CN103550769A - Production method of porcine circovirus type 2 - Google Patents

Abstract

The invention discloses a production process for producing porcine circovirus type 2 by culturing cells pK-15A1# in a bioreactor. The production process comprises the technical steps of (1) selecting pK-15A1# cells for multiplying circovirus type 2; (2) utilizing the bioreactor as a cell culture tool; (3) carrying out a virus culture process of synchronous virus inoculation; (4) culturing the pK-15A1# cells on micro-carriers in the bioreactor; (5) carrying out multiplication culture on viruses of porcine circovirus type 2 in the cells on the micro-carriers in the bioreactor; and (6) harvesting virus culturing materials of porcine circovirus type 2. The production process has the advantages that the production cost can be lowered, and compared with that of a traditional spinner bottle production process, the input-output ratio of the production process is increased by 5-10 times; the production cycle is short, the occupied area is small, the production scale can be easily and rapidly expanded, the environmental pollution is slight and is easy to avoid, the automation degree is high, few workers are required, the quality is stable, the cost can be remarkably lowered, and the yield and quality of vaccines can be remarkably improved.

Description

A kind of production method of porcine circovirus 2 type

Technical field

The present invention relates to a kind of applying biological reactor cell culture technology and produce the method for vaccine, can be used for suitability for industrialized production porcine circovirus type 2 vaccines, substitute traditional spinner culture method production technology.

Background technology

At present, mainly all adopt cell spinner bottle cultural method to realize in porcine circovirus 2 type inactivated vaccine manufacturing country, it is low that this traditional handicraft is produced semi-finished product antigen valence, is difficult to reach and joins Seedling requirement, need to carry out labor intensity large, and length consuming time, efficiency are low, and production cost is high; Easily by environmental pollution; Difference between different production batch or between the different bottles of same production batch is large; Be difficult to expanding production; The vaccine quality hidden danger that easily occurs being polluted by antibacterial or other virus and cause, relates to bio-safety and public health problem.

Summary of the invention

The object of the invention is to overcome the weak point that existing cell spinner bottle culture method technology exists, provide a kind of applying biological reactor cell microcarrier suspension culture technology to produce the method for porcine circovirus 2 type inactivated vaccine.The method can reduce production costs in a large number, and with short production cycle, and occupied ground is little, is easy to expand the scale of production fast, low in the pollution of the environment and be easy to process, and automaticity is high, and employment is few, and quality is easy to realize equalization stable.Can significantly improve vaccine output and quality.

For achieving the above object, the present invention has taked following technical scheme:

A production method for porcine circovirus 2 type is to select bioreactor as cultivation instrument; The growing carrier of selecting microcarrier to attach as cell; Select pK-15A1# cell as seedling cell;

(1), seedling going down to posterity and inoculating with cell

By the pK-15A1# cell of fine and close monolayer sampling counting after Digestive system digestion disperses, with the MEM growth-promoting media containing 10%NBCS, pK-15A1# cell is adjusted into 2-3 * 10 ⁵individual/ml, inoculates on bioreactor and cultivates; Described condition of culture is: temperature is 37.0 ℃ (± 0.2 ℃), and pH value is that 7.40, DO is 40%-80%, and rotating speed is 40rpm-80rpm;

(2), the poison that connects of cell for seedling

The pK-15A1# cell of cultivating in bioreactor, according to volume ratio 1%-5% inoculation PCV2 virus, is continued to cultivate according to described condition of culture after connecing poison;

(3), connect the cultivation after poison

After connecing poison, when cell culture 12h, add the essential amino acids of 200 μ mol/L; When the about 24h of cell culture, change containing the MEM growth-promoting media of 10%NBCS once; When cell culture 48h, cell covers with microcarrier 50%-60%, after sucking-off MEM growth-promoting media, by the aseptic PBS washed twice of preheating, then adds the MEM maintenance medium containing 2%NBCS and 1%D-glucosamine to continue to cultivate;

(4), results and the bioactivity of virus

Maintenance medium stops stirring after cultivating rear about 54h, after standing 5min, sucking-off supernatant is as receipts, after this add MEM maintenance medium to continue to cultivate 48h, standing rear sucking-off supernatant, results are two receipts, after this add MEM maintenance medium continue to cultivate 48h, now by microcarrier whole results together with culture fluid, after freeze thawing three times, as antigen, preserve.

