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Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation

A technology of nerve growth factor and determination method, which is applied in the field of determining the content of nerve growth factor in nerve growth factor preparations by gel chromatography, can solve the problems of difficult separation of NGF and albumin, inability to accurately quantify, etc., and achieve the reduction of solvent usage Small size, shortened detection time, and high degree of automation

Active Publication Date: 2015-09-23
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Simultaneously, contain the NGF preparation that uses human serum albumin as stabilizer, because the content of NGF in the preparation is low, and human serum albumin and NGF are both proteins, and the concentration in the preparation is obviously higher than NGF, thereby seriously affected Accurate quantification of NGF
At present, methods commonly used in the determination of protein content in biological products, such as Lowry method, ultraviolet absorption method, Bradford method, enzyme-linked immunosorbent assay (ELISA), SDS-PAGE method, etc., cannot effectively eliminate this interference, and cannot be accurately quantified
Publication No. CN1566941 discloses a method for measuring the content of nerve growth factor in the preparation. Although the method can accurately measure the content of NGF in the preparation, it is still difficult to make the NGF and albumin reach the same level. Completely separated, it is also difficult to accurately determine the content of the active ingredient NGF, which is not conducive to product quality control

Method used

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  • Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation
  • Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation
  • Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1: Determination of NGF content in the preparation containing human serum albumin NGF by external standard method

[0054] 1. Chromatographic conditions:

[0055] Instrument: Waters ACQUITY H-Class Bio ultra-high performance liquid chromatography;

[0056] Gel chromatography column: Waters UPLC SEC BEH125, 125 Angstrom (1.7μm), 4.6×150mm;

[0057] Detector: UV detector, the detector uses a titanium flow cell;

[0058] Mobile phase: acetonitrile: 0.25M phosphate buffer solution = 15:85, pH value: 7.0, flow rate: 0.2ml / min; column temperature: 40°C; wavelength: 214nm; injection volume: 2μl.

[0059] 2. Experimental steps:

[0060] 1. Determination of control spectrum

[0061] Get the NGF stock solution of known concentration, add mobile phase and be mixed with the solution of 50 μ g / ml, as NGF contrast solution; Get mobile phase and corresponding adjuvant solution (containing 0.1 volume % human serum albumin, 5.0 volume % % mannitol) as a blank control, ea...

Embodiment 2

[0073] Embodiment 2: Dealbumin-depleted nerve growth factor NGF content is determined by external standard method

[0074] 1. Chromatographic conditions:

[0075] With embodiment 1.

[0076] 2. Experimental steps:

[0077] 1. Determination of control spectrum

[0078] The experimental procedure is the same as in Example 1. Take 2 μl of the corresponding adjuvant solution (containing 10 mg / ml amino acid, 5 volume % mannitol, and 50 mM disodium hydrogen phosphate) in the NGF preparation without albumin as a blank control, and detect it by the above method to determine the adjuvant The chromatographic peak position of each component in . The result is as Figure 5 As shown, the peak retention time of the excipients of the preparation far exceeds the retention time of the NGF contrast, and the interference of the excipients can also be excluded.

[0079] 2. Dealbumin NGF preparation

[0080] Take the NGF stock solution with known concentration, add mobile phase to prepare a ...

Embodiment 3

[0090] Embodiment 3: the recovery test of NGF content in the preparation by external standard method

[0091] 1. Chromatographic conditions:

[0092] With embodiment 1.

[0093] 2. Experimental steps:

[0094] 1, the preparation of contrast solution: get the known NGF stoste of concentration (specific source is the same as embodiment 1), add mobile phase and be mixed with the solution of 50 μ g / ml, as NGF contrast solution, prepare 2 parts, shake up, stand-by.

[0095] 2, the preparation of sample solution: get the auxiliary material solution containing human albumin (0.1 volume %) and mannitol (5.0 volume %) and each 10ml of the auxiliary material solution containing amino acid (10mg / ml) and mannitol (5 volume %) and an appropriate amount of NGF stock solution to prepare NGF solutions containing human serum albumin and de-albumin, respectively, wherein the concentrations of NGF were 40, 50, and 60 μg / ml, respectively, and 3 parts were prepared for each concentration, shaken...

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Abstract

The invention discloses a method for determining content of a nerve growth factor in a nerve growth factor preparation. According to the method, the content of the nerve growth factor is determined by using gel chromatography in ultra-performance liquid chromatography (UPLC); gel chromatography conditions are presented as follows: a gel chromatography column is a UPLC molecular sieve chromatography column of which the molecular weight detection range is 1000 to 80000 Daltons and the pore diameter is 125 to 200 angstroms; the mobile phase is phosphate buffer of which the pH value is 6.5 to 7.2 and the concentration is 0.1 to 0.5M, and contains an organic phase of 5 to 20 percent by volume; the column temperature is 30 to 50 DEG C; the flow rate of the mobile phase is 0.1 to 0.5ml / min; a titanium flow cell is adopted by a detector, and the detection wavelength is 205 to 280nm. According to the method for determining the content of the nerve growth factor in the nerve growth factor preparation, disclosed by the invention, the NGF in the preparation can be accurately quantified.

Description

technical field [0001] The invention relates to a method for determining the content of nerve growth factor in nerve growth factor preparations, in particular to a method for determining the content of nerve growth factor in nerve growth factor preparations using gel chromatography in ultra-high performance liquid 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] At present, all kinds of NGF preparations produced and sold in China are in the for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 白海芬冯彩丽
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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