CN117907450A

CN117907450A - Method for detecting non-activated impurities in amine-activated polyethylene glycol

Info

Publication number: CN117907450A
Application number: CN202211282523.0A
Authority: CN
Inventors: 贺娇; 朱丹丹; 何平; 赵宣
Original assignee: Tianjin Jenkem Technology Co Ltd
Current assignee: Tianjin Jenkem Technology Co Ltd
Priority date: 2022-10-19
Filing date: 2022-10-19
Publication date: 2024-04-19

Abstract

The invention relates to a method for detecting inactive impurities in amine active polyethylene glycol, taking a sample to be detected, adding a dodecylamine solution for dissolution and derivatization, and taking the sample as a liquid to be detected after the reaction is complete; detecting by high performance liquid chromatography; the high performance liquid chromatography adopts the 120A pore size octadecylsilane chemically bonded silica gel as a stationary phase, water as a mobile phase A and methanol as a mobile phase B for gradient elution, and the high performance liquid chromatography adopts an evaporative light scattering detector for detection.

Description

Method for detecting non-activated impurities in amine-activated polyethylene glycol

Technical Field

The invention belongs to the technical field of chemical analysis, and particularly relates to a method for detecting non-activated impurities in amine-activated polyethylene glycol.

Background

Polyethylene glycol (PEG) and its derivatives are synthetic polymers with wide application in the biopharmaceutical industry, forming conjugates with peptides, proteins, lipids, oligonucleotides and small molecule drugs. The polyethylene glycol drug can improve the pharmacokinetic characteristics of the polyethylene glycol drug while retaining the original bioactivity of the polyethylene glycol drug, and improve the properties of water solubility, biocompatibility, stability and the like of the drug. Among these, PEGNHS esters are amine-reactive pegylation reagents that can react through primary amine groups of nucleophilic attack drugs to form amide linkages and release NHS. The existing amine-activated polyethylene glycol may contain a small amount of impurities, which have a structure similar to that of the activated polyethylene glycol, are difficult to separate, and have an influence on the quality thereof, and in order to control the quality of the amine-activated polyethylene glycol, a suitable method is required to separate the amine-activated polyethylene glycol from the impurities thereof.

Among these PEGNHS esters are common amine-active polyethylene glycols, which have a wide variety of configurations, wherein the y-type PEG NHS esters have a larger steric structure, such as exemplified by the following:

Compared with linear PEGNHS, the y-type PEGNHS has a plurality of end groups, further has a plurality of drug connection points, can load a plurality of drug molecules, is widely applied to PEGylation modification of polypeptides and small molecule drugs, has strong water solubility, enables proteins and other biological preparations containing lysine to be simply and effectively modified,

The prior art uses a number of methods, such as (ANALYTICAL METHODS TO QUALIFY AND QUANTIFY PEG AND PEGYLATED Biopharmaceuticals) developed an online 2D LC system to detect free polyethylene glycol reagent (Methyl-PEG-NHS-Easter) and pegylated protein, the first dimension using a special size exclusion column, capturing PEG in a 500 μl loop, and then loading the circulating content onto the C8 column of the assay using a second dimension gradient elution. DAD and CAD detector detection were used. But it requires high equipment and analysis costs.

The prior art (Isoform separation and binding site determination ofmono-PEGylated lysozyme with pH gradient chromatography) discloses a method for separating 5kDa and 10kDa m-PEG aldehyde modified monopolyethylene glycol lysozyme isomers by linear pH gradient chromatography, separating monopolyethylene glycol lysozyme subtypes by linear pH gradient on a cation exchange column, and analyzing the results by MALDI-MS, wherein the method has high requirement on instruments and cannot be applied to chromatographic methods.

