Method for detecting content of free PEG (polyethylene glycol) in pegylated human erythropoietin

Abstract

The invention provides a method for detecting free PEG in pegylated human erythropoietin, the free PEG is detected through high performance liquid chromatography, the high performance liquid chromatography detection method comprises the following steps: (1) preparing a mobile phase A and a mobile phase B, the mobile phase A is a 0.1% TFA-aqueous solution, and the mobile phase B is a 0.1% TFA-84% acetonitrile solution; (2) setting chromatographic conditions: carrying out gradient elution by using a C4 chromatographic column; the flow velocity of the mobile phase is 0.75-1.0 mL / min; 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.

Description

Technical Field

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

[0002] Recombinant human erythropoietin (EPO) is an effective drug for the treatment of renal anemia. As the cost of dialysis for renal failure in China decreases and the number of dialysis patients continues to increase, the demand for EPO will gradually increase in the future. However, EPO has a short half-life, and frequent injections of the drug bring great pain and safety risks to patients. PEGylated recombinant human erythropoietin (PEG-EPO) is a therapeutic drug modified by covalent reaction between the active group of the polyethylene glycol end group and the amino acid side chain group and the amino group of the N-terminus of EPO. Its half-life in the blood circulation is significantly prolonged, and its solubility and stability are improved. However, due to the excessive addition of polyethylene glycol (PEG) during the coupling reaction, it cannot be completely removed through the purification process, and there may be a risk of PEG shedding during the storage of PEG-EPO stock solution and finished products. Therefore, according to the guidelines and regulatory requirements of the International Council for Harmonization of Technical Registration of Pharmaceuticals for Human Use (ICH), for safety and efficacy considerations, it is necessary to control the free PEG content in the stock solution and monitor the stability of the free PEG content in the preparation.

[0003] Polyethylene glycol (PEG) is a class of compounds formed by the polymerization of ethylene oxide. It has a certain molecular weight distribution and hydrophobic characteristics. It is soluble in water, ethanol and many other organic solvents, but has no specific ultraviolet absorption. Therefore, based on the differences between PEG and the target modified protein and the coupled product, C18, C8, C4 and other reverse phase chromatographic columns or size exclusion (SEC) chromatographic columns are often selected for separation, and universal detectors such as evaporative light scattering detector (ELSD), charged aerosol detector (CAD) or differential refractometer (RI) are used for detection.

[0004] Some PEG-EPO products use linear PEG (30kD) to modify EPO. Because the spatial structure of PEG is large, the hydrophobicity of PEG and PEG-EPO, especially multi-PEG-EPO, is close, and it is difficult to separate them using conventional reverse phase chromatography. In addition, the non-ionic surfactant poloxamer (P188, also a polymer) is added to the injection of this product, which also brings certain challenges to the separation of PEG. In addition, the protein content in the PEG-EPO stock solution and preparation is low. According to the requirements of the ICH guidelines, the corresponding limit of the residual amount is also extremely low. It is necessary to use a highly sensitive method to detect PEG, or to use a pretreatment method to enrich the free PEG, and then detect PEG. The commonly used method of removing protein with an organic solvent and then vacuum rotary evaporation and drying and re-dissolving, even if a high concentration of organic solvent is used to dilute the sample, the protein cannot be completely precipitated and removed, and the free PEG content is originally a trace amount. When using this method, it will also partially precipitate, resulting in a recovery rate that does not meet the methodological requirements. When the SEC size exclusion method is used to directly analyze the sample, PEG and PEG-EPO cannot be effectively separated. For example, by using a series of ion exchange columns and SEC columns, the free PEG (A phase: 0.02 mol / L citric acid, pH 3.0, 20 min) and the target protein (B phase: 0.02 mol / L citric acid, 1 mol / L sodium chloride, pH 3.0, 7 min) are eluted isocratically by switching the mobile phase A and B phase (B has a high salt concentration), and quantitative analysis is performed by comparing with the PEG standard curve. However, due to the high concentration of non-volatile buffer salts in the mobile phase, it is impossible to use a highly sensitive ELSD or CAD detector for detection. Only a general-purpose differential refractive index detector (RI) with poor sensitivity can be used for quantification. In addition, the series chromatography column method is time-consuming (90 min / injection, the RI detector needs to balance each sample for about 1 h, isocratic elution detection). Even if volatile buffer salts are used for elution, the peak broadening and peak tailing caused by secondary interactions such as hydrophobic and ion pair interactions cannot be completely overcome, which affects integration and accurate quantification.

