Study On The Application Of Deep Filtration in The Production Process Of Foot-and-mouth Disease Vaccine

Oct 29, 2024

In the 14th century, there are written records of similar foot-and-mouth disease in Europe. Foot-and-mouth disease is an acute, febrile, highly contagious disease caused by foot-and-mouth disease virus.

The clinical symptoms of adult animals include: increased body temperature, blister and ulceration in the mouth, blister and ulceration in the hoof, and even hoof shell shedding, causing difficulty in eating and sleeping, weight loss and even loss of economic value in economic animals;

The clinical symptoms of young animals include: elevated body temperature, myocarditis and other symptoms, once onset, young animals have a high mortality.

Over the past 500 years, foot-and-mouth disease has repeatedly broken out around the world, and a large number of pigs, cattle, sheep and other domestic animals and wild even-toed ungulates have died or become ill, severely impacting the livestock and breeding industry.

In order to study the application of deep filtration technology in the production process of foot-and-mouth disease vaccine, the filterability performance of surface filtration and deep filtration was tested and compared, as well as the protective effect on bactericidal filtration. The advantages of high charge deep filtration in cell debris removal were studied. The corresponding filtration schemes for cell culture medium with different turbidity were analyzed.

The results showed that the deep filtration load was 13.9 times that of the surface filtration, the turbidity of the filter liquid was only 23.5% of the surface filtration, and the subsequent 0.2um debacterialization load was 7.2 times that of the surface filtration. By using its special resin ligand, the turbidity of the filter liquid is 38.6% of that of the ordinary deep filter with the same pore size, which is beneficial to protect the downstream process. The results showed that deep filtration, especially strong positive charge deep filtration, could be effectively applied to the production of FMD vaccine, and suitable filtration processes should be established for different culture media.

Foot-and-mouth disease (FMD) is an acute, febrile and contagious disease of cloven-hoofed animals caused by Foot-and-mouth disease virus (FMDV). The animals most susceptible to this disease include cattle, pigs and sheep. Foot-and-mouth disease is endemic in Asia, Africa, the Middle East and South America, with a very high incidence and rapid transmission. The World Organization for Animal Health (OIE) lists it as the first category A zoonotic disease, requiring member states to include foot-and-mouth disease in the list of notifiable animal diseases. Vaccination is an effective means of specific prevention of FMD. FMD inactivated vaccine, which is made by inactivating intact virus with inactivator, plays an important role in the prevention and control of FMD.

The antigenicity of FMDV itself is poor, and the inactivation of the virus has a great impact on its antigen. Therefore, in the current production of FMD vaccine, high-purity FMD vaccine can ensure the antigen content and purity of the unit dose, which is particularly important and is also the current vaccination strategy. This puts forward high requirements for the downstream purification of viruses. In addition, before 2009, the upstream of inactivated foot-and-mouth disease vaccines in China mostly adopted the culture method of rotating bottle and sticking to the wall. With the increasing demand for vaccines, the disadvantages of this culture method requiring a large number of rotating bottles, low efficiency and low expression level gradually emerged.

Therefore, in recent years, the cell culture technology has slowly changed from the previous adherent culture to suspension culture, and the virus production has been significantly improved. However, with the increase of virus production, the change of culture mode has brought greater pressure to downstream purification, because the cell debris generated by the culture process, the cell release of host proteins, host DNA, etc., has correspondingly increased, which puts further requirements on the downstream purification ability.

Membrane filtration technology is widely used in the production process of biological products such as vaccines, among which DC filtration technology is usually used in the harvest process of disease venom to separate insoluble impurities such as cell debris. In the early FMDV clarification, the surface filtration technology represented by the folding membrane filter element was mainly adopted. With the change of culture conditions, the increase of cell debris impurities, surface filtration is difficult to achieve the clarification effect, and because the surface filtration load is low, the cost of filter materials is becoming higher and higher.

Under the above background, deep filtration has been applied more and more in the process of cytoliquid separation of FMDV. The deep filter material is usually composed of long and fine fibers from natural sources, and the channel formed by the filter medium after forming a film is often very tortuous, similar to the labyrinth aperture structure. Impurity particles are often trapped inside the filter rather than on the surface. Moreover, the wall of the filter channel often absorbs a part of extremely fine particle impurities by electrostatic action or intermolecular force. In general, the filtration mechanism of deep filtration is characterized by mechanical retention and adsorption.

Install the deep filter plate to the constant current filter device, wash the filter plate with buffer PBS above 54 L/m2; After the washing is finished, start the peristaltic pump to slowly pump the liquid, slowly adjust the pump speed to about the expected flow rate, and filter the liquid. Record the filtration volume, time, pressure difference and turbidity during the filtration process. The filtrate was collected for antigen detection.

Surface filtration is a technology that captures particles on the surface of the filter material to achieve filtration. Filtration mechanism is mainly based on the size of particles in the fluid sieve, large particles are trapped on the surface of the filter membrane. Deep filtration utilizes internal channels for particle interception and adsorption at depth. The comparison diagram of the two is shown in Figure 1.

The inactivated solution of foot-and-mouth disease vaccine from a certain manufacturer was pre-filtered by 0.45um polypropylene surface membrane and deep filter plate respectively, and then filtered by 0.2um bactericidal filter. Filterability data are shown in Table 1.

