CN104072601A - Preparation method of FII sediment in blood product - Google Patents
Preparation method of FII sediment in blood product Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
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Abstract
The invention provides a preparation method of FII sediment in a blood product. The preparation method of the FII sediment in the blood product comprises the following steps: by taking plasma as a raw material, sequentially preparing FI+II+III sediment and FI+III supernatant by using a low-temperature alcohol method, then taking the FI+III supernatant, separating FII through a low-temperature alcohol method, carrying out pressure filtration, and taking out the sediment. Compared with the prior art, in the pressure filtration process after separating the FII, a PALL50 filtration plate is used; kieselguhr is taken as a filter aid; kieselguhr used in a pre-coated layer is 80-95g/m<2> of the filtration plate; the use amount of the kieselguhr in a liquid to be filtered is 0.2-0.3g/L of plasma; the kieselguhr consists of the following components in percentage by mass: 96.0-98.7% of SiO2, 0.6-1.2% of Al2O3, 0.3-0.4% of Fe2O3, 0.05-0.12% of acid soluble substances and 0.15-0.20% of water soluble substances. Researches show that globulin purity in the FII sediment prepared by the method is greatly improved, so that the guarantee is provided for the improvement of the quality of products; meanwhile, globulin prepared by the method is capable of improving the globulin yield, obviously increasing commodity profit and bringing inestimable commercial values for enterprises.
Description
Technical field
The present invention relates to the preparation method of FII precipitation in blood products.
Background technology
Sphaeroprotein is used for the treatment of primary immunoglobulin G deficiency disease, as X interlocking Immunodeficiency, common variability immunodeficient disease, IMMUNOGLOBULIN G SUBCLASS DEFICIENCY etc.; Treatment Secondary cases immunoglobulin G deficiency is sick, as severe infection, and septicemia of newborn, infantile bronchiolitis etc.; Treatment autoimmune disorder, as primary thrombocytopenic purpura, mucocutaneous lymphnode syndrome etc., are important clinical medicines.At present, cold ethanol method separation and purification sphaeroprotein from human normal plasma is mainly used in the large-scale production of domestic human blood goods, for example < < medical biotechnology goods are learned > > (second edition, People's Health Publisher), < < blood products is learned > > (second edition, People's Health Publisher), CN201110030778.3, CN201110223760.5, CN201210013227.0, CN201110030776.4 etc.Normally used separation process is shown in Fig. 1, wherein, component I (claiming again FI) mainly contains Fibrinogen, component I+III (claiming again FI+III) mainly contains the third ball, thrombogen and Profibrinolysin, component I I (claiming again FII) mainly contains sphaeroprotein, and component V (claiming again FV) mainly contains albumin.Cold ethanol method process is relatively simple, and output is higher, can produce on a large scale, is worldwide widely used.
Using pressure filtering technique to carry out Solid-Liquid Separation is that current most blood products producers are both at home and abroad for the method for cold ethanol separating technology component separation.Press filtration can be used flocculating aids conventionally; its intergranular gap also forms complicated labyrinth structure; when goods fluid challenge filter plate; mainly the mode by electrostatic adhesion and mechanical stop catches solids component, and the cake layer being formed by flocculating aids and deep layer filter plate become the important place that catches solid precipitation particle.Filter plate should have suitable aperture, to can hold back flocculating aids particle to bring minimum resistance to goods solution mobile when forming solid cake layer.
The main control parameters of filter-pressing process comprises the conditions such as selection of filter plate (filtration medium) type, flocculating aids type and consumption.
Wherein, flocculating aids is of a great variety, and its major function is adsorption precipitation particle, helps to filter, also can promote security and the quality of the finished product.The selection of flocculating aids can be considered turbidity requirement, throughput, the rate of recovery of product, the requirement of the aspects such as the stability of product and purity of filtered solution conventionally, and the needs of removing special composition in product solution.Blood products is produced conventional flocculating aids at present diatomite and perlite etc.Diatomite is unicellular diatom settlings in ancient times, has row premium propertiess such as light weight, porous, high-strength, wear-resisting, insulation, adiabatic, absorption and filling, has good chemical stability.The major advantage of super-cell is: there is good microvoid structure, absorption property and anti-compression properties, can not only make to be obtained good velocity ratio by filtrate body, and the fine suspended substance of energy filtering, guaranteed clarity.