[0005] 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

[0006] In view of the above problems, the present invention discloses a method for detecting free PEG in PEGylated human erythropoietin, wherein free PEG is detected by high performance liquid chromatography, and the high performance liquid chromatography detection method comprises the following steps:

[0007] (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;

[0008] (2) Setting the chromatographic conditions: Use a C4 column and gradient elution. The elution procedure is as follows:

[0009] Time ％A ％B Initial 70 30 1.0 70 30 3.0 45 55 6.0 45 55 16.0 25 75 17.0 25 75 17.1 10 90 20.0 10 90 20.1 70 30 25.0 70 30

[0010] The flow rate of the mobile phase is 0.75-1.0 mL / min;

[0011] The column temperature is 60℃~70℃;

[0012] The sample chamber temperature is 2-8°C;

[0013] (3) Detect free PEG in the sample to be tested.

[0014] This method is convenient and quick. It can accurately quantify free PEG in PEG-EPO stock solution and preparation in a short time by using a specific ratio of acetonitrile in mobile phase B and elution gradient. It can completely separate PEG-EPO and PEG peaks with similar hydrophobic properties, and avoid the interference of P188 peak with similar hydrophobic properties. The mobile phase is simple to prepare and the sample does not need to be processed. The method is applicable to the detection of both stock solution and preparation. The minimum detection amount of PEG is about 0.03μg, and the five-point linear reference can accurately quantify free PEG in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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:

[0016] Figure 1 PEG-EPO stock solution specificity overlay;

[0017] Figure 2 PEG-EPO stock solution sample picture;

[0018] Figure 3 PEG-EPO formulation specificity overlay;

[0019] Figure 4 Sample image of PEG-EPO preparation;

[0020] Figure 5 Double logarithmic standard curves were drawn for 3 / 5 / 7.5 / 10 / 12.5 μg / ml PEG peak area and concentration;

[0021] Figure 6 Double logarithmic standard curves were drawn for 4 / 7.5 / 10 / 15 / 20 μg / ml PEG peak area and concentration;

[0022] Figure 7 Overlay of PEG-EPO stock solution and spiked samples;

[0023] Figure 8 Overlay of PEG-EPO formulation and spiked samples. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The present invention discloses a method for detecting free PEG in PEGylated human erythropoietin. The free PEG is detected by high performance liquid chromatography. The high performance liquid chromatography detection method comprises the following steps:

[0027] (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;

[0028] (2) Setting the chromatographic conditions: Use a C4 column and gradient elution. The elution procedure is as follows:

[0029] Time ％A ％B Initial 70 30 1.0 70 30 3.0 45 55 6.0 45 55 16.0 25 75 17.0 25 75 17.1 10 90 20.0 10 90 20.1 70 30 25.0 70 30

[0030] The flow rate of the mobile phase is 0.75-1.0 mL / min;

[0031] The column temperature is 60℃~70℃;

[0032] The sample chamber temperature is 2-8°C;

[0033] (3) Detect free PEG in the sample to be tested.

[0034] Furthermore, in step (2),

[0035] The flow rate of the mobile phase was 0.8 mL / min;

[0036] The column temperature was 65°C;

[0037] The sample chamber temperature was 5°C.

[0038] Furthermore, the injection volume of the sample to be tested is 10 to 200 μl.

[0039] Furthermore, the sample to be tested is a PEG-EPO stock solution, and the injection volume is 15 μl.

