CN103436583B - Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell - Google Patents
Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell Download PDFInfo
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- CN103436583B CN103436583B CN201310397952.7A CN201310397952A CN103436583B CN 103436583 B CN103436583 B CN 103436583B CN 201310397952 A CN201310397952 A CN 201310397952A CN 103436583 B CN103436583 B CN 103436583B
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Abstract
The invention provides a method for producing a therapeutic monoclonal antibody of a canine distemper virus, a product thereof and a hybridoma cell. The method provided by the invention comprises the following steps: (1) carrying out amplification culture on the hybridoma cell which secretes and produces the therapeutic monoclonal antibody of the canine distemper virus in a cell culture solution; (2) then, inoculating the hybridoma cell into a cell culture solution in a bioreactor and culturing; (3) harvesting the therapeutic monoclonal antibody, produced by the hybridoma cell, of the canine distemper virus. The method provided by the invention has the advantages that the produced therapeutic monoclonal antibody of the canine distemper virus is high in purity, little in batch difference and easy in quality control, and the quality of the therapeutic monoclonal antibody can be improved remarkably.
Description
Technical field
The present invention relates to the technical field that monoclonal antibody is produced, be specifically related to a kind of method of manufacture of therapeutic canine distemper virus monoclonal antibody, its goods and hybridoma.
Background technology
Koehler and Milstein(Nature Vol.256, pp495-497 in 1975), adopt hybridoma technique, by the mouse boosting cell of sheep red blood cell (SRBC) (SRBC) immunity, merge with murine myeloma cell, have successfully been obtained the monoclonal antibody of anti-SRBC.Create the authentic monoclonal antibody technology be with historically new significance, for biotechnology opens a brand-new field.At present, the application of this technology has related to the various fields such as medical science, agricultural, food, environment, is widely used in the aspects such as fundamental research, medical diagnosis on disease, environment and food inspection, treatment, prevention, protein purification.Along with the application in monoclonal antibody in vivo Diagnosis and Treat, higher requirement be it is also proposed to monoclonal antibody product, thus facilitate the technical development of external large scale culturing hybridoma manufacture order clonal antibody.This technology is also day by day perfect along with the development of mechanical chemical industry, Computer Subject, is the every field of biomedical (comprising the mankind and animal doctor), especially in fundamental research and the clinical application field of the prevention and therapy of virus infection, plays the effect that it is important.
Canine distemper (Canine Distemper, CD) be by canine distemper virus (Canine Distemper virus, CDV) cause, infect high degree in contact infectivity, the lethal infectious diseases of the Canidae (especially pup) in the beast of prey, Mustelidae and a part of Procyonidae animal.With Early manifestation two-phase pattern of fever, acute coryza, and subsequently with bronchitis, bronchiolitis, serious gastro-enteritis and nervous symptoms for feature.There is the height angling of nose and foot pad in minority case.This disease transmission is strong, sickness rate is high, and often cause the animal morbidities such as large quantities of dog, ermine, fox, lethality rate 30% ~ 80%, snow leopard is up to 100%.In addition, this virus infection also threatens experimental dog.At present clinically to the treatment mainly symptomatic treatment of this virus infection, coordinate hyper-immune serum and mab treatment.The traditional method of preparation therapeutic canine distemper virus monoclonal antibody is working system in body.This method is inoculated in mouse (non-syngeneic animal) abdominal cavity by merging the canine distemper virus monoclonal hybridoma produced, hybridoma is grown in mouse peritoneal, induction produces a large amount of ascites, simultaneously, canine distemper virus monoclonal hybridoma secretory antibody in ascites, and obtains a large amount of ascites monoclonal antibodies.Generally speaking, be often mixed with the various foreign proteins (comprising Ig) of mouse in such ascites, could use after therefore will purifying; In addition, yielding poorly of this method, the production cycle is long, differences between batches are large, is not suitable for the production of the therapeutic monoclonal antibody carrying out mass.
At present, need badly and can produce that to have purity high, differences between batches are little, easy to control the quality, can significantly improve the method for the therapeutic canine distemper virus monoclonal antibody of therapeutic monoclonal antibodies quality.
Summary of the invention
According to demand and the deficiency in above-mentioned field, the invention provides a kind of method of manufacture of therapeutic canine distemper virus monoclonal antibody, the present invention also provides the therapeutic canine distemper virus directly obtained by the method monoclonal antibody.The therapeutic canine distemper virus monoclonal antibody purity that the inventive method is produced is high, and differences between batches are little, easy to control the quality, can significantly improve therapeutic monoclonal antibodies quality.In addition, the present invention also provides the hybridoma of secretion canine parvovirus prevention monoclonal antibody.
Technical scheme of the present invention is as follows:
A method for manufacture of therapeutic canine distemper virus monoclonal antibody, is characterized in that, comprise the steps:
(1) hybridoma of secretion manufacture of therapeutic canine distemper virus monoclonal antibody is carried out enlarged culturing in cell culture fluid;
(2) inoculate in the cell culture fluid in bio-reactor and cultivate;
(3) the therapeutic canine distemper virus monoclonal antibody that hybridoma generates is gathered in the crops.
The formula of described cell culture fluid is: 85%DMEM liquid, 15% calf serum, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep; Or Serum-free Hybridoma cell culture medium, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep.
Inoculum density is 1 × 105 ~ 1 × 106/ml.
Inoculum density is 2 × 105 ~ 8 × 105/ml, wherein adopts batch, continuously perfusion in step (2) or change liquid collecting mode in bio-reactor, cultivate described hybridoma.