The parameters such as in technique scheme, bioreactor is can A.T.C, pH, dissolved oxygen, mixing speed, are applicable to the bioreactor that microcarrier is cultivated, and volume is 3L-3000L.

In technique scheme, microcarrier microcarrier, microcarrier needs to clean before use, sterilizing, and method is as follows:

1) with PBS, soak microcarrier more than 2 hours;

2) with PBS, clean microcarrier 3 times;

3) with PBS, soak microcarrier, 121 ℃ of steam sterilizations 30 minutes.

In technique scheme, trypsinization formula of liquid is: mass fraction is respectively 0.25% pancreatin (1:250) Digestive system; The formula of cell growth medium is: the ratio that the volume ratio of MEM is 88%, NBCS is 10%, and the ratio that dual anti-ratio is 1%, L-Glutamin is 1%.

In technique scheme, planting malicious inoculum concentration is 1-5%, and Virus culture formula of liquid is: containing the MEM liquid of serum 1-2%, add appropriate dual anti-ly, pH is adjusted into 7.2-7.4.Cultivation temperature is 37.0 ± 0.2) ℃.

In technique scheme, bioreactor setup parameter is: 33-38 ℃ of cultivation temperature cultivation temperature, pH6.5-7.68, dissolved oxygen 30%-70%, mixing speed 40-80rpm.

The poison that connects of healthy cell adopts cells Synchronous to connect malicious technique, and cells Synchronous connects malicious technique and is:

(1) adopt the pK-15A1# cell that pollutes without mycoplasma and PCV2/PCV1 as seedling cell;

(2) to be inoculated into the density on microcarrier be 2-3 * 10 to cell ⁵individual/ml;

(3) condition that cell is cultivated on bioreactor is: temperature: 37.0 ℃ (± 0.2 ℃), pH value: 7.40, DO:40%-80%, rotating speed is 40rpm-80rpm;

(4) cell connects poison according to 1%-5%(volume ratio) inoculation PCV2 kind poison, continues to cultivate according to set before condition of culture after connecing poison;

(5) when cell culture 12h, should take the circumstances into consideration to add the necessary aminoacid of 200 μ mol/L; When the about 24h of cell culture, should change cell culture with growth-promoting media once;

(6) at cell culture, sample observation of cell when the about 48h and should cover with the about 60%-70% of microcarrier, now should sucking-off growth-promoting media, add the aseptic PBS washed twice of appropriate preheating and extract PBS out, adding the MEM maintenance medium containing 2%NBCS and 1%D-glucosamine to continue to cultivate;

(7) maintenance medium stops stirring after cultivating rear about 54h, and after standing 5min, sucking-off supernatant is as receipts;

(8) after this add appropriate MEM maintenance medium to continue to cultivate 48h, standing rear sucking-off supernatant, results are two receipts;

After this add appropriate MEM maintenance medium to continue to cultivate 48h, now by microcarrier whole results together with culture fluid, after freeze thawing three times, as antigen, save as three receipts.

Compared with prior art, the present invention has following beneficial effect:

(1) with bioreactor Microcarrier Cell Culture Techniques, replace rolling bottle cell culture technology to manufacture porcine circovirus 2 type inactivated vaccine, can solve that production efficiency is low, unstable product quality, problem that virus titer is low, by the change of production technology and production technology, General Promotion vaccine quality and output, the safety that improves vaccine.

(2) the present invention can reduce production costs in a large number, and with short production cycle, and each production cycle only needs 5-6 days, compares above obviously shortenings in 18-30 days of existing rolling bottle cell culture method.

(3) applying biological reactor carries out production of vaccine, has automaticity high, and employment is few, production technology simple and stable.The large occupied ground of easy to operate, output is little, is easy to expand the scale of production fast.Quality is easy to realize equalization stable.

(4) application this method is produced vaccine, and product virus titer improves 10-20 doubly than traditional rolling bottle cell culture method, and cost 3-4 doubly.Product quality homogeneous, is easy to large-scale production, and whole production process does not relate to other biological safety and the public health problem that conventional production methods has.

Accompanying drawing explanation

Fig. 1 is preparation flow figure of the present invention.

