors and do not necessarily represent the prior art in the field. Summary of the invention

[0007] In view of the above problems, the present invention discloses a method for detecting poloxamer 188 in pegylated human erythropoietin, wherein poloxamer 188 is detected by reversed-phase high performance liquid chromatography, and the reversed-phase high performance liquid chromatography detection method comprises the following steps:

[0008] (1) preparing mobile phase A and mobile phase B, wherein mobile phase A is 0.1% TFA-water solution, and mobile phase B is 0.1% TFA-84% acetonitrile solution;

[0009] (2) Setting the chromatographic conditions: Use a C4 column and gradient elution. The elution procedure is as follows:

[0010] time A％ B％ Initial 40 60 3.00 25 75 6.00 10 90 12.00 10 90 12.10 40 60 15.00 40 60

[0011] The flow rate of the mobile phase is 0.8-1.2 mL / min;

[0012] The column temperature is 60℃~70℃;

[0013] The sample chamber temperature is 2-8°C;

[0014] (3) Detecting the sample to be tested.

[0015] Furthermore, in step (3), an evaporative light scattering detector (ELSD) is used to detect the sample to be tested.

[0016] Further, the parameters of the evaporative light scattering detector are set as follows:

[0017] Gas Type: Compressed air Gas flow: 2.3L / min Gain: 1 led Not adjustable Atomizer temperature: 85℃ Drift tube temperature: 85℃ .

[0018] Furthermore, the method further comprises determining the content of poloxamer 188, wherein the determination comprises the following steps:

[0019] (a) Prepare water and poloxamer 188 standard solutions with mass concentrations of 0.005%, 0.0075%, 0.01%, 0.02% and 0.03%;

[0020] (b) detecting the poloxamer 188 standard solutions of different concentrations according to the method to obtain the detection peak areas respectively;

[0022] (c) making a double logarithmic curve based on the content of poloxamer 188 in the standard solution of different concentrations and the corresponding detection peak area;

[0024] (d) detecting the sample to be tested, and obtaining the detection peak area of ​​the sample to be tested;

[0025] (e) Substituting the detection peak area of ​​the sample to be tested into the double logarithmic curve in step (c) to calculate the content of the sample to be tested.

[0026] The present invention adopts a reversed-phase high-performance liquid method, and establishes a separation method for effectively separating P188 and free PEG by investigating and optimizing parameters such as mobile phase composition, flow rate, chromatographic column temperature, and elution gradient. A 15-minute gradient can effectively separate free PEG, PEG-EPO, and P188. Furthermore, by adjusting the injection volume and linear range, the injection volume and the gradient concentration of the linear point of the double logarithmic curve are finally determined, and P188 is accurately quantitatively analyzed by the standard curve method. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 P188 content detection chart in preparations;

[0029] Figure 2 Specificity graph (overlay of preparation and DP Buffer);

[0030] Figure 3 P188 0.005%-0.03% content range linear graph;

[0031] Figure 4 P188 0.005%-0.03% content range sample overlay;

[0032] Figure 5 Overlay of reproducible samples. DETAILED DESCRIPTION

[0033] In the following description, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0034] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms "one", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include all values ​​between the endpoints and the endpoints. The preferred embodiments of the present invention are described below, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0035] The present invention discloses a method for detecting poloxamer 188 in pegylated human erythropoietin. Poloxamer 188 is detected by reversed-phase high performance liquid chromatography. The reversed-phase high performance liquid chromatography detection method comprises the following steps:

[0036] (1) preparing mobile phase A and mobile phase B, wherein mobile phase A is 0.1% TFA-water solution, and mobile phase B is 0.1% TFA-84% acetonitrile solution;

[0037] (2) Setting the chromatographic conditions: Use a C4 column and gradient elution. The elution procedure is as follows:

[0038] time A％ B％ Initial 40 60 3.00 25 75 6.00 10 90 12.00 10 90 12.10 40 60 15.00 40 60

[0039] The flow rate of the mobile phase is 0.8 to 1.2 mL / min, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min;

[0040] The column temperature is 60-70°C, for example, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C;

[0041] The sample chamber temperature is 2-8°C;

[0042] (3) Detecting the sample to be tested.

