Optimization Study On Downstream Purification Process Of Chicken Embryo Harvest Broth For Type IV Influenza Virus ( Part 2 )

The Effects of Different Ultrafiltration Concentration Processes on the Quality of Clarified Fluid from Type IV Influenza Virus

The influenza virus concentrate has a large volume, low hemagglutinin content, and high levels of impurities and proteins, necessitating ultrafiltration concentration before subsequent processing . Ultrafiltration is a preferred method for mild, large-scale production concentration. Studies have shown that a 750kd hollow fiber system can significantly reduce the ovalbumin content of the concentrate, while another study indicates that a 1000kd membrane encapsulation system can significantly reduce the impurities and proteins in avian influenza concentrate. After reviewing extensive literature and comparing two commonly used ultrafiltration concentration devices, the 750kd hollow fiber system and the 1000kd membrane encapsulation system , with all conditions identical except for the operating system, the optimal ultrafiltration concentration process was determined based on the test results.

The test results are shown in Figure 4-9. UA1-1 represents the average of three batches of ultrafiltration concentrate filtered using a 750kd hollow fiber system, UA1-2 represents the average of three batches of ultrafiltration concentrate filtered using a 1000kd membrane system, and so on.

Figure 4. Detection results of influenza virus concentrate - total protein content

Figure 5. Detection results of influenza virus concentrate - ovalbumin content

Figure 6. Detection results of influenza virus concentrate - total protein removal rate

Figure 7. Detection results of influenza virus concentrate - hemagglutination titer

Figure 8. Detection results of influenza virus concentrate - concentration titer yield

Figure 9. Detection results of influenza virus concentrate - hemagglutinin content

 

The results showed that the bacterial endotoxin levels in all groups of the influenza virus concentrate were below 60 EU/mL. There were no statistically significant differences in ovalbumin content, hemagglutination titer, hemagglutinin content, and concentration titer yield, which may be related to the structure of the influenza virus. However, treatment with the 1000 kDa membrane system showed a reduction in the total protein content of influenza V and BY strains, as well as a decrease in the total protein removal rate of influenza H3N2 and BY strains. Furthermore, the 1000 kDa membrane system took less time to concentrate the same volume of clarified solution, demonstrating an advantage in terms of time cost. Therefore, the 1000 kDa membrane system was chosen for the ultrafiltration concentration of influenza virus concentrate of influenza V strains.

 

Effects of different concentration ratios on the quality of clarified influenza virus solution of type IV

Concentration factor is a crucial step in purification, closely related to the yield of the target protein, concentration time, and the safety of the concentration system. A 1000 kd membrane encapsulation system was chosen for ultrafiltration concentration studies . 60 L of allantoic fluid from each of four influenza virus types (UA1, UA3, UB1, and UB2) was collected and subjected to continuous flow centrifugation and multi-stage clarification to obtain 54 L of clarified solution from each. Each clarified solution was divided into nine groups of 6 L each, and concentration factors of 30, 40, and 50 were investigated, with each factor repeated three times to verify repeatability and stability. The corresponding final volumes were 0.2 L, 0.15 L, and 0.12 L, respectively.

, samples were taken to test the total protein content, total protein removal rate, hemagglutination titer, concentration titer yield, and hemagglutinin content. The test results are shown in Figure 10-14 .

 

Figure 1.0 Results of the concentration of influenza virus serotype 4 clarified solution at different concentration ratios - total protein content

Figure 1.1 Results of the concentration of the clarified solution of influenza virus type IV after different concentration ratios - total protein removal rate

Figure 12. Detection results of influenza virus serotype IV clarified fluid after concentration at different concentration ratios - hemagglutination titer

Figure 1.3 Results of concentration of the clarified solution of influenza virus of different types after different concentration ratios - concentration titer and yield

Figure 14. Detection results of hemagglutinin content in the clarified solution of influenza virus type IV after concentration at different concentration ratios.

 

The results showed that for different concentration ratios, lower concentration ratios indicated lower total protein content and total protein removal rate, as well as lower hemagglutination titers, concentration titer yields, and hemagglutinin content. Higher concentration ratios could avoid these issues, but they placed higher demands on the system, increased system operating pressure, and potentially increased losses. Furthermore, the increased pressure led to longer production times, thus increasing time costs. Considering all factors, a concentration ratio of 30-40 times was optimal .

 

The effect of different purification processes on the quality of concentrated influenza virus of type IV

The results showed that Capto Core 700 purification yielded higher total protein and ovalbumin removal rates, and lower total protein and ovalbumin contents, but lower hemagglutinin content and yield. Sucrose density gradient centrifugation, on the other hand, achieved higher hemagglutinin content and yield . Furthermore, the hemagglutinin yield of Capto Core 700 was consistently above 50%, slightly lower than that of sucrose density gradient centrifugation. However, considering that sucrose density gradient centrifugation requires desaccharification after purification, is more time-consuming, and results in greater losses, Capto Core 700 is the preferred method. The Core 700 purification method is superior as a purification method for concentrated influenza virus of type IV .

References: Huang, Zhentian. Optimization of downstream production process for quadrivalent influenza virus split vaccine [D]. Shenyang Agricultural University, 2024. DOI:10.27327/d.cnki.gshnu.2024.001595.