The specific embodiment

Below in conjunction with Figure of description and specific embodiment, the invention will be further described.

(1) select bioreactor as the means of cultivating: 3L-3000L bioreactor.

(2) select microcarrier as cell, to attach the carrier of growth

The cleaning of microcarrier, sterilizing methods:

1) weigh Cytodex microcarrier 3-5g/L, use appropriate PBS immersion bubble three hours.

2) with appropriate PBS liquid, clean 3 times at every turn.

3) add appropriate PBS immersion bubble microcarrier, 121 ℃ of steam sterilizations 30 minutes.

(3) select pK-15A1# cell as seedling cell:

(4) seedling going down to posterity and cultivating with cell:

Above-mentioned cell, through trypsinization liquid (containing 0.25% pancreatin (1:250)) had digestive transfer culture, continues to cultivate with growth-promoting media, and cultivation temperature is 37.0(± 0.2) ℃.While forming 50-60% monolayer, for continuing to go down to posterity or be inoculated in bioreactor, carry out microcarrier cultivation.

(5) breeding of cell seed culture of viruses:

With cell maintenance medium, porcine circovirus 2 type kind poison, in the well-grown above-mentioned cell monolayer of 1-5% ratio inoculation, is continued to cultivate, cultivation temperature is 33-38 ℃.Gather in the crops virus liquid when cultivating 50h left and right; Carry out steriling test and efficacy test, qualified after using this virus as producing with kind of a poison.

(6) microcarrier of pK-15A1# clone cell in bioreactor cultivated:

Get well-grown pK-15A1# clone cell on rolling bottle, through trypsinization liquid digestion, be prepared as cell suspension, after cell counting in reaction of inoculation device.Bioreactor is set as follows 37 ℃ of parameters of temperature, pH7.2-7.4, mixing speed 40-80rpm, dissolved oxygen content 40-80% to carry out reactor and automatically controls cultivation.

(7) breeding of seedling venom:

After cultivating, on microcarrier to be seen on the 3rd, cell covers with the maintenance medium being replaced by containing 1%D-glucosamine after 60% and connects poison operation.The culture parameters such as design temperature 33-38 ℃, pH7.2-7.6, mixing speed 40-80rpm, dissolved oxygen content 30-60%, carry out reactor and automatically control cultivation.After connecing poison, approximately about 50h, pay close attention to DO value, treat that DO value is obvious ascendant trend, stopped reaction device stirs, and until microcarrier, all sinks to after reactor bottom, gathers in the crops respectively supernatant and microcarrier.

(8) processing of results virus liquid:

After time multigelation centrifugal or filter remove cell debris after-20 ℃ of freezing preservations as semi-finished product.

1, the mensuration of results virus liquid poison valency:

By the rear sampling of cytopathy venom mixing of results above, measure malicious valency.Viral level answers>=10 ^5.5tCID ₅₀/ ml.

2, the inspection of semifinished product

Steriling test: carry out asepsis growth by 301 pages of the veterinary biologics quality standard > > of < < People's Republic of China (PRC) appendix.

3, product inspection

Vaccine is manufactured and product inspection is manufactured by < < porcine circovirus 2 type inactivated vaccine (DBN-SX07 strain) and inspection procedure > > requirement is carried out, and should meet the requirements.

Claims (4)

1. a production method for porcine circovirus 2 type, is characterized in that:

Select bioreactor as cultivation instrument; The growing carrier of selecting microcarrier to attach as cell; Select pK-15A1# cell as seedling cell;

(1), seedling going down to posterity and inoculating with cell

By the pK-15A1# cell of fine and close monolayer sampling counting after Digestive system digestion disperses, with the MEM growth-promoting media containing 10%NBCS, pK-15A1# cell is adjusted into 2-3 * 10 ⁵individual/ml, inoculates on bioreactor and cultivates; Described condition of culture is: temperature is 37.0 ℃ ± 0.2 ℃, and pH value is that 7.40, DO is 40%-80%, and rotating speed is 40rpm-80rpm;

(2), the poison that connects of cell for seedling

The pK-15A1# cell of cultivating in bioreactor, according to volume ratio 1%-5% inoculation PCV2 virus, is continued to cultivate according to described condition of culture after connecing poison;