[0043] Furthermore, in step (2),

[0044] The flow rate of the mobile phase was 1.0 mL / min;

[0045] The column temperature was 65°C;

[0046] The sample chamber temperature was 5°C.

[0047] Furthermore, the injection volume of the sample to be tested is 20-100 μl, for example, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl; preferably 50 μl.

[0048] Furthermore, in step (3), the sample to be tested is detected by using an evaporative light scattering detector (ELSD).

[0049] The detection of the sample to be tested in the present invention is not limited to the above detectors or detection methods. Since P188 has been effectively separated through steps (1) and (2), any detector or detection method that can detect P188 can be applied to the present invention. The performance and effect of CAD and ELSD detectors are equivalent and can be used in the present invention.

[0050] Further, the parameters of the evaporative light scattering detector are set as follows:

[0051] Gas Type: Compressed air Gas flow: 2.3L / min Gain: 1 led Not adjustable Atomizer temperature: 85℃ Drift tube temperature: 85℃ .

[0052] Furthermore, the method further comprises determining the content of poloxamer 188, wherein the determination comprises the following steps:

[0053] (a) Prepare water and poloxamer 188 standard solutions with mass concentrations of 0.005%, 0.0075%, 0.01%, 0.02% and 0.03%;

[0054] (b) detecting the poloxamer 188 standard solutions of different concentrations according to the method to obtain the detection peak areas respectively;

[0056] (c) making a double logarithmic curve based on the content of poloxamer 188 in the standard solution of different concentrations and the corresponding detection peak area;

[0058] (d) detecting the sample to be tested, and obtaining the detection peak area of ​​the sample to be tested;

[0059] (e) Substituting the detection peak area of ​​the sample to be tested into the double logarithmic curve in step (c) to calculate the content of the sample to be tested.

[0060] Example

[0061] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0062] Instrument reagents and consumables:

[0063] instrument:

[0064] name factory model High performance liquid chromatography Waters ACQUITY Arc ELSD detector AllChrom ELSD 6100 Electronic balance Mettler Toledo MS204TS

[0065] Reagents:

[0066]

[0067] Consumables:

[0068]

[0069] DP: PEG-EPO preparation, which is a finished injection solution made by diluting the PEG-EPO stock solution (DS), adding excipients, aseptically filling and packaging. P188 is an excipient added to PEG-EPO DP that is used as a surfactant.

[0070] Sample Information:

[0071]

[0072] (1) Preparation of mobile phase:

[0073] Mobile phase A (0.1% TFA-water solution): Measure 2000 ml of ultrapure water and add it into a 2L reagent bottle, add 2 ml of TFA and mix well, and degas by ultrasonication for 15 minutes.

[0074] Mobile phase B (0.1% TFA-84% acetonitrile solution): 1680 ml of acetonitrile and 320 ml of ultrapure water were added to a 2 L reagent bottle, and then 2 ml of TFA was added to mix well, and ultrasonic degassing was performed for 15 min.

[0075] (2) Sample preparation:

[0076] PEG-EPO preparations do not require any pretreatment and can be directly injected into the injection vial;

[0077] P188 mother solution (0.1% P188 solution): Weigh 10.0 mg of P188 solid particles, add 10 ml of ultrapure water to dissolve and mix, and prepare the P188 gradient concentration samples required for the standard curve according to the following table:

[0078] name Amount of P188 stock solution added (μl) Amount of water added (μl) Final concentration 0.03% standard solution 60 140 0.03％ 0.02% standard solution 40 160 0.02％ 0.01% standard solution 20 180 0.01％ 0.0075% standard solution 15 185 0.0075％ 0.005% standard solution 10 190 0.005％

[0079] (3) LC flow rate, ELSD parameters and elution gradient

[0080]

[0081]

[0082] a) Specificity testing

[0083] Two batches of PEG-EPO preparation and DP Buffer were injected with 50 μl respectively. The result spectrum showed that PEG-EPO, PEG and DP Buffer had no interference with the peak of P188. The single injection PEG-EPO preparation sample is shown in Figure 1 ; See the dedicated overlay Figure 2 .