Prior art (Towards improved characterisation of complex polyethylene glycol excipients using supercritical fluid chromatography-evaporative light scattering detection-mass spectrometry and comparison with size exclusion chromatography-triple detection array) describes functionalized polyethylene glycol (PEG) 2000 analogues using Supercritical Fluid Chromatography (SFC) in combination with Evaporative Light Scattering Detector (ELSD) and Mass Spectrometer (MS), m-PEG-OH2000, m-PEG-cm 2000 and cm-PEG-cm 2000 analogues (where cm=och ₃, cm=och2cooh), which disclose selective enhancement of homologous sequences in specific PEG analogues by SFC, more detailed assessment of material quality, but with SFC-ELSD-MS methods the instrument structure is complex, the number of mobile phases that can be selected is limited, and the flexibility of the method is not as good as liquid chromatography.

In the prior art (Evaluation of supercritical fluid chromatography for testing of PEG adducts in pharmaceuticals), supercritical Fluid Chromatography (SFC) is adopted, polyethylene glycol mixture with average molecular weight of 400-6000 Da is detected and separated by evaporative light scattering, a chromatographic column is BEH (ethyl ethylene bridging hybridization) column (150 mm multiplied by 3mm,1.7 m), a mobile phase is a mixture of carbon dioxide and methanol, PEG and PEG adducts can be separated from drug-excipient interaction by the method, the structural difference of the analyzed target substances is large, and the separation of amine active polyethylene glycol with similar structure and impurities of the amine active polyethylene glycol cannot be ensured.

The detection method has the advantages of simple operation, accurate result, high stability and good precision, and can well control the quality of the amine active polyethylene glycol.

Disclosure of Invention

The invention aims to provide a detection method of amine-activated polyethylene glycol, which solves the problem that the prior art proposed in the background art is almost blank in the detection method of polyethylene glycol capable of separating and detecting amine activity and impurities with similar structures.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

The invention provides a method for detecting inactive impurities in amine active polyethylene glycol, which comprises the steps of taking a sample to be detected, adding a dodecylamine solution for dissolution and derivatization, and taking the sample as a liquid to be detected after the reaction is completed; detecting by high performance liquid chromatography; the high performance liquid chromatography adopts 120A pore size octadecylsilane chemically bonded silica as stationary phase, water as mobile phase A, and methanol as mobile phase B for gradient elution, and adopts an Evaporative Light Scattering Detector (ELSD) for detection to obtain inactive impurity content.

The structure of the amine-activated polyethylene glycol is as follows:

the structure of the inactive impurities is as follows:

A is C or N;

R ₁ is a linking group selected from: single bond, C ₁-C₆ alkylene;

R ₂、R₃ is independently selected from: c ₁-C₆ alkylene, -O-, -S-, -C (O) -, -C (O) O-

/>Or a combination thereof;

R ₄ is selected from: -H, C _1-6 straight/branched alkyl;

The molecular weight of the PEG is 10 to 80KDa, for example, the molecular weight of the PEG may be 10KDa, 15KDa, 20KDa, 25KDa, 30KDa, 35KDa, 40KDa, 45KDa, 50KDa, 55KDa, 60KDa, 65KDa, 70KDa, 75KDa, 80KDa.

In the invention, the active ingredients in the sample are derived by adopting the dodecyl amine, so that the separation degree of the sample and the impurities is increased, and the separation degree of the sample and the impurities meets the requirements.

Further, the amine-activated polyethylene glycol is Y-NHS-40K, and the structure is as follows:

preferably, the PEG has a molecular weight of 40kDa

Further, the inactive impurity is Y-COOH-40K, and the structure is:

Preferably, the amine-activated polyethylene glycol has the same PEG molecular weight as the inactive impurities.

