Application of suspension cell culture technology in the field of veterinary vaccine

Cell suspension culture is the core technology for the large-scale cultivation of animal cells to produce biological products by using bioreactor. It is the mainstream mode of bioproduct production in the world. Its biggest advantage is to obtain the maximum yield through more accurate and effective process control. At the same time, it can steadily improve the quality of the products. However, the technology has not been widely used in China at present, and the production of biological products still mainly adopts the method of vibrating cell culture with low virus yield, high production cost and high labor intensity. With the development of modern biotechnology, the use of cell suspension culture technology for biological product production is an inevitable trend in the development of the biopharmaceutical industry.

1. Application history and current status of suspension culture technology in biopharmaceuticals

In 1962, Capstick et al. pioneered the suspension culture of BHK21 cells and used them for veterinary vaccine production. In 1967, Van Wezel developed microcarriers and achieved large-scale cultivation of adherent cells in bioreactors. By the 1980s, CHO cells achieved suspension culture, and the development of therapeutic antibody production technology greatly promoted the application of bioreactors in the biopharmaceutical industry. By the end of the 20th century, it had entered the scale of upgrading.

After 2000, with the development of technologies such as fluid culture, perfusion culture, and personalized cell culture media, the scale of bioreactors, which are the main equipment for large-scale cultivation, has also become larger and simpler. Now, there are more than 100 reactors with a volume of 10,000L and above, and the largest has reached 25000L. Almost all of them are mechanically agitated reactors that can be enlarged, mainly owned by companies such as Genetech, Amgen, Boehringer Ingelheim and Lonza. Of the six categories of biotech drugs with the highest global sales in 2007, five were produced by mammalian cells. Throughout the country, there are more than 110 human and veterinary vaccine manufacturers, and only four vaccines are produced using reactor suspension culture technology. In general, veterinary vaccine companies use the domestically developed 650 L and 1200 L reactors to produce foot-and-mouth disease vaccines.

2. Key technologies in suspension culture process <br>The suspension culture technology mainly includes the acquisition of high expression cell lines, the development of personalized culture media, the improvement of animal cell reactors and supporting facilities, and the optimization of production processes.

2.1 Domestication of cells and strains

In order to improve the productivity and safety of biological products, viruses and cells that are propagated in bioreactors need to be adapted to each other. Screening suitable host cells for different strains and their expression levels is of great significance for large-scale production of cells. .

In order to achieve the stability of domestication, it is usually transferred from high-density cell culture to low-density cell culture, and the serum concentration is gradually lowered by high-concentration serum culture. When cells are domesticated, care should be taken to maintain cell viability by more than 90%. The ability of cell proliferation and expression and secretion can be measured to avoid changes in the expression characteristics of cells after acclimation. After acclimation, the proliferative capacity, vigor and productivity of the cells should meet the needs of industrial large-scale production. Due to the different nutrient requirements of specific cells, the degree of difficulty in acclimation is also different. When domesticating, it is necessary to select appropriate cell culture medium, and specifically supplement certain nutrients to meet the specific needs of cells, so that domesticated cells can be made. It can maintain its suspension or serum-free growth characteristics.

2.2 Personalization of cell culture media

In large-scale culture techniques, maintaining high-density or even serum-free growth of cells, the nutrient content of cell culture medium is essential for cell proliferation and maintenance. In the suspension culture technique, the specificity of nutrient metabolism of different cells is different, the cell and virus culture processes are different, the shear resistance is required, the amplification function is satisfied, and the stability and expression amount of the target biological product are also required, which requires individualization. Support for the medium. The cell culture mediums sold in China are mostly MEM, DMEM, 199, RPMI1640 cell culture media invented in the 1950s. These catalogue products are difficult to meet the technical requirements for large-scale cultivation of animal cells in bioreactors, directly affecting the biopharmaceutical industry. Technology upgrades. From the long-term consideration of human and animal safety, the production of biological products is increasingly inclined to use serum-free and animal-free medium, because serum-free medium eliminates the exogenous contamination of serum and cytotoxicity, making the product easy Purification and recovery rate are high. More than 50% of biopharmaceutical production in the world has adopted serum-free cell culture medium.

2.3 Research and development of cell culture process

The development and application of suspension culture process technology is mainly the development of production process and process optimization, the optimal environmental control to maintain cell growth and virus reproduction, and the role in suspension culture technology is also crucial.

2.3.1 Suspension culture and microcarrier culture Suspension culture technique is divided into suspension cell culture and adherent cell microcarrier suspension culture according to cell adherence. The suspension cells can directly produce proliferation in the reactor, the cells grow freely, the culture environment is uniform, the sampling is simple, the culture operation is simple and controllable, the amplification is convenient, the pollution rate and the cost are low; and the adherent cells in the reactor need to be micro-suspended in the reactor. The nutrient environment of the carrier, cell and sphere contact parts is poor, and the cultivation, sampling observation and amplification process are complicated and the cost is high.