Inoculum density is 4 × 105 ~ 5 × 105/ml, cultivates the culture temperature of described hybridoma between 36 ~ 37 DEG C, and the pH value of described cell culture fluid is 7.0 ~ 7.2, and the dissolved oxygen scope of described bio-reactor is set to 50% ~ 90%.
When adopting continuous irrigation stream mode to cultivate hybridoma, it is tank volume every day 1 ~ 5 that stream adds volume.
The feature of aforesaid method is, the preserving number of described hybridoma is: CGMCC No.7613.
Also comprise the steps: that manufacture of therapeutic canine distemper virus monoclonal antibody step (3) gathered in the crops carries out concentrated and purifying, more aseptic, quantitative separating is in the container of sterilizing.
The therapeutic canine distemper virus monoclonal antibody that aforesaid method is produced.
A kind of hybridoma cell strain, its preserving number is CGMCC No.7613.
In the present invention, " bio-reactor " refers to and provides the biological reaction apparatus of good enclosed environment for incubation growth with the cell of on-fixed, is usually also called cell fermentation tank.There is multiple bio-reactor to go in the present invention, comprise such as stirring type bioreactor, airlift bioreactor, hollow-fiber bioreactor and fluidized bed aerosol generator etc.
Bioreactor culture cell mainly contains batch (Batch), stream adds formula (Fed-batch) and perfusion type (Perfusion) three kinds.In simple batch experiments, along with the accumulation of nutraceutical consumption and by product, cell stops growing and starts death, and product also stops producing, and production efficiency is very low.Stream adds formula and cultivates, and namely in culturing process, according to cellular metabolic demands, continuous or compartment of terrain adds specific fed-batch medium in reactor, supplements new nutritive substance, reduces by product.Whole culturing process does not flow out or reclaims, and usually stops after cell enters decline phase or decline phase, reclaims whole reaction system, isolated cell and cell debris, concentrated, protein of interest.After perfusion type is cultivated and referred to that a cell adds reactor together with substratum, in Growth of Cells and product forming process, constantly the substratum consumed is discharged, continuously pour into again the method for new substratum simultaneously.
In the method for the present invention by bio-reactor manufacture of therapeutic canine distemper virus monoclonal antibody, the training method of cell can be batch, stream adds in formula or perfusion type any one, these three kinds of cell cultures modes all can meet requirement of the present invention, realize goal of the invention of the present invention, consider the otherwise problems such as experiment condition, use the cell cultures mode of perfusion type in the embodiment of the present invention, this does not represent other two kinds of modes just can not realize goal of the invention of the present invention.
Specifically, the step of use bioreactor culture can be:
From working cardial cell storehouse, take out hybridoma, adopt fast melt method that seed cell is transferred to rapidly 37 DEG C of water-baths by liquid nitrogen and recover, with 85%DMEM liquid, 15% calf serum nutrient solution, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep; Or Serum-free Hybridoma cell culture fluid, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep carries out cell expansion cultivation.
Meanwhile, bio-reactor is thoroughly cleaned, sterilize after open air, oxygen, nitrogen and carbonic acid gas four gas air feeder, setting bioreactor culture condition: temperature, pH value, dissolved oxygen, stirring velocity etc. are for subsequent use.
When production cell seed propagation reaches suitable total amount, be inoculated in bio-reactor by the zone of reasonableness of final concentration of cells and cultivate, wherein inoculum density scope is 1 × 10
5~ 1 × 10
6/ ml can meet the basic inoculation requirement of the present invention, and inoculum density scope is 2 × 10
5~ 8 × 10
5/ ml, the inoculum density effect of this scope is better, and inoculum density scope is 4 × 10
5~ 5 × 10
5/ ml effect is best, and the inoculum density of this scope can ensure that cell fully adapts to from static cultivation to dynamic cultivation, can shorten again the time from being inoculated into results.When cell density reaches 1 × 10
6during/more than ml, can set and open the mode that peristaltic pump starts to pour into and gather in the crops culture supernatant.According to cell density and tiring, adjustment peristaltic pump rotating speed, changes perfusion volume.Preferably reach 2 × 10 at cell density
6during/more than ml, especially when cell density reaches 5 × 10
6during/more than ml, then the corresponding cell concn of tiring detected reaches Eligibility requirements in this density, progressively promotes setting speed, then with continuous perfusion or change liquid collecting mode obtain cells and supernatant be therapeutic canine distemper virus monoclonal antibody.Hybridoma seed cell can be inoculated in bio-reactor with any suitable concentration by the present invention.In the present invention, as long as the concentration being inoculated into the seed cell in bio-reactor is 10
5/ more than ml.In one embodiment of the invention, the concentration being inoculated into the seed cell in bio-reactor is preferably 2 × 10
5/ more than ml.In another two embodiments, the concentration being inoculated into the seed cell in bio-reactor is preferably 5 × 10 respectively
5/ ml or 8 × 10
5/ more than ml.Certainly, those of ordinary skill easily can determine that the zone of reasonableness of seed cell inoculum density is as above-mentioned scope.
The hybridoma of any suitable secretion therapeutic monoclonal antibodies can be adopted in the above-mentioned methods.This hybridoma can derive from the preserved material that existing preserved material such as derives from preservation mechanism, or buys from commercial channels, or prepares voluntarily.Those of ordinary skill knows the method building secretion therapeutic monoclonal antibodies hybridoma, such as canine distemper virus CDV MD-77 strain immune mouse can be used, application SPA-HRP immunohistochemistry technology detects immune serum and tires, after it reaches 1:640, get the highest Mouse spleen cells of serum titer to merge mutually with murine myeloma cell, 96 porocyte plates are cultivated after 10 days, draw cell conditioned medium liquid to be ELISA and to screen positive model fusion, for 1 time or obtain hybridoma cell strain after (such as 3 times) clone for several times.In addition, the immunohistochemical methods of described hybridoma secretion therapeutic monoclonal antibodies is tired within the scope of 1:32 ~ 1:128.Preferred antibody tires the hybridoma of more than 1:64 as production therapeutic monoclonal antibodies hybridoma cell strain.In the specific embodiment of the present invention, adopt what prepare voluntarily to be the mouse hybridoma cell system that CGMCC No.7613 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 17th, 2013 with preserving number.
