CN104644673B - A kind of preparation method of transfer factor and its preparation method of injection - Google Patents
A kind of preparation method of transfer factor and its preparation method of injection Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of transfer factor, it is characterised in that it includes pretreatment, homogenate, multigelation, centrifugation, ethanol precipitation, a ultrafiltration, inactivation of virus, second ultrafiltration, the invention also discloses a kind of preparation methods of transfer factor injection.The present invention can be effectively ensured the safety of product, reduce anaphylactogen, improve safety using not used ethanol precipitation removes all kinds of foreign proteins in traditional handicraft and viral inactivation method effectively removes various animal derived viruses；The load that subsequent ultrafiltration film ultrafiltration can be reduced after ethanol precipitation removes foreign protein simultaneously, extends the service life of ultrafiltration membrane.
Description
Technical field
The present invention relates to field of biological pharmacy, the preparation of preparation method and its injection in particular to a kind of transfer factor
Method.
Background technology
Transfer factor Transfer Factor (TF), also known as transmission factor are thin by the lymph with cellular immunity function
Born of the same parents generate, and pass through a kind of dialyzable factor obtained from the T cell of freeze thawing sensitization.It can reinforce the immunity of animal, can incite somebody to action
Delay allergic reaction ability is transferred to another individual from an individual (including humans and animals), they transport the anti-of father's lymphocyte
(delayed hypersensitivity reaction) is immunized to not exposure or primary lymphocyte in former specific cell.Transfer factor carries sensitization leaching
The specific immunity information of bar cell, can keep receptor inactive by specific immunity information submission to recipient lymphocytes
Lymphocyte is changed into specific sensitized lymphocyte, to the immune response for exciting recipient cell to mediate.Transfer factor has
Extensive immunology adjusts activity, on the one hand can induce activated immune cell, enhances body non-specific immunity.Another party
Specific immunity ability can be transmitted to other animals by face, and excitation animal generates specific immunity.
It is acted on since it is specific, transfer factor is clinically used for treating the uncontrollable viral or mould of certain antibiotic
Property intracellular infection (such as herpes zoster, Japanese Type-B encephalitis, candida albicans infection, vital myocarditis etc.)；To pernicious
Tumour can be used as auxiliary therapeutical agent (being mainly used for lung cancer, nasopharyngeal carcinoma, breast cancer, osteosarcoma etc.)；Immunologic deficiency disease (such as eczema,
Decrease of platelet repeatedly infects syndrome and chronic mucocutaneous nosomycosis has a better effect).Currently, it is published about
In the preparation process of transfer factor injection, following methods are generally included：(1) use meat grinder or homogenizer that pig spleen (ox spleen) is even
Slurry, freeze thawing, or ultrasound are with smudge cells；(2) it centrifuges；(3) transfer of ultrafiltration membrane ultrafiltration of the centrifugate through 10000 molecular weight or so
The factor；(4) water for injection is added to prepare ejection preparation according to the content of transfer factor.
Chinese patent application " a method of extracting transfer factor from cattle spleen " (number of patent application：
200810154335.3) disclosure production method：Freezing cattle spleen is crushed, flocculation, is dialysed, filtration sterilization, is made containing transfer
The stoste of the factor；A kind of " preparation method of pig spleen transfer factor injection " (number of patent application:201310136476.3) open
The preparation method of pig spleen transfer factor injection：Pig spleen after the assay was approved is cleaned, is homogenized, formalin-inactivated, freeze thawing
It is broken, stoste is obtained after filtering, ultrafiltration, and water for injection is added and obtains injection；A kind of " life of pig spleen transfer factor injection
Production method " (number of patent application:201110126248.9) it is crushed, centrifuged using homogenate, low temperature ultrasonic in, ultrafiltration technology extraction turns
Move the factor；" a kind of transfer factor solution and preparation method thereof " (number of patent application:201410306339.4) in pig spleen is carried out
Transfer factor solution is obtained after cleaning, homogenate, multigelation, adjusting pH, filtering, ultrafiltration；A kind of " preparation side of transfer factor
Method " (number of patent application:201310640313.9) new freshly-slaughtered poultry spleen cleaned, be homogenized, freeze thawing is crushed, through centrifugation pressurization take out
Transfer factor solution is obtained after filter, ultrafiltration；A kind of " Swine spleen transfer factor preparation method " (number of patent application：
201310755932.2) include tissue rubs, homogeneous is broken, micro-filtration, ultrafiltration, it is thin using high pressure homogenizer quick crashing
Born of the same parents.
