J Bacteriol Virol.  2006 Dec;36(4):271-278. 10.4167/jbv.2006.36.4.271.

Detection and Quantification of Residual Cellular DNA in the Production of Recombinant HPV-16 L1 Virus-Like Particles

• Son HK
• Jeong HS
• Kim YL
• Kim SN
• Shin JH
• Min HK
• Seong BL
• Park SN

• KMID: 2055036
• DOI: http://doi.org/10.4167/jbv.2006.36.4.271

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 microgram 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.

Keyword

HPV-16 L1 VLPs; Residual cellular DNA; Biopharmaceuticals

MeSH Terms

Centrifugation
Deoxyribonucleases
Detergents
Digoxigenin
DNA Probes
DNA*
Human papillomavirus 16*
Insects
Limit of Detection
Mycoplasma
Recombinant Proteins
Risk Factors
Sonication
Spodoptera
Sucrose
DNA
DNA Probes
Deoxyribonucleases
Detergents
Digoxigenin
Recombinant Proteins
Sucrose

Figure

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  Figure 1. Flowchart for the production of HPV-16 L1 VLPs. Each step was individually assessed for the ability to remove residual cellular DNA.

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  Figure 2. Immunoblot analysis of HPV-16 type L1 VLPs in each fraction after CsCl equilibrium density centrifugation. The fractions containing VLPs were identified by immunoblot analysis using HPV-16 L1 monoclonal antibody, CamVir-1 and anti-mouse IgG alkaline phosphatase.

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  Figure 3. Identification of HPV-16 L1 VLPs by electron micrograph. To confirm VLPs assembled, each preparation was applied to carbon-coated grids with an equal volume of VLPs suspension. After drying, the carbon-coated grids containing VLP were negatively stained with 2% uranyl acetate, and were observed at × 50,000 or × 60,000 electron microscope (JEOL.JP/JEM-1010, Japan). (A) CsCl ultracentrifuged sample; (B) DNase treated sample; and (C) final product. Arrows indicate examples of assembled HPV VLPs. Assembled VLPs were shown in all three samples (A, B, C).

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  Figure 4. Detection limit of residual cellular DNA. The limit of detection of the hybridization assay was evaluated at 0.1 ng with Random primed labeling kit. The figure shows 1000, 500, 100, 50, 10, 5, 1, 0.5, and 0.1 ng of targeted host cell derived-DNA (Sf9) spotted onto nylon membrane and assayed as described in materials and methods section. Detection limit of residual cellular DNA was 0.1 ng in this assay.

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  Figure 5. Reproducibility and repeatability studies of hybridization assay. In order to validate the detection limit at 0.1 ng of random primed labeling DNA using the host-derived DNA, the reproducibility test was carried out on three different days. For the repeatability test, the DNA of 0.1 ng was blotted four times onto two separate membranes.

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  Figure 6. Dot-blot hybridization assay for in process samples and final product. Compared to the standard DNA, the amount of residual cellular DNA in each process sample during purification process and in final product was determined (the amount of residual DNA in final product was 0.5 ng∼1 ng per 100 μg of protein). Vero 9013 cells and BHK-21 cells were used for controls of specificity test in the hybridization assay.

Reference

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