The turbidity of the foot-and-mouth disease vaccine inactivated solution is 77 NTU. If the bacteria is filtered directly, a large number of cell fragments will inevitably cause the 0.2um filter membrane to be blocked quickly, which will affect the transmission of 146S viparticles. Therefore, pre-filtration was designed before debacterialization filtration, and the two pre-filtration methods of deep filtration and surface filtration were compared. The double layer deep filter plate is selected for filtration, the load is 641 L/m2, and the deep filter reduces the material of 77 NTU to 9.65 NTU, which is clear and bright. The surface filtration load of 0.45um is only 46 L/m2, and the turbidity of the filter liquid is as high as 41 NTU, which is not suitable for pre-filtration. According to the comparison of the subsequent 0.2um debacterialized filtration load, the debacterialized filtration membrane load after deep filtration is 655 L/m2, which is 7.2 times of the corresponding surface filtration filtration load (only 91 L/m2).

146S test results show that the yield of deep filtration system is higher than that of surface filtration system. The deep filtration load was 13.9 times that of the surface filtration, the turbidity of the filter liquid was only 23.5% that of the surface filtration, and the subsequent 0.2um bacteria-removing filtration load was 7.2 times that of the surface filtration. Therefore, both from the cost point of view and from the yield point of view, the deep filtration system is significantly better than the surface membrane filtration system.

The positive charge deep filter plate and strong positive charge deep filter plate were used to compare the inactivated solution of foot-and-mouth disease vaccine from different sources. The filterability results are shown in Table 2, and the change of filtration velocity and pressure difference in the filtration process is shown in the color layout 2.

The two groups of deep filter plates used have the same aperture accuracy, and the performance is very consistent from the point of view of load and pressure difference change, and the pressure difference change curve in Figure 2 is basically identical.

However, from the turbidity of the filter liquid, the difference is very large. The filter liquid of the ordinary positively charged double layer filter plate is 25 NTU, while the filter liquid of the strongly positively charged deep layer filter plate with the same aperture is only 9.65 NTU, which is only 38.6% of the ordinary deep filter plate. This is mainly due to the special resin ligand in the strong positive charge deep filter plate filter material. The turbidity is reduced to less than 10NTU by the strongly positive double-layer filter plate, and the cell fragments are fully adsorbed or trapped, which is conducive to the subsequent ultrafiltration or bactericidal filtration process. In addition to the adsorption effect on insoluble cell debris,Yigzaw Y et al have also reported the removal effect of strongly positively charged deep filter plate on cell host protein, and Dorsey N et al have reported the removal effect of strongly positively charged deep filter plate on DNA.

Through the above comparative study of surface filtration and deep filtration, and the study of the clarification effect of the special strong positive charge deep filter plate, different filtration schemes should be matched to the inactivated supernatant of foot-and-mouth disease vaccine produced under different culture conditions. For centrifugal supernatant with 3 different turbidity, low turbidity, medium turbidity and high turbidity, the deep filtration scheme and corresponding filtration parameters are shown in Table 3.

For the centrifugal supernatant with low turbidity, the single layer deep filter plate can effectively clarify and remove a small amount of cell debris. For the centrifugal supernatant of medium turbidity, the double layer deep filter plate can effectively clarify, especially the strong positive charge deep filter plate can reduce the turbidity to less than 5 NTU; For centrifugal supernatant with higher turbidity, the combined filter plate can be effectively clarified. No matter which process, the processing capacity is 500 ~800 L/m2, the turbidity of the filter liquid is less than 20 NTU, and the target antigen is fully recovered, suitable for large-scale production.

Discussion

In the 1970s, the filter media was specified not to use asbestos materials, because of the presence of carcinogens in asbestos materials. The deep filter material does not contain asbestos and has a stronger positive charge. The filter material is made of wood fiber matrix and filter aid. In the production process, polymer resin is used to connect the basic components, and the positive charge functional groups in it effectively give the charge adsorption ability of the deep filter material. The excellent performance of adsorptive deep filtration on the capturing ability of fine particles is shown in Figure 3.

In this experiment, different resin components and different precision deep filter materials were selected to clarify the filtration test of FMDV culture solution, and the comparison test was carried out with surface filtration. The results showed that deep filtration was more suitable for the removal of cell debris than surface filtration, which was conducive to the subsequent microbial load control filtration unit (0.2um). In addition, by comparing the filtration effect of the strong positive charge deep filter plate and the ordinary positive charge deep filter plate, the strong positive charge filter plate shows a strong turbidity control ability, which can reduce the turbidity to less than 5 NTU, which is very conducive to improving the robustness of the entire filtration process.

The upstream culture process has a great influence on the harvesting steps of active ingredients. Through testing, this experiment finally established a scalable filtration scheme for cell culture media with different turbidity, which has certain guiding significance for large-scale production.

The depth filter to be launched by Guidling Technology is a disc type filter element, which is a kind of efficient filter element with deep filter cardboard as the filter material, fully meeting the regulatory requirements of biopharmaceutical, mainly designed and manufactured specifically for the removal of large solid contents such as cells and cell fragments, and can be applied to mammalian cell culture fluid; Clarification of lysate of bacteria and yeast cells; Removal of host cell protein (HCP); Remove virus and DN; Removal of endotoxins; The removal of defoamer.

The product has the advantages of small dead volume, linear amplification, disc capsule structure, compact structure design, and can be used in series with other equipment, small footprint, stable flow rate, long life and low cost.

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