In the present invention, any liquid nutrient medium being suitable for Growth of Hybridoma Cell can be adopted.In the embodiment of the present invention, preferred nutrient solution formula is: 85%DMEM liquid, 15% calf serum, adds 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep.In another embodiment, substratum used is Serum-free Hybridoma cell culture medium, adds 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep.
The present invention can cultivate seed cell at any suitable temperature, and temperature can meet basic fostering requirement at 20 ~ 38 DEG C, preferably 30 ~ 37 DEG C, is more preferably 33 ~ 37 DEG C.In a concrete embodiment, the temperature of cultivating seed cell is best results between 36 ~ 37 DEG C.The dissolved oxygen scope of preferred containers for culturing organisms used is located between 50% ~ 90%.In the present invention, in bioreactor culture process, the pH value of cultivating the nutrient solution of seed cell controls between 7.0 ~ 7.2.Nutrient solution pH can be controlled in suitable scope by adding appropriate acid or alkali in culturing process, such as carbonic acid gas and sodium bicarbonate etc.
Preferably, bioreactor culture temperature is located between 36 ~ 37 DEG C, and stream during bioreactor culture adds volume between every day 1 ~ 5 tank volume.
When after the Growth of Cells in bio-reactor to suitable density, in the present invention of results culture supernatant preferably, when cell density reaches 10
6during/more than ml, culture supernatant can be gathered in the crops.More preferably 2 × 10 are reached at cell density
6during/more than ml, especially reach 5 × 10
6during/more than ml, the cell concn of this point value is that product is tired the basic standard evaluated, results culture supernatant.Can by multiple suitable method results supernatant liquor, the methods such as such as centrifugal, filtration, precipitation, this is well known to those of ordinary skill in the art.
Method of the present invention comprises the step being carried out by the therapeutic monoclonal antibodies of results filtering and concentrating further.Specifically adopt with the following method: get steriling test and be negative, the monoclonal antibody supernatant that bioactivity is qualified, peristaltic pump is connected to monoclonal antibody receiving flask, 0.45 μm of pre-flock system is utilized to cross impurity such as filtering cell debris, then flat sheet membrane bag ultrafiltration system (molecular weight cut-off is 30kDa) is connected, open the ultrafiltration and concentration that peristaltic pump carries out monoclonal antibody, regulating opening for feed and discharge port pressure at 20 ~ 30psig, stopping ultrafiltration and concentration system when reaching the multiple of ultrafiltration and concentration.
Method of the present invention also comprises aseptic for concentrated monoclonal antibody, quantitative separating in the container of sterilizing.
Compared with prior art, the present invention has following beneficial effect:
1, instead of with bio-reactor in the present invention and utilize mouse to manufacture therapeutic canine distemper virus monoclonal antibody injection liquid, the problem that mouse source external source pathogeny is polluted can be solved, by the strict control of starting material and culture condition, ensure that the therapeutic canine distemper virus monoclonal antibody injection liquid produced is pure, guarantee the security of therapeutic canine distemper virus monoclonal antibody injection liquid;
2, adopt the inventive method manufacture of therapeutic canine distemper virus monoclonal antibody output high, cost is low, can significantly improve this product economy benefit;
3, production technique of the present invention is simple and stable, easy to operate, and antibody purity is high, and differences between batches are little, easy to control the quality, can significantly improve therapeutic monoclonal antibodies quality;
4, the therapeutic canine distemper virus monoclonal antibody that the inventive method is produced has good curative effect to treatment canine distemper virus disease.
Hybridoma preservation information:
Hybridoma cell strain title: therapeutic canine distemper virus monoclonal antibody hybridoma cell CDV1H2
Strain classification: mouse hybridoma cell system
Deposit number is: CGMCC No.7613
Preservation date: on May 17th, 2013
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC)
Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
Following embodiment is provided to be to understand the present invention further better; be not limited to described embodiment; content of the present invention and protection domain are not construed as limiting; anyone is under enlightenment of the present invention or any and the present invention the present invention being carried out combining with the feature of other prior aries and draw is identical or akin product, all drops within protection scope of the present invention.
Unreceipted specific experiment step or condition person in embodiment, can carry out according to the operation of the normal experiment step described by the document in this area or condition.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional reagent product of commercial acquisition.
Biomaterial:
Canine distemper virus CDV:CDV MD-77 strain, is provided by Ministry of Agriculture's Disease Diagnosis of Veterinary center;
Canine distemper virus 98017 strain, is provided by Ministry of Agriculture's Disease Diagnosis of Veterinary center;
Canine parvovirus AMS-1 strain, is provided by Ministry of Agriculture's Disease Diagnosis of Veterinary center;
Canine infectious hepatitis virus (ICHV), is provided by Ministry of Agriculture's Disease Diagnosis of Veterinary center;
Reovirus 3 type (Reo-3), is provided by Ministry of Agriculture's Disease Diagnosis of Veterinary center;
SP2/0 cell is so kind as to give by Military Medical Science Institute;
Balb/c mouse is purchased from Beijing company of dimension tonneau China;
Applicant states, above biomaterial all has preservation in applicant laboratory, can provide for proof test from the applying date in Two decades years to the public.
