CN1709506A - Horse anteserum preparing and purifying process - Google Patents
Horse anteserum preparing and purifying process Download PDFInfo
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- CN1709506A CN1709506A CN 200510026317 CN200510026317A CN1709506A CN 1709506 A CN1709506 A CN 1709506A CN 200510026317 CN200510026317 CN 200510026317 CN 200510026317 A CN200510026317 A CN 200510026317A CN 1709506 A CN1709506 A CN 1709506A
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Abstract
The present invention relates to a preparation and purification process of horse antiserum (antitoxin). Said process includes the following steps: immune plasma digestion treatment, double precipitation by adding ammonium sulfate, third adsorption precipitation and concentration. Said invention adopts ion exchanger column chromatography to make adsorption and purification, and can remove acidic heteroproteins, such as Fc fragment, aggregate and small-molecular peptide, etc, except for F(ab)2 fragment, so that it can effectively raise the purity of horse antiserum (antitoxin) preparation.
Description
Technical field:
The present invention relates to biological technical field, be specifically related to preparation, the purifying process of a kind of horse anteserum (antitoxin).
Background technology:
Horse anteserum (antitoxin) is the immune serum (blood plasma) by corresponding toxin or toxoid (comprising antibacterial, virus) immune animal gained, the immunoglobulin preparation that makes through the pepsin digestion ammonium sulfate precipitation.At present, the preparation method of horse anteserum (antitoxin) comprises the steps: 1, with distilled water with various sero-fast immune blood plasmas dilutions, and carry out digestion process with gastric enzyme and get Digestive system; 2, Digestive system is carried out precipitating the first time and heating processing, add ammonium sulfate in Digestive system, and regulate pH value, then Digestive system is heated and keep, cooling is again filtered and is collected supernatant, discarded precipitation; 3, above-mentioned clear liquid is carried out the sedimentation and filtration second time, promptly in above-mentioned supernatant, add ammonium sulfate, leave standstill the after-filtration taking precipitate, abandon supernatant; 4, alum precipitate; Be about to above-mentioned precipitate dilution, add Alumen again in diluent, leave standstill after-filtration and get supernatant, precipitate is discarded; 5, above-mentioned supernatant concentration can be made antiserum stock solution.But the weak point of this technology is that the antiserum purity [F (ab) 2 content, specific activity (IU/gp)] of preparation is lower, and the incidence rate of horse serum anaphylaxis rate and serum sickness is higher.
Summary of the invention:
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and the preparation technology of a kind of raising horse anteserum (antitoxin) preparation purity [F (ab) 2 content, specific activity (IU/gp)] is provided.
The invention provides the preparation purifying process of a kind of horse anteserum (antitoxin), this technology comprises the steps:
(1) digestion step: immune blood plasma is digested with the immune blood plasma digestion process;
(2) sedimentation and filtration treatment step for the first time: will add ammonium sulfate in the Digestive system of above-mentioned processing and form suspension, and to regulate pH value be 4.8-5.2 that then suspension is heated and keep, cooling is again filtered and collected supernatant, discarded precipitation;
(3) sedimentation and filtration treatment step for the second time, it is 7.0-7.4 that the above-mentioned clear liquid in above-mentioned (2) step is adjusted pH value, adds ammonium sulfate and carries out precipitating second time, leaves standstill the after-filtration taking precipitate, abandons supernatant;
(4) alum precipitate; The precipitate in soon above-mentioned (3) step is diluted to protein content and is no more than 2g/L, and adjusting the diluent pH value again is 7.7-7.9, adds Alumen and precipitates, and leaves standstill after-filtration and gets supernatant, and precipitate is discarded;
(5) above-mentioned supernatant is concentrated with ammonium sulfate precipitation method or ultrafiltration make antiserum stock solution;
(6) ion-exchanger adsorption and purification:
1) adorning post with anionite, is 7.0 ± 0.2 with pH value, 0.15M NaCl, and the 10mM phosphate buffer carries out balance;
2) will carry out the antiserum stock solution of clarification filtration with the film of 0.45 μ m by last sample pump adding cylinder top, with pH value is 7.0 ± 0.2,0.15M NaCl, the 10mM phosphate buffer is washed post, flow velocity is 3ml/min, collection penetrates liquid (acid impurities is attracted on the chromatography media, and F (ab) 2 streams are worn);
3) with behind the 2M NaCl cleaning cylinder, wash post, medium is soaked wherein preserve with 20% ethanol;
4) will penetrate the liquid ultrafiltration and concentration and adjust antiserum concentration, adjust protein content and be no more than 170g/L, by adjusting the back total product amount, adding sodium chloride, to make final content be 750-950g/L, adding chloroform, to make final content be no more than 5g/L or thimerosal 0.1g/L or metacresol 2.5g/L be antiseptic, regulate pH to 6.0-7.0, with the aseptic filtration of 0.22um filter membrane.
