Method for determining rhNGF protein content

A technology for protein content and protein content, applied in the measurement of color/spectral properties, etc., can solve the problems of complex, time-consuming, and unsuitable processes

Pending Publication Date: 2022-05-20
四川自豪时代药业有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

However, in the Kjeldahl nitrogen determination, each measurement requires precise preparation of standard sulfuric acid titration solution, digestion sample, distillation and titration, the whole process is more complicated and time-consuming (about 90 minutes to calibrate one sample), and each time requires The sample size is large (more than 25mg is required for each test)
Therefore, the Kjeldahl method is not suitable as a routine rapid determination of the protein content of rhNGF solution

Method used

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  • Method for determining rhNGF protein content
  • Method for determining rhNGF protein content
  • Method for determining rhNGF protein content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: Determination of protein concentration of rhNGF sample by Kjeldahl method

[0014] Using the Kjeldahl method (for specific methods, please refer to the first method of "Pharmacopoeia of the People's Republic of China" 2020 edition four parts 0731) to measure the protein concentration of samples 1 to 5, the measured data are as follows, see Table 1:

[0015] Table 1 Kjeldahl method to measure the concentration of each sample

[0016]

[0017] The absolute protein content of different rhNGF samples can be accurately determined by Kjeldahl method.

Embodiment 2

[0018] Embodiment 2: the establishment of standard curve

[0019] Dilute sample No. 1 to 1.0mg / ml, 0.9mg / ml, 0.8mg / ml, 0.7mg / ml, 0.6mg / ml, 0.5mg / ml, 0.4mg / ml, 0.3mg / ml, 0.2mg / ml , 0.1mg / ml a series of concentrations, the absorbance of each concentration sample was measured 5 times at 280nm, and the average value was calculated. The results are as follows, see Table 2:

[0020] Table 2 OD value under gradient dilution concentration

[0021]

[0022] According to the data in the table, use Excel to establish the concentration-OD value proportional relationship, and perform linear regression to obtain the standard curve, judge whether the standard curve is qualified according to the correlation coefficient of the standard curve, and then average the qualified standard curve to obtain the standard curve, see figure 1 .

[0023] Depend on figure 1 It can be seen that the obtained rhNGF standard curve equation is: y=0.9228x+0.0064, and its R 2 =0.9999, the applicable range ...

Embodiment 3

[0024] Embodiment 3: the determination of rhNGF sample content

[0025] Take rhNGF samples A, B and C with unknown concentrations, dilute them 16 times with buffer solution, measure their absorbance at 280 nm with a UV-Vis spectrophotometer, measure 3 times, and calculate the average value. Then the average value was substituted into the rhNGF standard curve equation obtained in Example 2 to calculate the concentration of the rhNGF sample. The results are shown in Table 3 below.

[0026] Table 3 Determination of rhNGF content

[0027]

[0028]

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Abstract

The invention relates to the field of biological pharmacy, in particular to a method for measuring the protein content of rhNGF. According to the method disclosed by the invention, the protein content of the rhNGF can be rapidly and accurately measured by utilizing an ultraviolet spectrophotometric method.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for accurately measuring the content of rhNGF protein. Background technique [0002] According to the third part of the 2015 edition of the "Chinese Pharmacopoeia", there are six methods for the determination of protein content of biotech drugs: Kjeldahl method, Lowry method and UV-visible spectrophotometry. Among them, the Kjeldahl method is an absolute protein quantitative method that calculates the protein content based on the nitrogen content in the protein by measuring the nitrogen content in the protein; the Lowry method and the biuret method are both determined by measuring the light absorption of the protein in the alkaline copper solution. The determination method for calculating the protein content, these two methods need to use the standard protein solution to do linear regression or control, and the protein content of the test product can be calculated by co...

Claims

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Application Information

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IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 魏琪蒋飞
Owner 四川自豪时代药业有限公司
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