阅读说明：本技术 一种大规模悬浮培养禽腺病毒的方法 (Method for large-scale suspension culture of avian adenovirus ) 是由 王玉红 韩建文 关秀春 李王强 刘岳 郑长虹 王鹏听 黄理进 贾建斌 贠鲁祥 黄 于 2021-09-01 设计创作，主要内容包括：本发明提供一种大规模悬浮培养禽腺病毒的方法,所提供的方法可以获得高效价的Ⅰ群4型、8b型、D11型、8a型等禽腺病毒；所述的方法中使用的一种驯化的LMH细胞,为鸡肝癌悬浮LMH细胞,其保藏号为CCTCC NO：C2021202。本发明采用逐步降血清法将贴壁LMH细胞驯化为可稳定连续传代的悬浮细胞。然后用生物反应器全悬浮培养LMH细胞,然后逐级放大悬浮培养LMH细胞,并在悬浮细胞上分别接种增殖禽腺病毒抗原的方法,本发明方法可以获得高效价的禽腺病毒悬浮培养工艺病毒抗原,抗原效价是现行疫苗规程抗原效价的5～10倍左右。(The invention provides a method for large-scale suspension culture of avian adenovirus, which can obtain high-titer avian adenovirus I of group 4, 8b, D11, 8a and the like; the domesticated LMH cell used in the method is a chicken liver cancer suspension LMH cell, and the preservation number of the domesticated LMH cell is CCTCC NO: C2021202. the invention adopts a gradual serum-reducing method to acclimate the adherent LMH cells into suspension cells which can be stably and continuously passaged. And then, using a bioreactor to culture LMH cells in a full suspension manner, then amplifying the LMH cells in a suspension manner step by step, and respectively inoculating and proliferating the avian adenovirus antigens on the suspension cells.)

1. The LMH cell is characterized in that the preservation number of the LMH cell is CCTCC NO: v202158.

2. The LMH cells of claim 1, wherein the LMH cells are acclimated using a stepwise decrease in serum content of the culture medium.

3. A method for culturing avian adenovirus, which comprises culturing the LMH cell of claim 1 in suspension and then amplifying the avian adenovirus.

4. The method of claim 3, wherein the suspension culture is performed by suspension culture of the LMH cells in a serum-free medium.

5. The method of claim 4, wherein the suspension culture is performed in a volume of 7L to 1500L.

6. The method of claim 4, wherein said suspension culture is performed under the following conditions: the inoculation density is 1.5X 10^6.0～2.0×10^6.0Culturing the cells/ml at 37 deg.C, dissolved oxygen DO of 40, and pH of 7.0 + -0.2; when the LMH cell density reaches 2.5X 10^6.0For each cell/ml, the group I type 4/8 b/D11 avian adenovirus production seed virus was inoculated at MOI of 0.2, and virus culture was carried out at 37 ℃, dissolved oxygen DO of 40 and pH of 7.2. + -. 0.2.

7. The method of claim 3, wherein the avian adenovirus is group I4, 8b, or D11.

Technical Field

The invention belongs to the technical field of virus culture, and particularly relates to a method for large-scale suspension culture of avian adenovirus, namely a method for suspension culture of avian adenovirus (I group 4, 8b and D11) by using domesticated suspension LMH cells (chicken liver cancer cells), which comprises the steps of suspension domestication of adherent LMH cells, culture of suspension cells and suspension culture amplification of avian adenovirus (I group 4, 8b, D11 and 8 a).

Background

In the past decades, the inactivated avian adenovirus vaccine in China mainly undergoes tissue inactivated vaccine, chick embryo inactivated vaccine and cell inactivated vaccine. The tissue inactivated vaccine is prepared by extracting liver homogenate of infected chicken with chloroform and inactivating formaldehyde, has simple and extensive process, and can not be applied to large-scale production at all and is gradually eliminated. The inactivated chick embryo vaccine is prepared by inoculating SPF chick embryos with adenovirus, harvesting allantoic fluid, and inactivating to prepare a finished vaccine. The inactivated chicken embryo fibroblast vaccine is also eliminated due to the complex process, high cost and low potency. At present, the virus solution is obtained by culturing on adherent LMH cells and inactivated to prepare the finished vaccine, but the process adopts traditional adherent production modes such as a rotary bottle or a cell factory and the like, so that the process is complicated, the titer is not ideal, the serum is depended on, the labor intensity is high, and the large-scale amplification cannot be realized due to the limitation of the culture space. In recent years, with frequent outbreak of the avian adenovirus epidemic disease, the harm is more serious, and vaccine workers urgently need to research a safe and efficient novel process to replace the existing production process.

