CN114252519B - Method for determining purity of nerve growth factor - Google Patents

Abstract

The invention provides a method for measuring the purity of nerve growth factor. The method uses reversed-phase chromatography. The chromatographic conditions include: (1) using a reversed-phase chromatographic column; (2) mobile phase: mobile phase A contains 0.06%-0.16 % volume ratio of formic acid aqueous solution; mobile phase B is (0.06% ~ 0.16%) formic acid aqueous solution - (40 ~ 90) acetonitrile / (10 ~ 60) isopropanol (V/V); or mobile phase B is (0.06% ~0.16%) formic acid aqueous solution - (80~100) acetonitrile/(0~20) water (V/V). The reverse chromatography method of the present invention can effectively separate the four shear forms of nerve growth factor and the main post-translational modification products. As a quality control indicator in the purification process of the original solution, it can better control the quality of nerve growth factor.

Description

A method for determining the purity of nerve growth factor

Technical Field

The invention relates to the technical field of purity determination, and in particular to a method for determining the purity of nerve growth factor by using reverse phase chromatography.

Background Art

Nerve growth factor (NGF) is one of the most important active ingredients in the nervous system. It can maintain the survival of sympathetic and sensory nerve cells, has the dual biological functions of neuronal nutrition and promoting neurite growth, and has an important regulatory effect on the development, differentiation, growth, regeneration and expression of functional characteristics of central and peripheral neurons, and plays an important role in protecting the normal function of the nervous system.

There are different molecular weight isomers in the stock solution of mouse nerve growth factor, but the existing methods (for example, the SEC-HPLC method used to determine the purity of the stock solution in the quality standard of mouse nerve growth factor included in the third volume of the 2015 edition of the Chinese Pharmacopoeia) do not have enough resolution to separate and accurately determine the different molecular weight isomers. Therefore, it is impossible to monitor the molecular weight isomers generated during the purification of the stock solution, and it is difficult to control the product quality.

In view of this, a method capable of effectively and accurately determining the purity of nerve growth factor is needed in the art.

Summary of the invention

The purpose of the present invention is to overcome the problems and defects in the prior art and provide a method for determining the purity of nerve growth factor. The method developed by the present invention utilizes reverse chromatography (such as RP-HPLC and/or RP-UPLC), and after selecting and adjusting various conditions and parameters in the chromatography, the nerve growth factor stock solution or the sample in the NGF purification process in the stock solution is separated and determined. The basic principle is to separate the various substances flowing through the chromatographic column according to their polarity differences. The greater the polarity, the shorter the retention time. Since each substance has a specific retention time under certain conditions, the position of the target peak can be known by the retention time on the chromatogram, and the purity of NGF in the NGF stock solution can be calculated by the peak area normalization method, and then the attribution of each chromatographic peak can be identified by mass spectrometry.

The method of the present invention can effectively separate nerve growth factor and its molecular weight isomers, and identify the attribution of each chromatographic peak by mass spectrometry. The method of the present invention can at least effectively separate and accurately determine the four shear forms and main post-translational modification forms of nerve growth factor, accurately monitor the purification process of the stock solution, and accurately control the quality of the product.

definition

As used herein, the term "chromatography" refers to any technique for chemically separating mixtures and components that relies on the selective attraction of components in a mixture to a stationary phase. Examples include adsorption chromatography, partition chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like.

As used herein, the term "RP-HPLC (Reversed Phase High Performance Liquid Chromatography)" refers to reverse phase high performance liquid chromatography. HPLC is used to separate compounds based on their polarity and the stationary phase of the column.

Reversed Phase Chromatography is an elution method used in liquid chromatography, in which the polarity of the mobile phase is significantly greater than that of the stationary phase.

As used herein, the term "RP-UPLC (Reversed Phase Ultra Performance Liquid Chromatography)" refers to reverse phase ultra-high performance liquid chromatography. UPLC is essentially the same as HPLC, except that: (1) small particles and high performance particulate stationary phases are used; HPLC columns usually use octadecyl silica gel bonded columns with a particle size of 5 μm, while ultra-high performance liquid chromatography columns have a particle size of 3.5 μm or even 1.7 μm; (2) the use of ultra-high pressure infusion pumps. As the particle size of the chromatographic column used decreases, the pressure generated during use naturally increases exponentially, so the infusion pump of liquid chromatography is also changed to an ultra-high pressure infusion pump; (3) sensitive detectors with high sampling speeds; (4) the use of low diffusion and low cross-contamination autosamplers equipped with needle injection probes and pressure-assisted injection technology; and (5) the overall system optimization design of the instrument.