Further, the dodecylamine solution is a dodecylamine methanol solution;

Preferably, the concentration of the dodecyl amine in the dodecylamine methanol solution is 1-5 mg/mL, which can be 1mg/m, 2mg/m, 3mg/m, 4mg/m or 5mg/m, preferably 3mg/mL;

further, the temperature of the stationary phase is 35-40 ℃;

Preferably, the stationary phase temperature includes, but is not limited to: 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃,40 ℃, preferably 40 DEG C

Further, the flow rate of the mobile phase of the high performance liquid chromatography is 0.8-1.2 mL/min,

Preferably, the flow rate includes, but is not limited to: 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, preferably at a flow rate of 1.0mL/min;

furthermore, the sample injection amount of the high performance liquid chromatography is 8-10 mu L;

preferably, the sample injection amount is 8. Mu.L, 8.5. Mu.L, 9. Mu.L, 9.5. Mu.L, 10. Mu.L, preferably 10. Mu.L;

Further, the chromatographic column parameters of the high performance liquid chromatography using octadecylsilane chemically bonded silica as a stationary phase are as follows: 4.6 x 250mm,5 μm; the pore diameter is 120A.

Further, in the gradient elution, the mobile phase A and the mobile phase B are calculated according to the volume percentage, and the elution procedure is as follows:

0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30;

0-2 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 70:30;

2-5 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 24:76 linearly;

5-20 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 20:80 linearly;

The volume ratio of the mobile phase A to the mobile phase B is gradually changed into 15:85 linearly for 20-21 min;

21-22 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 5:95 linearly;

22-23 min, the volume ratio of the mobile phase A to the mobile phase B is 5:95, and isocratic elution is kept;

23-24 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 70:30 linearly;

And (3) performing isocratic elution after 24-30 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 70:30.

Furthermore, the method adopts an external standard logarithmic linear method to calculate the content and carries out linear verification on the inactive impurities.

In the invention, since the peak area and the concentration of the evaporative light scattering detector are not directly linear in quantification, the logarithm of the peak area and the logarithm of the concentration are linear, and the external standard linear method is adopted to quantify impurities under the condition of having a reference substance. Thereby better solving the detection problem and the content control problem of the non-activated impurity Y-COOH-40K in the sample.

The invention also provides an application of the detection method of the inactive impurities in the amine active polyethylene glycol in the quality control of the amine active polyethylene glycol.

The invention has the beneficial effects that:

The Y-NHS-40K sample is difficult to completely separate from the Y-COOH-40K impurity with similar structure on a C18 chromatographic column after being directly dissolved, and the sample has no ultraviolet absorption and cannot be detected on an ultraviolet detector. According to the invention, the sample is derived by adopting the dodecyl amine, the derived Y-NHS-40K and Y-COOH-40K impurities are separated by adopting the 120A pore size octadecyl silane bonded silica gel column, the Y-NHS-40K sample and the Y-COOH-40K impurities can be well separated by adopting an Evaporative Light Scattering Detector (ELSD) for detection, and the ELSD detector can accurately quantify the derived sample. In the aspect of quantification, since the peak area and the concentration of the evaporative light scattering detector are not directly linear, the logarithm of the peak area and the logarithm of the concentration are linear, and the external standard linearity is adopted to quantify impurities under the condition of a reference substance. The method can conveniently separate the Y-NHS-40K sample from the non-activated impurity Y-COOH-40K, thereby effectively controlling the quantity of the non-activated impurity Y-COOH-40K, having high sensitivity and good stability, being capable of quickly and conveniently determining the content of the Y-COOH-40K and having wide application prospect in the quality control of the amine-activated polyethylene glycol.

Drawings

FIG. 1 shows a Y-NHS-40K pattern;

FIG. 2 shows a 3mg/mL solution of dodecylamine (blank solution);

FIG. 3 shows a Y-COOH-40K pattern;

FIG. 4 shows a Y-COOH-40K LOQ pattern (0.025 mg/mL)

FIG. 5 shows a Y-NHS-40K+0.5% Y-COOH-40K pattern;

FIG. 6 shows a linear relationship of Y-COOH-40K.

FIG. 7 shows a map of sample 1 of comparative example 1;

FIG. 8 is a graph showing sample 2 of comparative example 1;

FIG. 9 shows a Y-COOH-40K pattern using the chromatographic method of comparative example 2;

FIG. 10 shows the Y-NHS-40K pattern by chromatography according to comparative example 2.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

In the present invention, the term "stationary phase" is a phase that will be immobilized in chromatographic separation, resulting in retention of a sample.