[0084] b) P188 gradient concentration sample linear detection

[0085] Water, 0.005%, 0.0075%, 0.01%, 0.02%, 0.03% P188 standard solution were injected 50μl respectively, and the results were plotted as double logarithmic curves of P188 theoretical content and peak area. The 0.005% to 0.03% standard solution had good linearity, R 2 (Correlation coefficient) is 0.9997; see the curve graph Figure 3 , linear sample overlay Figure 4 .

[0086] Linear results table:

[0087] name Injection volume (μl) Theoretical content (%) Peak area (μV·S) 0.005％P188 50 0.005 873576 0.0075％P188 50 0.0075 1899515 0.01％P188 50 0.01 3021684 0.02％P188 50 0.02 10447574 0.03％P188 50 0.03 21471505

[0088] c) Repeatability test

[0089] For the same batch of PEG-EPO preparation, 6 samples were taken and transferred to 6 liquid phase vials respectively, and each vial was injected with 1 injection, and the injection was 50μl; the P188 test result of DP PEG-EPO was 0.0097%-0.0101%, the relative standard deviation (RSD) value of the results of the six-injection repeatable samples was 1.77%, and the RSD value of the peak area was 3.18%, with good repeatability. The six-injection repeatable sample overlay is shown in Figure 5 .

[0090] Repeatability sample results table:

[0091]

[0092]

[0093] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. In addition, although the elements of the present invention may be described or required in individual form, it is also conceivable to have multiple elements unless explicitly limited to a single element.

Claims

1. A method for detecting poloxamer 188 in PEGylated human erythropoietin, characterized in that: Poloxamer 188 is detected by reverse phase high performance liquid chromatography, and the reverse phase high performance liquid chromatography detection method comprises the following steps: (1) preparing mobile phase A and mobile phase B, wherein mobile phase A is 0.1% TFA-water solution, and mobile phase B is 0.1% TFA-84% acetonitrile solution; (2) Setting the chromatographic conditions: Use a C4 column and gradient elution. The elution procedure is as follows: The flow rate of the mobile phase is 0.8-1.2 mL / min; The column temperature is 60℃~70℃; The sample chamber temperature is 2-8°C; (3) Detecting the sample to be tested.

2. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 1, characterized in that: In the step (2), The flow rate of the mobile phase was 1.0 mL / min; The column temperature was 65°C; The sample chamber temperature was 5°C.

3. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 1, characterized in that: The injection volume of the sample to be tested is 20-100 μl.

4. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 3, characterized in that: The injection volume of the sample to be tested is 50 μl.

5. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 1, characterized in that: In the step (3), an evaporative light scattering detector (ELSD) is used to detect the sample to be tested.

6. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 5, characterized in that: The parameters of the evaporative light scattering detector are set as follows:

7. The method for detecting poloxamer 188 in PEGylated human erythropoietin according to claim 5 or 6, characterized in that: The method also includes determining the content of poloxamer 188, wherein the determination comprises the following steps: (a) Prepare water and poloxamer 188 standard solutions with mass concentrations of 0.005%, 0.0075%, 0.01%, 0.02% and 0.03%; (b) detecting the poloxamer 188 standard solutions of different concentrations to obtain detection peak areas respectively; (c) making a double logarithmic curve based on the content of poloxamer 188 in the standard solution of different concentrations and the corresponding detection peak area; (d) detecting the sample to be tested, and obtaining the detection peak area of ​​the sample to be tested; (e) Substituting the detection peak area of ​​the sample to be tested into the double logarithmic curve in step (c) to calculate the content of the sample to be tested.

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