Embodiment 1 secretes the acquisition of the hybridoma cell line of anti-CDV monoclonal antibody
1.1 use canine distemper virus CDV(CDV MD-77 strain, and HA tires as 1:5120, derives from Ministry of Agriculture's Disease Diagnosis of Veterinary center) immune Balb/c mouse.Virus liquid and Freund's complete adjuvant (CFA) equal-volume mixing and emulsifying, through limb muscle multi-point injection, every per injection 300ul.15d and 30d after first immunisation, adds Freund's incomplete adjuvant (IFA) with the virus liquid of same dosage respectively and carries out booster immunization.Second time is taken a blood sample after strengthening, and carries out ELISA bioactivity and immunoenzyme bioactivity to serum simultaneously.
The preparation of 1.2 hybridomas:
Reach 1:20000 when ELISA tires, after simultaneously immunoenzyme is tired and reached 1:640, get mouse spleen and do and merge.72h booster immunization again before merging, through tail vein injection virus liquid 1 time, 50ul/ only.Prepare 10 pieces and merge plate.
Merge: get seroimmunity groupization and detect the highest Mouse spleen cells of tiring and merge mutually with murine myeloma cell, first spleen grinding is obtained splenocyte suspension, then mix with the SP2/0 murine myeloma cell being in logarithmic phase of cell count low ten times, through PEG1500 effect 1min by two kinds of cytogamy together, then fused cell liquid 100ml is dispensed in 10 piece of 96 orifice plate and cultivates.Merging substratum is the complete screening culture medium of RPMI1640 containing HAT and 20%FBS.Antigen-specific sex clone, by ELISA experiment screening, after 3 time clonings, obtains stable cell strain of monoclonal antibody.
The screening of hybridoma: after merging, cell is cultivated after 10 days on 96 porocyte plates, draw cell conditioned medium and do ELISA and immunoenzyme detection, cloning is continued in positive hole, till the cell strain of secretory antibody can be stablized.The cell strain of secretory antibody can be stable judgement be after monoclonal antibody cytogamy, karyomit(e) number can be kept constant, and secretion capacity is constant.
The selection result: obtain 1 strain of hybridoma strain, by its called after CDV1H2, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 17th, 2013, its preserving number is CGMCC No.7613.
The qualification of hybridoma cell strain: meet therapeutic canine distemper virus monoclonal antibody hybridoma cell standard, cellular form is rounded, and karyomit(e) mean number is between 96 ~ 103, and caryogram should be identical.Carry out mycoplasma inspection by existing " People's Republic of China's veterinary drug allusion quotation ", should grow without mycoplasma.Carry out steriling test by existing " People's Republic of China's veterinary drug allusion quotation ", answer asepsis growth.When cell count reaches 5 × 10
5individual/more than ml time, the cell cultures film flying of supernatant liquor canine distemper virus 98017 strain, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), reovirus 3 type (Reo-3), carries out immunoenzyme test.Canine distemper virus monoclonal antibody and canine distemper virus cell cultures film flying are positive, are negative with other viral film flying.
Embodiment 210L(Bei Lang) bioreactor culture
(1) the CDV1H2 clone cell culture fluid piping and druming in embodiment 1, dispersion are gone down to posterity, continuing to cultivate in 37 DEG C in cell culture fluid, when forming good individual layer, going down to posterity for continuing; Total cellular score is made to reach 10
9/ more than 500ml.
The qualification of seed cell: when cell count reaches 5 × 10
5individual/more than ml time, the cell cultures film flying of supernatant liquor canine distemper virus 98017 strain, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), reovirus 3 type (Reo-3), carries out immunoenzyme test.Canine distemper virus monoclonal antibody and canine distemper virus cell cultures film flying are positive, are negative with other viral film flying; Secrete monoclonal antibody is IgG1 type;
(2) the cultivation manufacture of therapeutic monoclonal antibodies: take out secretion therapeutic monoclonal antibodies hybridoma from working cardial cell storehouse, adopt fast melt method that seed cell is transferred to rapidly 37 DEG C of water-baths by liquid nitrogen to recover, with 85%DMEM liquid, 15% calf serum, then the nutrient solution adding 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep carries out cell expansion cultivation.
Meanwhile, bio-reactor is thoroughly cleaned, sterilize after open air, oxygen, nitrogen and carbonic acid gas four gas air feeder, setting bioreactor culture condition: temperature 37 DEG C, pH value 7.2, dissolved oxygen 60%, stirring velocity 80rpm/min etc. are for subsequent use.
When production cell seed propagation reaches 10
9/ more than 500ml, is greater than 2 × 10 by final concentration of cells value
5/ ml is inoculated in the cell culture fluid in bio-reactor and cultivates, nutrient solution used is: 85%DMEM liquid, 15% calf serum, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep again, the pH value of this cell culture fluid is adjusted to 7.0 ~ 7.2, cultivates the temperature of this hybridoma between 36 ~ 37 DEG C.
When cell density reaches 1 × 10
6during/more than ml, can set and open the mode that peristaltic pump starts to pour into and cultivate described hybridoma.According to cell density and tiring, adjustment peristaltic pump setting speed, changes perfusion volume.What select in the present embodiment is reach 5 × 10 when cell density
6during/more than ml, progressively promote setting speed, cultivate with continuous irrigation stream mode, obtain cell culture supernatant, when bio-reactor continuously perfused culture is collected, stream adds volume every day between 1 ~ 5 tank volume.
The cell culture supernatant of results is tested by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Nutrient solution has no side effect safely to dog, results nutrient solution canine distemper Neutralizing titer >=1:128.
