Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical Products
Open accessAbstract
BACKGROUND: The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving. RESULTS: In this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3-105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065-0.452% and 0.471-1.312%, respectively. CONCLUSIONS: These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.
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qPCR has generally been considered one of the most practical methods for routine HCD content determination because of its sequence specificity, sensitivity, and rapid and high-throughput nature.
“In recent years, qPCR has generally been considered one of the most practical methods for routine HCD content determination because of its sequence specificity, sensitivity, and rapid and high-throughput nature [35,36].”
From Evaluation of the Efficacy of the Vaccine Production Process in Removing Residual Host Cell DNA from the Vero Cell Rabies Vaccine (2024) · cites this paper as [36]
The Background (page unavailable) calls qPCR the most practical method for residual DNA quantitation, citing sensitivity and time-saving features. The Results > Specificity section (page unavailable) shows that the assay amplified CHO DNA but not the other genomes tested. The paper does not identify high throughput as a reason for qPCR’s practicality. (LLM, conf=0.85)
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Cited by
Sorted by citations
Li, J., Pan, R., Yue, F., Gao, T., Wu, X., Shi, L., Wang, Y., Zhao, D., Lan, Z., Chen, H., et al. (2024) · Vaccines · doi:10.3390/vaccines12121379 · Cited by 8 · see citing works
- Authors
- Zheng, W., Jiang, L., Lei, Q., Yang, J., Gao, X., Wang, W., Zhang, Y., Kong, T., Chen, Q., & Li, G.
- Affiliations
- Lanzhou Institute of Biological Products (China) (CN)
- Venue
- Biological Procedures Online
- Published
- 2019-09-01
- Volume
- 21
- Issue
- 1
- Pages
- 17-17
- ISSN
- 1480-9222 (ISSN-L)
- PMC
- PMC6717637
- OpenAlex
- W2970649025
- Cited by
- 25
- References
- 0
- Citations by year
- 2026: 4 · 2025: 4 · 2024: 3 · 2023: 4 · 2022: 3 · 2021: 5 · 2020: 1
- FWCI
- 0.74
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Biological Procedures Online (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Immunology and Microbiology › Immunology › Biosimilars and Bioanalytical Methods
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- quantitative real-time PCR, biopharmaceutical products, sample preparation, primer and probe design, assay validation, detection limit, limit of quantification, ICH guidelines
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