CN1137529A - Production process for modified low temp. ethanolic human gammaglobulin - Google Patents
Production process for modified low temp. ethanolic human gammaglobulin Download PDFInfo
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- CN1137529A CN1137529A CN 95118470 CN95118470A CN1137529A CN 1137529 A CN1137529 A CN 1137529A CN 95118470 CN95118470 CN 95118470 CN 95118470 A CN95118470 A CN 95118470A CN 1137529 A CN1137529 A CN 1137529A
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Abstract
The present invention relates to an improved production process for preparing human gammaglobulin by using low-temp. ethyl alcohol. It is characterized by that the precipitate A+B+C which are separated out from reduce-cooling precipitated plasm is used as raw material, and after said precipitate is frozen for more than 48 hr., its components A and C are separated out, and then its component B further is obtained by means of further filtering process. The obtained precipitate is further passed through dissolving, filtering and concentrating treatment to obtain the invented purified refined gammaglobulin. Its yield is high, quality is good, operation is easy and cost is low.
Description
The present invention relates to proteinic separation, purification process, be specifically related to the production technique of human, particularly traditional cold ethanol human producing and manufacturing technique improvement.
After people such as Cohn found isolating the 5th method of cold ethanol human plasma protein fraction segmentation and the 6th method, people such as Oncley set up isolating the 9th method of the further segmentation of Cohn component I I+III (but also being called the Oncley method) not only in nineteen forty-four, Deutsch in 1945 etc. have improved and have reclaimed the technology of third ball and be used for suitability for industrialized production from component I I+III in the 9th method, this improved technology is called the Deutsch method, Nitschmann prepared third ball with the Deutsch method from the precipitate A (being equivalent to component I I+III) that improves the 10th method in 1954, and Kistler had carried out improvement to the Nitschmann method again and delivered in 1962 afterwards.In three more than ten years afterwards, CohnShi 9 methods, Deutsch method and Kistler rule become the common process of producing third ball.But these technologies still come with some shortcomings, and less stable often occurs as third ball product of producing, and therefore quality problems such as natural cracking and polymerization are necessary conventional third ball producing process of tradition is improved.
The objective of the invention is to set up the improvement cold ethanol human production technique of reliable matter third ball product of preparation.
Purpose of the present invention adopts following process implementing:
By testing repeatedly and long term production, recognize PH, alcohol concn, temperature, ionic strength, protein concn five factors are the principal elements that influence the gamma-globulin rate of recovery and quality.Search out an operational path and a processing parameter that reaches the object of the invention, this technological process spends the isolated component I+II+III of CPP and is precipitated as raw material, this precipitation is through after freezing more than 48 hours, through separated portion I, separated portion III again, component I I supernatant obtains the component precipitation again through dissolving dark filter through dark filter, concentrates and makes refining gamma-globulin.Its specific embodiment is:
Its condition of A, separated portion I is: 0.01MNaAc-0.0056MNa
2HPO
410 times of dissolvings of solution, PH7.6 ± 0.1, temperature 0～-2 ℃, ionic strength 0.033, protein 2.5% gets component I supernatant and component I precipitation through centrifugation.
The condition of B, separated portion III is: the dilution of times frozen water such as component I supernatant, and PH5 ± 0.05, ethanol 17%, temperature-4 ℃, ionic strength 0.014, protein 0.8% gets component III supernatant and component III precipitation through centrifugation.
The condition of C, the dark filter of component III supernatant is: 1MNaHCO
3Transfer to PH5.5 ± 0.1, be cooled to-7--8 ℃, 0.5% diatomite filter deeply the dark filtrate of component III.
The condition of D, separated portion II is: PH7.2, and ethanol 25%, temperature-7 ℃, ionic strength 0.05, protein 0.4% gets component I I precipitation and component I I supernatant (abandoning) through centrifugation.
E, component I I resolution of precipitate, filtering condition are: the dissolving of 4 times of frozen water, temperature 0-2 ℃, 0.5% diatomite, filter deeply filtrate.
F, the dark filtrate ultrafiltration of E step: dealcoholysis, concentrated.