(3) the concentrated and purifying of therapeutic monoclonal antibodies nutrient solution: gather in the crops supernatant nutrient solution ultrafiltration system and filter, tire according to therapeutic monoclonal antibodies nutrient solution and determine cycles of concentration.Remove cell debris by filtering system, the liquid of results puts less than-15 DEG C preservations;
The inspection of concentrated solution: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Nutrient solution has no side effect safely to dog, results nutrient solution canine distemper virus Neutralizing titer >=1:1024.
(4) packing: by therapeutic monoclonal antibodies that is concentrated, purifying, aseptic, quantitative separating is in the container of sterilizing.Get product after adding rapidly sterilizing plug, gland after packing, less than-15 DEG C preservations.
Inspection after construction: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Dog is had no side effect safely, canine distemper virus Neutralizing titer >=1:1024.
Embodiment 330L(Bei Lang) bioreactor culture
(1) the CDV1H2 clone cell culture fluid piping and druming in embodiment 1, dispersion are gone down to posterity, continuing to cultivate in 37 DEG C in cell culture fluid, when forming good individual layer, going down to posterity for continuing; Total cellular score is made to reach 3 × 10
9/ more than 500ml.
The qualification of seed cell: when cell count reaches 5 × 10
5individual/more than ml time, the cell cultures film flying of supernatant liquor canine distemper virus 98017 strain, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), reovirus 3 type (Reo-3), carries out immunoenzyme test.Canine distemper virus monoclonal antibody and canine distemper virus cell cultures film flying are positive, are negative with other viral film flying; Secrete monoclonal antibody is IgG1 type;
(2) the cultivation manufacture of therapeutic monoclonal antibodies: take out secretion therapeutic monoclonal antibodies hybridoma from working cardial cell storehouse, adopt fast melt method that seed cell is transferred to rapidly 37 DEG C of water-baths by liquid nitrogen to recover, with Serum-free Hybridoma cell culture medium, then add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep carries out cell expansion cultivation as nutrient solution.
Meanwhile, bio-reactor is thoroughly cleaned, sterilize after open air, oxygen, nitrogen and carbonic acid gas four gas air feeder, setting bioreactor culture condition: temperature 37 DEG C, pH value 7.2, dissolved oxygen 80%, stirring velocity 80rpm/min etc. are for subsequent use.
When production cell seed propagation reaches 3 × 10
9/ more than 500ml, is greater than 5 × 10 by the zone of reasonableness of final concentration of cells
5/ ml is inoculated in bio-reactor and carries out cell cultures, the formula of nutrient solution used is: Serum-free Hybridoma cell culture medium, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep again, pH value is adjusted to 7.0 ~ 7.2, cultivates the temperature of this hybridoma between 36 ~ 37 DEG C.
When cell density reaches 1 × 10
6during/more than ml, can set and open the mode that peristaltic pump starts to pour into and gather in the crops culture supernatant.According to cell density and tiring, adjustment peristaltic pump setting speed, changes perfusion volume.Preferably 2 × 10 are reached at cell density in the present embodiment
6during/more than ml, progressively promote setting speed, with continuous perfusion or change liquid collecting mode and obtain cell culture supernatant, when bio-reactor continuously perfused culture is collected, stream adds volume every day between 1 ~ 4 tank volume.
The cell culture supernatant of results is tested by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Nutrient solution has no side effect safely to dog, results nutrient solution canine distemper virus Neutralizing titer >=128.
(3) the concentrated and purifying of therapeutic monoclonal antibodies nutrient solution: harvested cell culture supernatant ultrafiltration system filters, and tires determine cycles of concentration according to therapeutic monoclonal antibodies nutrient solution.Remove cell debris by filtering system, the liquid of results puts less than-15 DEG C preservations;
The inspection of concentrated solution: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Cell culture supernatant has no side effect safely to dog, results nutrient solution canine distemper virus Neutralizing titer >=1024
(4) packing: by therapeutic monoclonal antibodies that is concentrated, purifying, aseptic, quantitative separating is in the container of sterilizing.Get product after adding rapidly sterilizing plug, gland after packing, less than-15 DEG C preservations.
Inspection after construction: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Dog is had no side effect safely, canine distemper virus Neutralizing titer >=1024.
Embodiment 4100L(Bei Lang) bioreactor culture
(1) the CDV1H2 clone cell culture fluid piping and druming in embodiment 1, dispersion are gone down to posterity, continuing to cultivate in 37 DEG C in cell culture fluid, when forming good individual layer, going down to posterity for continuing; Total cellular score is made to reach 10
10/ more than 500ml.
The qualification of seed cell: when cell count reaches 5 × 10
5individual/more than ml time, the cell cultures film flying of supernatant liquor canine distemper virus 98017 strain, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), reovirus 3 type (Reo-3), carries out immunoenzyme test.Canine distemper virus monoclonal antibody and canine distemper virus cell cultures film flying are positive, are negative with other viral film flying; Secrete monoclonal antibody is IgG1 type;
(2) the cultivation manufacture of therapeutic monoclonal antibodies: take out secretion therapeutic monoclonal antibodies hybridoma from working cardial cell storehouse, adopt fast melt method that seed cell is transferred to rapidly 37 DEG C of water-baths by liquid nitrogen to recover, carry out cell expansion cultivation with the nutrient solution of 85%DMEM liquid, 15% calf serum, 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep.
Meanwhile, bio-reactor is thoroughly cleaned, sterilize after open air, oxygen, nitrogen and carbonic acid gas four gas air feeder, setting bioreactor culture condition: temperature 37 DEG C, pH value 7.2, dissolved oxygen 90%, stirring velocity 80rpm/min etc. are for subsequent use.