Therefore, the cell suspension culture technology becomes a new technology worthy of development at present, is a new large-scale cell culture technology, is an ideal mode for large-scale cell culture, is easier to amplify compared with the full suspension cell culture technology in the adherent cell culture mode, has simple and rapid process, greatly reduces the cost and culture conditions (temperature, pH value and CO)₂Concentration, etc.) is easy to control, and the culture process is systematic and automatic and is not easy to be polluted; in addition, the full suspension culture can be carried out without serum, the animal components are greatly reduced, the obtained antigen titer is high, the adverse reaction is small, the labor production efficiency can be greatly improved, the labor intensity is reduced, and the like, so that the full suspension culture is favored.

In recent years, the avian adenovirus outbreak is frequent, the harm is large, the vaccine demand is huge, and several vaccine manufacturers are actively researching and developing suspension LMH cell culture avian adenovirus processes, and are in the test stage. The method of the invention is that the company of the applicant can firstly carry out suspension domestication of LMH cells and scale up the LMH cells to the manufacturer of 1500L reactor.

Disclosure of Invention

The invention aims to provide a method for large-scale suspension culture of avian adenovirus, which can obtain high-titer avian adenovirus (I group 4 type, 8b type, D11 type and 8a type) suspension culture process virus antigens and reach or exceed the titer of the existing adherent cell protocol.

The invention firstly provides domesticated LMH cells, which are chicken liver cancer suspension LMH cells, are preserved in China center for type culture Collection of Wuhan, Wuhan university in 2021, 8 months and 4 days, and the preservation number is CCTCC NO: C2021202.

the provided domesticated LMH cell is obtained by domesticating by gradually reducing the serum content in a culture medium;

the invention also provides a method for culturing the avian adenovirus, which comprises the steps of performing suspension culture on domesticated LMH cells and then amplifying the avian adenovirus;

the suspension culture adopts a serum-free culture medium to culture LMH cells in a suspension manner;

the suspension culture is carried out, wherein the volume of the culture medium is 7L-1500L

The suspension culture is carried out under the following culture conditions: the inoculation density is 1.5X 10^6.0～2.0×10^6.0Culturing the cells/ml at 37 deg.C, Dissolved Oxygen (DO) of 40, and pH of 7.0 + -0.2; when the LMH cell density reaches 2.5X 10^6.0Inoculating the virus for producing group I type 4/8 b/D11/8a avian adenovirus at MOI of 0.2-1.0 per ml, and culturing at 37 deg.C, Dissolved Oxygen (DO) of 40, and pH of 7.2 + -0.2.

The invention adopts a gradual serum-reducing method to acclimate the adherent LMH cells into suspension cells which can be stably and continuously passaged. And then, using a bioreactor to culture LMH cells in a full suspension manner, then amplifying the LMH cells in a suspension manner step by step, and respectively inoculating and proliferating avian adenovirus (I group 4 type, 8b type, D11 type and 8a type) virus antigens on the suspension cells.

Drawings

FIG. 1: a flow chart of the suspension domestication of LMH cells,

FIG. 2: photograph (200-fold) of suspended LMH cells,

FIG. 3: suspending LMH cell growth curve;

FIG. 4: comparing the immune antibody of suspension culture avian adenovirus group I4 and adherent culture virus liquid;

FIG. 5: a comparison graph of the immune antibodies of avian adenovirus 8b, D11 cultured in suspension and adherent culture virus liquid.

Detailed Description

The invention adopts a method for propagating avian adenovirus (I group 4, 8b, D11 and 8 a) antigens by LMH cells through full suspension culture in a bioreactor, compared with the traditional adherent cell culture method, the method can obtain the avian adenovirus (I group 4, 8b, D11 and 8 a) full suspension cell culture process virus antigens with high titer, and the antigen titer is about 5 times of the antigen titer of the current vaccine procedure; the virus content of the semi-finished product is not less than 10^8.0TCID₅₀The 0.1ml method can be used for preparing vaccine (the virus content of the existing regulation is not less than 10)^7.0TCID₅₀/0.1ml)。

The full-suspension LMH cells screened by the invention are the adherent LMH cells which are fed back from the center of Chinese animal health and epidemiology, and the cells are more sensitive to the avian adenovirus through molecular biological modification and optimization. Then the adherent cells are obtained by suspension domestication and screening; SF501 full suspension dry powder medium (exclusively used for LMH suspension cells) purchased from Shanghai Beiji Biotechnology Co., Ltd under the product number FG 0105701; DMEM (high-sugar) dry powder medium is produced by Hyclone company, and the product number is: SH 30045.05; fetal bovine serum is produced by Bailing corporation, Lanzhou, with a product number: SA 212.02; other common reagents are domestic analytical pure reagents.

