CN102526729A - Preparation process of antitoxic serum for viral inactivation treatment - Google Patents
Preparation process of antitoxic serum for viral inactivation treatment Download PDFInfo
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- CN102526729A CN102526729A CN201210088398XA CN201210088398A CN102526729A CN 102526729 A CN102526729 A CN 102526729A CN 201210088398X A CN201210088398X A CN 201210088398XA CN 201210088398 A CN201210088398 A CN 201210088398A CN 102526729 A CN102526729 A CN 102526729A
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Abstract
A preparation process of antitoxic serum for viral inactivation treatment comprises the steps of pepsin digestion, first precipitation and heating degeneration treatment, second precipitation treatment, alum precipitation treatment, ultrafiltration concentration treatment, and stock solution preparation. Two viral inactivation methods are utilized in the steps and can be respectively and independently implemented or implemented in a combined mode to enable an antitoxic serum product to be safe and inactivation to be complete. Furthermore, the two viral inactivation methods are physical methods, are free of addition of other matters, do not bring about other practical problems to preparation of antitoxic serum stock solution do not happen, and have no special requirements for plants, facilities and personnel, thereby being convenient and easy to operate.
Description
Technical field
The present invention relates to a kind of antitoxic serum preparation technology of viral inactivation treatment.
Background technology
Antitoxic serum; Can be described as antitoxin; Be also referred to as antiserum; Be the immune blood plasma (serum) by corresponding toxin or toxoid, antibacterial, virus immunity animal (being mainly equus) gained, through the immunoglobulin preparation that pepsin digestion, ammonium sulfate precipitation make, its preparation method is " Chinese biological goods rules " and " the disclosed technology of Chinese pharmacopoeia.At present, the method for preparing of antitoxic serum generally comprises following steps: 1, pepsin digestion,, and carry out digestion process with pepsin and obtain Digestive system various immune blood plasmas (serum) dilution with purified water or water for injection; 2, precipitate and the degenerative treatments of heating for the first time, add ammonium sulfate in the Digestive system, and regulate pH value, then Digestive system is heated and keep, cooling is again filtered and is collected supernatant, precipitate discarded; 3, precipitation process for the second time promptly adds ammonium sulfate in above-mentioned supernatant, leave standstill the after-filtration taking precipitate, discarded supernatant; 4, alum precipitate is handled, and with above-mentioned precipitate dilution, adds alum solution, leaves standstill after-filtration and gets supernatant, precipitate discarded; 5, ultrafiltration and concentration is handled, and above-mentioned supernatant is carried out ultrafiltration desalination and concentrated; 6, sodium chloride, antiseptic are added in stock solution preparation in ultrafiltration and concentration liquid, regulate pH value, and aseptic filtration promptly makes antitoxic serum stock solution.
The weak point of this technology is; Prepared stock solution derives from animal blood slurry (serum) raw material; And may have the virus of infectious diseases common to human beings and animals in the animal material; But the institute at present in the technical process does not all have the corresponding ability of removing virus in steps, thereby there is serious safety issue in the antitoxic serum goods when application.。
Summary of the invention
The object of the invention just provides a kind of antitoxic serum preparation technology of viral inactivation treatment, and this technology can effectively prevent in the goods the possible residual of virus, the safety when improving the application of antitoxic serum goods.
The antitoxic serum preparation technology of viral inactivation treatment of the present invention may further comprise the steps:
1, pepsin digestion uses existing common process;
2, precipitate and the degenerative treatments of heating for the first time, after digestion finishes,, regulate pH to 4.50-6.50 by the ammonium sulfate of every 100ml Digestive system adding 10-20g; Heat to 60 ± 1 ℃, be incubated 10 hours, insulation finishes; Be cooled to below 45 ℃, press the 100ml Digestive system and add 0.5-0.8g kieselguhr, after stirring; Digestive system is pumped into plate and frame type filter-press carry out isolated by filtration, filter and collect supernatant, precipitate discarded;
3, precipitation process is for the second time used existing common process;
4, alum precipitate is handled, and uses existing common process;
5, ultrafiltration and concentration is handled, and uses existing common process;
6, stock solution preparation, the ultrafiltration and concentration liquid pump that above-mentioned ammonium sulfate content is up to standard is gone into preparing tank, tires according to required stock solution and adds water for injection; Press 8.0-8.5g/L and add sodium chloride; Press 2.0-2.5g/L then and add metacresol as antiseptic, transfer pH to 4.1 ± 0.3, the clear liquid lucifuge left in 24 ± 1 ℃, 21 days after the aseptic filtration; Again above-mentioned clear liquid is transferred pH to 6.0-7.0; Aseptic filtration gets antitoxic serum stock solution, and described pH value is regulated, and the regulator that uses is sodium hydroxide, the hydrochloric acid of 1-2M.