G, ultrafiltration and concentration liquid transfer PH6.5 ± 0.1, add degerming behind the glycine 0.3M.
H, packing: the 10% the third ball solution.
The drawing explanation:
Fig. 1 is a process flow sheet of the present invention.
Fig. 2 is a gamma-globulin chromatography collection of illustrative plates of the present invention.
Goods of the present invention are listed in table 1, table 2 and Fig. 2 through every analyses such as electrophoresis purity analysis and gas phase, liquid chromatograies with the traditional handicraft comparing result.
Table 1: several technique third ball rate of recovery and recovery rate are relatively
| Separation method | The rate of recovery (%) | Recovery rate (grams per liter) |
| The present invention of Kistler-Nittschmann method Hao short cut technique | Be 9.8% 48.5 80 of albumen | 5.2 2.8 4.7 |
Table 2: the main quality of the third ball and Cohn5 method are relatively
| Item compared | The 10% the third ball solution (n=23) | |
| This technique | The Cohn9 method | |
| 57 ℃ of water-bath heat endurances of pH value (hr) Content of polymers (%) electrophoresis purity (%) cracking degree (TCA method OD value) plasmin activity (IU/ml) | 6.5 muddiness 1.24 ± 0.38＞98＜0.04＜0.003 after ± 0.1 4 hour | 6.4-7.4 gelation 4.22 ± 0.89＞95＞0.5＞0.05 after 4 hours |
From the result of chart as seen, technique of the present invention has following characteristics: the 1 gamma-globulin rate of recovery is 80%, recovery rate 4.7 grams per liter blood plasma. 2, good product quality, electrophoresis purity are more than 98%, and particularly heat endurance is very good, Content of polymer is low, and plasmin activity is below 6% of Cohn9 method only, and the cracking degree only is below 8% of Cohn9 method, that is to say that the third ball that technique of the present invention makes is almost without cracking. 3, with short production cycle, from being dosed into the ultra-filtration and separation operation as long as three day time. 4, easily grasp, this technological operation difficulty is little, and worker's parameter is easily grasped, batch good reproducibility. 5, cost is low, and the comprehensive production cost of this technique is low than conventional method. 6, be conducive to the comprehensive utilization of blood plasma.
Further specify the present invention below in conjunction with embodiment:
Embodiment 1:
(1) component I+II+III precipitation 25Kg adds 0.01M NaAc-0.0056M Na
2HPO
4Solution is dissolved to 250 liters, and adjusting PH is 7.6 ± 0.1, and temperature 0--2 ℃, centrifugal removal component I precipitation.(2) 240 liters of addings of component I supernatant liquor wait a times frozen water dilution, transfer PH to 5.0 ± 0.1, add 95% ethanol 94.8 to rise to concentration 17%, stir simultaneously and are cooled to-4 ℃, standing over night.(3) tubular-bowl centrifuge is centrifugal, removes the component III precipitation, gets 320 liters of component III supernatants, uses 1M NaHCO
3Transfer to PH5.5 ± 0.1, be cooled to-7--8 ℃, add diatomite by 0.5%, stir and filter deeply after 30 minutes.(4) dark filtrate is adjusted PH to 7.2, adds 95% ethanol to 25% concentration, stirs simultaneously, keeps-7 ℃ of temperature, quiet letting slip night.(5) centrifugal, obtain 9.7 kilograms of component precipitations, because of 4 times of frozen water dissolvings, temperature is controlled at 0-2 ℃, adds diatomite by 5% and filters deeply, and it is that 6.4-6.5 carries out the ultrafiltration dealcoholysis, concentrates that filtrate is adjusted pH value, press 0.3M and add the glycine stablizer, get 23.3 liters of 10.2% protein concentration liquid, filtration sterilization afterwards, packing.
The rate of recovery and the purity of 23 batches of gamma-globulins of embodiment 1 explained hereafter are described with table 3 below.