The main purpose of adding flocculating aids in blood products is to improve strainability, raising filtrate clarity, reduction filtrate turbidity, heat of adsorption protoplasm, lipid, proteolytic enzyme and metaprotein, thereby guarantees quality of item.Flocculating aids reaches the effect of drainage and absorption in the following manner:
1) mechanical retention effect: the filter cake that flocculating aids forms produces numerous holes and forms the wall between hole and hole, can hold back suspended particles wherein when liquid is flowed through filter cake, comprising:
A. surface filtration effect: Solid-Liquid Separation occurs in the surface of filtration medium, is trapped within flocculating aids surface when the particle diameter of suspended solids is greater than diatomite/perlitic aperture in liquid.
B. Depth Filtration effect: Solid-Liquid Separation occurs in the inside of filtration medium: when the particle diameter of suspended solids is less than diatomite/perlitic aperture in liquid, smaller solid particulate enters medium inside through filter cake surface, and the inner tortuous microporous channels of medium and more tiny hole can be held back these particles.
2) adsorption: produce adsorbing major cause and comprise Van der Waals force, Zeta potential and ion exchange, comprising:
A. the chain group that the suspended particles in liquid form adheres to diatomite/perlite and is trapped;
B. than the little suspended particles in the inner aperture of diatomite/perlite, entering into the inner surface of diatomite/perlite is trapped because opposite charges adsorbs.
Although diatomite is one of flocculating aids conventional in blood products, yet diatomite is various in style, due to differences such as the diatomaceous raw material of difference, processing modes, make different diatomaceous earth products in component content and performance, produce larger difference.
Summary of the invention
The object of the present invention is to provide the preparation method of FI+III supernatant in a kind of blood products.
Particularly, the invention provides the preparation method of FII precipitation in blood products, it is to take blood plasma as raw material, utilize cold ethanol method to prepare successively FI+II+III precipitation, FI+III supernatant, get FI+III supernatant through the separated FII of cold ethanol method, press filtration, gets precipitation and get final product again; Unlike the prior art, in the pressure-filtering process after the separated FII of the present invention, use PALL50 filter plate, the consumption of filter plate is 5～10m
2/ 1000L; Employing diatomite is flocculating aids, and precoated layer diatomite used is 80-95g/m
2filter plate, treats that in filtrate, diatomaceous consumption is 0.2～0.3g/L blood plasma; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
Describedly treat that filtrate being treat the sample of press filtration, after the separated FII of FI+III supernatant, treat press filtration sample.
Further, in described diatomite, SiO
2content is 96.0～96.2%, Al
2o
3content is 1.0～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
Further, described diatomaceous specific surface area is 2.2～2.3m
2/ g, is preferably 2.228m
2/ g.
Further, described diatomaceous centrifugal wet density is 0.23～0.24g/cm
2, be preferably 0.237g/cm
2.
Preferably, described diatomite model is CELPURE1000.
In order further to improve the quality of finished product, the present invention adopts following FI+III supernatant preparation method:
(1) preparation of FI+II+III precipitation: take blood plasma as raw material, through cold ethanol method separation and Extraction FI+II+III, press filtration, gets precipitation and get final product; In described pressure-filtering process, adopt PALL50P filter plate, diatomite is flocculating aids, and filter plate consumption is 10～13m
2/ 1000L blood plasma, diatomite consumption is 4.5～5.5g plasma proteins/1g diatomite; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%;
(2) preparation of FI+III supernatant: get FI+II+III precipitation, through cold ethanol method separation and Extraction FI+III, press filtration, gets supernatant and get final product; In described pressure-filtering process, adopt BECO Steril PR40 filter plate, diatomite is flocculating aids, and filter plate consumption is 10～13m
2/ 1000L blood plasma, diatomite consumption is 8～9g/L blood plasma; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
While limiting filter plate and diatomite consumption in step (2), described " blood plasma " is the starting raw material in step (1).
Wherein, in preparation method of the present invention, press filtration flow velocity and filter plate pressure in each step are:
The FII precipitation that the present invention also provides aforesaid method to prepare.
Further, in described FII precipitation, sphaeroprotein content, more than 99%, is preferably 99.0～100%.