On the one hand above-mentioned process sufficiently effective cannot remove all kinds of foreign proteins, so only with being centrifuged after freeze thawing
So that product easily causes allergic reaction, cannot be removed effectively all kinds of animal derived viruses, make preparation exist virus pollution and it is different
The underproof risk of normal toxicity；On the other hand it is exactly that existing preparation method is filtered using microfiltration membranes, ultrafiltration membrane, filter membrane
Load is big, short life, of high cost, limits further using medically.It is used in inactivation process and formaldehyde is added or is risen
Temperature processing, formaldehyde is carcinogenic substance, and generating system after remaining free formaldehyde injection human body swashs property reaction；Heating then easily makes transfer factor
Activity reduce and influence using effect.Amino acid, small peptide substance are easily polymerized to macromolecular substances and analyse in injection
Going out causes preparation unstable, also easily causes relevant side effect.
Invention content
Present invention aim to solve the deficiency of above-mentioned background technology, provide a kind of transfer factor preparation method and
The preparation method of its injection, to reduce the foreign protein anaphylactogen in transfer factor, effectively remove it is all kinds of it is animal derived virus and
Extend the service life of ultrafiltration membrane.
The technical scheme is that：A kind of preparation method of transfer factor：
(1) it pre-processes：Animal spleen is removed into its attachment fat, is eluted with water；
(2) it is homogenized：After the water for injection mixing of 1.5~2 times of animal spleen weight is added, it is placed in colloid mill and is twisted repeatedly
4~6 times, homogenate is made, moves at -16 DEG C and stores 3 days or more；
(3) multigelation：Multigelation will be homogenized 3~5 times；
(4) it centrifuges：It is centrifuged after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, 0~5
DEG C 12 hours or more are stood, centrifuging and taking supernatant reduced vacuum is concentrated into the 1/5~1/10 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 6.0~7.0, respectively after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration
Obtain solution of transfer factor；
(7) inactivation of virus：Albumin quality point in albumin to every milliliter of solution is added in solution of transfer factor
Number is 1~2 ‰, after 60 DEG C keep the temperature 10 hours, cools the temperature to 20 DEG C or less rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 3K~5K molecular weight.
Preferably, the homogenate solidified taking-up is placed in 33~37 DEG C of water-baths in the multigelation step, be stirred continuously
Make its fast melt, when homogenate all melt for liquid when, place into cryogenic freezing, 3~5 times repeatedly.
Preferably, it in the centrifugation step, centrifuges 30 minutes, takes at 4 DEG C, rotating speed 3000r/min after homogenate is thawed
It is homogenized supernatant.
Preferably, in the methanol precipitation step absolute ethyl alcohol and homogenate mixtures at 4 DEG C, rotating speed 3000r/min from
The heart 30 minutes.
Preferably, the vacuum degree that reduced vacuum concentrates in the methanol precipitation step is -0.03~-0.08MPa.
Preferably, the animal spleen is pig spleen or ox spleen.
The present invention also provides a kind of preparation method of transfer factor injection, step is：Measure the transfer factor solution
The content of interior polypeptide and nucleic acid is added water for injection and is adjusted to 100 μ g of 3mg containing polypeptide and ribose in every 2ml solution, chlorination is added
Sodium makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and the Tween-80 mass concentration being added in stabilizer Tween-80 to solution is 1
~2 ‰, it is aseptic subpackaged up to transfer factor injection after aseptic filtration.
Advantages of the present invention is：
1. removing all kinds of foreign proteins using not used ethanol precipitation in traditional handicraft, the peace of product can be effectively ensured
Quan Xing reduces anaphylactogen, improves safety；The negative of subsequent ultrafiltration film ultrafiltration can be reduced after ethanol precipitation removes foreign protein simultaneously
Lotus extends the service life of ultrafiltration membrane, reduces cost, it is made to have further application on biology.
2. removing foreign protein using ethanol precipitation, and successively by 50KD, 10KD, 3~5KD ultrafiltration membrane multistages are super
Filter, not only can guarantee the thorough removal of foreign protein, but also can efficiently control high molecular weight material, and product polymer substance is made and is less than
1.0%, it is less than 5.0% well below national Specification, can more effectively ensure product quality, ensure clinical safety.