Described horse anteserum (antitoxin) comprises tetanus antitoxin, diphtheria antitoxin, Gas Gangrene Antitoxin, botulinum Antitoxin, rabies antiserum, antivenin etc.
Described ion-exchanger is a kind of among SP, Q, CM, DEAE, the ANX Sepharose Fast Flow.
The present invention compared with prior art has following advantage:
1, effectively improve the purity of horse anteserum (antitoxin) preparation, be mainly reflected in F (ab) 2 content and specific activity (IU/gp), as following table (one), shown in the table (two):
Table one
| Quality index | F (ab) 2 content | The specific activity tetanus antitoxin | (IU/gp) rabies antiserum |
| Improve behind the purification before the purification | ??50-70％ ??70-90％ ??20-50％ | ??40000-60000 ??60000-120000 ??30-50％ | ??3000-5000 ??5000-10000 ??30-50％ |
Table two
| The goods title | State | F (ab) 2 content | Specific activity (IU/gp) |
| Tetanus antitoxin | Stock solution | ??61 | ??54000 |
| Behind the purification | ??85 | ??95000 | |
| Rabies antiserum | Stock solution | ??56 | ??3500 |
| Behind the purification | ??75 | ??7300 |
| Botulinum Antitoxin (A type) | Stock solution | ??65 | ??22000 |
| Behind the purification | ??81 | ??36000 |
2, effectively reduce horse anteserum (antitoxin) preparation because the anaphylaxis that foreign protein causes and the incidence rate of serum sickness.
Concrete embodiment:
Example 1
The preparation purifying process of horse anteserum (antitoxin):
1, digestion
With immune blood plasma doubly, regulate pH to 3.0-3.6, add an amount of gastric enzyme and toluene 0.2% (ml/ml), in 29-31 ℃ of digestion 1-1.5 hour with 1-2N hydrochloric acid with distilled water diluting 2-4.
2, precipitate and the processing of heating for the first time
To add ammonium sulfate 15g in every 100ml Digestive system of above-mentioned processing, diphtheria and tetanus antitoxin are regulated pH to 5.0-5.4 with the 1-2N sodium hydroxide; Antivenin, gas gangrene and botulinum Antitoxin are regulated pH to 4.8-5.2, and antivenin, diphtheria, tetanus, Gas Gangrene Antitoxin are heated to 57 ± 1 ℃; Botulinum Antitoxin is heated to 51 ± 1 ℃, all keeps 30 minutes.Heating finishes, and is cooled to as early as possible below 45 ℃, filters, and gets clear liquid.
3, precipitate for the second time
Above-mentioned clear liquid is regulated pH to 7.0-7.4 with the 1-2N sodium hydroxide, and every 100ml adds ammonium sulfate 20g; Stirred back static 30 minutes, and filtered, get precipitation.
4, alum precipitate
Above-mentioned precipitation is diluted with water to protein content and is no more than 2g/L, and every 100ml adds 10% alum steep 6-10ml, regulates pH to 7.7-7.9 with the 1-2N sodium hydroxide, filters, and gets clear liquid.
5, concentrate
Above-mentioned clear liquid is mixed, carry out ultrafiltration by ultrafiltration apparatus, being concentrated into ammonium sulfate content is below 0.1% (g/ml), is antiserum stock solution.
The ion-exchanger adsorption and purification
(1) with anionite dress posts such as DEAE Sepharose Fast Flow, be 7.0 ± 0.2 with pH value, 0.15M NaCl, 10mM phosphate buffer carry out balance;
(2) will carry out the antiserum stock solution of clarification filtration with the film of 0.45 μ m by last sample pump adding cylinder top, with pH value is 7.0 ± 0.2,0.15M NaCl, the 10mM phosphate buffer is washed post, flow velocity is 3ml/min, collection penetrates liquid (acid impurities is attracted on the chromatography media, and F (ab) 2 streams are worn);
(3) with behind the 2M NaCl cleaning cylinder, wash post, medium is soaked wherein preserve with 20% ethanol.
(4) will penetrate the liquid ultrafiltration and concentration and adjust antiserum concentration (antibody titer), and adjust protein content and be no more than 170g/L.By adjusting the back total product amount, adding sodium chloride, to make final content be 750-950g/L, adds chloroform and make final content be no more than 5g/L, and thimerosal 0.1g/L or metacresol 2.5g/L are antiseptic.Regulate pH to 6.0-7.0.With the aseptic filtration of 0.22um filter membrane.
