Experiment of testing rhGH by heating means
First: Experimental objective;
Protein in liquid meet the thermomolecular will degenerationand agglutination, the appearance will appear lacte like milk, but this is related to the protein’s variety and concentration in the liquid. Now some clients consider that dissolve freeze-drying rhGH product by injecting water, after heat 2 minutes, if the product become turbid and lay aside over 12 hours it won’t be clear, and then it can be approved it is the real rhGH. Therefore we collected samples which from many suppliers on the market, and compare molecular biology and the heating result so that to prove the scientificalness of this method.
Second: material and method
1. material
1.1 sample: rhGH sold on the market ?Kigtropin?10iu???Jintropin?10iu company one???Jintropin?10iu company two???Hygetropin?10iu???somatropin?10iu???Green top gh - growth hormone (somatropin) - ?10iu???Blue top gh - growth hormone (somatropin) - ?10iu company one???Blue top gh - growth hormone (somatropin) - ?10iu company two???rhGH?5iu?10iu?15iu?20iu?biosupercompany offer?
1.2 standard product: rhGH (10iu, biological products research institute)
1.3 water for injection: purchase from medicine company
1.4 SDS-PAGE material; import and split charging
2. method
2.1 dissolve sample by injecting water 1ml
2.2 take solution 15 microlitre, SDS PAGE test protein concentration, purity and protein molecular weight.
2.3 Surplus solution respectively 100 degree water bath 15 minutes
3. result
Sample no.
Sample name?specification?
SDS-PAGE testing result
Boiling test(boiling time)
0
Standard product
3,85mg/ml, molecular weight; 22.125KD?purity 98%
defecate?15min?
?
Kigtropin?10iu?
4.5 mg/ml?molecular weight; 69KD?is albumin
Milky white?2min?
?
Jintropin?10iu company one?
0 mg/ml?
defecate?15min?
?
Jintropin?10iu company two?
4.3 mg/ml?molecular weight; 69KD?is albumin
Milky white?2min?
?
Hygetropin?10iu?
0 mg/ml?
defecate?15min?
?
somatropin?10iu?
0 mg/ml?
defecate?15min?
?
Green top gh - growth hormone (somatropin) - (10iu)
0 mg/ml?
defecate?15min?
?
Blue top gh - growth hormone (somatropin) - (10iu company one)
3.82 mg/ml,molecular weight; 22.125KD?purity 96%
defecate?15min?
?
Blue top gh - growth hormone (somatropin) - (10iu company two)
4.87 mg/ml,molecular weight; 69KD,is albumin
Milky white?2min?
?-1
RhGH(5iu biosuper company )
1.86 mg/ml,molecular weight; 22.125KD?purity 98%
defecate?15min?
?-2
RhGH(10iu biosuper company )
3.80 mg/ml,molecular weight; 22.125KD?purity 98%
defecate?15min?
?-3
RhGH(15iu biosuper company )
4.92 mg/ml,molecular weight; 22.125KD?purity 98%
defecate?15min?
?-4
RhGH(20iu biosuper company )
7.10 mg/ml,molecular weight; 22.125KD?purity 98%
defecate?15min?
4?result and analysis?
4,1?SDS-PAGE test result indicate?sample????? don’t contain rhGH, it contains albumin. Sample ??????? test no albumin. Sample ???-1??-2??-3??-4 tesed out they contain different content rhGH.
4,2?boiling test sample ????? solution after 2minutes it immediate become milky white,and after 12 hours, it keeps milky white. Other samples which include standard product, they are all keep clear and transparent after boiling 15 minutes. Analysis: albumin is the micromolecule protein, they degeneration after heating, molecule crosslinking easily and make the solution become milky white(scientist named albumin because it is condesed and then become white easily after boiling), rhGH also will degeneration after heating, but under the low concentration(the maximum concentration is 0.71% in this test) doesn’t croslink easily, so solution still is transparent after heating 15miniutes.
5?conclusion
The method of testing real rhGH by solution become milky white in boiling test only can test if the product use albumin instead rhGH. At present there are many products are blended with albumin. But on the market, usually rhGH’ content will not over 15iu, the real rhGH tested by boiling test, the result will not become milky white. so it is wrong to test the content of rhGH by boiling. We consider it is still need to test by molecular biology in the normal laboratory, the result is authentic.