3. in preparation method be added albumin protective agent after use 60 DEG C, 10 hours can effective inactivation of viruses, after adding
Continuous ultrafiltration technology can fully ensure that the inactivation of animal derived virus, ensure the safety of formulation products.Albumin conduct simultaneously
Protective agent can guarantee that the activity of transfer factor under the conditions of higher temperatures will not reduce.Product vigor is made and presses T cell determination of activity
Method-takes off E receptor methods and measures is not less than 30%, and significantly larger than national Specification is not less than 10%.
4. ox spleen (or pig spleen) homogenate is used low temperature multigelation, the active principle of intraor extracellular can be made fully to release
It puts, ensures the abundant extraction of active ingredient.
5. Tween-80 is added in injection blending process makees stabilizer, must effectively preparation can be avoided to have during storage
Machine species amino acid, small peptide, nucleotide, purine etc. are polymerized to macromolecular substances precipitation, influence the clarity of preparation, ensure product
The stabilization of long-time storage process, while reducing allergic reaction incidence.
Specific implementation mode
With reference to specific embodiment, the present invention is described in further detail.
Embodiment 1
The preparation method of transfer factor, includes the following steps,
(1) it pre-processes：Pig spleen is removed into its attachment fat, is cleaned 2 times with drinking water, then cleaned 3 times with purified water；
(2) it is homogenized：It after the water for injection mixing of 1.5 times of pig spleen weight is added, is placed in colloid mill and is twisted repeatedly 4 times, make
At homogenate, moves at -16 DEG C and store 3 days or more；
(3) multigelation：The homogenate solidified taking-up is put into 33 DEG C of water-bath, being stirred continuously makes its fast melt,
When homogenate all melt for liquid when, place into freezing in low-temperature cold store, 3 times repeatedly；
(4) it centrifuges：It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, it is quiet at 5 DEG C
It sets 12 hours or more, 4 DEG C, centrifuge 30 minutes under rotating speed 3000r/min, takes supernatant reduced vacuum under -0.03MPa vacuum degrees
It is concentrated into the 1/5 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 6.0, must be turned after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration respectively
Move solution of the factor；
(7) inactivation of virus：Albumin quality point in albumin to every milliliter of solution is added in solution of transfer factor
Number is 1 ‰, after 60 DEG C keep the temperature 10 hours, cools the temperature to 20 DEG C or less rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 3K molecular weight；
Gained transfer factor solution is prepared into transfer factor injection.
Prepare injection：The content of polypeptide and nucleic acid in transfer factor solution is measured, water for injection is added and is adjusted to every 2ml
100 μ g of 3mg containing polypeptide and ribose in solution, sodium chloride, which is added, makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and stabilizer is added
Tween-80 mass concentration in Tween-80 to solution is 1 ‰, aseptic subpackaged up to transfer factor injection after aseptic filtration.
Embodiment 2
The preparation method of transfer factor, includes the following steps,
(1) it pre-processes：Pig spleen is removed into its attachment fat, is cleaned 2 times with drinking water, then cleaned 3 times with purified water；
(2) it is homogenized：After the water for injection mixing of 2 times of pig spleen weight is added, it is placed in colloid mill and is twisted repeatedly 6 times, be made
Homogenate is moved at -16 DEG C and is stored 3 days or more；
(3) multigelation：The homogenate solidified taking-up is put into 35 DEG C of water-bath, being stirred continuously makes its fast melt,
When homogenate all melt for liquid when, place into freezing in low-temperature cold store, 3 times repeatedly；
(4) it centrifuges：It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, it is quiet at 0 DEG C
It sets 12 hours or more, 4 DEG C, centrifuge 30 minutes under rotating speed 3000r/min, takes reduced vacuum under supernatant -0.05MPa vacuum degrees dense
It is reduced to the 1/10 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 7.0, must be turned after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration respectively
Move solution of the factor；
(7) inactivation of virus：Albumin quality point in albumin to every milliliter of solution is added in solution of transfer factor
Number is 2 ‰, after 60 DEG C keep the temperature 10 hours, cools the temperature to 20 DEG C or less rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 5K molecular weight.
Gained transfer factor solution is prepared into transfer factor injection.
Prepare injection：The content of polypeptide and nucleic acid in transfer factor solution is measured, water for injection is added and is adjusted to every 2ml
100 μ g of 3mg containing polypeptide and ribose in solution, sodium chloride, which is added, makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and stabilizer is added
Tween-80 mass concentration in Tween-80 to solution is 2 ‰, aseptic subpackaged up to transfer factor injection after aseptic filtration.
