Skip to main content

Advertisement

SpringerLink
Log in

Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 102 to 107 copies/μL (from 0.00064 to 6.4 pg/μL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10−5 pg/μL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Lok, A. S., & McMahon, B. J. (2009). Chronic hepatitis B: update 2009. Hepatology, 50(3), 661–662.

    Article  Google Scholar 

  2. Romano, L., et al. (2013). Hepatitis B virus infection among first-time blood donors in Italy: prevalence and correlates between serological patterns and occult infection. Blood Transfusion, 11(2), 281–288.

    Google Scholar 

  3. Wallace, J., et al. (2012). Challenges to the effective delivery of health care to people with chronic hepatitis B in Australia. Sexual Health, 9(2), 131–137.

    Google Scholar 

  4. Kao, J. H., & Chen, D. S. (2002). Global control of hepatitis B virus infection. Lancet Infectious Diseases, 2(7), 395–403.

    Article  Google Scholar 

  5. Liang, X., et al. (2009). Epidemiological serosurvey of hepatitis B in China—declining HBV prevalence due to hepatitis B vaccination. Vaccine, 27(47), 6550–6557.

    Article  Google Scholar 

  6. Bo, H., et al. (2005). Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody. Journal of Biochemistry and Molecular Biology, 38(6), 683–689.

    Article  Google Scholar 

  7. Salahuddin, S. Z., et al. (2007). The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell. Virology Journal, 4, 106.

    Article  Google Scholar 

  8. Beldarraín Iznaga, A., et al. (2007). DNA removal from a purification process of recombinant hepatitis B surface antigen. Electronic Journal of Biotechnology, 10(1), 37–47.

    Article  Google Scholar 

  9. Yang, H. and J. Zhang, A Bayesian approach to residual host cell DNA safety assessment. PDA J Pharm Sci Technol, 2016.

  10. Hardy, E., et al. (2000). Large-scale production of recombinant hepatitis B surface antigen from Pichia pastoris. Journal of Biotechnology, 77(2–3), 157–167.

    Article  CAS  Google Scholar 

  11. Ikeda, Y., Iwakiri, S., & Yoshimori, T. (2009). Development and characterization of a novel host cell DNA assay using ultra-sensitive fluorescent nucleic acid stain “PicoGreen”. Journal of Pharmaceutical and Biomedical Analysis, 49(4), 997–1002.

    Article  CAS  Google Scholar 

  12. Pripuzova, N., et al. (2012). Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens. PloS One, 7(8), e43246.

    Article  CAS  Google Scholar 

  13. Alizadeh, Z., Milani, S., & Sharifi, Z. (2014). Occult hepatitis B virus infection among Iranian blood donors: a preliminary study. Archives of Iranian Medicine, 17(2), 106–107.

    Google Scholar 

  14. Heiat, M., Ranjbar, R., & Alavian, S. M. (2014). Classical and modern approaches used for viral hepatitis diagnosis. Hepatitis Monthly, 14(4), e17632.

    Article  Google Scholar 

  15. Lee, D. H., et al. (2010). Quantitative detection of residual E. coli host cell DNA by real-time PCR. Journal of Microbiology and Biotechnology, 20(10), 1463–1470.

    Article  CAS  Google Scholar 

  16. Paryan, M., et al. Simultaneous detection and genotype determination of HSV 1 and 2 by real-time PCR using melting curve analysis and a unique pair of primers. Appl Immunohistochem Mol Morphol, 2015

  17. Riahi, F., et al. (2015). Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay. Applied Biochemistry and Biotechnology, 175(5), 2447–2455.

    Article  CAS  Google Scholar 

  18. Paryan, M., et al. (2012). Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples. Iran Journal Microbiology, 4(1), 8–14.

    CAS  Google Scholar 

  19. Andersen, C. B., et al. (2006). Equal performance of TaqMan, MGB, molecular beacon, and SYBR green-based detection assays in detection and quantification of roundup ready soybean. Journal of Agricultural and Food Chemistry, 54(26), 9658–9663.

    Article  CAS  Google Scholar 

  20. Lahijani, R., et al. (1998). Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay. Human Gene Therapy, 9(8), 1173–1180.

    Article  CAS  Google Scholar 

  21. Wolter, T., & Richter, A. (2005). Assays for controlling host-cell impurities in biopharmaceuticals. BioProcess International, 3(2), 40–46.

    CAS  Google Scholar 

  22. Ji, X., Lee, K., & DiPaolo, B. (2002). High-sensitivity hybridization assay for quantitation of residual E. coli DNA. Biotechniques, 32(5), 1162–1167.

    CAS  Google Scholar 

  23. Wang, K. Y., et al. (2006). 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA. Vaccine, 24(14), 2656–2661.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Ms. Arlene Haghverdian for kindly final revision of the manuscript. This work was funded by Pasteur Institute of Iran (Grant No: 718) and Iran National Science Foundation (INSF, Grant No: 93010904), Tehran, Iran. The authors appreciate Pasteur Institute of Iran, Tehran, Iran, for providing technical support.

Author information

Authors and Affiliations

  1. Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, P.O.Box: 3159915111, Tehran, Iran

    Mahdi Paryan & Hooman Kaghazian

  2. Department of Cellular and Molecular Biology, Tehran East Branch, Islamic Azad University, Tehran, Iran

    Mana Khodayar

  3. Department of Medical Biotechnology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

    Vahid Kia

  4. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Samira Mohammadi–Yeganeh

  5. Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Samira Mohammadi–Yeganeh

Authors
  1. Mahdi Paryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mana Khodayar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vahid Kia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Samira Mohammadi–Yeganeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hooman Kaghazian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mahdi Paryan.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Paryan, M., Khodayar, M., Kia, V. et al. Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine. Appl Biochem Biotechnol 179, 375–382 (2016). https://doi.org/10.1007/s12010-016-2000-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2000-4

Keywords

Search

Navigation