7, result
| The goods title | State | F (ab) 2 content | Specific activity (IU/gp) |
| Tetanus antitoxin | Stock solution | ??61 | ??54000 |
| Behind the purification | ??85 | ??95000 | |
| Rabies antiserum | Stock solution | ??56 | ??3500 |
| Behind the purification | ??75 | ??7300 | |
| Botulinum Antitoxin (A type) | Stock solution | ??65 | ??22000 |
| Behind the purification | ??81 | ??36000 |
Claims (3)
1, a kind of horse anteserum preparing and purifying process, this technology comprises the steps:
(1) digestion step: immune blood plasma is digested with the immune blood plasma digestion process;
(2) sedimentation and filtration treatment step for the first time: will add ammonium sulfate in the Digestive system of above-mentioned processing and form suspension, and to regulate pH value be 4.8-5.2 that then suspension is heated and keep, cooling is again filtered and collected supernatant, discarded precipitation;
(3) sedimentation and filtration treatment step for the second time: the above-mentioned clear liquid pH value in above-mentioned (2) step is adjusted into 7.0-7.4, adds ammonium sulfate and carry out precipitating second time, leave standstill the after-filtration taking precipitate, abandon supernatant;
(4) alum precipitate: the precipitate in soon above-mentioned (3) step is diluted to protein content and is no more than 2g/L, and adjusting the diluent pH value again is 7.7-7.9, adds Alumen and precipitates, and leaves standstill after-filtration and gets supernatant, and precipitate is discarded;
(5) above-mentioned supernatant is concentrated with ammonium sulfate precipitation method or ultrafiltration make antiserum stock solution;
(6) ion-exchanger adsorption and purification:
1) adorning post with anionite, is 7.0 ± 0.2 with pH value, 0.15M NaCl, and the 10mM phosphate buffer carries out balance;
2) the antiserum stock solution that will carry out clarification filtration with the film of 0.45 μ m is 7.0 ± 0.2 by last sample pump adding cylinder top with pH value, 0.15M NaCl, the 10mM phosphate buffer is washed post, and flow velocity is 3ml/min, collects to penetrate liquid, acid impurities is attracted on the chromatography media, and F (ab) 2 streams are worn;
3) with behind the 2M NaCl cleaning cylinder, wash post, medium is soaked wherein preserve with 20% ethanol;
4) will penetrate the liquid ultrafiltration and concentration and adjust antiserum concentration, adjust protein content and be no more than 170g/L, by adjusting the back total product amount, adding sodium chloride, to make final content be 750-950g/L, adding chloroform, to make final content be no more than 5g/L or thimerosal 0.1g/L or metacresol 2.5g/L be antiseptic, regulate pH to 6.0-7.0, with the aseptic filtration of 0.22um filter membrane.
2, horse anteserum preparing and purifying process as claimed in claim 1 is characterized in that wherein said horse anteserum comprises tetanus antitoxin, diphtheria antitoxin, Gas Gangrene Antitoxin, botulinum Antitoxin, rabies antiserum or antivenin.
3, the preparation purifying process of horse anteserum as claimed in claim 1 (antitoxin) is characterized in that described anionite is SP, Q, CM, DEAE or ANX Sepharose Fast Flow.
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| CN 200510026317 CN1709506A (en) | 2005-05-31 | 2005-05-31 | Horse anteserum preparing and purifying process |
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| CN 200510026317 CN1709506A (en) | 2005-05-31 | 2005-05-31 | Horse anteserum preparing and purifying process |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102512449A (en) * | 2012-01-09 | 2012-06-27 | 玉溪九洲生物技术有限责任公司 | Method for removing viruses in preparation of antitoxin and antiserum |
| CN102532306A (en) * | 2012-01-09 | 2012-07-04 | 玉溪九洲生物技术有限责任公司 | Method for inactivating viruses in process of preparing antitoxin and antiserum |
| CN102639554A (en) * | 2009-11-18 | 2012-08-15 | Lfb生物制药公司 | Method for treating blood plasma including a step of washing by means of dispersion |
| CN113801221A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake poison resisting serum nano-membrane filtration method |
| 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 |
-
2005
- 2005-05-31 CN CN 200510026317 patent/CN1709506A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102639554A (en) * | 2009-11-18 | 2012-08-15 | Lfb生物制药公司 | Method for treating blood plasma including a step of washing by means of dispersion |
| CN102512449A (en) * | 2012-01-09 | 2012-06-27 | 玉溪九洲生物技术有限责任公司 | Method for removing viruses in preparation of antitoxin and antiserum |
| CN102532306A (en) * | 2012-01-09 | 2012-07-04 | 玉溪九洲生物技术有限责任公司 | Method for inactivating viruses in process of preparing antitoxin and antiserum |
| CN113801221A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake poison resisting serum nano-membrane filtration method |
| CN113801221B (en) * | 2020-06-16 | 2024-07-05 | 上海赛伦生物技术股份有限公司 | Sea snake venom resisting serum nanometer film filtering method |
| 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 |
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