When production cell seed propagation reaches 10
10/ more than 500ml, is greater than 8 × 10 by final concentration of cells value
5/ ml is inoculated in the cell culture fluid in bio-reactor and cultivates, the formula of nutrient solution used is: 85%DMEM liquid, 15% calf serum, 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep, the pH value of this cell culture fluid is adjusted to 7.0 ~ 7.2, cultivates the temperature of this hybridoma between 36 ~ 37 DEG C.
When cell density reaches 1 × 10
6during/more than ml, can set and open the mode that peristaltic pump starts to pour into and cultivate described hybridoma.According to cell density and tiring, adjustment peristaltic pump setting speed, changes perfusion volume.What select in the present embodiment is reach 1 × 10 when cell density
6during/more than ml, progressively promote setting speed, with continuous perfusion or change liquid collecting mode and cultivate, obtain cell culture supernatant, when bio-reactor continuously perfused culture is collected, stream adds volume every day between 1 ~ 3 tank volume.
The cell culture supernatant of results is tested by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Nutrient solution has no side effect safely to dog, results nutrient solution canine distemper virus Neutralizing titer >=128
(3) the concentrated and purifying of therapeutic monoclonal antibodies nutrient solution: harvested cell culture supernatant ultrafiltration system filters, and tires determine cycles of concentration according to therapeutic monoclonal antibodies nutrient solution.Remove cell debris by filtering system, the liquid of results puts less than-15 DEG C preservations;
The inspection of concentrated solution: test by existing " People's Republic of China's veterinary drug allusion quotation " annex page, should without bacterium, mould, mycoplasma growth.Nutrient solution has no side effect safely to dog, results nutrient solution canine distemper virus Neutralizing titer >=1024
(4) packing: by therapeutic monoclonal antibodies that is concentrated, purifying, aseptic, quantitative separating is in the container of sterilizing.Get product after adding rapidly sterilizing plug, gland after packing, less than-15 DEG C preservations.
Inspection after construction: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without bacterium, mould, mycoplasma growth.Dog is had no side effect safely, canine distemper virus Neutralizing titer >=128.
The canine distemper virus monoclonal antibody injection liquid neutralization test that embodiment 5 the present invention prepares
This test adopts fixed virus-diluted blood heat-clearing method neutralization test, for the detection of canine distemper virus monoclonal antibody Neutralizing titer.
1 materials and methods
1.1 new-born calf serum
1.2DMEM
1.2.1DMEM mother liquor: 1L measures DMEM powder and is dissolved in 100ml autoclaving deionized water, G6 frit.
1.2.2 serum-free DMEM liquid:
1.2.3Vero cell nutrient solution: containing the serum-free DMEM liquid of 10% bovine serum;
1.2.4Vero cell maintenance medium: containing the serum-free DMEM liquid of 2% bovine serum;
1.3 canine distemper virus: CDV MD-77 strain;
1.4Vero cell;
1.5 Tissue Culture Plate.
2 test methods
2.1 by Vero cell cultures in 25cm2 cell bottle, to grow up to after individual layer by every bottle of 0.1ml amount inoculation CDV until cell, cultivate after 5 days for 33 DEG C, by compared with control cells with connect poison cell and use trysinization respectively, receive poison and cell suspension, measure TCID
50;
2.2 virus dilution: viral suspension is diluted to 100TCID
50/ 0.1ml;
2.3 dilution monoclonal antibodies: canine distemper virus monoclonal antibody injection liquid to be measured is made 2 times of doubling dilutions; 3 batches of canine distemper virus monoclonal antibodies prepared by embodiment 2; 3 batches of canine distemper virus monoclonal antibodies prepared by embodiment 3; 3 batches of canine distemper virus monoclonal antibodies prepared by embodiment 4.
2.4 external neutralization: 0.25ml each extent of dilution canine distemper virus monoclonal antibody injection liquid mixes with 0.25ml virus liquid, and 37 DEG C act on 1 hour;
2.5 on 96 porocyte culture plates every hole add 40 μ l Vero cell suspensions;
2.6 every holes add 100 μ l monoclonal antibody-viral mixed solutions, and each extent of dilution adds 6 holes.5%CO2 incubator, 37 DEG C of cultivations;
2.7 tests establish 0.1 simultaneously, 1,100TCID50 virus control, Vero cell controls, the contrast of canine distemper virus monoclonal antibody injection liquid stoste, each 4 holes;
2.8 treat that cell grows up to individual layer, change serum-free DMEM nutrient solution, every day observation of cell pathology, by Reed and MuenchShi method calculation result.
3 results:
9 batches of canine distemper monoclonal antibody Neutralizing titer
| Batch | Neutralizing titer | |
| Example 1 | 20120301 | 1﹕2048 |
| Example 1 | 20120302 | 1﹕1024 |
| Example 1 | 20120303 | 1﹕1024 |
| Example 2 | 20120401 | 1﹕1024 |
| Example 2 | 20120402 | 1﹕2048 |
| Example 2 | 20120403 | 1﹕1024 |
| Example 3 | 20120501 | 1﹕1024 |
| Example 3 | 20120502 | 1﹕1024 |
| Example 3 | 20120503 | 1﹕1024 |
Through fixed virus-diluted blood heat-clearing method, the detection of Neutralizing titer is carried out to each 3 batches of canine distemper virus monoclonal antibodies that example 2,3,4 is produced, result shows embodiment 2, the canine distemper virus monoclonal antibody Neutralizing titer of 3,4 preparations all reaches 1:1024, tires qualified.
The canine distemper virus monoclonal antibody injection liquid that embodiment 6 the present invention prepares is to the effectiveness study of the strong malicious artificial challenge dog of distemper virus
1 experiment material
1.1 canine distemper virus kind poison: CDV98017 virus-culturing fluid, virus titer is 10
6tCID
50/ ml.