In the present invention, the term "mobile phase" refers to a substance that carries a component to be measured forward during chromatography, referred to as a mobile phase. And the other phase is in an equilibrium state with the stationary phase and drives the sample to move forward.

In the present invention, the term "separation degree" is also called resolution, and in order to determine the separation condition of a separation substance on a chromatographic column in the chromatographic column, the separation degree is generally used as an index of total separation efficiency of the column, and is denoted by R. R is equal to the ratio of the difference between the retention time of adjacent chromatographic peaks to the peak-to-average value of the two chromatographic peaks, and represents the separation degree of the two adjacent peaks, and the larger R represents the better separation of the two adjacent components. Generally when R <1, the two peaks overlap partially; when r=1.0, the degree of separation can reach 98%; when r=1.5, the degree of separation can reach 99.7%. R=1.5 is typically used as an indicator that two adjacent components have been completely separated. When r=1, called 4σ separation, the two peaks are substantially separated, the bare peak area is 95.4%, and the inner peak groups overlap by about 2%. R=1. At 5, referred to as 6σ separation, the bare peak area was 99.7%. R is more than or equal to 1.5 and is called complete separation, and the rule of Chinese pharmacopoeia is that R should be more than 1.5.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are not specific to the manufacturer and are commercially available conventional products

Example 1 HPLC detection of Y-NHS-40K

Preparing a sample to be tested:

Test solution: a50 mg sample of Y-NHS-40K (purity: more than or equal to 95% of Beijing key Kai technology Co., ltd.) is precisely weighed and placed in a 5mL measuring flask, and a diluent is added to completely dissolve the sample, and the sample is uniformly shaken to a constant volume to be used as a Y-NHS-40K sample solution (the concentration of Y-NHS-40K is 10 mg/mL).

A diluent: 3mg/mL of dodecylamine in methanol.

Blank solution: a3 mg/mL solution of dodecylamine in methanol was used as a blank solution.

Chromatographic conditions:

Instrument: shimadzu LC-20 high performance liquid chromatograph

A detector: ALLChromeELSD 6100 evaporative light scattering detector

Chromatographic column: octadecylsilane chemically bonded silica as filler (ChromeCore 120 C18 4.6*250mm;5 μm)

Mobile phase a: water was used as mobile phase a.

Mobile phase B: methanol is used as mobile phase B

Gradient elution was performed as follows:

TABLE 1 gradient elution procedure

Time (min)	Mobile phase a (v%)	Mobile phase B (v%)
			0.01	70	30
2	70	30
			5	24	76
20	20	80
			21	15	85
22	5	95
			23	5	95
24	70	30
			30	-	stop

Column temperature: 40 ℃;

Flow rate: 1.0mL/min;

Sample injection amount: 10. Mu.L;

work station: LC-Solution;

The test solution was analyzed by the chromatographic condition method described above.

Detection result

The chromatogram is shown in figure 1, three peaks appear in the sample solution, the peak emergence time of the peak responding to the highest is 23.5-24.5 min, the peak emergence time of the peak responding to the second highest is 18-19 min, and the peak emergence time of the peak responding to the third highest is 15.7-17.1 min.

The blank solution was analyzed according to the chromatographic conditions method described above.

The chromatogram of the blank solution is shown in FIG. 2, and no chromatographic peak appears in FIG. 2, which shows that 3 chromatographic peaks in FIG. 1 are substances in the solution to be tested after the Y-NHS-40K sample is derived.