The present invention also provides the preparation method of sphaeroprotein, and it is to be precipitated as raw material with above-mentioned FII, after refining, gets final product to obtain sphaeroprotein.
The present invention's research shows, the FII precipitation that adopts the inventive method to prepare, and wherein sphaeroprotein purity significantly improves, and guarantee is provided for improving the quality of products; Meanwhile, the sphaeroprotein that adopts the inventive method to prepare, can improve sphaeroprotein yield, obviously increases commodity profit, and the commercial value of bringing for enterprise is inestimable.
In addition, in the inventive method gained sphaeroprotein finished product, IgA content obviously reduces, and can reduce the probability that the untoward reaction of IgA antigen-antibody occurs patient, thereby improves Product Safety.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The embodiment of form, is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
The conventional production scheme of Fig. 1 blood plasma product
Fig. 2 production scheme of the present invention
Fig. 3 is used the prior art production scheme of C503 type diatomite drainage
Fig. 4 FII precipitates electrophoretogram, and wherein, A is the resulting FII precipitation of the inventive method electrophoretogram, the FII precipitation electrophoretogram that B obtains for using the existing method of C503 diatomite drainage.
Fig. 5 FI+II+III precipitates electrophoretogram, and wherein, A is the resulting FI+II+III precipitation of the inventive method electrophoretogram, the FI+II+III precipitation electrophoretogram that B obtains for using the existing method of C503 diatomite drainage.
Fig. 6 FI+III supernatant liquor electrophoretogram, wherein, A is the resulting FI+III supernatant liquor of the inventive method electrophoretogram, the FI+III supernatant liquor electrophoretogram of B for using the existing method of C503 diatomite drainage to obtain.
Embodiment
The present invention's diatomite used is all purchased from Imerys Perlite China, Inc.
Embodiment 1 preparation method of the present invention
Take blood plasma as raw material, adopt ordinary method to prepare successively FI+II+III precipitation, FI+III supernatant, get FI+III supernatant, through cold ethanol method, FII is carried out to separation, re-use PALL50 filter plate, take CELPURE1000 diatomite as flocculating aids simultaneously, carry out press filtration, get precipitation and obtain FII precipitation.Wherein, the consumption of diatomite and filter plate is:
| Treat filtrate diatomite used | 0.2～0.3g/L blood plasma |
| Diatomite used in precoated layer | 80-95g/m 2Filter plate |
| Filter plate | 5～10m 2/1000L |
The present invention's diatomaceous detected result used is as follows:
(1) per-cent that burning scope is used XRF to measure
(2) the calcining product acid-soluble material matter (2.0% upper limit) of fluxing
(3) the water-soluble material of calcining product (2.0% upper limit) of fluxing
(4) specific surface area
(5) centrifugal wet density
The preparation method of the supernatant of FI+III described in the present invention can adopt prior art, but more preferably makes to prepare with the following method:
(1) preparation of FI+II+III precipitation: get blood plasma, through cold ethanol method, FI+II+III is carried out to separation, re-use PALL50P filter plate, take CELPURE1000 diatomite simultaneously as flocculating aids and treat filtrate mixing, carry out press filtration, gained precipitation is FI+II+III precipitation.Wherein, the consumption of diatomite and filter plate is:
| Diatomite | 4.5～5.5g plasma proteins/1g diatomite |
| Filter plate | 10～13m 2/ 1000L blood plasma |
(2) preparation of FI+III supernatant: get FI+II+III precipitation, through cold ethanol method, FI+III is carried out to separation, re-use BECO Steril PR40 filter plate, take CELPURE1000 diatomite simultaneously as flocculating aids and treat filtrate mixing, carry out press filtration, get supernatant liquor and obtain FI+III supernatant.Wherein, the consumption of diatomite and filter plate is:
| Diatomite | 8～9g/L blood plasma |
| Filter plate | 10～13m 2/ 1000L blood plasma |
Adopt the preparation method of FI+II+III precipitation, FI+III supernatant herein, compare with the FI+II+III precipitation, the FI+III supernatant that use C503 diatomite to prepare, can find out:
(1) in aforesaid method gained FI+II+III precipitation, sphaeroprotein content is about 81% (Fig. 5 A), and in the FI+II+III precipitation that use C503 diatomite drainage obtains, sphaeroprotein content is about 76% (Fig. 5 B).Contrast knownly, adopt sphaeroprotein purity in FI+II+III precipitation prepared by above-mentioned preferred method obviously to increase approximately 5 percentage points.