The present invention will be further described with reference to the following examples.

Example 1: acclimatization of LMH cells

Adopting a bioreactor to culture LMH cells in a full suspension manner to proliferate the avian adenovirus (I group 4, 8b, D11 and 8 a) antigens: namely, recovering the full-suspension LMH cells and culturing the LMH cells in a 500ml shake flask; cell culture in a small reactor (5L-25L), cell amplification culture in a bioreactor (100-1500L), virus inoculation, harvesting and inactivation of virus liquid, preparing a semi-finished product into a multi-connected inactivated vaccine post-immune 35-day-old SPF chicken, and respectively measuring immune antibodies for 21 days and 28 days.

1. Suspension acclimation of LMH cells

Recovering adherent LMH cells P3 with DMEM culture solution containing 10% fetal calf serum, and subculturing to P4 after growth is dense. The cells with good growth of P4 generation were selected and adapted to culture gradually with DMEM medium containing different serum concentrations. The domestication of suspension culture cells was performed by using a method of gradually reducing the serum content, as shown in FIG. 1.

According to the method shown in figure 1, 54 generations of adherent LMH cells fed back by the center of Chinese animal health and epidemiology are suspended and domesticated successfully to form a novel cell strain which can be subjected to high-density suspension culture under the serum-free condition. The P54 generation was identified as the first generation of primary cells of the suspended LMH cells, i.e. F1.

The cells screened are round and uniform in size, the suspension is uniformly distributed in the culture solution, the culture solution between cells is clear, the cells are transparent and have slight agglomeration (figure 2). The cell has low requirement on nutrients, low sugar consumption and low acid production, no extra supplementary material is needed in the whole cell culture and virus culture process, no external acid and alkali is needed to adjust the pH, and the culture process is stable and easy to control. The culture process ensures that the inoculation density is 1.5 multiplied by 10^6.0～2.0×10^6.0Adjusting the appropriate rotation speed of the reactor (the rotation speed can be only achieved by slight rotation of the liquid level, and the rotation speed cannot be too large), setting the temperature at 37 ℃, the Dissolved Oxygen (DO) value at 40 and the pH value at 7.0 +/-0.2, and multiplying by about 50-75% every 24 hours.

Cell growth curves were prepared according to the above-mentioned culture conditions, and the cell growth curves were plotted with the horizontal axis representing the culture time and the vertical axis representing the cell density and cell viability (table 1 and fig. 3).

Table 1: suspension LMH cell growth table

The suspension LMH cells after cloning and purification are systematically identified. The result shows that the cell is pure and has no pollution of bacteria, mould, mycoplasma and exogenous virus; the karyotype of each generation of cells is basically the same; suspension cells with high sensitivity to avian adenoviruses (group I4, 8b, D11, 8 a).

2. Preparation of whole suspension LMH cells:

the extraction density was 5.0X 10⁷And (3) freezing 4.0ml of LMH, placing in a water bath at 37 ℃, shaking for dissolving for 2-3 min, adding 100ml of suspension cell culture solution, transferring into a 500ml triangular shake flask, fully and uniformly mixing, sampling, counting cells, counting cell viability, and recording data. Culturing in a shaking table at the temperature of 37 ℃ and the rotating speed of 120 r/min, sampling after every 24h, counting cells and counting the cell viability, proliferating the cells at the speed of 60-90%, culturing in bottles when the liquid is supplemented to 150ml, proliferating the cells, inoculating when the minimum required number of inoculated 5L-25L reactors is reached, ensuring the initial cell density of the inoculation to be more than 150 ten thousand/ml, and being too low to be beneficial to the rapid proliferation of the cells.