As used herein, the term "mobile phase" refers to the solvent that is introduced into the column.

As used herein, the term "stationary phase" refers to a stationary phase used in chromatography for which the mobile phase components exhibit selective affinity.

In one aspect, the present invention provides a method for determining the purity of nerve growth factor, wherein the method uses reverse phase chromatography, and the chromatographic conditions include:

(1) Using a reverse phase chromatography column;

(2) Mobile phase: Mobile phase A is an aqueous solution containing 0.06% to 0.16% by volume of formic acid; mobile phase B is (0.06% to 0.16%) formic acid aqueous solution-(40 to 90) acetonitrile/(10 to 60) isopropanol (V/V); or mobile phase B is (0.06% to 0.16%) formic acid aqueous solution-(80 to 100) acetonitrile/(0 to 20) water (V/V). In a preferred embodiment of the present invention, mobile phase A is an aqueous solution containing 0.08% to 0.14% by volume of formic acid; mobile phase B is (0.08% to 0.14%) formic acid aqueous solution-(40 to 90) acetonitrile/(10 to 60) isopropanol (V/V); or mobile phase B is (0.08% to 0.14%) formic acid aqueous solution-(90 to 100) acetonitrile/(0 to 10) water (V/V).

In a preferred embodiment of the present invention, the mobile phase is: mobile phase A is 0.09% to 0.11% by volume formic acid aqueous solution, and mobile phase B is (0.09% to 0.11%) formic acid aqueous solution-50 acetonitrile/50 isopropanol (V/V).

More preferably, mobile phase A is 0.1% by volume formic acid in water, and mobile phase B is 0.1% by volume formic acid in water-50% acetonitrile/50% by volume isopropanol (V/V).

In a preferred embodiment of the present invention, the method adopts gradient elution, and the eluent is a mixture of mobile phase A and mobile phase B, wherein the volume ratio of mobile phase A in the mixture is reduced from an initial volume ratio of more than 78% to a volume ratio of less than 66%-76% to complete peak elution, and then the volume ratio of mobile phase A is further reduced to elute impurities; wherein the sum of the volume ratios of mobile phase A and mobile phase B in the mobile phase is 100%.

In a preferred embodiment of the present invention, the reverse phase chromatographic column uses tetraalkylsilane bonded silica as a filler. Preferably, the chromatographic column has a particle size of 1.7 μm to 3.5 μm, a length of 5 cm to 15 cm, and an inner diameter of 2.1 mm to 4.6 mm; more preferably, the reverse phase chromatographic column is 15 cm×4.6 mm, with a particle size of 3.5 μm.

In a preferred embodiment of the present invention, the reverse phase chromatography column is TSK Protein C4-300, Agilent AdvanceBio RP-mAb C4, or Waters Protein BEH C4.

In a preferred embodiment of the present invention, the reverse phase chromatography is reverse phase high performance liquid chromatography (RP-HPLC) or reverse phase ultra high performance liquid chromatography (RP-UPLC).

In a preferred embodiment of the present invention, the chromatographic conditions also include: a flow rate of 0.1ml/min to 1.2ml/min; preferably, the flow rate of the reversed-phase HPLC is 0.2ml/min to 1.2ml/min, preferably, the flow rate of the reversed-phase HPLC is 0.2ml/min to 0.8ml/min, more preferably, the flow rate of the reversed-phase HPLC is 0.2ml/min, 0.3ml/min, 0.4ml/min, 0.5ml/min, 0.6ml/min, 0.7ml/min, 0.8ml/min; the flow rate of the reversed-phase ultra-high performance liquid chromatography is 0.1ml/min to 0.5ml/min, preferably, the flow rate of the reversed-phase ultra-high performance liquid chromatography is 0.2ml/min to 0.4ml/min; more preferably, the flow rate of the reversed-phase ultra-high performance liquid chromatography is 0.2ml/min, 0.3ml/min, 0.4ml/min.

In a preferred embodiment of the present invention, the chromatographic conditions also include: a column temperature of 25°C to 70°C, a detection wavelength of 210nm to 285nm, and an injection volume of 1μg to 100μg; preferably, the column temperature is 30°C to 45°C, the detection wavelength is 275nm to 285nm, and the injection volume is 5μg to 35μg; more preferably, the column temperature is 35°C, the detection wavelength is 280nm, the flow rate is 0.2ml/min, and the injection volume is 10μg.