(2) in aforesaid method gained FI+III supernatant electrophoretogram (Fig. 6 A), sphaeroprotein peak area accounts for 98.5%, and the FI+III supernatant sphaeroprotein peak area that uses C503 diatomite drainage to obtain only accounts for 93.2% (Fig. 6 B).Contrasting both can find out, in FI+III supernatant prepared by the inventive method, sphaeroprotein purity has obviously increased 5 percentage points.
Therefore,, in order to improve the quality of finished product, in the present invention, preferably use above-mentioned FI+II+III precipitation, FI+III supernatant preparation method.
In addition, the press filtration flow velocity of aforesaid method and filter plate pressure are:
The impact of embodiment 2 preparation method of the present invention on pressure-filtering process
Below test, by indexs such as filtering traffic, pressure and filtrate turbidity, compares (filter plate consumption 5～10m to the present invention with using the existing preparation method of C503 type diatomite, PALL ECO1000 filter plate
2/ 1000L blood plasma, C503 diatomite consumption 110～130g/m in precoated layer
2filter plate, treats C503 diatomite consumption 0.3～0.5g/L blood plasma in filtrate), its result is as follows:
1, filtering traffic
By conventional means, detected and filtered speed, its result is as follows:
The contrast of table 1 filtering rate
As shown in Table 1, compare with the diatomaceous preparation method of existing use C503, in the inventive method pressure-filtering process, unit time inner filtration flow is suitable.
2, filter pressure
By conventional means, detected and filtered pressure, its result is as follows:
The contrast of table 2 filter pressure
As shown in Table 2, compare with the diatomaceous preparation method of existing use C503, the pressure load that in the inventive method pressure-filtering process, unit surface filter plate bears is suitable.
3, filtrate turbidity
By conventional means, detect filtrate turbidity, its result is as follows:
The contrast of table 3 filtrate turbidity
As shown in Table 3, compare with the diatomaceous preparation method of existing use C503, the inventive method gained filtrate turbidity value is suitable.
The impact of embodiment 3 preparation method of the present invention on product
By electrophoretic technique, detect sphaeroprotein purity in FII precipitation, the present invention is compared with using the existing preparation method of C503 type diatomite, ECO1000 filter plate, the results are shown in Figure 4.Wherein, two kinds of methods are except filter-pressing process difference, and other operations are all identical with raw material, more the difference on effect that two kinds of drainage techniques are brought can be described with this understanding.
As seen from the figure: in the inventive method gained FII precipitation collection of illustrative plates (Fig. 4 A), sphaeroprotein peak area accounts for 99.3%; And sphaeroprotein peak area only accounts for 97.9% (Fig. 4 B) in the FII precipitation that use C503 diatomite drainage obtains.Contrasting both can find out, in FII precipitation prepared by the inventive method, sphaeroprotein purity obviously improves, and before refining without finished product, sphaeroprotein purity has just surpassed 99%.
The above results shows, the inventive method is obviously better than prior art to the extraction separating effect of composition in FII precipitation.
The preparation method of embodiment 4 sphaeroprotein
According to the method for embodiment 1, prepare II precipitation, after refining according to ordinary method, obtain sphaeroprotein.
Get with batch raw material, the sphaeroprotein of preparing according to the inventive method compares with the globulin products that uses C503 diatomite traditional technology (process for purification is identical) to prepare, and its result is as follows:
(1) sphaeroprotein yield and purity
Table 4 test batch albumin, sphaeroprotein yield and purity
| ? | Sphaeroprotein yield (n=3) | Sphaeroprotein purity (n=3) |
| Technique products obtained therefrom yield of the present invention and purity mean value | 5.46g/L blood plasma | 99.73％ |
| The product yield and the purity mean value that use C503 diatomite traditional technology to obtain | 5.0g/L g/L blood plasma | 99.2％ |
As seen from the above table, for using the diatomaceous traditional technology of C503, the sphaeroprotein finished product that utilizes the inventive method to prepare, its sphaeroprotein yield and purity all improve.