Example 2 HPLC detection of Y-COOH-40K

Preparing a sample to be tested:

preparation of Y-COOH-40K at different concentrations:

Preparing standard stock solution of a reference substance: weighing 25mg to 10mL volumetric flask of Y-COOH-40K (Beijing key Kai technology Co., ltd., purity: not less than 95%) sample, adding diluent (3 mg/mL dodecylamine/methanol solution) to dissolve the sample completely, fixing the volume to scale, mixing uniformly, and preparing control solution step by step as control stock solution according to the following linear solution preparation method. (Y-COOH-40K concentration was 2.5 mg/mL)

1. Control solution 1 (Y-COOH-40K concentration 0.5 mg/mL)

Precisely measuring 2mL of reference stock solution, placing in a 10mL measuring flask, adding diluent (3 mg/mL of dodecylamine/methanol solution) to a scale, and shaking uniformly.

2. Control solution 2 (Y-COOH-40K concentration 0.25 mg/mL)

Precisely measuring 1mL of reference stock solution, placing in a 10mL measuring flask, adding diluent (3 mg/mL of dodecylamine/methanol solution) to a scale, and shaking uniformly.

3. Control solution 3 (Y-COOH-40K concentration 0.125 mg/mL)

Precisely measuring 5mL of reference substance solution 2, placing in a 10mL measuring flask, adding diluent (3 mg/mL of dodecylamine/methanol solution) to a fixed volume to scale, and shaking uniformly.

4. Control solution 4 (Y-COOH-40K concentration 0.05 mg/mL)

Precisely measuring 2mL of reference substance solution 2, placing in a 10mL measuring flask, adding diluent (3 mg/mL of dodecylamine/methanol solution) to a fixed volume to scale, and shaking uniformly.

5. Control solution 5 (Y-COOH-40K concentration 0.025 mg/mL)

Precisely measuring 1mL of reference substance solution 2, placing in a 10mL measuring flask, adding diluent (3 mg/mL of dodecylamine/methanol solution) to a fixed volume to scale, and shaking uniformly.

Chromatographic conditions: same as in example 1

Detection result

The control solution 3 was analyzed under the chromatographic conditions of example 1, and the analysis result is shown in FIG. 3, wherein when the concentration of the sample is 0.125mg/mL of Y-COOH-40K, the chromatographic peak time is 15.7-17.2 min, and the response value reaches 54mv.

The control solution 6 was analyzed under the chromatographic conditions of example 1, and the analysis result is shown in FIG. 4, wherein the peak chromatographic time is 15.7-17.2 min and the response value reaches 20.2mv when the concentration of the Y-COOH-40K sample is 0.025 mg/mL.

From the comprehensive views of FIGS. 1 to 4, the chromatographic peak with the peak time of 15.7 to 17.2min is the peak of Y-COOH-40K, and the two chromatographic peaks with the peak time of 23.5 to 24.5min and 18 to 19min are the chromatographic peaks after the derivatization of Y-NHS-40K, and as can be seen from FIG. 1, the method can completely separate Y-COOH-40K with similar structure from Y-NHS-40K, and the separation degree can reach complete separation.

Example 3 detection of Y-NHS-40K+0.5% Y-COOH-40K

Preparing a sample to be tested:

To the sample solution (concentration of Y-NHS-40K: 10 mg/mL) in example 1 was added Y-COOH-40K at a volume fraction of 0.5%.

Chromatographic conditions:

Detection was performed as described in example 1

Detection result:

the results are shown in FIG. 5, wherein the peak time is 15.7-17.2 min and is Y-COOH-40K, the peak time is 18-19 min, and the two chromatographic peaks are the chromatographic peaks after Y-NHS-40K derivatization, which show that the method can effectively separate Y-NHS-40K from Y-COOH-40K.

Comparative example 1 sample was not derived

Preparing a sample to be tested:

sample 1: the sample of Y-COOH-40K was weighed 25mg into a 10mL volumetric flask, and the other steps were conducted in accordance with the procedure of reference example 2, except that the steps of example 2 were different: the diluent is a pure solution of methanol, i.e. the sample is not derivatized.