In a preferred embodiment of the present invention, the gradient elution procedure of the reverse phase high performance liquid chromatography (RP-HPLC) is as follows:

Time (minutes) A (volume %) B (volume %) 0 78-84 16-22 20 65-70 30-35 25 20-65 35-80 30 78-84 16-22 45 78-84 16-22

In one embodiment of the present invention, the gradient elution procedure of the reverse phase ultra performance liquid chromatography (RP-UPLC) is as follows:

Time (minutes) A (volume %) B (volume %) 0 83-84 16-17 twenty two 66 34 25 20 80 25.01 83-84 17 40 83-84 17

In a specific embodiment of the present invention, the existence form of the nerve growth factor includes but is not limited to: complete nerve growth factor, nerve growth factor with 1 amino acid missing at the C-terminus, nerve growth factor with 8 amino acids missing at the N-terminus, nerve growth factor with 1 amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus, and nerve growth factor glycosylation products and/or nerve growth factor oxidation products.

On the other hand, the present invention provides a nerve growth factor obtained according to the above method, wherein the existence form of the nerve growth factor includes but is not limited to: a complete nerve growth factor, a nerve growth factor with one amino acid missing from the C-terminus, a nerve growth factor with eight amino acids missing from the N-terminus, a nerve growth factor with one amino acid missing from the C-terminus and eight amino acids missing from the N-terminus, and a nerve growth factor glycosylation product and/or a nerve growth factor oxidation product. The present invention can measure the purity of the nerve growth factor. In some embodiments of the present invention, the nerve growth factor is a nerve growth factor of mouse or human; preferably, the nerve growth factor is a nerve growth factor extracted from the submandibular gland of a natural mouse or a submandibular gland of a transgenic mouse; further, the transgenic mouse is a transgenic mouse expressing human nerve growth factor.

The sources of samples that can be detected using the method of the present invention include, but are not limited to, samples of nerve growth factor stock solution or the purification process of the stock solution. In some embodiments of the present invention, the nerve growth factor is a nerve growth factor of mouse or human; preferably, the nerve growth factor is a nerve growth factor extracted from the submandibular gland of natural mice or the submandibular gland of transgenic mice; further, the transgenic mouse is a transgenic mouse expressing human nerve growth factor.

The method of the present invention adopts reverse chromatography to select and adjust various conditions and parameters in the chromatography. The method has good system applicability, strong specificity, good intermediate precision and strong durability. Compared with the previous nerve growth factor determination method (for example, SEC-HPLC method), it can at least effectively separate and determine the four shear forms and main post-translation products of nerve growth factor, which can be used as quality control indicators in the purification process of the stock solution, and can better control the quality of the nerve growth factor product. Specifically, the method of the present invention can effectively separate and determine nerve growth factors including but not limited to the following forms: complete nerve growth factor, nerve growth factor with 1 amino acid missing at the C-terminus, nerve growth factor with 8 amino acids missing at the N-terminus, nerve growth factor with 1 amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus, and nerve growth factor glycosylation products and/or nerve growth factor oxidation products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a chromatogram of the purity of NGF in NGF stock solution determined by SEC-HPLC;

FIG2 shows a chromatogram of the purity of NGF in the NGF stock solution determined by RP-HPLC (TSK column, mobile phase A: 0.1% formic acid-water solution, B: 0.1% formic acid-90% acetonitrile/10% water solution);

FIG3 shows a chromatogram of the purity of NGF in the NGF stock solution determined by RP-HPLC (Agilent column, mobile phase A: 0.14% formic acid-water solution, B: 0.14% formic acid-90% acetonitrile/10% isopropanol);

FIG4 shows a chromatogram of the purity of NGF in the NGF stock solution determined by RP-HPLC (TSK column, mobile phase A: 0.08% formic acid-water solution, B: 0.08% formic acid-40% acetonitrile/60% isopropanol);

FIG5 shows a specific chromatogram of the purity of NGF in NGF stock solution determined by RP-HPLC (mobile phase A: 0.1% formic acid-water solution, B: 0.1% formic acid-50% acetonitrile/50% isopropanol);

FIG6 shows a chromatogram of the purity of NGF in the NGF stock solution determined by RP-UPLC (Waters column, mobile phase A: 0.1% formic acid-water solution, B: 0.1% formic acid-acetonitrile solution);

FIG7 shows a chromatogram of the purity of NGF in NGF stock solution determined by RP-UPLC (Agilent column, mobile phase A: 0.1% formic acid-water solution, phase B: 0.1% formic acid-90% acetonitrile/10% water);