Method in the above-mentioned steps 2 is the principle that has adopted Pasteur's method inactivation of virus: different microorganisms has different optimum growth temperatures and heat-resisting, cold-resistant ability.In certain temperature range, temperature is low more, and microbial reproduction is slow more; Temperature is high more, breeds fast more; But temperature is too high, and microorganism will be dead.Pasteur's method inactivation of virus is exactly to utilize the various viruses heat labile characteristics that resist cold in fact, handles with certain temperature and temperature retention time, and the higher structure of virus protein is damaged; Albumen no longer includes physiologically active; So lose infection, cause a disease and fertility, reach the effect of inactivation of viruses.
Method in the above-mentioned steps 6 is to have adopted low pH to incubate the principle of putting the method inactivation of virus: low pH can make the cellular antigens electric charge of virus surface change; Irreversible degeneration takes place in proteinic space structure; Thereby make virus forfeiture and the bonded ability of cell receptor; Can not get into the cell completion and infect, reach the effect of inactivation of viruses.
Described antitoxic serum comprises the liquid preparation and the lyophilized formulations of tetanus antitoxin, diphtheria antitoxin, Gas Gangrene Antitoxin, botulinum Antitoxin, rabies antiserum, all kinds of antivenins.
Described various animal immune blood plasma (serum) comprise following animal origin: horse, mule, donkey, pig, sheep, cattle, rabbit, camel.
The antitoxic serum preparation technology of viral inactivation treatment of the present invention has adopted two kinds of virus inactivating methods, and two kinds of virus inactivating methods can be to implement or combine enforcement respectively separately, and following advantage is compared with prior art arranged:
1, the present invention has increased the step of inactivation of virus in antitoxic serum preparation technology, makes the antitoxic serum product safer;
2, the present invention has adopted the combination of two methods to carry out inactivation of virus, makes deactivation more thorough;
3, the virus inactivating method that adopts of the present invention is physical method, does not add any other material, and the preparation of antitoxic serum stock solution is not brought other possible problems;
4, the virus inactivating method of the present invention's employing does not have specific (special) requirements to Factory Building, facility, personnel, and is convenient, easy to operate.
The specific embodiment
A kind of antitoxic serum preparation technology of viral inactivation treatment comprises following step:
1, pepsin digestion; With the immune blood plasma of assay approval, extraordinarily go into the purified water dilution by the 2-4 of plasma volume, transfer pH to 2.90-3.50; The gastric enzyme that adds 3-10 unit of activity according to the diluent total amount by every ml diluent; Add 0.2% (ml/ml) toluene (or not adding), control Digestive system temperature digested 1-1.5 hour in 30 ± 1 ℃;
2, precipitate and the degenerative treatments of heating for the first time, after digestion finishes,, regulate pH to 4.50-6.50 by the ammonium sulfate of every 100ml Digestive system adding 10-20g; Heat to 60 ± 1 ℃, be incubated 10 hours, insulation finishes; Be cooled to below 45 ℃, press the 100ml Digestive system and add 0.5-0.8g kieselguhr, after stirring; Digestive system is pumped into plate and frame type filter-press carry out isolated by filtration, filter and collect supernatant, precipitate discarded;
3, precipitation process for the second time pumps into above-mentioned clear liquid in the sterilized digestion Agitation Tank, transfers pH to 7.20-7.40; Every 100ml adds 19-21g ammonium sulfate, after stirring, presses 100ml filtrating and adds 0.8-1g kieselguhr; Stir; Filtrate is pumped into plate and frame type filter-press carry out isolated by filtration, filter the collecting precipitation thing, discarded supernatant;