[0040] Furthermore, the sample to be tested is a PEG-EPO preparation, and the injection volume is 200 μl.

[0041] Furthermore, in step (3), an evaporative light scattering detector (ELSD) is used to detect the sample to be tested, and the parameters of the evaporative light scattering detector are set as follows:

[0042]

[0043]

[0044] Further, the parameters of the evaporative light scattering detector are set as follows:

[0045] Gas Type: Nitrogen Gas flow: 1L / min Gain: 1 led 100％ Atomizer temperature: 90℃ Drift tube temperature: 100℃

[0046] Further, the evaporative light scattering detector parameters are set as:

[0047] Gas Type: Compressed air Gas flow: 2.5L / min Gain: 1 led Not adjustable Atomizer temperature: 95℃ Drift tube temperature: 95℃ .

[0048] Further, the method also includes determining the content of free PEG, and the determination comprises the following steps:

[0049] (a) Prepare water and free PEG standard solutions with mass concentrations of 3.0 μg / ml, 5.0 μg / ml, 7.5 μg / ml, 10.0 μg / ml, and 12.5 μg / ml;

[0050] (b) detecting the free PEG standard solutions of different concentrations to obtain detection peak areas respectively;

[0051] (c) making a double logarithmic curve based on the free PEG content in the standard solution of different concentrations and the corresponding detection peak area;

[0052] (d) detecting free PEG in the sample to be tested to obtain a detection peak area of ​​free PEG;

[0053] (e) Substituting the detection peak area of ​​free PEG into the double logarithmic curve in step (c) to calculate the content of free PEG in the sample to be tested.

[0054] Further, the method also includes determining the content of free PEG, and the determination comprises the following steps:

[0055] (a) preparing water and free PEG standard solutions with mass concentrations of 3.0 μg / ml, 4.0 μg / ml, 7.5 μg / ml, 10.0 μg / ml, 15 μg / ml, and 20 μg / ml;

[0056] (b) detecting the free PEG standard solutions of different concentrations to obtain detection peak areas respectively;

[0057] (c) making a double logarithmic curve based on the free PEG content in the standard solution of different concentrations and the corresponding detection peak area;

[0058] (d) detecting free PEG in the sample to be tested to obtain a detection peak area of ​​free PEG;

[0059] (e) Substituting the detection peak area of ​​free PEG into the double logarithmic curve in step (c) to calculate the content of free PEG in 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] (1) Instruments and equipment

[0063] name factory model High performance liquid chromatography Waters ACQUITY Arc ELSD detector AllChrom ELSD 6100 High performance liquid chromatography Agilent 1260 ELSD detector Agilent G4260B Precision balance Mettler Toledo MS105DU Balance Mettler Toledo MS204TS

[0064] (2) Reagents and consumables:

[0065] DP: (Drug Product) preparation, mainly used in the field of biopharmaceuticals, to prepare and dilute the DS (Drug Substance) biological stock solution, add excipients, aseptically fill and package it into finished products.

[0066] In this application, "DP PEG-EPO" refers to PEG-EPO preparations, and "DS PEG-EPO" refers to PEG-EPO stock solutions. In addition, in this application, PEG-EPO stock solutions are sometimes referred to as "stock solutions" and represented by DS; PEG-EPO stock solutions are diluted and excipients are added to prepare PEG-EPO preparations, which are sometimes referred to as "preparations" and represented by DP.

[0067] Sample Information:

[0068]

[0069] Reagents:

[0070]

[0071]

[0072] Consumables:

[0073] name factory model Part Number Chromatographic columns Waters XBridge Protein BEH C4 186004505

[0074] (3) Mobile phase preparation:

[0075] 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.

[0076] Mobile phase B (0.1% TFA-84% acetonitrile solution): add 1680 ml of acetonitrile and 320 ml of ultrapure water into a 2 L reagent bottle, then add 2 ml of TFA and mix well, and perform ultrasonic degassing for 15 min.