3. Small reactor (5L-25L) for cell culture

(1) Sterilizing a small reactor (5L-25L) bioreactor, and connecting a corrected pH electrode, a DO probe, a liquid inlet pipe, a liquid discharge pipe and a sampling pipe with the reactor; adding PBS (the concentration is 0.01mmol/L, the pH value is 7.2) which does not exceed the volume of the pH electrode, ensuring that the probe ends of the pH electrode and the DO are below the liquid level, ensuring that the pipe orifice ends are sealed, only leaving the exhaust end of the tank top to be connected with an air filter to be communicated with the outside, placing the tank body in an autoclave after checking that the whole tank body is sealed and airtight, and autoclaving for 30-40 min at 121 ℃.

(2) Connecting the bioreactor with a control system one day before cell inoculation by culture medium pretreatment, discharging PBS (concentration is 0.01mmol/L, pH value is 7.2) in a reactor tank body, adding sterile suspension cell nutrient solution, adjusting the appropriate rotating speed of the reactor (the liquid level slightly rotates, the rotating speed can damage cells) to set the temperature at 37 ℃, the Dissolved Oxygen (DO) value at 40 and the pH value at 7.0 +/-0.2, and pre-incubating for 1 day.

(3) Inoculating and culturing cells, namely inoculating 1.5L-2L of LMH (dendritic cell) full suspension cells prepared from the cells 1 into a small bioreactor, and ensuring that the inoculation density is 1.5 multiplied by 10^6.0～2.0×10^6.0Each cell/ml, adjusting the proper rotating speed (liquid) of the reactorThe surface is slightly rotated), culturing at 37 deg.C, Dissolved Oxygen (DO) value of 40, and pH value of 7.0 + -0.2, sampling every 24 hr, observing cell growth, and counting cells. When the cell density reaches 6.0X 10⁶When the concentration is higher than the above range, the cells can be cultured in an enlarged manner.

4. Bioreactor amplification (100-1500L) cell culture

(1) And (3) sterilizing the bioreactor (100-1500L), connecting the corrected pH electrode and DO probe with the bioreactor, ensuring the sealing of each port T-shaped valve, only leaving an exhaust end to be connected with an air filter to be communicated with the outside, and performing online high-pressure steam sterilization at 121 ℃ for 30-40 min.

(2) The day before the culture medium is pretreated and inoculated with cells, a sterile filter is used for filtering and sterilizing, sterile suspension cell nutrient solution is added, a pH electrode and a stirring paddle are submerged in the liquid level, and the pre-incubation is carried out for 1 day under the conditions that the temperature is set to be 37 ℃, the Dissolved Oxygen (DO) value is 40 and the pH value is 7.0 +/-0.2.

(3) Cell inoculation and culture cells in a small reactor (5L-25L) were inoculated in a large bioreactor to ensure an inoculation density of 1.5X 10^6.0～2.0×10^6.0Adjusting the appropriate rotation speed of the reactor (the liquid level can be slightly rotated) for each cell/ml, culturing at 37 deg.C, Dissolved Oxygen (DO) value of 40, and pH value of 7.0 + -0.2, sampling every 24h, observing cell growth condition, and counting cells. When the cell density reaches 6.0X 10⁶When the concentration is more than ml, the cell amplification culture or the inoculation culture can be performed.

5. Preparation of virus liquid

(1) Virus inoculation is carried out by transferring cell suspension into a reactor which is sterilized in advance and pre-incubated with cell culture solution, and uniformly stirring is carried out to ensure that the cell density reaches 2.5 multiplied by 10^6.0Inoculating the virus for I group 4 type/8 b/D11 avian adenovirus production at MOI of 0.2 per ml, adjusting the rotation speed of the reactor (the liquid surface is slightly rotated), and culturing the virus at 37 deg.C, Dissolved Oxygen (DO) of 40, and pH of 7.2 + -0.2.

(2) After 24 hours of virus inoculation, the samples were taken 1 time a day to observe cytopathic conditions, and at the same time, cell counts were performed to observe and record cytopathic (cell enlargement, cell disruption) conditions.

(3) When the vitality of the harvested cells is reduced to 70-80%, the virus liquid is harvested and sampled for intermediate product inspection. Storing at below-15 deg.C for no more than 30 days.

The DMEM culture solution is prepared by taking 13.54g of DMEM (high-sugar) dry powder culture medium (special for LMH adherent cells) and preparing 1000ml of water for injection.

The cell nutrient solution is prepared by taking 23.3g SF501 full suspension dry powder culture medium (special for LMH suspension cells) and preparing 1000ml with water for injection.

The PBS solution with pH of 7.2 is prepared by collecting 8.0g of sodium chloride, 0.2g of potassium chloride, 1.25g of disodium hydrogen phosphate, and 0.2g of potassium dihydrogen phosphate, and making into 1000ml with water for injection.