4, alum precipitate is handled, and water for injection is injected sterilized digestion Agitation Tank, is cooled to below 30 ℃; Then dissolved dilution to protein content in the above-mentioned deposition adding water for injection is no more than 2% (g/ml), adds alum solution, regulate pH to 7.70-7.90 by the concentration that is not less than 0.8% (g/ml); Behind the stirring and adsorbing 0.5-1h; Pump into plate and frame type filter-press and carry out isolated by filtration, filter and collect supernatant, precipitate discarded;
5, ultrafiltration and concentration, the clear liquid that above-mentioned Alumen is adsorbed after-filtration pumps into the ultrafiltration jar behind pre-flock, carry out ultrafiltration and concentration through ultrafilter again, and the ammonium sulfate content in making ultrafiltration and concentration liquid is below 1.0g/L;
6, stock solution preparation, the ultrafiltration and concentration liquid pump that above-mentioned ammonium sulfate content is up to standard is gone into preparing tank, tires according to required stock solution and adds water for injection; Press 8.0-8.5g/L and add sodium chloride; Press 2.0-2.5g/L then and add metacresol as antiseptic, use the hydrochloric acid of 1-2M to transfer pH to 4.1 ± 0.3, the clear liquid lucifuge left in 24 ± 1 ℃, 21 days after the aseptic filtration; Use the sodium hydroxide of 1-2M to transfer pH to 6.0-7.0 above-mentioned clear liquid again, aseptic filtration gets antitoxic serum stock solution.
Antitoxin stock solution should place 2-8 ℃ of lucifuge place, preserves at least 1 month as stable phase.
The inactivation of virus effect
Requirement method by " blood products removal/inactivation of viruses technical method and verification guide principle " verifies that the inactivation of virus effect of above-mentioned virus inactivating method can reach and descend more than 4 log, meets the requirements.
| The inactivation of virus effect | Pseudorabies virus (PRV) | Encephalitis b virus (Sindbis) |
| 60 ℃, 10 hours | Do not detect | Do not detect |
| PH4.1,21 days | Do not detect | Do not detect |
Product quality indicator
By " Chinese pharmacopoeia (version in 2010) standard method is measured, the antitoxic serum stock solution of new technology preparation, and quality index has corresponding raising
| Project | IgG content % | F (ab) 2 content % | Than IU/g alive |
| Former technology | 5.0 | 62 | 55813 |
| New technology | 0.1 | 64 | 64583 |
Claims (4)
1. the antitoxic serum preparation technology of a viral inactivation treatment comprises following conventional steps: (1), pepsin digestion; (2), precipitate and the degenerative treatments of heating for the first time; (3), precipitation process for the second time; (4), alum precipitate is handled; (5), ultrafiltration and concentration is handled; (6), stock solution preparation, it is characterized in that: the concrete technology of said step (2) is after step (1) digestion finishes, to add the ammonium sulfate of 10-20g by every 100ml Digestive system; Regulate pH to 4.50-6.50, heat to 60 ± 1 ℃, be incubated 10 hours, insulation finishes; Be cooled to below 45 ℃, press the 100ml Digestive system and add 0.5-0.8g kieselguhr, after stirring; Digestive system is pumped into plate and frame type filter-press carry out isolated by filtration, filter and collect supernatant, precipitate discarded.
2. the antitoxic serum preparation technology of a viral inactivation treatment comprises following conventional steps: (1), pepsin digestion; (2), precipitate and the degenerative treatments of heating for the first time; (3), precipitation process for the second time; (4), alum precipitate is handled; (5), ultrafiltration and concentration is handled; (6), stock solution preparation, it is characterized in that: the concrete technology of said step (6) is, will (5) in the ultrafiltration and concentration liquid pump of ultrafiltration and concentration processing go into preparing tank; Tire according to required stock solution and to add water for injection, press 8.0-8.5g/L and add sodium chloride, press 2.0-2.5g/L adding metacresol then as antiseptic; Transfer pH to 4.1 ± 0.3; The clear liquid lucifuge left in 24 ± 1 ℃, 21 days after the aseptic filtration, more above-mentioned clear liquid was transferred pH to 6.0-7.0, and aseptic filtration gets antitoxic serum stock solution.