8A and 8B show chromatograms of NGF purity in NGF stock solution measured at different organic phase ratios (the mobile phases of FIG. 8A are A: 0.1% formic acid-water solution, B: 0.1% formic acid-40% acetonitrile/60% isopropanol solution; the mobile phases of FIG. 8B are A: 0.1% formic acid-water solution, B: 0.1% formic acid-90% acetonitrile/10% isopropanol solution);

Figures 9A-9B show chromatograms of NGF purity in NGF stock solution measured at different column temperatures. The result shown in Figure 9A is at a column temperature of 30°C, and the result shown in Figure 9B is at a column temperature of 45°C;

Figures 10A-10B show chromatograms of NGF purity in NGF stock solution measured at different formic acid concentrations. The mobile phases of Figure 10A are A: 0.08% formic acid-water solution (v/v), B: 0.08% formic acid-50% acetonitrile/50% isopropanol solution (v/v), and the mobile phases of Figure 10B are A: 0.14% formic acid-water solution (v/v), B: 0.14% formic acid-50% acetonitrile/50% isopropanol solution (v/v);

FIG. 11 shows a specific chromatogram of the purity of NGF in NGF stock solution determined by RP-UPLC method.

DETAILED DESCRIPTION

In order to make the technical problems to be solved, the technical solutions and advantages adopted by the present invention clearer, the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The nerve growth factor stock solution used in the following comparative examples and examples was prepared from the mouse submandibular gland by the method disclosed in Example 5 of patent CN109762056A.

Unless otherwise specified, the reagents used in the following comparative examples and examples were prepared by conventional methods or obtained from commercial sources.

Unless otherwise specified, the experimental methods used in the following comparative examples and embodiments are conventional methods.

Unless otherwise specified, the materials, instruments, etc. used in the following comparative examples and examples were obtained from commercial sources.

Comparative Example 1: Determination of NGF purity in NGF stock solution by SEC-HPLC

1. Chromatographic conditions:

Instrument: SHIMADZU LC-20AD high performance liquid chromatograph;

Gel chromatography column: TSK G3000SWxl SEC, 300 angstroms (5 μm), 7.8 × 300 mm;

Mobile phase: 0.25 mol/L phosphate buffer (containing 0.15 mol/L disodium hydrogen phosphate solution and 0.1 mol/L sodium dihydrogen phosphate solution)-acetonitrile (85:15, volume ratio, v/v)

Flow rate: 0.6 ml/min; column temperature: 30°C; detection wavelength: 280 nm; injection volume: not less than 20 μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, obtain the position and related parameters of target peak by retention time on chromatogram, and calculate the content of target substance by peak area. The results are shown in Table 1 and Figure 1.

Table 1 Results of NGF purity in NGF stock solution determined by SEC-HPLC

sample Retention time (min) Peak area purity NGF stock solution 16.115 2140232 100%

The results showed that the purity of NGF in the NGF stock solution measured by SEC-HPLC was 100%, but NGF and its molecular weight isomers could not be separated.

Example 1: Determination of the purity of NGF in NGF stock solution by RP-HPLC

1. Chromatographic conditions:

Instrument: Waters e2695 high performance liquid chromatograph;

Column: TSK Protein C4-300 3μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (volume ratio, v/v), B: 0.1% formic acid-90% acetonitrile/10% water solution (v/v).

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 80 20 25 65 35 30 20 80 32 80 20 40 80 20

Column temperature: 40°C, detection wavelength: 280nm, flow rate: 0.8ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml), take 10 μg from it and inject it into liquid chromatograph, detect at 280 nm, record the chromatogram, and calculate the purity by area normalization method. The results are shown in Table 2 and Figure 2.

Table 2 Results of NGF purity in NGF stock solution determined by RP-HPLC

From the results in Table 2 and FIG. 2 , it can be seen that the RP-HPLC method of this embodiment separated 5 known chromatographic peaks and one unknown peak, and the total purity of peaks 1 to 5 was 100.00%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing from the C-terminus, peak 4 was NGF with 8 amino acids missing from the N-terminus, and peak 5 was NGF with one amino acid missing from the C-terminus and 8 amino acids missing from the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 57.92%.

Example 2: Determination of the purity of NGF in NGF stock solution by RP-HPLC

1. Chromatographic conditions:

Instrument: Waters e2695 high performance liquid chromatograph;

Column: Agilent AdvanceBio RP-mAb C4 3.5 μm, 2.1 × 150 mm

Mobile phase: A: 0.14% formic acid-water solution (volume ratio, v/v), B: 0.14% formic acid-90% acetonitrile/10% isopropanol solution (v/v).

