CN110066762A - The method of industrialized production new born bovine purified blood serum - Google Patents
The method of industrialized production new born bovine purified blood serum Download PDFInfo
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- CN110066762A CN110066762A CN201810058613.9A CN201810058613A CN110066762A CN 110066762 A CN110066762 A CN 110066762A CN 201810058613 A CN201810058613 A CN 201810058613A CN 110066762 A CN110066762 A CN 110066762A
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- C—CHEMISTRY; METALLURGY
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The present invention relates to animal blood serum purification process, feeding crosses new born bovine raw material serum pre-flock removal fibrin of the colostrum containing impurity components such as a large amount of lipids, endotoxin and hemoglobins；Serum is separated into retention part (macromolecular complex) and dialysis part (free molecule) through molecular cut off 100-300kd ultrafiltration column；Retention part is added the de- albumen aquation film medium of solid phase and the impurity such as lipid (containing chylomicrons and lipoprotein), immunoglobulin, endotoxin, hemoglobin is promoted to aggregate into flocculent deposit, obtains supernatant liquid through continuous flow centrifugation, clarification filtration；Supernatant liquid merges with ultrafiltration column dialysis part, and new born bovine purified blood serum is made after degerming.Using this method, feeding crosses that colostrum, endotoxin and the exceeded newborn bovine serum of hemoglobin can be processed to meet vaccine cell culture medium, enzyme exempts from the serum of kit quality requirement.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of methods of industrialized production new born bovine purified blood serum.
Background technique
Newborn bovine serum is that vaccine for man and live vaccine produce most important cell culture medium, while being also that enzyme is excused from an examination agent
The important auxiliary material of box, the market demand are huge.Newborn bovine serum is that aseptic collection is just born the stot artery that do not feed in China
The serum that blood is fabricated to, " pharmacopeia " require endotoxin clear and bright lower than 200mg/mL, appearance lower than 10EU/ml, hemoglobin.New west
The sources newborn bovine serum such as orchid, Australia, new born stot feeding cross colostrum 4-14 days, the serum of neck bloodletting production,
Containing a large amount of lipid components, turbid appearance, sticky, it is difficult to aseptic filtration；Usual haemolysis is serious, also pollution has endotoxin, BVDV etc.,
Further limit use value.
The present invention provides a kind of low-cost industrial processing method, feeding is crossed into colostrum containing a large amount of lipids (including chyle
Particle and lipoprotein), the newborn bovine serum of the impurity components such as endotoxin and hemoglobin, be processed into and meet the training of production of vaccine cell
It supports base or kit enzyme exempts from the serum of protective agent quality requirement.This technology fills up international blank, and realizes industry metaplasia
It produces；The serum purification technique for removing the impurity components such as lipid, endotoxin and hemoglobin, both at home and abroad without report.
Summary of the invention
The object of the present invention is to provide a kind of methods of industrialized production new born bovine purified blood serum.
The method of industrialized production new born bovine purified blood serum provided by the present invention, comprising: the defrosting of raw material serum is melted
Change, pre-flock removes fibrin；Pretreated serum is separated into retention part through molecular cut off 100-300kd ultrafiltration column
(macromolecular complex) and dialysis part (free molecule)；Retention part is added the de- albumen aquation film medium of solid phase and promotes lipid
The impurity such as (containing chylomicrons and lipoprotein), immunoglobulin, endotoxin, hemoglobin aggregate into flocculent deposit, through continuous flow
The supernatant liquid that centrifugation, clarification filtration obtain；Supernatant liquid merges with the dialysis part, and new born bovine purifying blood is made after degerming
Clearly.
Preferably, raw material serum is New Zealand, the new born stot feeding of Australia spends colostrum 4-14 days containing a large amount of
Lipid, endotoxin and the exceeded serum of hemoglobin.This method is also suitable for feeding and crosses the new life that colostrum only needs to remove lipid
Ox raw material serum, or individually haemolysis needs to remove the new born bovine raw material serum of hemoglobin.
Preferably, 25m polypropylene material bag-type strainer is selected in pre-filtering.