Sample 2: the procedure of example 2 was followed except for weighing about 25mg of M-CM-20K into a 10mL volumetric flask and the other steps were conducted in accordance with the procedure of example 2, except that: the diluent is a pure solution of methanol, i.e. the sample is not derivatized. Wherein M-CM-20K is a common impurity in Y-NHS-40K, and refers to a compound with a methyl group at one end (M refers to methyl group) and a carboxyl group at one end (CM refers to carboxyl group) and with a molecular weight of 20K, and Y-COOH-40K can be better quantified by separating from M-CM-20K.

Sample 1 and sample 2 were each tested using the following chromatographic conditions, the results for sample 1 are shown in FIG. 7, and the results for sample 2 are shown in FIG. 8.

Chromatographic conditions:

Instrument: shimadzu LC-20 high performance liquid chromatograph

A detector: ALLChromeELSD 6100 evaporative light scattering detector

Mobile phase a: water is used as mobile phase A

Mobile phase B: methanol is used as mobile phase B

TABLE 2 gradient elution conditions

Time (min)	Mobile phase a (v%)	Mobile phase B (v%)
			0	70	30
2	70	70
			12	20	80
18	17	83
			23	5	95
28	5	95
			30	70	30
40	-	Stop

Column temperature: 40 ℃;

Flow rate: 1.0mL/min;

Sample injection amount: 10. Mu.L;

work station: LC-Solution;

Detection result:

As can be seen from the peak time of the chromatographic peaks of FIGS. 7 and 8, the peak time of sample 1 is 20.8 min-22.5 min, the peak time of sample 2 is 21.5 min-22.75 min, and the retention time of Y-COOH-40K and common impurity M-CM-20K are coincident, which means that Y-COOH-40K cannot be separated from other impurities in the sample if the sample is not derived.

Comparative example 2 comparison of different elution conditions

Preparing a sample to be tested:

Sample 3: the sample Y-COOH-40K25mg to 10mL was weighed into a volumetric flask and prepared to a concentration of 2.5mg/mL by the method of example 2.

Sample 4: the sample Y-NHS-40K was weighed into a volumetric flask of about 50mg to 5mL and prepared as described in example 1 to give a 10mg/mL sample.

Sample 3 and sample 4 were tested by the following chromatographic method:

Instrument: shimadzu LC-20 high performance liquid chromatograph

A detector: ALLChromeELSD 6100 evaporative light scattering detector

Mobile phase a: water is used as mobile phase A

Mobile phase B: methanol is used as mobile phase B

TABLE 3 gradient elution conditions

Time (min)	Mobile phase a (v%)	Mobile phase B (v%)
			0.01	70	30
2	70	30
			5	25	75
18	20	80
			21	15	85
22	5	95
			23	5	95
24	70	30
			30	—	Stop

Column temperature: 40 ℃;

Flow rate: 1.0mL/min;

Sample injection amount: 10. Mu.L;

work station: LC-Solution;

The results are shown in FIG. 9 and FIG. 10, FIG. 9 shows the chromatogram of sample 3, FIG. 10 shows the chromatogram of sample 4, the retention time of Y-COOH-40K in FIG. 9 is 15.5 min-18.1 min, and the retention times of 3 characteristic peaks of Y-NHS-40K in FIG. 10 are 13.25 min-15 min, 18 min-19 min, and 22.5-24 min, which indicate that under the mobile phase gradient, Y-COOH-40K cannot be completely separated from other impurities in the main peak.

Example 4 method verification

Preparing a reference substance solution:

The preparation method of the Y-COOH-40K reference solution is the same as that of example 2, and reference solutions 1 to 5 are obtained;

limit of quantitation (LOQ) solution: as can be seen from FIG. 4, the control solution of 0.025mg/mL is a limit of quantitation (LOQ) solution.

Establishment of a standard curve:

The linear solution control of each concentration was tested according to the chromatographic conditions described above and subjected to linear regression, the results and linear equations are shown in Table 4 and FIG. 6.