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 80 20 10 75 25 25 68 32 28 20 80 30 80 20 40 80 20

Column temperature: 40°C, detection wavelength: 280nm, flow rate: 0.6ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml), take 10 μg from it and inject it into liquid chromatograph, detect at 280 nm, record the chromatogram, and calculate the purity by area normalization method. The results are shown in Table 3 and Figure 3.

Table 3 Results of NGF purity in NGF stock solution determined by RP-HPLC

As shown in Table 3 and the results of FIG3 , the RP-HPLC method of this embodiment separated 5 known chromatographic peaks and one unknown peak, and the total purity of peaks 1 to 5 was 100.00%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing from the C-terminus, peak 4 was NGF with 8 amino acids missing from the N-terminus, and peak 5 was NGF with one amino acid missing from the C-terminus and 8 amino acids missing from the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 56.70%.

Example 3: RP-HPLC method to determine the purity of nerve growth factor stock solution

1. Chromatographic conditions:

Instrument: Waters e2695 high performance liquid chromatograph;

Chromatographic column: TSKProtein C4-300 3μm, 15cm×4.6mm

Mobile phase: A: 0.08% formic acid-water solution (volume ratio, v/v), B: 0.08% formic acid-40% acetonitrile/60% isopropanol solution (v/v).

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 84 16 20 66 34 25 20 80 30 84 16 35 84 16

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml), take 10 μg from it and inject it into liquid chromatograph, detect at 280 nm, record the chromatogram, and calculate the purity by area normalization method. The results are shown in Table 4 and Figure 4.

Table 4 Results of NGF purity in NGF stock solution determined by RP-HPLC

As shown in Table 4 and the results of FIG4 , the RP-HPLC method of this embodiment separated 5 known chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing from the C-terminus, peak 4 was NGF with 8 amino acids missing from the N-terminus, and peak 5 was NGF with one amino acid missing from the C-terminus and 8 amino acids missing from the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 57.81%.

Example 4: Verification of the method for determining the purity of NGF in NGF stock solution by RP-HPLC

1. Chromatographic conditions:

Instrument: Waters e2695 high performance liquid chromatograph;

Chromatographic column: TSK ProteinC4-300 3μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (volume ratio, v/v), B: 0.1% formic acid-50% acetonitrile/50% isopropanol solution (v/v).

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 78 twenty two 20 65 35 25 20 80 30 78 twenty two 35 78 twenty two

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. System applicability

NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml) was injected 6 times continuously, and the peak order was peak 1, peak 2, peak 3, peak 4, peak 5. The relative standard deviation (RSD) of the retention time of each peak should be no more than 1.0%, the sum of the percentages of peak 2 and peak 3 should be no less than 50.0%, and the RSD should be no more than 2.0%. The sum of the percentages of peaks 1 to 5 should be no less than 98.0%, and the RSD should be no more than 2.0%; the separation degree of peak 3 and peak 4 should be greater than 1.5, and the theoretical plate number of peak 2 should be no less than 2000. The results are shown in Table 5.

Table 5 System suitability results

The results showed that after 6 consecutive injections of NGF stock solution, the RSD of the retention time of peaks 1 to 5 was less than 1.0%, the RSD of the sum of the percentages of peaks 2 and 3 was 0.27%, and the RSD of the sum of the percentages of peaks 1 to 5 was 0.00%; the separation degree of peaks 3 and 4 was greater than 1.5, and the theoretical plate number of peak 2 was greater than 2000. This showed that the system applicability of this method was good.

2. Exclusivity

Blank solvent buffer and NGF stock solution from batch Y20171001 were injected in sequence, and the chromatograms were recorded. The results are shown in Figure 5.

Conclusion: The RP-HPLC method for determining the purity of NGF in NGF stock solution has good specificity and can accurately determine the amount of each component. It can be used to determine the purity of NGF in NGF stock solution.

3. Intermediate precision

On different inspection dates, different testers conducted purity tests on three batches of NGF stock solutions (batch numbers: Y20171001, Y20171002, and Y20171101) according to determined chromatographic conditions, and the results were statistically analyzed. The results are shown in Table 6.

Table 6 Intermediate precision results

Conclusion: The purity of three batches of NGF stock solutions was determined by different testers on different inspection dates. The RSD of the purity of each batch of samples was 0.00, which was less than 2.0%. This shows that the intermediate precision of RP-HPLC method for determining the purity of NGF in NGF stock solution is good.