[0077] (4) Sample and standard curve sample preparation:

[0078] Dilution (10% acetonitrile): Add 10 ml acetonitrile to 90 ml water and mix well.

[0079] PEG mother solution (1.0 mg / ml): Accurately weigh 100.3 mg of PEG solid powder into a 100 ml volumetric flask, add ultrapure water to dissolve and dilute to 100 ml, and shake well.

[0080] 50μg / ml PEG: 100μl PEG stock solution + 1900μl 10% acetonitrile

[0081] Table 1 Standard solution dilution table

[0082] Standard curve sample concentration Add 50 μg / ml PEG (μl) Add 10% acetonitrile (μl) Final concentration (μg / ml) 20.0μg / ml 400 600 20.0 15.0μg / ml 300 700 15.0 12.5μg / ml 250 750 12.5 10.0μg / ml 200 800 10.0 7.5 μg / ml 150 850 7.5 5.0μg / ml 100 900 5.0 4.0μg / ml 80 920 4.0 3.0μg / ml 60 940 3.0

[0083] Stock solution and spiked samples:

[0084] PEG-EPO stock solution was directly injected;

[0085] The preparation of PEG-EPO stock solution spiked samples is shown in the table below. For the stock solution, the limit of PEG residue is less than 5μg / mg, and samples with 80%, 100%, and 120% of the stock solution spike level were prepared respectively, and 3 parallel samples were prepared for each spike level.

[0086] The “fortification level” refers to the percentage content corresponding to the specified limit of the component to be tested (free PEG) added to the sample to be tested.

[0087] Table 2 Preparation of PEG-EPO stock solution spiked samples

[0088] name Spiking level Add 50μl / ml PEG Amount (μl) Add stock solution volume (μl) DS PEG-EPO + 4.0 μg / ml PEG 80％ 4 50 DS PEG-EPO + 5.0 μg / ml PEG 100％ 5 50 DS PEG-EPO + 6.0 μg / ml PEG 120％ 6 50

[0089] PEG-EPO preparations and spiked samples:

[0090] PEG-EPO preparation was injected directly;

[0091] The preparation of PEG-EPO spiked samples is shown in the table below. For the preparation, the limit of PEG residue is less than 0.5μg / mg. Prepare samples with 50%, 100%, and 150% of the preparation spike level, and prepare 3 parallel samples for each spike level:

[0092] Table 3 Preparation of spiked samples

[0093] name Spiking level Add 50μl / ml PEG Amount (μl) Added dose (μl) DP PEG-EPO + 0.25 μg / ml PEG 50％ 1.5 300 DP PEG-EPO + 0.5 μg / ml PEG 100％ 3.0 300 DP PEG-EPO + 0.75 μg / ml PEG 150％ 4.5 300

[0094] (5) Method parameters

[0095] PEG-EPO stock solution injection volume: 15 μl, corresponding to 15 μl standard solution injection;

[0096] The injection volume of PEG-EPO preparation was 200 μl, corresponding to the injection volume of 10 μl of standard solution.

[0097] Table 4 Parameters of the best method

[0098]

[0099]

[0100] Table 5 ELSD detector method parameters

[0101]

[0102] Different instruments and detectors can achieve the same results by adjusting parameters and can be replaced by equivalent instruments or equipment.

[0103] (5) Experimental process and results

[0104] a) Specificity:

[0105] Experimental process:

[0106] PEG-EPO stock solution: transfer water, 5.0 μg / ml PEG standard solution, DS Buffer, and DS PEG-EPO to liquid phase vials, inject 15 μl into each vial, and record the spectrum;

[0107] PEG-EPO preparation: Take water, 10.0μg / ml PEG standard solution, DP Buffer, and DP PEG-EPO and transfer them to liquid phase vials. Make one injection for each vial. Inject 10μl of water and 10.0μg / ml PEG standard solution, 200μl of DP Buffer and DP PEG-EPO, and record the spectrum.

