300kd Hollow Fiber Ultrafiltration: Analysis Of A High-Efficiency Concentration And Purification Process For Foot-and-Mouth Disease O/A Virus
Sep 03, 2026| Introduction
Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious animal disease caused by the foot-and-mouth disease virus (FMDV). This virus affects cloven-hoofed animals, with over 70 susceptible species. It spreads rapidly and has a high morbidity rate, typically affecting major livestock such as pigs, sheep, and cattle, where the incidence can reach 100%. The FMDV belongs to the genus Foot-and-Mouthvirus of the family MicroRNAviridae. The particles are spherical, approximately 27-30 nm in diameter, with a sedimentation coefficient of 146S for intact viral particles. Currently, various FMD vaccines are available, including attenuated live virus vaccines, live virus vector vaccines, inactivated vaccines, nucleic acid vaccines, synthetic peptide vaccines, and subunit vaccines. Inactivated FMD vaccines generally have fewer side effects in animals, and multivalent vaccines offer better immunization efficacy, making them the mainstay of FMD prevention in China.
Tangential Flow Ultrafiltration
Currently, tangential flow ultrafiltration is a commonly used purification and concentration method in the large-scale production of foot-and-mouth disease multivalent inactivated vaccines. This method not only achieves the desired concentration factor but also removes a large amount of foreign impurities. Tangential flow ultrafiltration is mainly used for purification, concentration, and filtration (desalting and buffer replacement). In addition, it can also be used for the clarification of fermentation broth or cell culture medium using tangential flow microfiltration, thus replacing centrifugation or primary clarification.
In conventional vertical filtration, large molecules tend to aggregate on the membrane surface, clogging the filter. Tangential flow filtration, however, allows the flow to proceed parallel to the membrane, avoiding this clogging problem. During tangential flow filtration, the solvent and substances smaller than the membrane pore size that permeate through the membrane are called the permeate, while substances larger than the pore size are retained as the concentrate. Combined with optimized washing and filtration processes, the separation and purification of molecules of varying sizes can be achieved. This technology offers advantages such as low cost, mild operating conditions, convenient cleaning and replacement, high membrane permeate flow rate, and simple operation, making it particularly suitable for the large-scale concentration and purification of bioactive substances.
Materials and Methods
Hollow Fiber Column: Molecular weight cutoff of 300kd, membrane area of 1600cm².
Materials: 5.6L each of the supernatant from FMDV type O and type A after centrifugation and clarification.
Process Parameters: During ultrafiltration concentration, the shear force was adjusted to approximately 3000⁻¹, and the TMP was controlled to be no higher than 10 psi. When the concentration factor approached 11 times, the sample was washed with 6CV Buffer solution. After washing, sterile air was introduced into the inlet tube, and all the concentrated sample in the tubing was injected into the sample concentration bottle. Then, sterile PBS was added to rinse the tubing until the final volume of the concentrated liquid was 500 mL, thereby improving the overall recovery rate.
Results and Discussion
In terms of initial virus purification, ultrafiltration concentration achieves a removal rate of over 90% for impurities such as proteins that do not contain viruses. In terms of volume, it can achieve high concentrations of 10-50 times, providing strong support for subsequent purification. A hollow fiber column with a molecular weight cutoff of 300kd is selected. This pore size, while retaining over 85% of the virus, can remove over 90% of impurity proteins. The membrane area is 1600 cm², and the throughput is 2-5 mL/cm², enabling ultrafiltration concentration to be completed within an effective time (within 2 hours). Detailed ultrafiltration concentration and purification data are shown in Table 1.
Table 1. Results of total detection of foot-and-mouth disease antigens (types O and A)
| Sample Name | Liquid Feed | Volume (mL) | 146S Content (μg/mL) | 146S Recovery Rate (%) | Protein Concentration (μg/mL) | Protein Removal Rate (%) |
|---|---|---|---|---|---|---|
| O-type | Original solution | 5600 | 2.53 | 87.94 | 5040 | 94.38 |
| O-type | Concentrate | 500 | 24.92 | 87.94 | 3170 | 94.38 |
| A-type | Original solution | 5600 | 3.37 | 94.80 | 5980 | 93.55 |
| A-type | Concentrate | 500 | 35.78 | 94.80 | 4318 | 93.55 |
Purification Method Considerations
Currently, there are various purification methods, but even with the same method for the same antigen, the purification effect is often unsatisfactory for many reasons, including antigen characterization and process parameters. For example, changes in upstream molecular design or fermentation culture systems can alter the viral molecular structure, leading to unsatisfactory purification results. Inappropriate clarification methods can increase raw material costs or the amount of heterologous protein, increasing the pressure on downstream purification. Improper ultrafiltration concentration, such as different ultrafiltration membrane pore sizes, concentration and elution factors, inlet flow rates, filtration rates, and TMP, can result in low antigen recovery rates. Extensive practice shows that poor purification can cause significant adverse reactions in animals after immunization, potentially leading to death.
Conclusion: This study shows that, in tangential flow processes, by selecting appropriate membrane pore size (300kd), washing ratio, inlet flow rate, and TMP, both high recovery rate and maximum impurity removal can be ensured.