4. Durability

Under the determined chromatographic conditions, the formic acid (FA) ratio changed by ±10%, the column temperature changed by ±2°C, the detection wavelength changed by ±5nm, the relative flow rate changed by ±10%, and three chromatographic columns with different batches were used to measure the purity of NGF stock solution (batch number: Y20171001, concentration: 0.45mg/ml). The results showed that the RSD of NGF purity measured under each condition was no more than 2.0%. The results are shown in Table 7.

Table 7 Durability test results

The results show that there is no significant difference in the purity of the product when the formic acid ratio, flow rate, column temperature, and detection wavelength are changed to a certain extent, and when three chromatographic columns of different batches are used for the determination of the stock solution. The RSD of NGF purity under each condition is less than 2.0%, indicating that the RP-HPLC method of the present invention has good durability in determining the purity of NGF in NGF stock solution.

Example 5: Determination of NGF purity in NGF stock solution by RP-UPLC

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 1.7μm, 5cm×2.1mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-acetonitrile solution (v/v)

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 80 20 3 81 19 10 79.5 20.5 13 77 twenty three 20 75 25 twenty three 50 50 25.01 80 20 30 80 20

Column temperature: 30°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μl (about 5μg).

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45 mg/ml), take 10 μg from it and inject it into liquid chromatograph, detect at 280 nm, record the chromatogram, and calculate the purity by area normalization method. The results are shown in Table 8 and Figure 6.

Table 8 Results of NGF purity in NGF stock solution determined by RP-UPLC method

From the results in Table 8 and FIG. 6 , it can be seen that the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 60.50%.

Example 6: Determination of NGF purity in NGF stock solution by RP-UPLC

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Agilent AdvanceBio RP-mAb C4 3.5 μm, 2.1 × 150 mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-90% acetonitrile/10% water (v/v)

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 82 18 14 68 32 20 20 80 25 20 80 25.01 82 18 35 82 18

Column temperature: 30°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 9 and Figure 7.

Table 9 Results of NGF purity in NGF stock solution determined by RP-UPLC method

As shown in Table 9 and the results of FIG. 7 , the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 61.57%.

Example 7: Determination of NGF purity in NGF stock solution by different organic phase ratios in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-40% acetonitrile/60% isopropanol solution (v/v) Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 84 16 twenty two 66 34 25 20 80 25.01 84 16 35 84 16

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 10 and Figure 8A.

Table 10 NGF purity results in NGF stock solution determined by different organic phase ratios

From the results in Table 10 and FIG8 , it can be seen that the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 60.44%.

Example 8: Determination of NGF purity in NGF stock solution by different organic phase ratios in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-90% acetonitrile/10% isopropanol solution (v/v)

Gradient elution program:

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 11 and Figure 8B.

Table 11 NGF purity results in NGF stock solution determined by different organic phase ratios

As shown in Table 11 and the results of FIG. 8B , the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 59.82%.

Example 9: Determination of NGF purity in NGF stock solution at different column temperatures in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-50% acetonitrile/50% isopropanol solution (v/v)

Gradient elution program:

Column temperature: 30°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (stock solution provided by Shutaishen (Beijing) Biopharmaceutical Co., Ltd., batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 12 and Figure 9A.

Table 12 Results of NGF purity in NGF stock solution measured at different column temperatures

From the results of Table 12 and FIG. 9A , it can be seen that the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 60.79%.

Example 10: Determination of NGF purity in NGF stock solution at different column temperatures in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-50% acetonitrile/50% isopropanol solution (v/v)

Gradient elution program:

Column temperature: 45°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (stock solution provided by Shutaishen (Beijing) Biopharmaceutical Co., Ltd., batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 13 and Figure 9B.

Table 13 Results of NGF purity in NGF stock solution measured at different column temperatures

As shown in Table 13 and the results of FIG. 9B , the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 55.86%.

Example 11: Determination of NGF purity in NGF stock solution by different formic acid concentrations in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.08% formic acid-water solution (v/v), B: 0.08% formic acid-50% acetonitrile/50% isopropanol solution (v/v)

Gradient elution program:

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (stock solution provided by Shutaishen (Beijing) Biopharmaceutical Co., Ltd., batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 14 and Figure 10A.

Table 14 NGF purity results in NGF stock solution measured at different formic acid concentrations

From the results of Table 14 and FIG. 10A , it can be seen that the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 59.81%.

