Detection and quantification of residual cellular DNA in the production of recombinant HPV-16 L1 virus-like particles

Hwa Kyung Son, Hye Sung Jeong, Young Lim Kim, Soon Nam Kim, Jin Ho Shin, Hong Ki Min, Baik Lin Seong, Sue Nie Park

Research output: Contribution to journalArticle

Abstract

A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng∼1 ng per 100 μg of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.

Original languageEnglish
Pages (from-to)271-278
Number of pages8
JournalJournal of Bacteriology and Virology
Volume36
Issue number4
DOIs
Publication statusPublished - 2006 Jan 1

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Human papillomavirus 16
Virion
DNA
Centrifugation
Recombinant Proteins
Sf9 Cells
Papillomavirus Vaccines
Digoxigenin
Subunit Vaccines
Sonication
Recombinant DNA
Deoxyribonucleases
Mycoplasma
DNA Probes
Endotoxins
Uterine Cervical Neoplasms
Detergents
Insects
Sucrose
Limit of Detection

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Virology

Cite this

Son, Hwa Kyung ; Jeong, Hye Sung ; Kim, Young Lim ; Kim, Soon Nam ; Shin, Jin Ho ; Min, Hong Ki ; Seong, Baik Lin ; Park, Sue Nie. / Detection and quantification of residual cellular DNA in the production of recombinant HPV-16 L1 virus-like particles. In: Journal of Bacteriology and Virology. 2006 ; Vol. 36, No. 4. pp. 271-278.
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Detection and quantification of residual cellular DNA in the production of recombinant HPV-16 L1 virus-like particles. / Son, Hwa Kyung; Jeong, Hye Sung; Kim, Young Lim; Kim, Soon Nam; Shin, Jin Ho; Min, Hong Ki; Seong, Baik Lin; Park, Sue Nie.

In: Journal of Bacteriology and Virology, Vol. 36, No. 4, 01.01.2006, p. 271-278.

Research output: Contribution to journalArticle

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