CN1472333A - Inspection of endotoxin of collagenase cell by limulus reagent - Google Patents
Inspection of endotoxin of collagenase cell by limulus reagent Download PDFInfo
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- CN1472333A CN1472333A CNA021258791A CN02125879A CN1472333A CN 1472333 A CN1472333 A CN 1472333A CN A021258791 A CNA021258791 A CN A021258791A CN 02125879 A CN02125879 A CN 02125879A CN 1472333 A CN1472333 A CN 1472333A
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- collagenase
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- endotoxin
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Abstract
A process for detecting the bacterial endotoxin of collagenase by limulus reagent includes such steps as diluting the collagenase with the water for injection or sodium chloride injection, and detecting its bacterial endotoxin by limulus reagent.
Description
Technical field
The invention belongs to field of biological pharmacy, specifically, is a kind of inspection of endotoxin of collagenase cell by limulus reagent of using.
Background technology
The state quality standard of collagenase preparation process is to adopt the rabbit temperature-raising method to detect its finished product pyrogen.Because this method requires to have qualified animal housing, need big with expense, complicated operation, on approbation rabbit will select in test strictness in preceding 7 days, prepares on request 1-2 day before the official testing, liquid medicine injection begins to finish to detection, take and reach more than 4 hours, recheck then need reach more than 8 hours as need and just can measure the result, therefore, be difficult to that collagenase is prepared intermediates and carry out quality monitoring, only be used for finished product detection.The collagenase preparation process needs the two weeks time from fermenting to finished product, just does pyrogen test, if defective, then causes the financial loss of scrapping by the gross.Because the essence of pyrogen is intracellular toxin, the bacterial endotoxin that detects collagenase intermediates in the collagenase preparation process just is equivalent to detect pyrogen, but does not also have simple suitable method at present.
Summary of the invention
The purpose of this invention is to provide a kind of simple to operately, expense is low, and detection time is short, and what approximately need to go out in more than 1 hour the result detects the method for collagenase bacterial endotoxin in the collagenase preparation process with limulus reagent test.
The present invention is achieved in that it is after the collagenase that will make in the preparation process is diluted to finite concentration with water for injection or sodium chloride injection, uses tachypleus amebocyte lysate to detect its bacterial endotoxin.
It is an amount of that main points of the present invention also are to get the collagenase crude product of tiring greater than 300u/mg, is diluted to 1.2-1.6 μ g/ml with water for injection or sodium chloride injection, uses the bacterial endotoxins test inspection, and containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Another main points of the present invention are, get refining collagenase solution, be diluted to 1200-1600u with water for injection earlier, be diluted to 3.0-4.0u/ml with water for injection or sodium chloride injection again, use the bacterial endotoxins test inspection, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Another main points of the present invention are, it is an amount of to get the injection collagenase, and every 1020-1440u is diluted to 2.55-3.6u/ml with water for injection or sodium chloride injection, uses the bacterial endotoxins test inspection, and containing the intracellular toxin amount among every 1ml should be less than 0.5EU.The invention effect
Owing to adopted above-mentioned detection method, as long as after the collagenase in the preparation process diluted, pressing the Pharmacopoeia of the People's Republic of China (version in 2000) appendix XI E bacterial endotoxins test detects, whether the content that just can judge collagenase intermediates and finished product bacterial endotoxin simply, easily meets the requirements, qualifiedly can enter next process, thereby the pyrogen index that has guaranteed final finished is qualified.
Embodiment
Embodiment 1
Is 37 ℃-41 ℃ through fermention medium in temperature with bacterial classification, and after cycle 40-45 hour static fermentation, Sterile Filtration adds ammonium sulfate precipitation, is dissolved in Tris-Ca
2+Damping fluid, centrifugal, ultrafiltration, lyophilize, get the collagenase crude product, take by weighing the crude product 25.7mg that tires to 400u/mg, be diluted to 1.5 μ g/ml with water for injection, press the inspection of the Pharmacopoeia of the People's Republic of China (version in 2000) appendix XI E bacterial endotoxins test, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Embodiment 2
The collagenase crude product is through DEAE cellulose column and Sephadex G-25 column chromatography, obtain refining collagenase solution, get the collagenase solution of tiring and be diluted to 1500u/ml with water for injection earlier to 4500u/ml, be diluted to 4.0u/ml with water for injection again, press the inspection of the Pharmacopoeia of the People's Republic of China (version in 2000) appendix XI E bacterial endotoxins test, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Embodiment 3
Refining collagenase solution adds auxiliary material, sterile filtration, and can, lyophilize gets the injection collagenase.Get and tire to 1 of the injection collagenase of 1350u, be diluted to 3.6u/ml with water for injection, press the inspection of the Pharmacopoeia of the People's Republic of China (version in 2000) appendix XI E bacterial endotoxins test, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Claims (4)
1. use inspection of endotoxin of collagenase cell by limulus reagent for one kind, it is characterized in that: after the collagenase that makes in the preparation process is diluted to finite concentration with water for injection or sodium chloride injection, use tachypleus amebocyte lysate to detect its bacterial endotoxin.
2. the inspection of endotoxin of collagenase cell by limulus reagent of using according to claim 1, it is characterized in that: it is an amount of to get the collagenase crude product of tiring greater than 300u/mg, be diluted to 1.2-1.6 μ g/ml with water for injection or sodium chloride injection, use the bacterial endotoxins test inspection, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
3. the inspection of endotoxin of collagenase cell by limulus reagent of using according to claim 1, it is characterized in that: get refining collagenase solution, earlier be diluted to 1200-1600u/ml with water for injection, be diluted to 3.0-4.0u/ml with water for injection or sodium chloride injection again, use the bacterial endotoxins test inspection, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
4. the inspection of endotoxin of collagenase cell by limulus reagent of using according to claim 1, it is characterized in that: it is an amount of to get the injection collagenase, every 1020-1440u is diluted to 2.55-3.6u/ml with water for injection or sodium chloride injection, use the bacterial endotoxins test inspection, containing the intracellular toxin amount among every 1ml should be less than 0.5EU.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021258791A CN1472333A (en) | 2002-08-01 | 2002-08-01 | Inspection of endotoxin of collagenase cell by limulus reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021258791A CN1472333A (en) | 2002-08-01 | 2002-08-01 | Inspection of endotoxin of collagenase cell by limulus reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1472333A true CN1472333A (en) | 2004-02-04 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA021258791A Pending CN1472333A (en) | 2002-08-01 | 2002-08-01 | Inspection of endotoxin of collagenase cell by limulus reagent |
Country Status (1)
| Country | Link |
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| CN (1) | CN1472333A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1721853B (en) * | 2004-07-12 | 2011-04-20 | 湛江安度斯生物有限公司 | Preparation method of bacterial endotoxins rapid detecting reagent case |
-
2002
- 2002-08-01 CN CNA021258791A patent/CN1472333A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1721853B (en) * | 2004-07-12 | 2011-04-20 | 湛江安度斯生物有限公司 | Preparation method of bacterial endotoxins rapid detecting reagent case |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |