Evaluation of the Efficacy of the Vaccine Production Process in Removing Residual Host Cell DNA from the Vero Cell Rabies Vaccine

Abstract

Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product.

Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal.

Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product.

Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production.

Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process.

Keywords: DNA fragment size distribution; HCD removal; Vero cells; rabies virus.

Figure 1

Process steps and samples at different stages of rabies vaccine purification.

Figure 2

Electrophoretic profiles of the distribution of residual HCD fragments from Vero cells in each process intermediate sample of batch 202304, as determined by the CGE method. (A): DNA ladder 100, 200, 500, and 1000 bp; a: virus harvesting solution; b: virus ultrafiltration concentrate solution; c: virus ion-exchange chromatography solution; d: virus inactivation solution; e: virus gel filtration chromatography solution; (B): enlarged views of c, d, and e. The dashed lines divide fragments with different lengths.

Figure 3

Electrophoretic profiles of the size distribution of residual HCD fragments from Vero cells in each process intermediate sample of batch 202305 as determined by the CGE method. (A): DNA ladder 100, 200, 500, and 1000 bp; a: virus harvesting solution; b: virus ultrafiltration concentrate solution; c: virus ion-exchange chromatography solution; d: virus inactivation solution; e: virus gel filtration chromatography solution; (B): enlarged views of c, d, and e. The dashed lines divide fragments with different lengths.

Figure 4

Trend analysis of residual HCD content in multiple batches of virus gel filtration chromatography-purified solutions.

Figure 5

Trend analysis of glycoprotein antigen content in multiple batches of virus gel filtration chromatography-purified solutions.

Figure 6

The electrophoretic profiles of residual HCD fragment size distribution in multiple batches of virus gel filtration chromatography-purified solutions, determined using the CGE method. (A): DNA ladder 100, 200, 500, and 1000 bp, where a to g represent the detection results of different batches of virus gel chromatography-purified solutions; (B): enlarged views of a to g. The dashed lines divide fragments with different lengths.