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Method for measuring purity of nerve growth factor

A nerve growth factor and purity technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inability to separate different molecular weight isomers, inability to monitor molecular weight isomers, difficulty in controlling product quality, etc., to achieve good quality , good intermediate precision and strong specificity

Pending Publication Date: 2022-03-29
STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] There are different molecular weight isomers in the stock solution of mouse nerve growth factor, but the method of prior art (for example in the quality standard of mouse nerve growth factor included in " Chinese Pharmacopoeia " 2015 edition three, the SEC that the purity of determination stock solution adopts - HPLC method) resolution is not enough, different molecular weight isomers cannot be separated and accurately measured, so it is impossible to monitor the molecular weight isomers produced during the purification process of the stock solution, and it is difficult to control product quality

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  • Method for measuring purity of nerve growth factor
  • Method for measuring purity of nerve growth factor
  • Method for measuring purity of nerve growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: RP-HPLC method measures the purity of NGF in the NGF stoste

[0061] 1. Chromatographic conditions:

[0062] Instrument: Waters e2695 high performance liquid chromatography;

[0063] Chromatographic column: TSK Protein C4-300 3μm, 15cm×4.6mm

[0064] 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).

[0065] Gradient elution program:

[0066] time (min) A(%)(v / v) B(%)(v / v) 0 80 20 25 65 35 30 20 80 32 80 20 40 80 20

[0067] Column temperature: 40°C, detection wavelength: 280nm, flow rate: 0.8ml / min, injection volume: 10μg.

[0068] 2. Experimental steps:

[0069] 1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45mg / ml), take 10μg from it and inject it into a liquid chromatograph, detect at 280nm, record the chromatogram, and calculate the purity by area normalization method. The results are shown in...

Embodiment 2

[0073] Embodiment 2: RP-HPLC method measures the purity of NGF in the NGF stoste

[0074] 1. Chromatographic conditions:

[0075] Instrument: Waters e2695 high performance liquid chromatography;

[0076] Chromatographic column: Agilent AdvanceBio RP-mAb C4 3.5μm, 2.1×150mm

[0077] 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).

[0078] Gradient elution program:

[0079] 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

[0080] Column temperature: 40°C, detection wavelength: 280nm, flow rate: 0.6ml / min, injection volume: 10μg.

[0081] 2. Experimental steps:

[0082] 1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45mg / ml), take 10μg from it and inject it into a liquid chromatograph, detect at 280nm, record the chromatogram, and calculate the purity by area normalization...

Embodiment 3

[0086] Embodiment 3: RP-HPLC method measures the purity of nerve growth factor stoste

[0087] 1. Chromatographic conditions:

[0088] Instrument: Waters e2695 high performance liquid chromatography;

[0089] Chromatographic column: TSKProtein C4-300 3μm, 15cm×4.6mm

[0090] 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).

[0091] Gradient elution program:

[0092] time (min) A(%)(v / v) B(%)(v / v) 0 84 16 20 66 34 25 20 80 30 84 16 35 84 16

[0093] Column temperature: 35°C, detection wavelength: 280nm, flow rate: 0.2ml / min, injection volume: 10μg.

[0094] 2. Experimental steps:

[0095] 1. Take NGF stock solution (batch number: Y20171001, concentration: 0.45mg / ml), take 10μg from it and inject it into a liquid chromatograph, detect at 280nm, record the chromatogram, and calculate the purity by area normalization method. The results ...

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Abstract

The invention provides a method for determining the purity of a nerve growth factor, which uses a reversed-phase chromatography, and the chromatographic conditions comprise: (1) using a reversed-phase chromatographic column; (2) mobile phase: a mobile phase A is a formic acid aqueous solution with the volume ratio of 0.06%-0.16%; a 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 reverse chromatography can effectively separate four shearing forms and main post-translational modification products of the nerve growth factor, and the four shearing forms and the main post-translational modification products are used as quality control indexes in the purification process of the stock solution, so that the quality of the nerve growth factor can be better controlled.

Description

technical field [0001] The invention relates to the technical field of purity determination, in particular to a method for determining the purity of nerve growth factor by reversed-phase chromatography. Background technique [0002] Nerve growth factor (NGF) is one of the most important active components of the nervous system. It can maintain the survival of sympathetic nerve and sensory nerve cells, has dual biological functions of neuron nutrition and neurite growth promotion, and plays an important role in the development of central and peripheral neurons. , differentiation, growth, regeneration, and expression of functional properties all have important regulatory roles, and play an important role in protecting the normal function of the nervous system. [0003] There are different molecular weight isomers in the stock solution of mouse nerve growth factor, but the method of prior art (for example in the quality standard of mouse nerve growth factor included in " Chinese...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/36G01N30/74G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/36G01N30/74G01N30/86
Inventor 白海芬冯彩丽
Owner STAIDSON BEIJING BIOPHARMACEUTICALS CO LTD
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