Research On Concentration And Purification Process Of Feline Trivalent Inactivated Vaccine: Feline Rhinotracheitis Virus

Feline rhinotracheitis, calicivirus disease, and feline panleukopenia (feline distemper) are the three major infectious diseases threatening the health of pet cats in China. Feline rhinotracheitis, caused by herpesviruses, is highly contagious, with a morbidity rate as high as 100% and a mortality rate of 20%-50% in kittens. Calicivirus infection is characterized by oral ulcers and respiratory symptoms; although the mortality rate is lower, it easily leads to chronic inflammation and joint lesions. Feline panleukopenia is characterized by acute gastroenteritis and severe leukopenia, with a mortality rate exceeding 50% in kittens. Therefore, the development of a trivalent inactivated vaccine can better protect the health of the feline population.

Feline rhinotracheitis virus, also known as feline herpesvirus type 1 (FHV-1), belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae and has typical herpesvirus morphological characteristics. The FHV-1 genome is a linear double-stranded DNA, approximately 134 kb in length, with a virus particle diameter of approximately 150-160 nm.

This study, based on locally prevalent FHV-1, FCV, and FPV strains, utilized hollow fiber tangential flow filtration (TFF) technology to systematically evaluate the virus recovery and protein removal rates of hollow fiber columns with different pore sizes, determining the optimal pore size for concentrating and purifying the three viruses. The study compared virus recovery and protein removal effects under different shear rates, providing a basis for determining and optimizing throughput indicators in subsequent large-scale production.

 

Experimental methods

 

according to Figure 1 .

 

Figure 1 shows the assembly of the hollow fiber operating system.

First, the system is pretreated by disinfecting with 0.1M NaOH solution, running permeate for 30 minutes without back pressure, and then circulating alkali solution for 30 minutes with the permeate end closed to complete sterilization. Then, it is permeated and rinsed with sterile water for injection for 30 minutes, and the pH is checked to confirm that there is no alkali residue. Finally, Tris buffer is introduced and permeated and rinsed with at least 200 mL to complete column equilibration.

After pretreatment, the virus solution was concentrated 10-fold and washed 20-fold. After the clarified solution was loaded onto the instrument, 55 mL of the system dead volume was first discharged, and the system was circulated in a closed loop for 5 minutes. Then, the permeate valve was opened for concentration, with the transmembrane pressure not exceeding 3 psi throughout the process. After concentration, the solution was washed twice with 10-fold volumes of buffer, adding buffer to restore the original volume each time before permeate washing. After washing, the system was circulated in a closed loop for 5 minutes, and virus solution A was collected. Then, virus solution B was harvested by head washing with 150 mL of buffer. Finally, the viral titer and total protein content of the original solution, solution A, and solution B were determined.

After the experiment, the membrane column was maintained. First, it was rinsed with sterile water at 1000 mL/min for 30 min; then rinsed with 0.5 M sodium hydroxide at 500 mL/min for 30 min, and statically soaked for 2-6 h to restore membrane performance; after thoroughly removing alkali with clean water, 0.1 M sodium hydroxide was introduced to fill the system, and the membrane column was sealed and preserved.

 

Experimental results

Comparison of TFF ultrafiltration concentration and purification of nasotracheitis virus fluid using hollow fiber columns with pore sizes of 500 kd and 750 kd

1800 mL of nasal tracheitis virus solution , centrifuge and clarify, then divide it into two 800 mL bottles. Use hollow fiber columns with 500 kd and 750 kd pore sizes for 10-fold ultrafiltration concentration and washing, respectively. Set the tangential flow rate to a shear rate of 2000 ^s⁻¹ , and ensure that the transmembrane pressure does not exceed 3 psi throughout the process.

The results showed that after 10-fold concentration and purification of 800 mL of virus solution using a 500 kd hollow fiber column, the virus recovery rate was 65.46% and the total protein removal rate was 94.89%. After purification of 800 mL of virus solution using a 750 kd hollow fiber column, the virus recovery rate was 84.69% and the total protein removal rate was 96.78% (Table 1).

In comparison, both hollow fiber columns with different pore sizes showed good removal efficiency for total protein, but the 750 kd pore size had a higher virus recovery rate than the 500 kd pore size.

Table 1. Results of cat rhinotracheitis virus concentration and purification using hollow fiber at 500 kd and 750 kd.

 

 

Comparison of the effects of different shear rates on the TFF ultrafiltration concentration and purification of nasotracheitis virus fluid

2700 mL of nasal tracheitis virus fluid , centrifuge and clarify it, then divide it into 3 bottles of 800 mL each. Use a hollow fiber column with a pore size of 750 kd to perform ultrafiltration concentration and washing at a shear rate of 2000 S -1, 4000 S-1 and 6000 S-1 respectively. No pressure is ^required throughout ^the process ^.

The results showed that when the virus solution was concentrated and purified at a shear rate of 2000 ^s⁻¹ , the virus recovery rate was 84.69% and the total protein removal rate was 96.78 %. When the virus solution was concentrated and purified at a shear rate of 4000 ^s⁻¹ , the virus recovery rate was 0.56% and the total protein removal rate was 96.31%. When the virus solution was concentrated and purified at a shear rate of 6000 ^s⁻¹ , the virus recovery rate was 0% and the total protein removal rate was 95.74% (Table 2 ).

The results showed that shear force had a significant impact on the recovery rate of feline rhinotracheitis virus. When using a 750 kd hollow fiber column for concentration and purification, it is not advisable to set the shear rate too high; a setting of 2000 ^{s⁻¹ is recommended} .

Table 2. Results of concentrating and purifying feline rhinotracheitis virus solution at different shear rates using a 750 kDa hollow fiber column.

 

 

References: Li, Xin. Process optimization of feline viral trivalent inactivated vaccine [D]. Jiangxi Agricultural University, 2025. DOI:10.27177/d.cnki.gjxnu.2025.000595.