Preferably, ultrafiltration uses in U.S. Spectrum company molecular cut off 100-300kd ultrafiltration column or ultrafiltration membrane
One kind.
Preferably, clarification filtration medium is Minnesota Mining and Manufacturing Company 90sp membrane stack.
Lipid and immunoglobulin, complement, hemoglobin, endotoxin etc. aggregate into the particle greater than 100nm in serum, lead to
Reason condition 100-300kd ultrafiltration membrane, which is difficult to give, to be come.Ultrafiltration column can effectively by above-mentioned serum impurity enriched in retention part, Gu
Can preferentially the polymerization of bulky grain ingredient, formation in serum be precipitated by mutually taking off albumen aquation film medium, be removed using centrifugation.Tool
Body, retention part takes off albumen aquation film medium with solid phase and mixes in proportion, and mixed proportion is that retention part presses raw material serum
1000L is added the Nanjing 25L Ao Fan Biotechnology Co., Ltd solid phase and takes off albumen aquation film medium DHF01.
After ultrafiltration membrane retention part removes impurity, merge with ultrafiltration membrane dialysis part, new born bovine purifying blood is made after degerming
Clearly.
Serum finished product after this method processing, immunoglobulin reduce by 40% or more, endotoxin and reduce from 30-100EU/ml
Within to 1.0EU/ml, hemoglobin be reduced within 80mg/L from 250-300mg/L, the detection of BVDV antigen ELISA is from the positive
Become negative, appearance is clear liquid.The newborn bovine serum of the method for the present invention production, culture BHK-21 and VERO cell effect connect
Nearly domestic newborn Serology Quality, endotoxin and hemoglobin, indicators are better than domestic serum.
The advantage of the invention is that entire production process, does not use chemical solvent, pollution will not be generated to serum；Solid phase
De- albumen aquation film medium, which only precipitates, is greater than 100nm particle in serum, will not cause to lose to serum effective component, solid phase takes off egg
Plain boiled water film medium also easily removes.This method production new born bovine purified blood serum only generates solid waste, without liquid waste
Object, environmental protection measure are easily achieved, and are suitble to industrialized production.
Specific embodiment
In order to better understand the present invention, it describes in detail by taking 1000L raw material serum as an example to this method.Following examples
Described in method be unless otherwise specified conventional method, the reagent and material commercially may be used unless otherwise specified
It obtains.
Specific implementation method is as follows.
(1) serum melts: feeding crosses colostrum containing lipid, endotoxin and the exceeded newborn bovine serum feedstock capture of hemoglobin
- 10 DEG C or less freezen protectives afterwards, 4-10 DEG C thaws, merges after thawing.
(2) it pre-filtering: is crossed with 25 μm of polypropylene bag-type strainers and filters out the particles such as fibrin (original), it is therefore an objective to protect ultrafiltration
Column.One No. 2 cloth bag of usual situation can smoothly filter 1000L serum.
(3) ultrafiltration: sufficiently being dialysed with Spectrum company, U.S. molecular cut off 100-300kd ultrafiltration column, serum point
At retention part (macromolecular complex) and dialysis part (free molecule), impurity component is enriched in retention part.Serum viscosity in rabbits
Very big, ultrafiltration column filter area should be not less than 50m2。
(4) contamination precipitation: retention part presses former serum volume 1000L, is added with stirring 25 liters of Nanjing Australia model biotechnologies
The solid phase of company takes off albumen aquation film medium DHF01, in serum the impurity such as lipid, immunoglobulin, endotoxin, hemoglobin into
One-step polymerization forms precipitating.
(5) continuous flow centrifugation: being precipitated serum with continuous flow centrifuge and removed together with the de- albumen aquation film medium of solid phase,
Centrifugal force should reach 8000g just and can efficiently separate supernatant precipitating, and centrifugal process temperature controls within the scope of 4-10 DEG C, be centrifuged into
Liquid speed degree need to be adjusted accordingly according to centrifugal effect, and centrifugation supernatant is answered bright.