1.2 laboratory animal
Wean 8 week age beasle dog 90, and the mental status and diet are for normally.
1.3 test medicine and other reagent
1.3.1 in embodiment 2 preparation 3 batches of canine distemper virus monoclonal antibody injection liquid, lot number is respectively: 20120301(Neutralizing titer 1 ﹕ 2048), 20120302 (Neutralizing titer 1 ﹕ 1024), 20120303 (Neutralizing titer 1 ﹕ 1024).Through physical behavior, pH value, activity, aseptic, mycoplasma, exogenous virus, safety etc. after the assay was approved for examination.Thered is provided by Anheal Laboratories Co., Ltd.
1.3.2 in embodiment 3 preparation 3 batches of canine distemper virus monoclonal antibody injection liquid, lot number is respectively: 20120401(Neutralizing titer 1 ﹕ 1024), 20120402 (Neutralizing titer 1 ﹕ 2048), 20120403 (Neutralizing titer 1 ﹕ 1024).Through physical behavior, pH value, activity, aseptic, mycoplasma, exogenous virus, safety etc. after the assay was approved for examination.Thered is provided by Anheal Laboratories Co., Ltd.
1.3.3 in embodiment 4 preparation 3 batches of canine distemper virus monoclonal antibody injection liquid, lot number is respectively: 20120501(Neutralizing titer 1 ﹕ 1024), 20120502 (Neutralizing titer 1 ﹕ 1024), 20120503 (Neutralizing titer 1 ﹕ 1024).Through physical behavior, pH value, activity, aseptic, mycoplasma, exogenous virus, safety etc. after the assay was approved for examination.Thered is provided by Anheal Laboratories Co., Ltd.
1.3.4 canine distemper virus antibody ELISA detection kit, is provided by Anheal Laboratories Co., Ltd.
1.3.5 physiological saline, is bought by medical pharmacy.
2 experimental techniques
2.1 inoculations: 90 qualified wean beasle dogs of health in 8 week age, the serum sample gathering dog before experiment carries out the test of canine distemper virus antibody ELISA, and detected result is negative.Press 5ml/ dosage abdominal injection only, 0.5ml/ dosage collunarium only with CDV98017 virus-culturing fluid, inoculate the beasle dog that weans 8 week age, Continuous Observation clinical symptom after inoculation, early, middle and late each measurement body temperature, and observe clinical symptom, comprise the mental status, appetite etc., perform record.Treat that intermittent or persistent fever appear in infected dogs, body temperature reaches more than 39.5 DEG C, lassitude, drink appetite stimulator, eye nose has serosity or purulent secretion, the respiratory symptom such as to cough, breathe heavily, or have a convulsion, tremble, the nervous symptoms such as paralysis, and confirm to detect positive through canine distemper virus antibody ELISA detection kit.
2.2 groupings: the morbidity dog after inoculation between the 3rd to 9 is selected 60 and is divided into 12 groups at random, often organize each 5.To fall ill every day dog random number, randomly draw morbidity dog numbering and carry out random packet, wherein the 4th group of morbidity dog in contrast, will not be treated again.
2.3 treatment
2.3.1. the monoclonal antibody injection liquid of embodiment 2 preparation
2.3.1.1 the 1st group of direct injection canine distemper virus monoclonal antibody injection liquid, by 1ml/kg dosage intramuscular injection 20120301 batches (Neutralizing titer is 1 ﹕ 2048), every day 1 time, is used in conjunction 3.
2.3.1.2 the 2nd group of direct injection canine distemper virus monoclonal antibody injection liquid, by 0.5ml/kg dosage intramuscular injection 20120302 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, is used in conjunction 3.
2.3.1.3 the 3rd group canine distemper virus monoclonal antibody injection liquid and physiological saline are pressed 1 ﹕ 1 volume mixture, by 0.5ml/kg dosage intramuscular injection 20120303 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, be used in conjunction 3.
2.3.1.4 the 4th group of morbidity dog in contrast, will not be treated.
2.3.2 the monoclonal antibody injection liquid of embodiment 3 preparation
2.3.2.1 the 5th group of direct injection canine distemper virus monoclonal antibody injection liquid, by 1ml/kg dosage intramuscular injection 20120401 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, is used in conjunction 3.
2.3.2.2 the 6th group of direct injection canine distemper virus monoclonal antibody injection liquid, by 0.5ml/kg dosage intramuscular injection 20120402 batches (Neutralizing titer is 1 ﹕ 2048), every day 1 time, is used in conjunction 3.
2.3.2.3 the 7th group canine distemper virus monoclonal antibody injection liquid and physiological saline are pressed 1 ﹕ 1 volume mixture, by 0.5ml/kg dosage intramuscular injection 20120403 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, be used in conjunction 3.
2.3.2.4 contrast same 2.3.1.4
2.3.3 the monoclonal antibody injection liquid of embodiment 4 preparation
2.3.3.1 the 8th group of direct injection canine distemper virus monoclonal antibody injection liquid, by 1ml/kg dosage intramuscular injection 20120501 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, is used in conjunction 3.
2.3.3.2 the 9th group of direct injection canine distemper virus monoclonal antibody injection liquid, by 0.5ml/kg dosage intramuscular injection 20120502 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, is used in conjunction 3.
2.3.3.3 the 10th group canine distemper virus monoclonal antibody injection liquid and physiological saline are pressed 1 ﹕ 1 volume mixture, by 0.5ml/kg dosage intramuscular injection 20120503 batches (Neutralizing titer is 1 ﹕ 1024), every day 1 time, be used in conjunction 3.
