Downstream purification process of porcine diarrhea vaccine

Diarrhea is one of the three major diseases that have caused great economic losses to the pig industry in China. There are many factors causing diarrhea in pigs, and the viral factors are the most serious. Pigs of all ages can be infected, especially Suckling piglets, and the mortality rate after infection can reach more than 50%. Porcine epidemic diarrhea virus (PEDV) and porcine Transmissible gastroenteritis virus (TGEV) are widespread in the world, and the two viruses can be infected singly or in combination. Currently, there are no specific drugs to treat PEDV and TGEV, and the prevention and control of porcine diarrhea are mainly commercial vaccines.

Traditional vaccines of porcine epidemic diarrhea mainly include whole virus inactivated vaccine and live attenuated vaccine. Clinically, PEDV and TGEV tandem inactivated vaccine or attenuated vaccine, and PEDV, TGEV and porcine rotavirus (RV) triple inactivated vaccine or attenuated vaccine are commonly used, mostly PEDV and TGEV tandem vaccine.

At present, there are many vaccines against porcine epidemic diarrhea in China.

The production process of inactivated and attenuated vaccines is similar, mainly including five steps of pathogen culture, pathogen inactivation or attenuated, pathogen extraction and purification, vaccine preparation and packaging, and vaccine quality control. This article mainly introduces the procedure of pathogen purification.

Clarification

The purpose of clarification is to ensure the removal of cell debris and other large particle aggregates. In the cell culture production of a variety of venom, in addition to containing a large number of virus particles, there are many cell tissue debris and metabolic products and other impurities, making the venom in the state of cloudy or semi-cloudy, the direct use of this venom concentration operation will soon make the ultrafiltration membrane blocked, and can not complete the concentration of the venom, and resulting in the ultrafiltration membrane scrap. Therefore, before the venom is concentrated, it must first be clarified, which is usually done by low-speed centrifugation and membrane filtration. It should be noted that when using membrane separation technology, due to the membrane material is often with electronegativity or hydrophobic groups, it is necessary to investigate whether the material of the membrane has adsorption to the virus particles.

An efficient clarification step requires a combination of high solid particle removal capacity and high product yield, easy amplification, and protection of later downstream operating units. At present, deep filter is used for clarification operation.

However, when the cultivation scale is too large, a large number of deep filter membranes will be required, resulting in a substantial increase in the cost of consumables. At the same time, the high density of cell culture will reduce the load of the deep filter membrane, resulting in increased costs and excessive product dilution.

The microfiltration method mainly uses tangential flow filtration device, and the membrane components are flat membrane cassstte and hollow fiber. Tangential flow filtration (TFF) is driven by the transmembrane pressure difference. Substances and impurities smaller than the membrane pore size pass through the membrane, while impurities such as cells with larger particles are trapped. The pore size of the membrane used for microfiltration is 0.45/0.22μm. Hollow fiber tangential flow microfiltration can directly process high solid content liquid, such as high-density cell culture medium, can eliminate centrifugation and pre-filtration steps with fewer steps and simple operation, membrane can be used repeatedly through cleaning, reducing equipment investment and operating costs, in line with the requirements of modular automated production.

Concentration and purification

After preliminary clarification, the virus culture medium still contains a large number of impurities, including defective particles, viral shell, cytotoxin, medium, serum and cell fragments, etc. If these impurities are injected into animals, they will not only affect the efficacy of the vaccine, but also cause a strong immune response of the host, and even lead to the death of the vaccinated animals. Therefore, concentration and purification are needed to remove impurities such as heteroproteins and cell fragments, enhance and induce effective immune responses, reduce the amount of vaccine used, and reduce adverse reactions.

The principle of virus purification: to maintain the biological activity and infectivity of the virus as the premise, to remove the virus impurities and extract the high purity of the virus.

At present, the main concentration and purification methods used in large-scale production are ultrafiltration.

Ultrafiltration method, mainly use tangential flow ultrafiltration device, hollow fiber filtration system and other filtration equipment, under normal circumstances, the application of ultrafiltration method can make the virus concentrate more than 100 times, the removal rate of heteroprotein up to 99%. Among them, hollow fiber membrane filtration technology has the advantages of mild and low shear force, easy plugging, flexible operation, long life and low cost, and easy amplification, so it is recommended to choose hollow fiber for virus concentration and purification.

When ultrafiltration method is used for concentration and purification, it is very important to select the appropriate membrane pore size, which directly determines the efficiency and quality of the concentration. On the one hand, it is necessary to select the membrane aperture to effectively trap the target molecules to ensure the yield, and on the other hand, the removal effect and processing speed of heteroproteins should be fully considered. Therefore, the best principle is to select the membrane with the largest pore size that can trap the target molecule, and try to select the filter membrane with uniform pore size distribution.

Porcine Epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are both coronaviruses, which are polymorphic spheres, with upside-down spik-like spikes, ranging in diameter from 60 to 190nm. Rotavirus disease is a zoonotic disease in which rotavirus (RV) has a wheel-like appearance and is about 70nm in diameter. These three viruses can generally be intercepted using 100-750kd aperture ultrafiltration membranes.