(6) clarify: centrifugation supernatant is filtered with Minnesota Mining and Manufacturing Company's 90sp membrane stack and is clarified, and filtered fluid lamp inspection should be without opalescence.1000L
Raw material serum, the filtering of the usual situation step are needed using 1.8 m2Membrane stack.
(7) degerming: 90sp membrane stack filtered fluid and ultrafiltration membrane dialysis part merge, after degerming as new born bovine purified blood serum at
Product.
Claims (2)
1. a kind of method of industrialized production new born bovine purified blood serum, comprising: feeding crosses colostrum containing a large amount of lipids, endotoxin and blood
The new born bovine raw material serum pre-flock of the impurity components such as Lactoferrin removes fibrin；Through molecular cut off 100-300kd ultrafiltration column
Serum is separated into retention part (macromolecular complex) and dialysis part (free molecule)；Retention part is added solid phase and takes off albumen
Aquation film medium promotes the impurity such as lipid (containing chylomicrons and lipoprotein), immunoglobulin, endotoxin, hemoglobin to aggregate into
Flocculent deposit, the supernatant liquid obtained through continuous flow centrifugation, clarification filtration；Supernatant liquid merges with the dialysis part, degerming
After new born bovine purified blood serum is made.
2. method described in claim 1, it is characterised in that: the raw material is that feeding crosses colostrum containing lipid, endotoxin and blood red
The exceeded newborn bovine serum raw material of albumen, this method are also suitable for feeding and cross the new born bovine raw material blood that colostrum only needs to remove lipid
Clearly, or individually haemolysis needs to remove the new born bovine raw material serum of hemoglobin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810058613.9A CN110066762A (en) | 2018-01-22 | 2018-01-22 | The method of industrialized production new born bovine purified blood serum |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810058613.9A CN110066762A (en) | 2018-01-22 | 2018-01-22 | The method of industrialized production new born bovine purified blood serum |
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| CN110066762A true CN110066762A (en) | 2019-07-30 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804578A (en) * | 2019-11-19 | 2020-02-18 | 内蒙古维克生生物技术股份有限公司 | Production method for removing IgG from bovine serum |
| CN112083158A (en) * | 2020-08-29 | 2020-12-15 | 赵峻岭 | Preparation method of bovine serum for confining liquid |
| CN112662610A (en) * | 2020-12-14 | 2021-04-16 | 五河县鑫盛生物技术有限公司 | Process for removing newborn bovine serum endotoxin |
| CN114894911A (en) * | 2022-03-18 | 2022-08-12 | 辽宁成大生物股份有限公司 | Method for controlling bovine serum product quality |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268401A (en) * | 2011-06-08 | 2011-12-07 | 庞观龙 | Production method of newborn calf serum with immunoglobulin removed |
| CN104099288A (en) * | 2014-07-17 | 2014-10-15 | 武汉三利生物技术有限公司 | Producing technology for new-born bovine serum |
-
2018
- 2018-01-22 CN CN201810058613.9A patent/CN110066762A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268401A (en) * | 2011-06-08 | 2011-12-07 | 庞观龙 | Production method of newborn calf serum with immunoglobulin removed |
| CN104099288A (en) * | 2014-07-17 | 2014-10-15 | 武汉三利生物技术有限公司 | Producing technology for new-born bovine serum |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804578A (en) * | 2019-11-19 | 2020-02-18 | 内蒙古维克生生物技术股份有限公司 | Production method for removing IgG from bovine serum |
| CN112083158A (en) * | 2020-08-29 | 2020-12-15 | 赵峻岭 | Preparation method of bovine serum for confining liquid |
| CN112662610A (en) * | 2020-12-14 | 2021-04-16 | 五河县鑫盛生物技术有限公司 | Process for removing newborn bovine serum endotoxin |
| CN114894911A (en) * | 2022-03-18 | 2022-08-12 | 辽宁成大生物股份有限公司 | Method for controlling bovine serum product quality |
| CN114894911B (en) * | 2022-03-18 | 2023-10-24 | 辽宁成大生物股份有限公司 | Method for controlling quality of bovine serum products |
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Application publication date: 20190730 |