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Method for measuring polysorbate content in protein solution

A polysorbate and protein technology, which is applied in the biological field, can solve the problems of easy loss of samples, long time, affecting the experimental results, etc., and achieves the effect of overcoming the long detection time interval, simple operation and good repeatability.

Inactive Publication Date: 2010-02-03
DONGGUAN TAILI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires precipitation, washing, and separation of proteins in the sample, and air purging is also required. The sample is easily lost during processing, which will cause large deviations; at the same time, this method needs to be placed overnight at room temperature, and the time long, and dichloromethane is volatile, which will also affect the experimental results

Method used

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  • Method for measuring polysorbate content in protein solution
  • Method for measuring polysorbate content in protein solution
  • Method for measuring polysorbate content in protein solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Determination of Tween-20 (polysorbate 20) content in embodiment 1 recombinant human erythropoietin (Fc) fusion protein [rhEPO-Fc] injection

[0029] The rhEPO-Fc injection (Lot. 20060301) was used for testing, and the content of Tween-20 in the injection was 0.01%, ie about 100 μg / ml. The test steps are:

[0030] (1) Take 0, 20, 40, 60, 80, and 100 μl of Tween-20 standard solution (1mg / ml) into glass test tubes with stoppers, and add water to 1ml;

[0031] (2) Add 2ml of chloroform and 3ml of ammonium thiocyanate cobaltammonium aqueous solution to each tube (weigh 6.0g of cobalt nitrate and 40.0g of ammonium thiocyanate, add water to dissolve and dilute to 200ml), mix well; shake fully at intervals of 15 minutes Mix once, 4 times in total;

[0032] (3) Stand in a 60°C water bath for 10 minutes;

[0033] (4) Remove the upper layer solution with a fine pipette, and take the lower layer solution to measure OD 620 Value, and the concentration as the abscissa, the corre...

Embodiment 2

[0039] Comparative test of embodiment 2 conventional method and improved method

[0040]Take rhEPO-Fc injection (Lot.20060301) (containing 50 μg / ml Tween-20) and self-prepared 100 μg / ml BSA (bovine serum albumin) solution (containing 50, 80 μg / ml Tween-20 respectively) as test samples. The improved method is implemented according to Example 1. The general method is as follows:

[0041] (1) Take 1.0ml of the above samples respectively in a plugged centrifuge tube, add 5ml of ethanol-sodium chloride saturated solution, shake well, and centrifuge at 3000rpm for 10 minutes;

[0042] (2) Take the supernatant, then carefully wash the tube wall with 1.0ml ethanol-sodium chloride saturated solution, combine the washing solution and the supernatant, and centrifuge at 3000rpm for 10 minutes;

[0043] (3) Take the supernatant and put it in a 55°C water bath, blow it with a hair dryer, concentrate it to 0.1-0.5ml, add 1ml of water to dissolve;

[0044] (4) Add 2.0ml of dichloromethane ...

Embodiment 3

[0051] The impact of embodiment 3 different proteins on the determination of polysorbate content

[0052] Different concentrations of rhEPO-Fc, recombinant human lymphocyte-associated factor (Fc) fusion protein [rhLFA-Fc], recombinant human parathyroid hormone 1-34 [rhPTH1-34], Exendin-4 and BSA were detected by the improved method above. Effect of solution on determination of polysorbate content. The experimental results are shown in Table 3.

[0053] Table 3: Comparison of polysorbate content determination results in several protein solutions

[0054]

protein name

protein content

(μg / ml)

Tween-20 content

Theoretical value (μg / ml)

Tween-20 content

Measured value (μg / ml)

deviation

(%)

rhEPO-Fc

50

50

48.63

2.74

rhEPO-Fc

100

50

45.68

8.64

rhEPO-Fc

150

50

Unable to sample for detection

-

rhLFA-Fc

50

50...

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Abstract

The invention relates to a method for measuring polysorbate content in a protein solution. The method comprises the following steps: (1) mixing a measured sample with methenyl chloride and an ammoniumcobalt thiocyanate aqueous solution to obtain a mixed solution; (2) demixing the mixed solution; (3) measuring the absorbance of the layered methenyl chloride layer under a ultraviolet-visible wavelength; and (4) calculating the concentration of the polysorbate in the measured sample according to the measured absorbance. The method performs the measurement without sedimentation of the protein inthe preparation or an overnight reaction of the sample, and overcomes the differences caused by the overlong measuring time interval because methylene chloride is volatile easily. The method can be used for controlling the polysorbate content in the protein solution.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for determining the content of polysorbate, in particular to a method for determining the content of polysorbate in a protein solution. Background technique [0002] Surfactants are more and more widely used in systems that come into contact with the human body, such as drugs, food, cosmetics, and personal hygiene products. However, surfactants and their metabolites may cause toxic and side effects on the body, including acute toxicity, subacute toxicity, Chronic toxicity, effects on fertility and reproduction, embryotoxicity, teratogenicity, mutagenicity, carcinogenicity, sensitization, hemolysis, etc. Therefore, it is necessary to control the content of the surfactant in the product according to the use of the product, the type of the surfactant, and the like. [0003] Nonionic surfactants are less toxic, and polysorbate nonionic surfactants are widely used as emulsifiers...

Claims

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Application Information

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IPC IPC(8): G01N21/33
Inventor 阳勇何凯罗兰兵张仁怀粟建辉韩为跃闻亚磊
Owner DONGGUAN TAILI BIOTECH
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