[0108] Experimental results:

[0109] The results show that blank and buffer (DS Buffer and DP Buffer) have no interference with PEG peak; protein peak does not affect PEG integration; PEG-EPO stock solution specific sample overlay is attached Figure 1 , the corresponding PEG-EPO stock solution sample is attached Figure 2 PEG-EPO preparation specific sample overlay is attached Figure 3 , the corresponding PEG-EPO preparation sample picture is attached Figure 4 .

[0110] b) Standard curve linearity and system suitability:

[0111] For PEG-EPO stock solution and PEG-EPO preparation, due to different protein concentrations and specifications, the acceptable standard limits of PEG residues formulated according to ICH regulations are different (stock solution less than 5μg / mg, preparation less than 10μg / mg). Based on the PEG response value and injection volume, two standard curve concentration ranges were formulated for the stock solution and preparation, respectively.

[0112] Experimental process:

[0113] PEG-EPO stock solution detection: For the standard curve, 15 μl of 3.0, 5.0, 7.5, 10.0, and 12.5 μg / ml PEG standard solutions were injected respectively. After the stock solution sample run was completed, 15 μl of 5.0 μg / ml standard solution was injected as a system suitability sample (quality control product).

[0114] PEG-EPO preparation detection: For the standard curve, 10 μl of 3.0, 4.0, 7.5, 10.0, 15, and 20 μg / ml PEG standard solutions were injected respectively. After the preparation sample run was completed, 10 μl of 10.0 μg / ml standard solution was injected as a system suitability sample (quality control product).

[0115] Experimental results:

[0116] A double logarithmic standard curve (hereinafter referred to as standard curve or linear curve) was made based on the free PEG content in the standard solution of different concentrations and the corresponding detection peak area. 2 A value greater than 0.98 indicates accurate quantification within the curve range. The detection concentration of the quality control product is calculated using the curve, and the recovery rate is calculated based on the ratio of the concentration to the theoretical concentration. The accuracy of the curve is further determined using the recovery rate range.

[0117] PEG-EPO stock solution: Standard curve R 2 =0.9970, greater than 0.98; the signal-to-noise ratio (S / N) value of the 3.0μg / ml PEG standard solution is approximately equal to 10, which meets the quantitative limit standard; the recovery rate of the quality control product is 100.0%, within the range of 90% to 110%; the recovery rate of the detected free PEG is also within the range of 90% to 110%. See the attached standard curve Figure 5 The test results are shown in Table 6.

[0118] Table 6 Corresponding standard curves and system suitability sample results for stock solution detection

[0119]

[0120] PEG-EPO preparation: Standard curve R 2 =0.9988, greater than 0.98; the S / N value of the 3.0μg / ml standard solution is greater than 3, meeting the detection limit standard; the recovery rate of the quality control product is 101.6%, within the range of 90% to 110%. See the attached standard curve Figure 6 The test results are shown in Table 7.

[0121] Table 7 Corresponding standard curves and system suitability sample results for preparation testing

[0122]

[0123] c) Accuracy and repeatability results:

[0124] Experimental process:

[0125] PEG-EPO stock solution: Take DS PEG-EPO and DS spiked samples, prepare three copies of each sample in parallel, inject one needle each, and inject 15μl; corresponding standard curve samples and system suitability samples for stock solution detection.

[0126] PEG-EPO preparation: Take DP PEG-EPO and DP spiked samples, prepare three copies of each sample in parallel, inject one needle each, and inject 200 μl; inject the corresponding standard curve samples and system suitability samples for the corresponding preparation test.

[0127] Experimental results:

[0128] PEG-EPO stock solution: 80% spiked level content, RSD (relative standard deviation)% is 3.59%; 100% spiked level content RSD% is 4.07%; 120% spiked level content RSD% is 3.20%; each concentration spiked level content RSD% ≤ 15.0%. The spiked recovery of different concentrations of the stock solution is 104.9% to 124.5%, which is within the range of 70% to 130%. See the attached sample and the overlay of each spiked sample. Figure 7 The results are shown in Table 8.

[0129] In the following table, ND stands for “not detected” and NA stands for “not applicable”.