Example 12: Determination of NGF purity in NGF stock solution using different formic acid concentrations in RP-UPLC method

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Mobile phase: A: 0.14% formic acid-water solution (v/v), B: 0.14% formic acid-50% acetonitrile/50% isopropanol solution (v/v)

Gradient elution program:

Time (min) A(%) (v/v) B(%) (v/v) 0 83.0 17.0 twenty two 66.0 34.0 25 20.0 80.0 25.01 83.0 17.0 35 83.0 17.0

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min, injection volume: 10μg.

2. Experimental steps:

1. Take NGF stock solution (stock solution provided by Shutaishen (Beijing) Biopharmaceutical Co., Ltd., batch number: Y20170803, concentration: 0.47 mg/ml), inject 20 μg, inject into liquid chromatograph, detect at 280 nm, record chromatogram, and calculate purity by area normalization method. The results are shown in Table 15 and Figure 10B.

Table 15 Results of NGF purity in NGF stock solution measured at different formic acid concentrations

From the results of Table 15 and FIG. 10B , it can be seen that the RP-UPLC method of the embodiment separated 5 chromatographic peaks, and the total purity of peaks 1 to 5 was 100%. The attribution of each chromatographic peak was identified by mass spectrometry, and peaks 1 to 5 were: peak 1 was NGF glycosylation and/or oxidation product, peak 2 was intact NGF, peak 3 was NGF with one amino acid missing at the C-terminus, peak 4 was NGF with 8 amino acids missing at the N-terminus, and peak 5 was NGF with one amino acid missing at the C-terminus and 8 amino acids missing at the N-terminus. Among them, the total purity (%) of peaks 2 and 3 was 58.20%.

Through experiments, by comparing chromatographic columns of different specifications and brands, organic phase ratios, column temperatures, and formic acid concentrations, a preferred embodiment of the present invention was finally found, wherein the reverse phase chromatographic column was selected from Waters Protein BEH C4 3.5 μm, 15 cm × 4.6 mm; mobile phase A was 0.1% formic acid-water solution (v/v), and mobile phase B was 0.1% formic acid-50% acetonitrile/50% isopropanol solution (v/v), and gradient elution was used; column temperature: 35°C, detection wavelength: 280 nm, flow rate: 0.2 ml per minute, and injection volume was 10 μg.

Example 13: Verification of the RP-UPLC method for determining the purity of NGF in NGF stock solution

1. Chromatographic conditions:

Instrument: Waters H-Class Bio ultra-high performance liquid chromatograph;

Column: Waters Protein BEH C4 3.5μm, 15cm×4.6mm

Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml/min

Mobile phase: A: 0.1% formic acid-water solution (v/v), B: 0.1% formic acid-50% acetonitrile/50% isopropanol solution (v/v)

Gradient elution program:

2. Experimental steps:

1. System applicability

NGF stock solution (batch number: Y20171002, concentration: 0.52 mg/ml) was injected 6 times continuously, and the peak order was peak 1, peak 2, peak 3, peak 4, peak 5. The relative standard deviation (RSD) of the retention time of each peak should be no more than 1.0%, the sum of the percentages of peak 2 and peak 3 should be no less than 50.0%, and the RSD should be no more than 2.0%. The sum of the percentages of peaks 1 to 5 should be no less than 98.0%, and the RSD should be no more than 2.0%; the separation degree of peak 3 and peak 4 should be greater than 1.5, and the theoretical plate number of peak 2 should be no less than 2000. The results are shown in Table 16.

Table 16 System suitability results

The results showed that the stock solution was injected 6 times continuously, the retention time RSD of peaks 1 to 5 was less than 1.0%, the sum of the percentages of peaks 2 and 3 was 0.12%, and the sum of the percentages of peaks 1 to 5 was 0.00%; the separation degree of peaks 3 and 4 was greater than 1.5, and the theoretical plate number of peak 2 was greater than 2000. This shows that the RP-UPLC method has good system applicability for determining the purity of NGF in NGF stock solution.

2. Exclusivity

Blank solvent buffer and NGF stock solution from batch Y20171002 were sampled in sequence, and the chromatograms were recorded. The results are shown in Figure 11.

Conclusion: The RP-UPLC method has good specificity for determining the purity of NGF in NGF stock solution and can accurately determine the amount of each component. It can be used to determine the purity of NGF in NGF stock solution.

3. Intermediate precision

On different inspection dates, different testers conducted purity tests on three batches of NGF stock solutions (batch numbers: Y20171001, Y20171002, and Y20171101) according to determined chromatographic conditions, and the results were statistically analyzed. The results are shown in Table 17.