The method of the invention verifies 1:

the virus contents of virus solutions after inoculating avian adenovirus (group I4, 8b, D11 and 8 a) into whole suspension LMH cells and adherent cells were compared, and the results are shown in tables 2, 3, 4 and 5.

Table 2: comparison of virus content of suspension LMH cells inoculated with avian adenovirus group I type 4 seed virus and adherent cells

From the above table data, it is found that: the virus content of virus liquid obtained by inoculating the avian adenovirus I group 4 type seed virus into the suspension LMH cells is 0.4-1.0 titer higher than that of virus liquid of adherent cells, namely the virus content of suspension culture is 5-10 times that of the adherent culture. In addition, the whole process of suspension culture is serum-free culture, and serum dependence is eliminated, so that the influence of exogenous viruses caused by serum can be avoided, and side effects caused by serum are reduced. The product quality can be greatly improved.

Table 3: comparison table of virus content of avian adenovirus 8b type seed virus inoculated suspension LMH cells and adherent cells

From the above table data, it is found that: the virus content of virus liquid obtained by inoculating the avian adenovirus 8b type seed virus into the suspension LMH cells is higher than that of virus liquid of adherent cells by about 1.0-1.5 titer, namely the virus content of suspension culture is more than 10 times of that of adherent culture. In addition, the whole process of suspension culture is serum-free culture, and serum dependence is eliminated, so that the influence of exogenous viruses caused by serum can be avoided, and side effects caused by serum are reduced. The product quality can be greatly improved.

Table 4: comparison table of virus content of suspension LMH cells inoculated with avian adenovirus D11 type seed virus and adherent cells

From the above table data, it is found that: the virus content of virus liquid obtained by inoculating the avian adenovirus D11 type seed virus into the suspension LMH cells is higher than that of virus liquid of adherent cells by about 1.0-1.4 titer, namely the virus content of suspension culture is about 10 times of that of adherent culture. In addition, the whole process of suspension culture is serum-free culture, and serum dependence is eliminated, so that the influence of exogenous viruses caused by serum can be avoided, and side effects caused by serum are reduced. The product quality can be greatly improved.

Table 5: comparison table of virus content of suspension LMH cells inoculated with avian adenovirus type 8a seed virus and adherent cells

From the above table data, it is found that: the virus content of virus liquid obtained by inoculating the avian adenovirus 8a type seed virus into the suspension LMH cells is higher than that of virus liquid of adherent cells by about 1.0-1.4 titer, namely the virus content of suspension culture is about 10 times of that of adherent culture. In addition, the whole process of suspension culture is serum-free culture, and serum dependence is eliminated, so that the influence of exogenous viruses caused by serum can be avoided, and side effects caused by serum are reduced. The product quality can be greatly improved.

Example 2: preparation of vaccines

1. After the avian adenovirus I group 4 type seed virus is respectively inoculated with fully suspended LMH cells and adherent cells, virus liquid is inactivated, and the inactivated semi-finished product is emulsified to prepare the triple inactivated vaccine of Newcastle disease, avian influenza (H9 subtype) and avian adenovirus (I group 4 type), which are respectively used for immunizing SPF chickens of 35 days old, and 0.2ml of intramuscular injection is injected into each chicken. Collecting blood 21 days after immunization, separating serum, measuring antibody by agar-agar gel method, immunizing for 21 days while performing challenge, and injecting avian adenovirus group I4 type YBAV-4 strain virus solution into muscles of all immunized chickens and control chickens 0.2ml (containing 2 × 10 each)^5.5TCID₅₀). And 7 days after the challenge, counting the challenge protection rate. The test results are shown in table 5 and fig. 4.

Table 5: suspension culture of avian adenovirus group I4 and adherent culture of virus liquid immune antibody and comparison of virus attack protection

Numbering	Antigens	21d antibodies	GMT	Protection against toxic substances
					1(C4 original times adherent culture)	C4	1,1,1,1,1,1,1	1	6/7 health promoting effect
2 (C42 times concentration adherent wall culture)	C4	1,4,2,2,2,2,2	2	7/7 health promoting effect
					3 (LMH-C4 original times suspension culture)	C4	16,2,4,8,4,8	5.66	7/7 health promoting effect
3 (LMH-C42 times concentration suspension culture)	C4	8,8,32,32,8,4,8	10.78	7/7 health promoting effect

The result of the immune challenge test shows that:

(1) the immune antibody of the avian adenovirus group I4 type original-fold virus liquid cultured in suspension culture is 5 times of the original-fold virus liquid cultured in adherence culture and 2 times of the concentrated virus liquid cultured in adherence culture

(2) The immune antibody of the avian adenovirus I group 4 type 2-fold virus liquid cultured in a suspension way is 5 times of that of the concentrated virus liquid cultured in an adherence way by 2 times;

(3) the poultry adenovirus I group 4 type original virus liquid and 2 times virus liquid which are cultured in a suspension way reach 7/7 protection, and only 2 times concentrated virus liquid is cultured in an adherence way to obtain 100 percent (7/7) protection. Therefore, the traditional adherent culture vaccine poultry adenovirus I group 4 virus liquid needs 2 times concentration, while the suspension culture virus liquid does not need concentration.

To sum up: the suspension culture of the avian adenovirus group I4 can obtain high antibody titer and exact protection efficiency, simplify the process and greatly reduce the production cost. The suspension culture process is a relatively high-efficiency production mode.

2. After the avian adenovirus 8b, 8a and D11 type seed viruses are respectively inoculated to fully suspended LMH cells and adherent cells, virus liquid is inactivated, and the inactivated semi-finished product is emulsified to prepare the triple inactivated vaccine of Newcastle disease, avian influenza (H9 subtype) and avian adenovirus (8b, 8a and D11 types), and the triple inactivated vaccine is used for respectively immunizing SPF chickens of 35 days old, and 0.2ml of intramuscular injection is injected into each chicken. Immunization was carried out for 21 days, blood was collected 28 days later, and serum was separated and antibodies were measured by the agarose gel electrophoresis method. Since the avian adenovirus 8b and D11 type challenge does not cause disease, the evaluation can only be carried out by measuring antibodies by using the agar amplification method. The test results are shown in table 6 and fig. 5.

Table 6: comparison table of immune antibodies of suspension culture avian adenovirus 8b and D11 types and adherent culture virus liquid

The result of the immune challenge test shows that:

(1) the 21-day and 28-day immune antibodies of the avian adenovirus 8b virus liquid cultured in suspension are about 2 times of those of the virus liquid cultured in adherence.

(2) The 21-day and 28-day immune antibodies of the poultry adenovirus D11 virus liquid cultured in suspension are more than 3 times of those of the virus liquid cultured in adherence.

(3) The 21-day and 28-day immune antibodies of the avian adenovirus 8a virus solution cultured in suspension are about 2 times of those of the virus solution cultured in adherence.

(4) The adherent culture vaccine avian adenovirus 8b and D11 virus liquid needs 3 times concentration in actual large-scale production, and the adherent culture virus liquid can achieve the effect of 3 times concentration without concentration.

To sum up: high antibody titer can be obtained by suspension culture of avian adenovirus 8b, 8a and D11, and the production process is greatly simplified and the production cost is greatly reduced. The suspension culture process is a relatively high-efficiency production mode.

In summary of all the experimental results, the advantages of the suspension culture method of avian adenovirus (I group 4, 8b, D11, 8 a) are as follows:

(1) the avian adenovirus (I group 4 type, 8b type, D11 type and 8a type) is cultured on LMH full suspension cells, and the titer of the semi-finished product can reach a higher level (the titer of the semi-finished product)>8.0TCID₅₀0.1 ml). From the titer of the semi-finished product, the titer of the I group 4 is improved by nearly 0.4-1.0 compared with the original titer, and the titer of the 8b type, the 8a type and the D11 type is improved by more than 1.0 compared with the original titer.

(2) From the aspect of cell technology, the operation flow is simplified, the operation is easy, the advantages of personnel and time are far more than those of adherent cells, and the operator can save half.

(3) The floor space of a production workshop is saved, the limitation of the workshop area is avoided, and the large scale is easy to realize.

(4) The whole process of suspension culture is serum-free culture, and serum dependence is eliminated, so that the influence of exogenous viruses caused by serum can be avoided, and side effects caused by serum can be reduced. The product quality can be greatly improved.

(5) From the production cost, under the condition of producing 100L of semi-finished products, the cost of culture medium and serum required by the original adherent cell process is about 10800 yuan, while the cost of the suspension cell production process is only 8000 yuan. The raw material cost is saved by 30 percent. The adherent culture antigen needs 3 times concentration during seedling preparation, and the suspension culture process is not concentrated, so the cost is saved by more than 50 percent integrally.