(3), connect the cultivation after poison

After connecing poison, when cell culture 12h, add the essential amino acids of 200 μ mol/L; When the about 24h of cell culture, change containing the MEM growth-promoting media of 10%NBCS once; When cell culture 48h, cell covers with microcarrier 50%-60%, after sucking-off MEM growth-promoting media, by the aseptic PBS washed twice of preheating, then adds the MEM maintenance medium containing 2%NBCS and 1%D-glucosamine to continue to cultivate;

(4), results and the bioactivity of virus

Maintenance medium stops stirring after cultivating rear about 54h, after standing 5min, sucking-off supernatant is as receipts, after this add MEM maintenance medium to continue to cultivate 48h, standing rear sucking-off supernatant, results are two receipts, after this add MEM maintenance medium continue to cultivate 48h, now by microcarrier whole results together with culture fluid, after freeze thawing three times, as antigen, preserve.

2. the production method of porcine circovirus 2 type according to claim 1, it is characterized in that: described bioreactor volume is 3L-3000L, the microcarrier adopting is Cytodex series, the working concentration of microcarrier is 3-5g/L, bioreactor needs to carry out electrode correction before use, sterilizing, balance, method is as follows;

(1) preparation of bioreactor:

According to the volume of cultivating, select suitable bioreactor, connect various pipelines and feed supplement, change the interface of liquid, connect T, pH, DO electrode, the method for consulting and using in description is carried out the correction of electrode;

(2) preparation of microcarrier:

According to the volume of cultivating and selected microcarrier concentration, take appropriate Cytodex-1 microcarrier in the vial of silicidation or in rustless steel container, (as 10g microcarrier adds the PBS of the 500ml) 30min that adds the PBS buffer of approximately 50 times of microcarrier quality to soak, after this changes fresh PBS and continues to soak 2h; The PBS that changes 10 times of volumes prepares sterilizing in bioreactor subject to sterilization;

(3) sterilizing:

Before sterilizing, conscientiously check that each pipeline and joint guarantee to be connected intact laggard row air tightness test, after test passes, can prepare to carry out sterilizing; According to the scale of bioreactor, the following general off-line sterilizing that adopts of 5L and 5L, 121 ℃ of high pressure 30min in pressure cooker, sterilizing finishes rear taking-up natural cooling; 10L and bioreactor more than 10L adopt the mode sterilizing of sterilizing in place conventionally.

3. the production method of porcine circovirus 2 type according to claim 1, is characterized in that: seedling adopts cells Synchronous to connect malicious technique with the poison that connects of cell.

4. the production method of porcine circovirus 2 type according to claim 3, is characterized in that: cells Synchronous connects malicious technique and is:

(1) adopt the pK-15A1# cell that pollutes without mycoplasma and PCV2/PCV1 as seedling cell;

(2) to be inoculated into the density on microcarrier be 2-3 * 10 to cell ⁵individual/ml;

(3) condition that cell is cultivated on bioreactor is: temperature: 37.0 ℃ (± 0.2 ℃), pH value: 7.40, DO:40%-80%, rotating speed is 40rpm-80rpm;

(4) cell connects poison according to 1%-5%(volume ratio) inoculation PCV2 kind poison, continues to cultivate according to set before condition of culture after connecing poison;

(5) when cell culture 12h, should take the circumstances into consideration to add the necessary aminoacid of 200 μ mol/L; When the about 24h of cell culture, should change cell culture with growth-promoting media once;

(6) at cell culture, sample observation of cell when the about 48h and should cover with the about 60%-70% of microcarrier, now should sucking-off growth-promoting media, add the aseptic PBS washed twice of appropriate preheating and extract PBS out, adding the MEM maintenance medium containing 2%NBCS and 1%D-glucosamine to continue to cultivate;

(7) maintenance medium stops stirring after cultivating rear about 54h, and after standing 5min, sucking-off supernatant is as receipts;

(8) after this add appropriate MEM maintenance medium to continue to cultivate 48h, standing rear sucking-off supernatant, results are two receipts;

After this add appropriate MEM maintenance medium to continue to cultivate 48h, now by microcarrier whole results together with culture fluid, after freeze thawing three times, as antigen, save as three receipts.

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