Table 17 Intermediate precision results

Conclusion: The purity of three batches of stock solutions was determined by different testers on different inspection dates. The RSD of the purity of each batch of samples was less than 2.0%, indicating that the intermediate precision of the RP-UPLC method for determining the purity of NGF in NGF stock solution was good.

4. Durability

Under the determined chromatographic conditions, the formic acid (FA) ratio changed by ±10%, the column temperature changed by ±2°C, the detection wavelength changed by ±5nm, the relative flow rate changed by ±10%, and three chromatographic columns with different batches were used to measure the purity of NGF stock solution (batch number: Y20171002, concentration: 0.52 mg/ml). The results showed that the RSD of NGF purity measured under each condition was no more than 2.0%. The results are shown in Table 18.

Table 18 Durability test results

The results show that when the formic acid ratio, flow rate, column temperature, and detection wavelength undergo certain changes and three chromatographic columns of different batches are used to determine the NGF stock solution, there is no significant difference in the determination of the NGF purity in the NGF stock solution. The RSD of the NGF purity under each condition is less than 2.0%, indicating that the RP-UPLC method of the present invention has good durability in determining the NGF purity in the NGF stock solution.

In summary, the method system of the present invention has good applicability, strong specificity, good intermediate precision and strong durability.

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (8)

1. A method of determining nerve growth factor purity using reverse phase chromatography, the chromatographic conditions comprising:

(1) Using a reverse phase chromatographic column with tetraalkylsilane bonded silica gel as filler;

(2) Mobile phase: the mobile phase A is formic acid aqueous solution containing 0.06-0.16% of volume ratio; the mobile phase B is (0.06% -0.16%) formic acid aqueous solution- (40-90) acetonitrile/(10-60) isopropanol (V/V); or the mobile phase B is (0.06% -0.16%) formic acid aqueous solution- (80-100) acetonitrile/(0-20) water (V/V);

the method adopts gradient elution, and the eluent is a mixed solution of a mobile phase A and a mobile phase B;

the reversed phase chromatography is reversed phase high performance liquid chromatography or reversed phase ultra high performance liquid chromatography;

the gradient elution procedure for the reversed phase high performance liquid chromatography was as follows:

time/minute A/vol% B/vol% 0 78-84 16-22 20 65-70 30-35 25 20-65 35-80 30 78-84 16-22 45 78-84 16-22

，

The gradient elution procedure for the reversed-phase ultra-high performance liquid chromatography was as follows:

time/minute A/vol% B/vol% 0 83-84 16-17 22 66 34 25 20 80 25.01 83-84 17 40 83-84 17

，

The nerve growth factor is extracted from the submandibular gland of a natural mouse or the submandibular gland of a transgenic mouse.

2. The method according to claim 1, wherein mobile phase a is an aqueous solution containing 0.08 to 0.14% by volume of formic acid; the mobile phase B is (0.08% -0.14%) formic acid aqueous solution- (40-90) acetonitrile/(10-60) isopropanol (V/V); or the mobile phase B is (0.08% -0.14%) formic acid aqueous solution- (90-100) acetonitrile/(0-10) water (V/V).

3. The method of claim 1, wherein the chromatographic column has a particle size of 1.7 to 3.5 μm, a length of 5cm to 15cm, and an inner diameter of 2.1mm to 4.6mm.

4. The method of claim 1, wherein the chromatographic conditions further comprise: the flow rate is 0.1 ml/min-1.2 ml/min.

5. The method according to claim 1, wherein the flow rate of the reverse phase high performance liquid chromatography is 0.2ml/min to 1.2ml/min, and the flow rate of the reverse phase ultra high performance liquid chromatography is 0.1ml/min to 0.5ml/min.

6. The method of claim 1, wherein the chromatographic conditions further comprise: the column temperature is 25-70 ℃, the detection wavelength is 210-285 nm, and the sample injection amount is 1-100 mug.

7. The method according to claim 6, wherein the column temperature is 30-45 ℃, the detection wavelength is 275-285 nm, and the sample injection amount is 5-35 μg.

8. The method of any one of claims 1 to 7, wherein the nerve growth factor comprises, but is not limited to, the following forms: a complete nerve growth factor, a nerve growth factor with 1 amino acid deleted at the C-terminus, a nerve growth factor with 8 amino acids deleted at the N-terminus, a nerve growth factor with 1 amino acid deleted at the C-terminus and 8 amino acids deleted at the N-terminus, and a nerve growth factor glycosylation product and a nerve growth factor oxidation product.