Sphaeroprotein market price is about 200 yuan/g at present, as described herein, its yield improves about 0.5g/L, with the average each about 1000L plasma calculated of turnout of enterprise, each production can increase income 500g sphaeroprotein, according to marketable value, estimate, each production can improve approximately 100,000 yuan of profits, and its commercial value is extremely remarkable.
(2) IgA content in sphaeroprotein finished product
IgA content in table 6 sphaeroprotein
| Traditional technology sphaeroprotein finished product IgA content (μ g/ml) | The inventive method sphaeroprotein finished product IgA content (μ g/ml) |
| 105.79 | 57.51 |
| 92.3 | 61.06 |
| 85.91 | 67.45 |
| 88.75 | 58 |
| 81.65 | 61.1 |
| 89.46 | 65.1 |
| 97.27 | ? |
| 93.72 | ? |
| Mean value: 91.8 | Mean value: 61.7 |
As seen from the above table, in the inventive method gained sphaeroprotein finished product, IgA content obviously reduces, and can reduce the probability that the untoward reaction of IgA antigen-antibody occurs patient, thereby improves Product Safety.
Claims (10)
1. the preparation method that in blood products, FII precipitates, it is to take blood plasma as raw material, utilize cold ethanol method to prepare successively FI+II+III precipitation, FI+III supernatant, get again FI+III supernatant through the separated FII of cold ethanol method, press filtration, gets precipitation and get final product, and it is characterized in that: in the pressure-filtering process after separated FII, use PALL50 filter plate, the consumption of filter plate is 5～10m
2/ 1000L; Employing diatomite is flocculating aids, and precoated layer diatomite used is 80-95g/m
2filter plate, treats that in filtrate, diatomaceous consumption is 0.2～0.3g/L blood plasma; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
2. preparation method according to claim 1, is characterized in that: in described diatomite, and SiO
2content is 96.0～96.2%, Al
2o
3content is 1.0～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
3. preparation method according to claim 1 and 2, is characterized in that: described diatomaceous specific surface area is 2.2～2.3m
2/ g, is preferably 2.228m
2/ g.
4. preparation method according to claim 1 and 2, is characterized in that: described diatomaceous centrifugal wet density is 0.23～0.24g/cm
2, be preferably 0.237g/cm
2.
5. according to the preparation method described in claim 1～4 any one, it is characterized in that: described diatomite model is CELPURE1000.
6. according to the preparation method described in claim 1～5 any one, it is characterized in that: in described pressure-filtering process, use PALL50 filter plate; Preferably, the consumption of described filter plate is 5～10m
2/ 1000L blood plasma.
7. according to the preparation method described in claim 1～6 any one, it is characterized in that: described FI+III supernatant is adopted preparation with the following method:
(1) preparation of FI+II+III precipitation: take blood plasma as raw material, through cold ethanol method separation and Extraction FI+II+III, press filtration, gets precipitation and get final product; In described pressure-filtering process, adopt PALL50P filter plate, diatomite is flocculating aids and treats that filtrate mixing, filter plate consumption are 10～13m
2/ 1000L blood plasma, diatomite consumption is 4.5～5.5g plasma proteins/1g diatomite; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%;
(2) preparation of FI+III supernatant: get FI+II+III precipitation, through cold ethanol method separation and Extraction FI+III, press filtration, gets supernatant and get final product; In described pressure-filtering process, adopt BECO Steril PR40 filter plate, diatomite is flocculating aids and treats that filtrate mixing, filter plate consumption are 10～13m
2/ 1000L blood plasma, diatomite consumption is 8～9g/L blood plasma; In described diatomite, SiO
2content is 96.0～98.7%, Al
2o
3content is 0.6～1.2%, Fe
2o
3content is 0.3～0.4%, and acid-soluble material matter content is 0.05～0.12%, and water-soluble substances content is 0.15～0.20%.
8. the FII precipitation that described in claim 1～7 any one prepared by method.
9. FII precipitation according to claim 8, is characterized in that: in described FII precipitation, sphaeroprotein content, more than 99%, is preferably 99.0～100%.
10. the preparation method of sphaeroprotein, is characterized in that: it is to be precipitated as raw material with FII claimed in claim 8, after refining, obtains sphaeroprotein.
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Application publication date: 20141001 |