Wuhan Institute of Biological Products Co., Ltd. and Huzhou Shenke Biotechnology Co., Ltd. (HZSKBIO^®) have jointly conducted research and published the core paper Establishment, Validation and Application of an Analytical Method for Size Analysis of Host Cell Residual DNA Fragments in Inactivated Whole-Virus Pandemic Influenza Vaccine (H5N1) in the Chinese Journal of Biologicals (Vol. 38, No. 12, December 2025), providing technical support for the quality and safety control of influenza vaccines.

In vaccine production, mammalian cell substrates such as Vero and MDCK cells have become the mainstream choice. However, the associated risk of host cell residual DNA (HCD) has long been a global regulatory focus. Large residual DNA fragments (>200 bp) may carry oncogenes or latent viral sequences, posing potential risks such as tumorigenesis and infection. Authorities including the WHO and FDA have explicitly mandated strict control over the fragment size and total amount of HCD. Previously, the lack of a standardized analytical method for HCD fragment size in the field of H5N1 vaccines became a key bottleneck for process optimization and regulatory compliance submissions.

In this study, HCD was extracted from samples using the magnetic bead method. By designing specific primers and probes, a real-time fluorescent quantitative polymerase chain reaction (qPCR) analytical method was established for the quantitative detection of DNA fragments of 84 bp, 142 bp, 204 bp and 504 bp respectively.

Systematic validation showed that the four fragment standards exhibited a good linear relationship with Ct values in the range of 0.003–300 pg/µL (all R² > 0.99). The limit of quantitation (LOQ) was 3 fg/µL for 84 bp, 142 bp and 204 bp fragments, and 1 fg/µL for the 504 bp fragment. The recoveries of spiked samples at high, medium and low concentrations ranged from 50% to 150%, and the coefficient of variation (CV) for precision, robustness and specificity validation was ≤ 40%, demonstrating excellent detection performance. Application of this method to samples during vaccine production revealed that large HCD fragments ≥ 204 bp accounted for as high as 81.0% of the total HCD in the harvest fluid, with a total HCD content of 689.9 ng/mL. After enzymatic hydrolysis and purification processes, the proportion of fragments ≥ 204 bp in the bulk vaccine decreased to 8.9%, no fragments ≥ 504 bp were detected, and the total HCD content was reduced to 10.2 ng/mL.

These data fully demonstrate that the method can accurately reflect the removal effect of the production process on HCD. The successful application of this method not only improves the safety margin of H5N1 vaccines, but its technical concept can also be applied to the quality control of various cell substrate vaccines such as rabies, polio and rotavirus vaccines, providing a referable standardized pathway for HCD risk control in the entire vaccine industry. It offers reliable technical support for quality control during vaccine production.

References

[1] Qiu R, Zhang QM, Le Y, et al. Establishment, Validation and Application of an Analytical Method for Size Analysis of Host Cell Residual DNA Fragments in Inactivated Whole-Virus Pandemic Influenza Vaccine (H5N1)[J]. Chinese Journal of Biologicals, 2025, 38(12): 1451-1456, 1468.