[0130] Table 8 Test results of stock solution and spiked samples

[0131]

[0132]

[0133] PEG-EPO preparation: RSD% of preparation content is 11.74%, RSD% of 50% spiked level content is 5.13%; RSD% of 100% spiked level content is 3.86%; RSD% of 150% spiked level content is 3.30%; RSD% of spiked level content of each concentration is ≤15.0%. The recovery rate of spiked preparation at different concentrations is 102.8% to 115.5%, which is within the range of 70% to 130%. See attached for the sample and the overlay of each spiked sample Figure 8 The results are shown in Table 9.

[0134] Table 9 Test results of preparations and spiked samples

[0135]

[0136]

[0137] 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 free PEG in PEGylated human erythropoietin, characterized in that: Free PEG is detected by high performance liquid chromatography, and the 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 0.75-1.0 mL / min; The column temperature is 60℃~70℃; The sample chamber temperature is 2-8°C; (3) Detect free PEG in the sample to be tested.

2. The method for detecting free PEG in PEGylated human erythropoietin according to claim 1, characterized in that: In the step (2), The flow rate of the mobile phase was 0.8 mL / min; The column temperature was 65°C; The sample chamber temperature was 5°C.

3. The method for detecting free PEG in PEGylated human erythropoietin according to claim 1, characterized in that: The injection volume of the sample to be tested is 10 to 200 μl.

4. The method for detecting free PEG in PEGylated human erythropoietin according to claim 3, characterized in that: The sample to be tested is PEG-EPO stock solution, and the injection volume is 15 μl.

5. The method for detecting free PEG in PEGylated human erythropoietin according to claim 3, characterized in that: The sample to be tested is a PEG-EPO preparation, and the injection volume is 200 μl.

6. The method for detecting free PEG in PEGylated human erythropoietin according to claim 5, characterized in that: In the step (3), an evaporative light scattering detector (ELSD) is used to detect the sample to be tested, and the parameters of the evaporative light scattering detector are set as follows: 。 7. The method for detecting free PEG in PEGylated human erythropoietin according to claim 6, characterized in that: The parameters of the evaporative light scattering detector are set as follows: 。 8. The method for detecting free PEG in PEGylated human erythropoietin according to claim 6, characterized in that: The parameters of the evaporative light scattering detector are set as follows: 。 9. The method for detecting free PEG in PEGylated human erythropoietin according to claim 6, characterized in that: Also included is a determination of the content of free PEG, the determination comprising the following steps: (a) preparing water and free PEG standard solutions with concentrations of 3.0 μg / ml, 5. μg / ml, 7.5 μg / ml, 10.0 μg / ml, and 12.5 μg / ml; (b) detecting the free PEG standard solutions of different concentrations to obtain detection peak areas respectively; (c) making a double logarithmic standard curve based on the free PEG content in the standard solutions of different concentrations and the corresponding detection peak area; (d) detecting free PEG in the sample to be tested to obtain a detection peak area of ​​free PEG; (e) Substituting the detection peak area of ​​free PEG into the double logarithmic curve in step (c) to calculate the content of free PEG in the sample to be tested.

10. The method for detecting free PEG in PEGylated human erythropoietin according to claim 6, characterized in that: Also included is a determination of the content of free PEG, the determination comprising the following steps: (a) preparing water and free PEG standard solutions with concentrations of 3.0 μg / ml, 4.0 μg / ml, 7.5 μg / ml, 10.0 μg / ml, 15 μg / ml, and 20 μg / ml; (b) detecting the free PEG standard solutions of different concentrations to obtain detection peak areas respectively; (c) making a double logarithmic standard curve based on the free PEG content in the standard solutions of different concentrations and the corresponding detection peak area; (d) detecting free PEG in the sample to be tested to obtain a detection peak area of ​​free PEG; (e) Substituting the detection peak area of ​​free PEG into the double logarithmic curve in step (c) to calculate the content of free PEG in the sample to be tested.

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