Sterile pharmaceutical products can be classified into terminally sterilized products, which undergo terminal sterilization during manufacturing, and aseptically manufactured products, in which part or all of the production process is performed under aseptic conditions.

Whenever possible, sterile products should be manufactured using terminal heat sterilization. However, for heat-sensitive products, aseptic processing or sterile filtration may be adopted as alternative sterilization methods.

The trivalent rotavirus vaccine is a live attenuated vaccine that cannot undergo terminal sterilization. To ensure product efficacy, the entire manufacturing process is carried out under aseptic conditions. This significantly increases operational complexity and requires highly skilled manufacturing personnel.

The preparation of the vaccine intermediate is one of the most critical stages in vaccine formulation. The sterility of this process directly affects both the safety of the final product and overall manufacturing costs. Therefore, incorporating a sterile filtration step during intermediate preparation is of great significance for ensuring product quality while reducing contamination risks.

Efficient downstream processing has become one of the major challenges facing the rapidly growing biopharmaceutical industry. The relatively large particle size of viruses and virus-like particles (approximately 100–400 nm) further increases the complexity of sterile filtration.

 

 

 

This study consisted of two phases of laboratory-scale filtration experiments on trivalent rotavirus vaccine intermediates to preliminarily evaluate the impact of introducing a sterile filtration step during intermediate preparation.

During the first phase, virus titers before and after filtration were evaluated using different membrane pore sizes and membrane materials.

 

Based on the favorable results obtained in Phase I, the second phase employed sterile filters with the same membrane material and pore size while expanding both the experimental scale and the number of production batches. Virus titers and physicochemical properties before and after filtration were evaluated. In addition, bacterial retention tests were performed on the selected membrane to determine whether the trivalent rotavirus vaccine would affect the bacterial retention performance of the filter.

The filtration membrane used in this study was a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The effects of sterile filtration on virus titer, appearance, pH, osmolality, and clarity were investigated, while bacterial retention performance was also evaluated.

The results demonstrated that 0.22 μm PVDF sterile filters had no significant effect on virus titer, appearance, pH, or osmolality of the trivalent rotavirus vaccine intermediate. However, they significantly improved product clarity, indicating that sterile filtration can effectively enhance product quality without compromising vaccine potency.

 

Table 1. Comparison of Virus Titers Before and After Filtration in Six Intermediate Batches (Phase I)

 

 

Table 2. Statistical Analysis of Virus Titers Before and After Filtration in Sixteen Intermediate Batches (Phase II)

 

 

 

Table 3. Summary of Parameters for the Bacterial Retention Test

 

 

Note:1.Total filtration volume = Minimum flow rate × Recirculation time + Final filtration volume.

 

Table 4. Results of the Bacterial Retention Test

 

 

Notes:

1.Membrane challenge level = Final bacterial suspension concentration × Final filtration volume / Effective filtration area (13.8 cm²).

2.TNTC indicates that the number of colonies was too numerous to count (TNTC).

 

Discussion

Filtration media, process conditions, and sterile filters are the three primary factors influencing sterilizing filtration performance. Selecting a membrane material that matches the characteristics of the trivalent rotavirus vaccine intermediate and the corresponding process conditions is the fundamental prerequisite for successful sterile filtration.

Hydrophilic polyvinylidene fluoride (PVDF) membranes are widely used in the biopharmaceutical industry for terminal sterilizing filtration because of their high permeability, high flow rate, low protein-binding properties, minimal extractables, and excellent chemical compatibility. The results of this study further demonstrate that the selected PVDF membrane is well suited to the characteristics of trivalent rotavirus vaccine intermediates. Sterile filtration using this membrane had no significant effect on virus titer or physicochemical properties, including appearance, pH, and osmolality, while significantly improving product clarity, thereby contributing to enhanced vaccine quality.

 

Virus titer is one of the most critical quality attributes for viral vaccines, serving as a direct indicator of product potency and overall quality. Consequently, it represents the most important parameter when evaluating the impact of sterile filtration.

 

The results obtained from a total of 22 intermediate filtration batches demonstrated that the preliminary filtration process employing a 0.22 μm PVDF membrane fully satisfied the product requirements for the titers of G2, G3, and G4 viral strains, as well as the total combined virus titer.

 

Previous studies have reported that sterile filtration can be an important cause of virus titer loss and that viral concentration is one of the factors affecting filtration performance. In the present study, intermediates with different virus titer ranges were subjected to sterile filtration. The results indicated that filtration produced no statistically significant reduction in virus titer within the following concentration ranges:

• Phase I: 4.50–7.80 lgCCID₅₀/mL
• Phase II: 4.70–8.10 lgCCID₅₀/mL

These findings suggest that sterile filtration can be implemented without compromising vaccine potency across the evaluated virus concentration ranges.

The trivalent rotavirus vaccine exhibited no bactericidal activity against Brevundimonas diminuta ATCC 19146. Based on this observation, a bacterial retention study was conducted.

The results demonstrated that the rotavirus vaccine did not adversely affect the performance of the 0.22 μm hydrophilic PVDF sterilizing-grade membrane. The membrane successfully achieved the sterilizing-grade filtration standard of retaining ≥1.0 × 10⁷ Brevundimonas diminuta ATCC 19146 cells per cm² of effective filtration area, confirming that the vaccine formulation does not compromise the bacterial retention capability of the sterilizing filter.

 

 

Reference

Tong, Y., Chen, T., Fu, S., et al. Preliminary Application of Sterile Filtration in the Production Process of Trivalent Rotavirus Vaccine. Chinese Journal of Pharmaceuticals, 2024, 55(1):120–124. DOI:10.16522/j.cnki.cjph.2024.01.016.