3. the antitoxic serum preparation technology of a viral inactivation treatment comprises following conventional steps: (1), pepsin digestion; (2), precipitate and the degenerative treatments of heating for the first time; (3), precipitation process for the second time; (4), alum precipitate is handled; (5), ultrafiltration and concentration is handled; (6), stock solution preparation; It is characterized in that: the concrete technology of said step (2) is after step (1) digestion finishes, by the ammonium sulfate of every 100ml Digestive system adding 10-20g, to regulate pH to 4.50-6.50; Heat to 60 ± 1 ℃, be incubated 10 hours, insulation finishes; Be cooled to below 45 ℃, press the 100ml Digestive system and add 0.5-0.8g kieselguhr, after stirring; Digestive system is pumped into plate and frame type filter-press carry out isolated by filtration, filter and collect supernatant, precipitate discarded; The concrete technology of said step (6) is, the ultrafiltration and concentration liquid pump that ultrafiltration and concentration in (5) is handled is gone into preparing tank, tires according to required stock solution and adds water for injection; Press 8.0-8.5g/L and add sodium chloride; Press 2.0-2.5g/L then and add metacresol as antiseptic, transfer pH to 4.1 ± 0.3, the clear liquid lucifuge left in 24 ± 1 ℃, 21 days after the aseptic filtration; Again above-mentioned clear liquid is transferred pH to 6.0-7.0, aseptic filtration gets antitoxic serum stock solution.
4. according to the antitoxic serum preparation technology of the described viral inactivation treatment of claim 1-3, it is characterized in that: the pH value in the said step (6) is regulated, and the regulator that uses is sodium hydroxide, the hydrochloric acid of 1-2M.
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| CN201210088398XA CN102526729A (en) | 2012-03-30 | 2012-03-30 | Preparation process of antitoxic serum for viral inactivation treatment |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107789647A (en) * | 2016-09-05 | 2018-03-13 | 上海赛伦生物技术股份有限公司 | It is a kind of to inactivate method viral in animal blood serum or blood plasma |
| CN114456258A (en) * | 2021-12-29 | 2022-05-10 | 江西生物制品研究所股份有限公司 | Purifying method of antitoxin antiserum product |
| CN116355085A (en) * | 2023-03-31 | 2023-06-30 | 西安麦博泰克生物科技有限公司 | Tetanus antitoxin production method |
| CN117126272A (en) * | 2023-07-11 | 2023-11-28 | 江西生物制品研究所股份有限公司 | Antitoxin serum and preparation method thereof |
Citations (2)
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| CN1483476A (en) * | 2002-09-17 | 2004-03-24 | 兰州生物制品研究所 | Process for preparing antitoxin toxinicide |
| CN1947727A (en) * | 2005-10-11 | 2007-04-18 | 上海赛伦生物技术有限公司 | Anti-echidnotoxin blood serum and its preparing method |
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2012
- 2012-03-30 CN CN201210088398XA patent/CN102526729A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483476A (en) * | 2002-09-17 | 2004-03-24 | 兰州生物制品研究所 | Process for preparing antitoxin toxinicide |
| CN1947727A (en) * | 2005-10-11 | 2007-04-18 | 上海赛伦生物技术有限公司 | Anti-echidnotoxin blood serum and its preparing method |
Non-Patent Citations (1)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107789647A (en) * | 2016-09-05 | 2018-03-13 | 上海赛伦生物技术股份有限公司 | It is a kind of to inactivate method viral in animal blood serum or blood plasma |
| CN114456258A (en) * | 2021-12-29 | 2022-05-10 | 江西生物制品研究所股份有限公司 | Purifying method of antitoxin antiserum product |
| CN116355085A (en) * | 2023-03-31 | 2023-06-30 | 西安麦博泰克生物科技有限公司 | Tetanus antitoxin production method |
| CN117126272A (en) * | 2023-07-11 | 2023-11-28 | 江西生物制品研究所股份有限公司 | Antitoxin serum and preparation method thereof |
| CN117126272B (en) * | 2023-07-11 | 2024-05-28 | 江西生物制品研究所股份有限公司 | Antitoxin serum and preparation method thereof |
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Application publication date: 20120704 |