Embodiment 3
The preparation method of transfer factor, includes the following steps,
(1) it pre-processes：Pig spleen is removed into its attachment fat, is cleaned 2 times with drinking water, then cleaned 3 times with purified water；
(2) it is homogenized：It after the water for injection mixing of 1.8 times of pig spleen weight is added, is placed in colloid mill and is twisted repeatedly 5 times, make
At homogenate, moves at -16 DEG C and store 3 days or more；
(3) multigelation：The homogenate solidified taking-up is put into 33 DEG C of water-bath, being stirred continuously makes its fast melt,
When homogenate all melt for liquid when, place into freezing in low-temperature cold store, 3 times repeatedly；
(4) it centrifuges：It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, it is quiet at 5 DEG C
It sets 12 hours or more, 4 DEG C, centrifuge 30 minutes under rotating speed 3000r/min, takes reduced vacuum under supernatant -0.08MPa vacuum degrees dense
It is reduced to the 1/5 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 6.5, must be turned after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration respectively
Move solution of the factor；
(7) inactivation of virus：Albumin quality point in albumin to every milliliter of solution is added in solution of transfer factor
Number is 1.5 ‰, after 60 DEG C keep the temperature 10 hours, cools the temperature to 20 DEG C or less rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 4K molecular weight.
Gained transfer factor solution is prepared into transfer factor injection.
Prepare injection：The content of polypeptide and nucleic acid in transfer factor solution is measured, water for injection is added and is adjusted to every 2ml
100 μ g of 3mg containing polypeptide and ribose in solution, sodium chloride, which is added, makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and stabilizer is added
Tween-80 mass concentration in Tween-80 to solution is 1.5 ‰, aseptic subpackaged up to transfer factor injection after aseptic filtration.
Embodiment 4
The preparation method of transfer factor, includes the following steps,
(1) it pre-processes：Ox spleen is removed into its attachment fat, is cleaned 2 times with drinking water, then cleaned 3 times with purified water；
(2) it is homogenized：It after the water for injection mixing of 1.5 times of ox spleen weight is added, is placed in colloid mill and is twisted repeatedly 4 times, make
At homogenate, moves at -16 DEG C and store 3 days or more；
(3) multigelation：The homogenate solidified taking-up is put into 37 DEG C of water-bath, being stirred continuously makes its fast melt,
When homogenate all melt for liquid when, place into freezing in low-temperature cold store, 3 times repeatedly；
(4) it centrifuges：It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, it is quiet at 3 DEG C
It sets 12 hours or more, 4 DEG C, centrifuge 30 minutes under rotating speed 3000r/min, takes reduced vacuum under supernatant -0.06MPa vacuum degrees dense
It is reduced to the 1/8 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 6.0, must be turned after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration respectively
Move solution of the factor；
(7) inactivation of virus：Albumin quality point in albumin to every milliliter of solution is added in solution of transfer factor
Number is 1 ‰, after 60 DEG C keep the temperature 10 hours, cools the temperature to 20 DEG C or less rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 3K molecular weight.
Gained transfer factor solution is prepared into transfer factor injection.
Prepare injection：The content of polypeptide and nucleic acid in transfer factor solution is measured, water for injection is added and is adjusted to every 2ml
100 μ g of 3mg containing polypeptide and ribose in solution, sodium chloride, which is added, makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and stabilizer is added
Tween-80 mass concentration in Tween-80 to solution is 1 ‰, aseptic subpackaged up to transfer factor injection after aseptic filtration.
Inactivation of virus effect detection：
By in solution of transfer factor obtained in embodiment be added Pseudorabies virus (Pseudorabies virus,
PRV) be indicator virus, be added albumin protective agent after after heat treatment in 60 DEG C × 10 hours, using IBRS-2 cells as
Culture cell, using the titre of 96 hole cytopathy political reform detection viruses, using cytopathy (CPE) as existing for differentiation virus
Index carries out blind passage three generations to not occurring the sample of cytopathy with cell.It is shown by virus control cultivation results, in use
Heat treatment method is stated, as shown in table 1, Pseudorabies virus titre can be made to decline PRV7.4 ㏒ TCID50/0.1ml, such as 2 institute of table
Show, sample is showed no special cytopathic effect (CPE) through a blind passage generation, two generations, three generations.
PRV verification results are added in 1 heating of table (60 DEG C, 10 hours) inactivation treatment transfer factor
" 0 " indicates all acellular lesion of all dilutions of the group.