Table 3: with the 23 batches third ball rate of recovery of improved technology production
| Batch (criticizing) | Blood plasma volume (liter) | Blood plasma total protein (grams per liter) | Purity (%) | Extraction yield (grams per liter) | Packing yield (grams per liter) | The rate of recovery (%) |
| ????4 ????4 ????3 ????4 ????4 ????1 ????3 | ????8400 ????7680 ????6300 ????9700 ????7300 ????1180 ????2000 | ?54.2±2.3 ?52.8±1.6 ?48.4±2.0 ?50.6±1.6 ?54.8±2.4 ?54.5 ?54.6±2.0 | ?98.6±1.40 ?98.5±1.14 ?99.0±0.60 ?98.4±0.71 ?98.5±0.18 ?98.7 ?99.0±0.22 | ?4.9±0.17 ?4.6±0.21 ?4.3±0.42 ?4.4±0.51 ?5.1±0.44 ?4.86 ?4.91±0.32 | ?4.6±0.3 ?4.5±0.3 ?4.2±0.4 ?4.2±0.2 ?4.9±0.3 ?-- ?-- | ?81.1±2.3 ?78.6±1.3 ?79.1±1.7 ?77.6±1.1 ?83.3±1.6 ?80.0 ?80.9±1.2 |
| ????23 | ????43060 | ?52.7±3.1 | ?98.7±0.87 | ?4.71±0.47 | ?4.48＝0.4 | ?80.1±2.5 |
As known from Table 3, the test that this technology stands to produce illustrates that this technology is stable, and its rate of recovery and purity are better than traditional technology.
Claims (5)
1, a kind of improvement cold ethanol human blood gamma-globulin production technique, the invention is characterized in: spend the isolated component I+II+III of CPP and be precipitated as raw material, this precipitation is through after freezing more than 48 hours, through separated portion I, separated portion III again, obtain component I I precipitation through dark filter again,, concentrate and make the purification refine gamma-globulin again through dissolving dark filter.
2, technology according to claim 1 is characterized in that, the processing condition of separated portion I are: with 10 times to the sedimentary 0.01 More's sodium-acetate of I+II+III, 0.0056 the dissolving of More's sodium dihydrogen phosphate is transferred PH7.6 ± 0.1, temperature 0-2 ℃, ionic strength 0.033, protein 2.5%.
3, technology according to claim 1 is characterized in that, the processing condition of separated portion III are: with a times frozen water dilution such as resetting and adding on the component I, be adjusted to PH5.0 ± 0.05, ethanol 17%, temperature-4 ℃, ionic strength 0.014, protein 0.8%.
4, technology according to claim 1, it is characterized in that the processing condition of separated portion II are: use the component III supernatant, add 1 More's sodium bicarbonate and transfer PH to 5.5 ± 0.1, be cooled to-7--8 ℃, filter deeply with 0.5% diatomite, get the dark filtrate of component III, transfer PH to 7.2 again, ethanol 25%, temperature-7 ℃, ionic strength 0.05, protein 0.4%.
5, technology according to claim 1, it is characterized in that the processing condition of purified components II are: component I I precipitation is added 4 times of frozen water dissolvings, controlled temperature 0-2 ℃, 0.5% diatomite is filtered deeply, filtrate is transferred PH to 0.4 6.5, and dealcoholysis concentrates, ultrafiltration and concentration liquid again, transfer concentrated solution pH value to 6.5 ± 0.1, add 0.3 More's glycine, make the 10% the third ball solution through degerming, packing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95118470 CN1137529A (en) | 1995-10-31 | 1995-10-31 | Production process for modified low temp. ethanolic human gammaglobulin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95118470 CN1137529A (en) | 1995-10-31 | 1995-10-31 | Production process for modified low temp. ethanolic human gammaglobulin |
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| CN1137529A true CN1137529A (en) | 1996-12-11 |
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| CN 95118470 Pending CN1137529A (en) | 1995-10-31 | 1995-10-31 | Production process for modified low temp. ethanolic human gammaglobulin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591391B (en) * | 2009-06-30 | 2012-07-11 | 同路生物制药有限公司 | Enterovirus 71 type human immunoglobulin and preparation method thereof |
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1995
- 1995-10-31 CN CN 95118470 patent/CN1137529A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591391B (en) * | 2009-06-30 | 2012-07-11 | 同路生物制药有限公司 | Enterovirus 71 type human immunoglobulin and preparation method thereof |
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