Although microcarrier culture is complex compared to suspension culture, it still has great advantages compared with rotary flask culture, which can improve production scale, product quality and labor efficiency, and is therefore the main means of suspension culture of adherent cells. Common microcarriers are solid Cytodex microcarriers, flaky DISK microcarriers, and porous Cytopore microcarriers. When DISK or Cytopore is used, cells are attached to the inside of the carrier for growth, and there are few surface cells. It is difficult to separate the carrier and the cells by cell digestion, and the amplification process of the reactor is difficult, and it is not suitable for the culture of non-release virus. Cytodex is more suitable for the reactor amplification process of cans, and the best form of the reported adherent cell production process is the mechanical agitated reactor solid microcarrier suspension culture system, which can achieve the most effective optimization process and the most suitable process. Design, the best process design is reflected in high-density continuous perfusion culture.

2.3.2 Suspension culture method The reactor suspension culture method needs to consider several factors such as the relationship between cells and viruses, product stability, cell culture medium or additive selection, and cell culture scale. According to the culture method, it is divided into batch culture, fed-batch culture and perfusion culture.

Batch culture can directly reflect the growth and metabolism of cells in the bioreactor , and the operation is simple, but the initial metabolic waste products are more, inhibit cell growth, and the cell growth density is not high; the flow-adding culture operation is simple, the yield is high, and the magnification is easy to enlarge. It is widely used, but it needs to design the fed medium. The culture volume of perfusion culture is small, the recovery volume is large, and the product stays in the tank for a short time. It can be stored in low temperature for timely storage, which is beneficial to maintain the activity of the product, but the operation is compared. It is complicated, the cell culture medium is inefficient, and the rotary filter is easily clogged. Different viruses adopt different culture methods. For example, for biological products with secreted type and rapid activity reduction, perfusion culture is preferred, and perfusion culture is more suitable for microcarrier culture.

The same biological product may change its production process due to its different nutrient environment. For example, BHK21 cells produce foot-and-mouth disease virus, low-serum culture uses batch culture, and serum-free maintenance solution needs to be replaced before poisoning; There is no need to change the fluid, and it may be better to work with the fed-batch culture.

3. Application of cell suspension culture in livestock and poultry vaccines Vaccine immunization is currently one of the important measures to control animal infectious diseases. How to improve the quality of vaccines and reduce the cost of vaccines has become the focus of major animal vaccine manufacturers. Industrial cell culture production of vaccines is an effective means to improve vaccine quality and reduce costs.

3.1 Porcine virus disease vaccine Rabies virus is propagated in a 2 L bioreactor. The maximum titer of the virus can reach 1.38×108 pfu/mL, and the quality of the produced vaccine meets the requirements of WHO. Serum-free microcarrier cell culture facilitates enteritis virus propagation and vaccine production. There are also many reports on the large-scale production of vaccines for pig pseudorabies, blue ear disease, swine foot and mouth disease, swine fever, and porcine parvovirus disease.

3.2 Avian virus disease vaccine The poultry vaccine is mainly produced by SPF chicken embryos. However, chicken embryo production requires a large amount of SPF chicken embryos, which has a long production cycle and is easy to be contaminated. Only one virus strain can be injected per SPF chicken embryo at a time. Therefore, the efficiency of SPF chicken embryo production vaccines is very low. China has been able to produce attenuated vaccines of chicken-transmitted H120 cells that meet the "Quality Standards for Veterinary Biological Products of the People's Republic of China" through cell culture. Yang Tao et al also reported the large-scale proliferation of avian influenza virus in MDCK cells. There are many reports on cell culture for the production of vaccines for avian Marek virus, infectious bursal disease, infectious laryngotracheitis, and egg drop syndrome.

4, Outlook <br> It should be recognized that the rapid implementation of suspension culture technology in vaccine production is only to build a platform for upgrading the process, rather than the ultimate goal, the real driving force for industry and enterprise development is new veterinary drugs, new vaccines, new Innovation in process and supporting technology.

For example, the development of serum-free medium and the development of serum-free culture virus production technology; the use of appropriate medium and transfection or domestication technology to achieve suspension culture construction of adherent cells; new host cell expression variety development, such as the same product adopts new The host cell expresses or develops a highly expressed host cell to adapt to the expression of several products. All in all, integrating the strength of the industry and rapidly realizing the upgrading of suspension culture technology in vaccine production is the focus of the development of China's biopharmaceutical industry in the next few years. This process should pay attention to mature technology, success rate, reduce and avoid waste of time, capital, manpower and other resources, reduce opportunity costs, and seize market opportunities.

The upgrade of suspension culture technology will lead to industry integration and restructuring, and the bio-pharmaceutical industry will have an obvious intensive trend. The powerful enterprises will continue to build on the innovation and new drugs and new technologies, and at the same time, with the financial strength to integrate manufacturing and The marketing system has become the leader in the true sense.

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