In the invention, since the peak area and the concentration of the evaporative light scattering detector are not directly linear, the logarithm of the peak area and the logarithm of the concentration are linear, and the external standard linearity is adopted to quantify impurities under the condition of a reference substance

Table 4 Linear results of Y-COOH-40K

As can be seen from FIG. 6, Y-COOH-40K was in the range of 0.025-0.5 mg/mL, the response was linear, and R ² was 0.999. The linearity of the analysis method was demonstrated to be good.

And (3) recovery rate detection:

the concentration of 10mg/mL of the sample (Y-NHS-40K) was taken as 100%, and Y-COOH-40K samples having mass concentrations of 1.25%, 0.5% and 0.25% were prepared.

As shown in table 5:

table 5 recovery sample formulation:

Weighing 4 parts of 50mg Y-NHS-40K, sequentially adding 0mL, 2.5mL, 1mL and 0.5mL of Y-COOH-40K reference substances with mass concentration of 0.25mg/mL, diluting with 3mg/mL of dodecylamine/methanol solution, and fixing the volume into a 5mL volumetric flask after diluting to obtain a liquid to be detected, wherein the mass concentration of the Y-COOH-40K of the 4 parts of liquid to be detected is as follows: 0% (sample blank), 1.25%, 0.5%, 0.25% and 4 parts of the liquid to be tested are detected by the chromatographic method of example 1, 30 minutes of chromatograms are recorded, and the recovery rates of Y-COOH-40K with different concentrations are calculated. The formulation of the liquid to be tested is shown in Table 5, and the sample recovery rate results are shown in FIG. 6:

TABLE 5 formulation of the test solution

TABLE 6 recovery results

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A detection method of inactive impurities in amine active polyethylene glycol is characterized in that a sample to be detected is taken, a dodecylamine solution is added for dissolution and derivatization, and the sample is taken as a liquid to be detected after the reaction is completed;

detecting by high performance liquid chromatography; the high performance liquid chromatography adopts octadecylsilane chemically bonded silica with 120A aperture as a stationary phase, water as a mobile phase A and methanol as a mobile phase B for gradient elution, and the high performance liquid chromatography adopts an evaporative light scattering detector for detection to obtain the content of inactive impurities;

The structure of the amine-activated polyethylene glycol is as follows:

the structure of the inactive impurities is as follows:

A is C or N;

R ₁ is a linking group selected from: single bond, C ₁-C₆ alkylene;

R ₂、R₃ is independently selected from: c ₁-C₆ alkylene, -O-, -S-, -C (O) -, -C (O) O- />Or a combination thereof;

R ₄ is selected from: -H, C _1-6 straight/branched alkyl;

The molecular weight of the PEG is 10-80 kDa.

2. The method of claim 1, wherein the amine-activated polyethylene glycol is Y-NHS-40K having the structure:

Preferably, the molecular weight of the PEG is 40KDa.

3. The method of claim 2, wherein the inactive impurity structure is:

4. The method of claim 1, wherein the dodecylamine solution is a dodecylamine methanol solution;

Preferably, the concentration of the dodecylamine in the dodecylamine methanol solution is 1-5 mg/mL, preferably 3mg/mL.

5. The method according to claim 1, wherein the stationary phase has a temperature of 35-40 ℃, preferably 40 ℃.

6. The method according to claim 1, wherein the flow rate of the mobile phase of high performance liquid chromatography is 0.8-1.2 mL/min, preferably 1.0mL/min.

7. The method according to claim 1, wherein the sample injection amount of the high performance liquid chromatography is 8-10 μl, preferably 10 μl.

8. The method of claim 1, wherein the high performance liquid chromatography column parameters with octadecylsilane chemically bonded silica as stationary phase are: 4.6 x 250mm,5 μm.

9. The method of claim 1, wherein in the gradient elution, mobile phase a and mobile phase B are in volume percent, and the elution procedure is:

0min, the volume ratio of the mobile phase A to the mobile phase B is 70:30;

10. The method of claim 1, wherein the method uses an external standard log-linear method to calculate the content and performs linear verification on the inactive impurities.