Table 2 does not occur PRV-CPE sample blind passage three generations's results
"-" is not occur specific C PE.
3 batches of products are made according to the above embodiments, carry out coherent detection respectively, the results are shown in Table 3：
3 product testing result table of table
Claims (7)
1. a kind of preparation method of transfer factor, which is characterized in that include the following steps,
(1) it pre-processes：Animal spleen is removed into its attachment fat, is eluted with water；
(2) it is homogenized：After the water for injection mixing of 1.5~2 times of animal spleen weight is added, it is placed in colloid mill and is twisted 4~6 repeatedly
It is secondary, homogenate is made, moves at -16 DEG C and stores 3 days or more；
(3) multigelation：Multigelation will be homogenized 3~5 times；
(4) it centrifuges：It is centrifuged after homogenate is thawed, takes homogenate supernatant；
(5) ethanol precipitation：It will be homogenized supernatant under constant stirring, absolute ethyl alcohol is added by 1: 2 volume ratio, it is quiet at 0~5 DEG C
It sets 12 hours or more, centrifuging and taking supernatant reduced vacuum is concentrated into the 1/5~1/10 of original volume, recycles ethyl alcohol；
(6) ultrafiltration：Concentrate pH value is adjusted to 6.0~7.0, must be turned after 50K molecular weight and 10k ultrafiltration membrane ultrafiltration respectively
Move solution of the factor；
(7) inactivation of virus：It is 1 that albumin mass fraction in albumin to every milliliter of solution is added in solution of transfer factor
~2 ‰, after 60 DEG C keep the temperature 10 hours, 20 DEG C or less are cooled the temperature to rapidly；
(8) second ultrafiltration：Transfer factor solution is obtained after the ultrafiltration membrane ultrafiltration of 3K~5K molecular weight.
2. the preparation method of transfer factor as described in claim 1, which is characterized in that will have been solidified in the multigelation step
Homogenate taking-up be placed in 33~37 DEG C of water-baths, being stirred continuously makes its fast melt, when homogenate all melt for liquid when, place into low
It is freezed at -16 DEG C of temperature, 3~5 times repeatedly.
3. the preparation method of transfer factor as described in claim 1, which is characterized in that in the centrifugation step, homogenate is thawed
It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min afterwards, takes homogenate supernatant.
4. the preparation method of transfer factor as described in claim 1, which is characterized in that absolute ethyl alcohol in the methanol precipitation step
It is centrifuged 30 minutes at 4 DEG C, rotating speed 3000r/min with homogenate mixtures.
5. the preparation method of transfer factor as described in claim 1, which is characterized in that reduced vacuum in the methanol precipitation step
The vacuum degree of concentration is -0.03~-0.08MPa.
6. the preparation method of transfer factor as described in claim 1, which is characterized in that the animal spleen is pig spleen or ox spleen.
7. a kind of preparation method for the injection preparing any transfer factor solutions of claim 1-6, which is characterized in that
Further include the content that step (9) measures polypeptide and nucleic acid in transfer factor solution, water for injection is added and is adjusted in every 2ml solution
100 μ g of 3mg containing polypeptide and ribose, sodium chloride, which is added, makes a concentration of 0.9g/ml of Chlorine in Solution sodium, and stabilizer tween-is added
Tween-80 mass concentration in 80 to solution is 1~2 ‰, aseptic subpackaged up to transfer factor injection after aseptic filtration.
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| CN107007841A (en) * | 2017-03-10 | 2017-08-04 | 石家庄石牧动物药业有限公司 | A kind of specific Transfer Factor- porcine preparation method of cyclodextrin encapsulated multivalence |
| CN114917251A (en) * | 2022-05-27 | 2022-08-19 | 福建农业职业技术学院 | Preparation method of pig placenta transfer factor |
| CN115006426A (en) * | 2022-07-28 | 2022-09-06 | 康普药业股份有限公司 | Preparation method of transfer factor and transfer factor |
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| US4180627A (en) * | 1978-08-17 | 1979-12-25 | The United States Of America As Represented By The Secretary Of Agriculture | Process for in vivo transfer of cell-mediated immunity in mammals with alcoholic precipitates of Bovine Transfer factor |
| CN102357102A (en) * | 2011-09-30 | 2012-02-22 | 浙江华尔成生物药业股份有限公司 | Preparation method of transfer factor soluble powder for animals |
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