2.3.3.4 contrast same 2.3.1.4
Observe 10.Calculate morbidity dog rehabilitation ratio.With experimental dog spirit, appetite, temperature recovery normally for rehabilitation standard.
3 results
After artificial challenge, three four groups test dogs all fall ill, and by the treatment of 3 batches of canine distemper virus monoclonal antibody injection liquid, its curative ratio reaches 100%.Treatment after the 5th day the results are shown in Table 4,5,6.
Canine distemper virus monoclonal antibody injection liquid result for the treatment of measurement result prepared by table 4 embodiment 2
| Group | Test dog number | Infective dose | Treatment monoclonal antibody lot number | Cure |
| 1st group | 5 | 10 6TCID 50/1.0ml | 20120301 | 5/5 |
| 2nd group | 5 | 10 6TCID 50/1.0ml | 20120302 | 5/5 |
| 3rd group | 5 | 10 6TCID 50/1.0ml | 20120303 | 5/5 |
| 4th group | 5 | 10 6TCID 50/1.0ml | Physiological saline | 0/5 |
1st group, the 2nd group and the 3rd group of dog are cured and reach 100%, and the 4th group of blank dog symptom is aggravated gradually, and wherein 1 dog is dead in latter 7 days of infection, and 1 dead in latter 10 days of infection.
Canine distemper virus monoclonal antibody injection liquid result for the treatment of measurement result prepared by table 5 embodiment 3
1st group, the 2nd group and the 3rd group of dog are cured and reach 100%, and the 4th group of blank dog symptom is aggravated gradually, and wherein 2 dogs are dead in latter 7 days of infection, and 1 dead in latter 10 days of infection.
Canine distemper virus monoclonal antibody injection liquid result for the treatment of measurement result prepared by table 6 embodiment 4
The embodiment of the present invention 2,3,3 groups of dogs healings in the canine distemper virus monoclonal antibody injection liquid Experiment on therapy dog of 4 preparations all reach 100%, and the 4th group of blank dog symptom is aggravated gradually, and wherein 1 dog is in infection death in latter 7 days, and 3 dead in infecting latter 10 days.
4 analysis and thinking
Test-results shows, after 3 batches of canine distemper virus monoclonal antibody injection liquid that three embodiment methods are prepared respectively treat 3 days to the dog that distemper virus disease occurs, clinical symptom is obviously alleviated, and cures and reaches 100%.
Explain the present invention by specific embodiment above, but the present invention is not limited thereto.The equivalent transformation that all spirit according to the present invention are done or modification, all should be encompassed within protection scope of the present invention.
Claims (9)
1. a method for manufacture of therapeutic canine distemper virus monoclonal antibody, is characterized in that, comprises the steps:
(1) hybridoma of secretion manufacture of therapeutic canine distemper virus monoclonal antibody is carried out enlarged culturing in cell culture fluid;
(2) inoculate in the cell culture fluid in bio-reactor and cultivate;
(3) the therapeutic canine distemper virus monoclonal antibody that hybridoma generates is gathered in the crops;
The preserving number of described hybridoma is: CGMCC No.7613.
2. method according to claim 1, is characterized in that, the formula of described cell culture fluid is: 85%DMEM liquid, 15% calf serum, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep; Or Serum-free Hybridoma cell culture medium, add 200 units/ml benzylpenicillin sodium and 200 units/ml Vetstrep.
3. method according to claim 2, is characterized in that, inoculum density is 1 × 10
5~ 1 × 10
6/ ml.
4. method according to claim 3, is characterized in that, inoculum density is 2 × 10
5~ 8 × 10
5/ ml, wherein adopts batch, continuously perfusion or changes liquid collecting mode in bio-reactor, cultivate described hybridoma in step (2).
5. method according to claim 4, is characterized in that, inoculum density is 4 × 10
5~ 5 × 10
5/ ml, cultivates the culture temperature of described hybridoma between 36 ~ 37 DEG C, and the pH value of described cell culture fluid is 7.0 ~ 7.2, and the dissolved oxygen scope of described bio-reactor is set to 50% ~ 90%.
6. method according to claim 5, is characterized in that, when adopting continuous irrigation stream mode to cultivate hybridoma, it is tank volume every day 1 ~ 5 that stream adds volume.
7. method according to claim 6, is characterized in that, also comprises the steps: that manufacture of therapeutic canine distemper virus monoclonal antibody step (3) gathered in the crops carries out concentrated and purifying, more aseptic, quantitative separating is in the container of sterilizing.
8. the therapeutic canine distemper virus monoclonal antibody of the arbitrary described method production of claim 1-7.
9. a hybridoma cell strain, its preserving number is CGMCC No.7613.
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| CN109897880A (en) * | 2019-03-18 | 2019-06-18 | 郑州伊美诺生物技术有限公司 | In vitro culture prepares the method and its application of AFP monoclonal antibody |
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| CN112980909A (en) * | 2021-02-26 | 2021-06-18 | 通用生物系统（安徽）有限公司 | Method for preparing antibody by hybridoma cell serum-free culture |
| CN112725285B (en) * | 2021-03-27 | 2023-11-14 | 哈尔滨元亨生物药业有限公司 | Method for preparing canine distemper virus monoclonal antibody by utilizing bioreactor |
| CN112852749A (en) * | 2021-03-27 | 2021-05-28 | 哈尔滨元亨生物药业有限公司 | Hybridoma cell strain C68 capable of efficiently secreting canine parvovirus monoclonal antibody and production method thereof by using bioreactor |
| CN114181305B (en) * | 2021-12-28 | 2024-01-26 | 山东畜牧兽医职业学院 | Canine distemper virus H protein nano antibody and preparation process thereof |
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